Larsen, Jens Erik Pontoppidan; Lund, Ole; Nielsen, Morten
Background B-cell epitopes are the sites of molecules that are recognized by antibodies of the immune system. Knowledge of B-cell epitopes may be used in the design of vaccines and diagnostics tests. It is therefore of interest to develop improved methods for predicting B-cell epitopes. In this paper, we describe an improved method for predicting linear B-cell epitopes. Results In order to do this, three data sets of linear B-cell epitope annotated proteins were constructed. A data set was co...
EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant
The identification and characterization of B-cell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting linear B-cell epitopes are highly desirable. We evaluated Support Vector Machine (SVM) classifiers trained utilizing five different kernel methods using fivefold cross-validation on a homology-reduced data set of 701 linear B-cell epitopes, extracted from Bcipep database, and 701 non-epitopes, randomly extracted from SwissProt sequences. Based on the results of our computational experiments, we propose BCPred, a novel method for predicting linear B-cell epitopes using the subsequence kernel. We show that the predictive performance of BCPred (AUC = 0.758) outperforms 11 SVM-based classifiers developed and evaluated in our experiments as well as our implementation of AAP (AUC = 0.7), a recently proposed method for predicting linear B-cell epitopes using amino acid pair antigenicity. Furthermore, we compared BCPred with AAP and ABCPred, a method that uses recurrent neural networks, using two data sets of unique B-cell epitopes that had been previously used to evaluate ABCPred. Analysis of the data sets used and the results of this comparison show that conclusions about the relative performance of different B-cell epitope prediction methods drawn on the basis of experiments using data sets of unique B-cell epitopes are likely to yield overly optimistic estimates of performance of evaluated methods. This argues for the use of carefully homology-reduced data sets in comparing B-cell epitope prediction methods to avoid misleading conclusions about how different methods compare to each other. Our homology-reduced data set and implementations of BCPred as well as the APP method are publicly available through our web-based server, BCPREDS, at: http://ailab.cs.iastate.edu/bcpreds/. PMID:18496882
Identification of epitopes that invoke strong responses from B-cells is one of the key steps in designing effective vaccines against pathogens. Because experimental determination of epitopes is expensive in terms of cost, time, and effort involved, there is an urgent need for computational methods for reliable identification of B-cell epitopes. Although several computational tools for predicting B-cell epitopes have become available in recent years, the predictive performance of existing tools remains far from ideal. We review recent advances in computational methods for B-cell epitope prediction, identify some gaps in the current state of the art, and outline some promising directions for improving the reliability of such methods. PMID:21067544
Jespersen, Martin Closter; Peters, Bjoern; Nielsen, Morten
for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from......Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex...... and immunology community....
Lian, Yao; Ge, Meng; Pan, Xian-Ming
B-cell epitopes have been studied extensively due to their immunological applications, such as peptide-based vaccine development, antibody production, and disease diagnosis and therapy. Despite several decades of research, the accurate prediction of linear B-cell epitopes has remained a challenging task. In this work, based on the antigen's primary sequence information, a novel linear B-cell epitope prediction model was developed using the multiple linear regression (MLR). A 10-fold cross-validation test on a large non-redundant dataset was performed to evaluate the performance of our model. To alleviate the problem caused by the noise of negative dataset, 300 experiments utilizing 300 sub-datasets were performed. We achieved overall sensitivity of 81.8%, precision of 64.1% and area under the receiver operating characteristic curve (AUC) of 0.728. We have presented a reliable method for the identification of linear B cell epitope using antigen's primary sequence information. Moreover, a web server EPMLR has been developed for linear B-cell epitope prediction: http://www.bioinfo.tsinghua.edu.cn/epitope/EPMLR/ .
Kringelum, Jens Vindahl; Lundegaard, Claus; Lund, Ole
evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version...... biomedical applications such as; rational vaccine design, development of disease diagnostics and immunotherapeutics. However, experimental mapping of epitopes is resource intensive making in silico methods an appealing complementary approach. To date, the reported performance of methods for in silico mapping...... of B-cell epitopes has been moderate. Several issues regarding the evaluation data sets may however have led to the performance values being underestimated: Rarely, all potential epitopes have been mapped on an antigen, and antibodies are generally raised against the antigen in a given biological...
Greenbaum, Jason A.; Andersen, Pernille; Blythe, Martin
of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools. (c) 2007 John Wiley & Sons, Ltd....
Full Text Available The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371. Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224 and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.
Background The incomplete ground truth of training data of B-cell epitopes is a demanding issue in computational epitope prediction. The challenge is that only a small fraction of the surface residues of an antigen are confirmed as antigenic residues (positive training data); the remaining residues are unlabeled. As some of these uncertain residues can possibly be grouped to form novel but currently unknown epitopes, it is misguided to unanimously classify all the unlabeled residues as negative training data following the traditional supervised learning scheme. Results We propose a positive-unlabeled learning algorithm to address this problem. The key idea is to distinguish between epitope-likely residues and reliable negative residues in unlabeled data. The method has two steps: (1) identify reliable negative residues using a weighted SVM with a high recall; and (2) construct a classification model on the positive residues and the reliable negative residues. Complex-based 10-fold cross-validation was conducted to show that this method outperforms those commonly used predictors DiscoTope 2.0, ElliPro and SEPPA 2.0 in every aspect. We conducted four case studies, in which the approach was tested on antigens of West Nile virus, dihydrofolate reductase, beta-lactamase, and two Ebola antigens whose epitopes are currently unknown. All the results were assessed on a newly-established data set of antigen structures not bound by antibodies, instead of on antibody-bound antigen structures. These bound structures may contain unfair binding information such as bound-state B-factors and protrusion index which could exaggerate the epitope prediction performance. Source codes are available on request. PMID:26681157
Full Text Available Abstract Background Antigen-antibody interactions are key events in immune system, which provide important clues to the immune processes and responses. In Antigen-antibody interactions, the specific sites on the antigens that are directly bound by the B-cell produced antibodies are well known as B-cell epitopes. The identification of epitopes is a hot topic in bioinformatics because of their potential use in the epitope-based drug design. Although most B-cell epitopes are discontinuous (or conformational, insufficient effort has been put into the conformational epitope prediction, and the performance of existing methods is far from satisfaction. Results In order to develop the high-accuracy model, we focus on some possible aspects concerning the prediction performance, including the impact of interior residues, different contributions of adjacent residues, and the imbalanced data which contain much more non-epitope residues than epitope residues. In order to address above issues, we take following strategies. Firstly, a concept of 'thick surface patch' instead of 'surface patch' is introduced to describe the local spatial context of each surface residue, which considers the impact of interior residue. The comparison between the thick surface patch and the surface patch shows that interior residues contribute to the recognition of epitopes. Secondly, statistical significance of the distance distribution difference between non-epitope patches and epitope patches is observed, thus an adjacent residue distance feature is presented, which reflects the unequal contributions of adjacent residues to the location of binding sites. Thirdly, a bootstrapping and voting procedure is adopted to deal with the imbalanced dataset. Based on the above ideas, we propose a new method to identify the B-cell conformational epitopes from 3D structures by combining conventional features and the proposed feature, and the random forest (RF algorithm is used as the
Salvador Eugenio C. Caoili
Full Text Available To better support the design of peptide-based vaccines, refinement of methods to predict B-cell epitopes necessitates meaningful benchmarking against empirical data on the cross-reactivity of polyclonal antipeptide antibodies with proteins, such that the positive data reflect functionally relevant cross-reactivity (which is consistent with antibody-mediated change in protein function and the negative data reflect genuine absence of cross-reactivity (rather than apparent absence of cross-reactivity due to artifactual masking of B-cell epitopes in immunoassays. These data are heterogeneous in view of multiple factors that complicate B-cell epitope prediction, notably physicochemical factors that define key structural differences between immunizing peptides and their cognate proteins (e.g., unmatched electrical charges along the peptide-protein sequence alignments. If the data are partitioned with respect to these factors, iterative parallel benchmarking against the resulting subsets of data provides a basis for systematically identifying and addressing the limitations of methods for B-cell epitope prediction as applied to vaccine design.
Huang, Yan Xin; Bao, Yong Li; Guo, Shu Yan; Wang, Yan; Zhou, Chun Guang; Li, Yu Xin
The prediction of conformational B-cell epitopes is one of the most important goals in immunoinformatics. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues of interaction between an antigen and an antibody. Consequently, this area of research has received considerable attention from immunologists, structural biologists and computational biologists. Phage-displayed random peptide libraries are powerful tools used to obtain mimotopes that are selected by binding to a given monoclonal antibody (mAb) in a similar way to the native epitope. These mimotopes can be considered as functional epitope mimics. Mimotope analysis based methods can predict not only linear but also conformational epitopes and this has been the focus of much research in recent years. Though some algorithms based on mimotope analysis have been proposed, the precise localization of the interaction site mimicked by the mimotopes is still a challenging task. In this study, we propose a method for B-cell epitope prediction based on mimotope analysis called Pep-3D-Search. Given the 3D structure of an antigen and a set of mimotopes (or a motif sequence derived from the set of mimotopes), Pep-3D-Search can be used in two modes: mimotope or motif. To evaluate the performance of Pep-3D-Search to predict epitopes from a set of mimotopes, 10 epitopes defined by crystallography were compared with the predicted results from a Pep-3D-Search: the average Matthews correlation coefficient (MCC), sensitivity and precision were 0.1758, 0.3642 and 0.6948. Compared with other available prediction algorithms, Pep-3D-Search showed comparable MCC, specificity and precision, and could provide novel, rational results. To verify the capability of Pep-3D-Search to align a motif sequence to a 3D structure for predicting epitopes, 6 test cases were used. The predictive performance of Pep-3D-Search was demonstrated to be superior to that of other similar programs
Janahi, Essam Mohammed; Dhasmana, Anupam; Srivastava, Vandana; Sarangi, Aditya Narayan; Raza, Sana; Arif, Jamal M; Bhatt, Madan Lal Bramha; Lohani, Mohtashim; Areeshi, Mohammed Yahya; Saxena, Anand Murari; Haque, Shafiul
Zika virus (ZIKV) is a mosquito-borne flavivirus distributed all over Africa, South America and Asia. The infection with the virus may cause acute febrile sickness that clinically resembles dengue fever, yet there is no vaccine, no satisfactory treatment, and no means of evaluating the risk of the disease or prognosis in the infected people. In the present study, the efficacy of the host's immune response in reducing the risk of infectious diseases was taken into account to carry out immuno-informatics driven epitope screening strategy of vaccine candidates against ZIKV. In this study, HLA distribution analysis was done to ensure the coverage of the vast majority of the population. Systematic screening of effective dominant immunogens was done with the help of Immune Epitope & ABCPred databases. The outcomes suggested that the predicted epitopes may be protective immunogens with highly conserved sequences and bear potential to induce both protective neutralizing antibodies, T & B cell responses. A total of 25 CD4+ and 16 CD8+ peptides were screened for T-cell mediated immunity. The predicted epitope "TGLDFSDLYYLTMNNKHWLV" was selected as a highly immunogenic epitope for humoral immunity. These peptides were further screened as non-toxic, immunogenic and non-mutated residues of envelop viral protein. The predicted epitope could work as suitable candidate(s) for peptide based vaccine development. Further, experimental validation of these epitopes is warranted to ensure the potential of B- and T-cells stimulation for their efficient use as vaccine candidates, and as diagnostic agents against ZIKV.
May 1, 2012 ... Outer membrane protein U (OmpU), an adhesion protein of Vibrio mimicus, is a good antigen, but its epitopes are still unclear. In order to locate the epitopes of OmpU protein, epitope prediction was performed using the amino acid sequence of OmpU protein of V. mimicus HX4 strain that was isolated.
Outer membrane protein U (OmpU), an adhesion protein of Vibrio mimicus, is a good antigen, but its epitopes are still unclear. In order to locate the epitopes of OmpU protein, epitope prediction was performed using the amino acid sequence of OmpU protein of V. mimicus HX4 strain that was isolated from the diseased ...
Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.
Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara
Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.
An, Mengting; Zhang, Fengbo; Zhu, Yuejie; Zhao, Xiao; Ding, Jianbing
Cystic echinococcosis, as a zoonosis, seriously endangers humans and animals, so early diagnosis of this disease is particularly important. Therefore, this study is to predict B-cell epitopes of EgAgB1 and EgAgB3 proteins by bioinformatics software. B-cell epitopes of EgAgB1 and EgAgB3 proteins are predicted using DNAStar and IEDB software. The results suggest that there are two potential B-cell epitopes in EgAgB1, which located in the 8-15 and 31-37 amino acid residue segments. And two potential B-cell epitopes in EgAgB2, located in the 20∼27 and 47∼53 amino acid residue segments. This study predicted the B-cell epitopes of EgAgB1 and EgAgB3 proteins, which laid the foundation for the early diagnosis of Cystic echinococcosis.
Full Text Available The prediction of conformational b-cell epitopes plays an important role in immunoinformatics. Several computational methods are proposed on the basis of discrimination determined by the solvent-accessible surface between epitopes and non-epitopes, but the performance of existing methods is far from satisfying. In this paper, depth functions and the k-th surface convex hull are used to analyze epitopes and exposed non-epitopes. On each layer of the protein, we compute relative solvent accessibility and four different types of depth functions, i.e., Chakravarty depth, DPX, half-sphere exposure and half space depth, to analyze the location of epitopes on different layers of the proteins. We found that conformational b-cell epitopes are rich in charged residues Asp, Glu, Lys, Arg, His; aliphatic residues Gly, Pro; non-charged residues Asn, Gln; and aromatic residue Tyr. Conformational b-cell epitopes are rich in coils. Conservation of epitopes is not significantly lower than that of exposed non-epitopes. The average depths (obtained by four methods for epitopes are significantly lower than that of non-epitopes on the surface using the Wilcoxon rank sum test. Epitopes are more likely to be located in the outer layer of the convex hull of a protein. On the benchmark dataset, the cumulate 10th convex hull covers 84.6% of exposed residues on the protein surface area, and nearly 95% of epitope sites. These findings may be helpful in building a predictor for epitopes.
The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...
Abstract. The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepa- titis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology model- ling and ...
on machine learning techniques. Several MHC class I binding prediction algorithms have been developed and due to their high accuracy they are used by many immunologists to facilitate the conventional experimental process of epitope discovery. However, the accuracy of these methods depends on data defining...... the NetMHCIIpan-3.0 predictor based on artificial neural networks, which is capable of giving binding affinities to any human MHC class II molecule. Chapter 4 of this thesis gives an overview of bioinformatics tools developed by the Immunological Bioinformatics group at Center for Biological Sequence...
Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara
Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction app...
Madrigal, M; Alape-Girón, A; Barboza-Arguedas, E; Aguilar-Ulloa, W; Flores-Díaz, M
Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein-kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms. Copyright © 2017 Elsevier Ltd. All rights reserved.
Gangwar, Roopesh Singh; Shil, Pratip; Sapkal, Gajanan N; Khan, Siraj A; Gore, Milind M
West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines. Copyright © 2011 Elsevier B.V. All rights reserved.
Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins
Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006
Yu, Tian-Fei; Ma, Bo; Wang, Jun-Wei
Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. Copyright © 2016. Published by Elsevier B.V.
Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.
Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong
Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.
Full Text Available BACKGROUND: Human bocavirus species 1-4 (HBoV1-4 have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs. METHODOLOGY/PRINCIPAL FINDINGS: We generated HBoV1-3 VP2 gene fragment phage display libraries (GFPDLs and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides--P1 (¹MSDTDIQDQQPDTVDAPQNT²⁰, and P2 (¹⁶²EHAYPNASHPWDEDVMPDL¹⁸⁰--that are conserved among HBoV1-4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1-4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4. Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide. CONCLUSIONS/SIGNIFICANCE: The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools.
Chenzhang, Yuwei; Wen, Qiang; Ding, Xianping; Cao, Man; Chen, Zuyi; Mu, Xuemei; Wang, Tao
Cervical cancers almost are infected by human papillmavirus (HPV), encoding E6 and E7 oncoproteins which are regard as ideal targets on the mechanism of this disease and development of vaccines. HLA (human leukocyte antigen) participates in the local immune response to prevent tumor invasion and progression. But due to highly polymorphism of HLA, prediction shows its importance in this study. More effective immunoinformatics was used for predicting epitopes from HPV-16 E6 and E7, including T- and B-cell epitopes. Eight substitutions are detected. Specifically speaking, for HLA-I, HLA-A*33:03 (26), HLA-B*13:01 (14), HLA-C*03:02 (5) for E6 and HLA-A*02:01 (6), HLA-B*40:01 (5), HLA-C*03:04 (4) for E7 are most frequency. Epitope 41-48 EVYDFAFR for HLA-A*33:03 (0.1) for E6 has best binding affinity, as well as HLA*02:01 and HLA-B*40:01 (0.2) for E7. The mutations of D25E and L83V of E6 and N29S of E7 produce new epitopes, and the percentile values change with them. For HLA-II, seventeen epitopes in the reference at percentile value from 0.22 to 4.76, while in variant from 0.22 to 4.96. For the B-cell epitopes, three most potent epitopes for E6 were listed, and N29S lead the growth of score from 0.81 to 0.83. In summary, E6 40-55 REVYDFAFRDLCIVYR and E7 11-22 YMLDLQPETTDL are the important regions, containing the majority of predicted epitopes. E6 72-83 for HLA-A*02:01 and E6 74-84 for HLA-B*15:02 maybe are the new direct for therapeutic vaccine aimed at L83V variants. HLA-DRB1*15:02 is better binder with T cell in our HLA class II. It is a systematic, detail recognition for T- and B-cell epitopes of HPV-16 E6 and E7 from Southwest China, which may be helpful to design vaccines specifically for women in Southwest China and testing methods specifically for this region. The results of our study may contribute to future researches on vaccines improvement, or screening methods for a particular population. Copyright © 2016 European Federation of Immunological Societies
Full Text Available The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60, is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft, the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.
Full Text Available Abstract Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV. Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865 and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716, were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine than that of mono-epitope peptide(rE2-a or rE2-b. Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.
Yu Hua; Jiang Lifang; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo
Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed
Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong
Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.
Conclusion: The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.
Full Text Available The autoantigen of anti-glomerular basement membrane (GBM disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IVNC1. Our previous study revealed a peptide on α3(IVNC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150. This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA.We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001. P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001 of antibody reaction after each residue replacement.We defined GFxF (α3142, 143,145 as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.
Nixon, Christina E; Park, Sangshin; Pond-Tor, Sunthorn; Raj, Dipak; Lambert, Lynn E; Orr-Gonzalez, Sachy; Barnafo, Emma K; Rausch, Kelly M; Friedman, Jennifer F; Fried, Michal; Duffy, Patrick E; Kurtis, Jonathan D
Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes. Copyright © 2017 American Society for Microbiology.
Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.
We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11...... high antibody titers against the ORF4 ES, In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES, This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library......-term viremic pigs towards some ES, The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed....
DeSheng, Kong; HuaiRan, Liu; JiaSen, Liu; Zuo, Yu; Qian, Jiang; DongChun, Guo; XiaoLiang, Hu; FengJie, Wang; QianQian, Huang; LianDong, Qu
The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.
da Silva, Andréa N M Rangel; Nascimento, Eduardo J M; Cordeiro, Marli Tenório; Gil, Laura H V G; Abath, Frederico G C; Montenegro, Silvia M L; Marques, Ernesto T A
Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65), 27 and 28 (aa131-150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.
Sharma, P; Gaur, S N; Arora, N
Osmotin, a protein from the pathogenesis-related family (PR-5), has been identified as an allergen based on in-silico and in-vitro studies. In the present study, three B cell epitopes of osmotin with single and double amino acid modifications were studied for immunotherapy in a murine model. The single-modification peptides (P-1-1, P-2-1 and P-3-1) and double-modification peptides (P-1-2, P-2-2 and P-3-2) showed significantly lower immunoglobulin (Ig)E binding with patients' sera compared to osmotin (P mice. The sera of mice group treated with peptides showed a significant increase in IgG2a level and a significant decrease in IgE and IgG1 levels (P mice that received peptide immunotherapy showed a shift from a T helper type 2 (Th2) to Th1 type where interferon (IFN)-γ and interleukin (IL)-10 levels were elevated, with a significant increase in groups treated with peptides P-3-1 and P-3-2 (P bronchoalveolar lavage fluid (BALF) in the peptide-treated mice groups. Total cell count and eosinophil count in BALF of the peptide-treated groups was also reduced compared to the phosphate-buffered saline (PBS)-treated group. Lung histology showed a significant reduction in cellular infiltrate in mice treated with P-2-2 and P-3-2 compared to PBS. In conclusion, peptides P-2-2 and P-3-2 lowered inflammatory responses and induced a Th1 response in mice. © 2014 British Society for Immunology.
Ghochikyan, Anahit; Petrushina, Irina; Davtyan, Hayk; Hovakimyan, Armine; Saing, Tommy; Davtyan, Arpine; Cribbs, David H; Agadjanyan, Michael G
Immunotherapeutic approaches reducing α-synuclein deposits may provide therapeutic benefit for Dementia with Lewy Bodies (DLB). Immunization with full-length human α-synuclein (hα-Syn) protein in a Parkinson's disease mouse model decreased the accumulation of the aggregated forms of this protein in neurons and reduced neurodegeneration. To enhance the immunogenicity of candidate vaccines and to avoid the risk of autoreactive anti-hα-Syn T-helper (Th) cell responses, we generated three peptide-based epitope vaccines composed of different B-cell epitopes of hα-Syn fused with a "non-self" Th epitope from tetanus toxin (P30). Immunization of mice with these epitope vaccines produced high titers of anti-hα-Syn antibodies that bound to Lewy bodies (LBs) and Lewy neurites (LNs) in brain tissue from DLB cases and induced robust Th cell responses to P30, but not to hα-Syn. Further development of these first generation epitope vaccines may facilitate induction of anti-hα-Syn immunotherapy without producing potentially harmful autoreactive Th cell responses. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)
textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific
Sabrina de Almeida Lima
Full Text Available Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1 (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV and two for hyaluronidase (LiHYAL (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA of sphingomyelinase D (SMase D SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox. We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.
MISAKI, Y; YAMAMOTO, K; YANAGI, K; MIURA, H; ICHIJO, H; KATO, T; MATO, T; WELLINGWESTER, S; NISHIOKA, K; ITO, K
The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the
Wu, Han-Chung; Huang, Yue-Ling; Chao, Ting-Ting; Jan, Jia-Tsrong; Huang, Jau-Ling; Chiang, Hsien-Yuan; King, Chwan-Chuen; Shaio, Men-Fang
Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among th...
Sun, Jing; Kudahl, Ulrich J.; Simon, Christian
of tens of thousands of HA sequences. The detailed description of B-cell epitopes, measurement of epitope area similarity among different strains, and estimation of antibody neutralizing coverage provide insights into cross-reactivity status of existing nAbs against influenza virus. We have developed...... that share 100% identity with experimentally verified neutralized strains. By cataloging influenza strains and their B-cell epitopes for known bnAbs, our method provides guidance for selection of representative strains for further experimental design. The knowledge of sequences, their B-cell epitopes......Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylactic...
Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta
Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.
Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.
Abbott, Jeffrey R; Palmer, Guy H; Howard, Chris J; Hope, Jayne C; Brown, Wendy C
Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.
U.S. Department of Health & Human Services — This repository contains antibody/B cell and T cell epitope information and epitope prediction and analysis tools for use by the research community worldwide. Immune...
Full Text Available Abstract Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.
Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C
The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production. Copyright © 2014 Elsevier Ltd. All rights reserved.
Zhao, Yingdong; Sung, Myong-Hee; Simon, Richard
Identifying epitopes that elicit a major histocompatibility complex (MHC)-restricted T-cell response is critical for designing vaccines for infectious diseases and cancers. We have applied two artificial intelligence approaches to build models for predicting T-cell epitopes. We developed a support vector machine to predict T-cell epitopes for an MHC class I-restricted T-cell clone (TCC) using synthesized peptide data. For predicting T-cell epitopes for an MHC class II-restricted TCC, we built a shift model that integrated MHC-binding data and data from T-cell proliferation assay against a combinatorial library of peptide mixtures.
Full Text Available The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease.
Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer. Copyright © 2012 Elsevier Ltd. All rights reserved.
Full Text Available Abstract Background The West Nile virus (WNV nonstructural protein 1 (NS1 is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934. Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.
Wang, Fen; Ye, Bin
Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.
Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O.; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen
Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR. PMID:24244326
Full Text Available Epitope-antibody-reactivities (EAR of intravenous immunoglobulins (IVIGs determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM analysis. Machine learning slightly outperformed PWM with area under the curve (AUC of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.
Song, Yanhua; Wang, Fang; Fan, Zhiyu; Hu, Bo; Liu, Xing; Wei, Houjun; Xue, Jiabin; Xu, Weizhong; Qiu, Rulong
Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.
Xiao, Ning; Cao, Ji; Zhou, Hao; Ding, Shu-Quan; Kong, Ling-Yan; Li, Jin-Nian
Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish. Copyright © 2016 Elsevier B.V. All rights reserved.
Wu, Han-Chung; Jung, Mei-Ying; Chiu, Chien-Yu; Chao, Ting-Ting; Lai, Szu-Chia; Jan, Jia-Tsrong; Shaio, Men-Fang
In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.
Menezes-Souza, Daniel; de Oliveira Mendes, Tiago Antônio; de Araújo Leão, Ana Carolina; de Souza Gomes, Matheus; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella Castanheira
The correct and early identification of humans and dogs infected with Leishmania are key steps in the control of leishmaniasis. Additionally, a method with high sensitivity and specificity at low cost that allows the screening of a large number of samples would be extremely valuable. In this study, we analyzed the potential of mitogen-activated protein kinase 3 (MAPK3) and mitogen-activated protein kinase 4 (MAPK4) proteins from Leishmania braziliensis to serve as antigen candidates for the serodiagnosis of human visceral and tegumentary leishmaniasis, as well as canine visceral disease. Moreover, we mapped linear B-cell epitopes in these proteins and selected those epitopes with sequences that were divergent in the corresponding orthologs in Homo sapiens, in Canis familiaris, and in Trypanosoma cruzi. We compared the performance of these peptides with the recombinant protein using ELISA. Both MAPK3 and MAPK4 recombinant proteins showed better specificity in the immunodiagnosis of human and canine leishmaniasis than soluble parasite antigens and the EIE-leishmaniose-visceral-canina-bio-manguinhos (EIE-LVC) kit. Furthermore, the performance of this serodiagnosis assay was improved using synthetic peptides corresponding to B-cell epitopes derived from both proteins.
Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang
The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide...
Full Text Available Abstract Background The major structural protein of coronaviruses, the membrane (M protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV. Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein. Results One hybridoma cell line secreting anti-M protein monoclonal antibody (McAb was generated and designated 4D4. To map the epitopes on the PEDV M protein, a total of 17 partially overlapping fragments covering the C-terminus of M protein were expressed as fusion proteins with a 6×His tag or a GST tag. A linear motif, 193TGWAFYVR200, was identified by enzyme-linked immunosorbent assay (ELISA and western blot (WB analysis using McAb 4D4. The motif 195WAFYVR200 was the minimal requirement for reactivity, as demonstrated by removing amino acids individually from both ends of the motif 193TGWAFYVR200. The result of WB analysis showed that the 4D4-defined epitope could be recognized by PEDV-positive serum, but not transmissible gastroenteritis virus (TGEV-positive serum. Furthermore, this epitope was highly conserved among different PEDV strains, as shown by alignment and comparison of sequences. Conclusion A McAb, 4D4, directed against the M protein of PEDV, was obtained, and the 4D4-defined minimal epitope sequence was 195WAFYVR200. The McAb could serve as a candidate for development of a McAb-based antigen capture ELISA for detection of PEDV. The epitope identified provides a basis for the development of epitope-based differential diagnostic techniques and may be useful in the design of epitope-based vaccines.
Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Tong, Mingwei; Wang, Jianke; Zhao, Hang; Lin, Peng; Cheng, Shipeng
Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses. Copyright © 2015 Elsevier B.V. All rights reserved.
Wheatley, Adam K; Whittle, James R R; Lingwood, Daniel; Kanekiyo, Masaru; Yassine, Hadi M; Ma, Steven S; Narpala, Sandeep R; Prabhakaran, Madhu S; Matus-Nicodemos, Rodrigo A; Bailer, Robert T; Nabel, Gary J; Graham, Barney S; Ledgerwood, Julie E; Koup, Richard A; McDermott, Adrian B
Because of significant viral diversity, vaccines that elicit durable and broad protection against influenza have been elusive. Recent research has focused on the potential of highly conserved regions of the viral hemagglutinin (HA) as targets for broadly neutralizing Ab responses. Abs that bind the highly conserved stem or stalk of HA can be elicited by vaccination in humans and animal models and neutralize diverse influenza strains. However, the frequency and phenotype of HA stem-specific B cells in vivo remain unclear. In this article, we characterize HA stem-specific B cell responses following H5N1 vaccination and describe the re-expansion of a pre-existing population of memory B cells specific for stem epitopes. This population uses primarily, but not exclusively, IGHV1-69-based Igs for HA recognition. However, within some subjects, allelic polymorphism at the ighv1-69 locus can limit IGHV1-69 immunodominance and may reduce circulating frequencies of stem-reactive B cells in vivo. The accurate definition of allelic selection, recombination requirements, and ontogeny of neutralizing Ab responses to influenza will aid rational influenza vaccine design. Copyright © 2015 by The American Association of Immunologists, Inc.
Ditlev, Sisse B; Nielsen, Morten A; Resende, Mafalda
capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope...
Klatser, P. R.; de Wit, M. Y.; Kolk, A. H.; Hartskeerl, R. A.
Using synthetic peptides representing overlapping sequences of the 100-amino-acid-long N-terminal region of the proline-rich antigen of Mycobacterium leprae (PRA), we have mapped the epitopes in the primary structure of PRA recognized by four monoclonal antibodies. The M. leprae-specific monoclonal
Full Text Available The impact of epidemic Staphylococcus aureus (S. aureus on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of S. aureus is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of S. aureus, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response in vivo. However, the B-cell epitopes of S. aureus GapC have not been well identified. Here we used the bioinformatics tools to analyze the sequence of GapC, and we generated protective anti-GapC monoclonal antibodies (mAbs. A protective mAb (1F4 showed strong specificity to GapC and the ability to induce macrophages to phagocytose S. aureus. We screened the motif 272GYTEDEIVSSD282, which was recognized by mAb 1F4, using a phage display system. Then, we used site-directed mutagenesis to identify key amino acids in the motif. Residues G272 D276 E277 I278 and V279 formed the core of the 272GYTEDEIVSSD282 motif. In addition, we showed that this epitope peptide induced a protective humoral immune response against S. aureus infection in immunized mice. Our results will be useful for the further study of epitope-based vaccines against S. aureus infection.
Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio
The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis. We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis. The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.
Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.
Andréa N M Rangel da Silva; Eduardo J M Nascimento; Marli Tenório Cordeiro; Laura H V G Gil; Frederico G C Abath; Silvia M L Montenegro; Ernesto T A Marques
Background Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. Met...
Nielsen, Morten; Lund, Ole; Buus, S
in the predictions. All attempts to make ab initio predictions based on protein structure have failed to reach predictive performances similar to those that can be obtained by data-driven methods. Thousands of different MHC-II alleles exist in humans. Recently developed pan-specific methods have been able to make...... reasonably accurate predictions for alleles that were not included in the training data. These methods can be used to define supertypes (clusters) of MHC-II alleles where alleles within each supertype have similar binding specificities. Furthermore, the pan-specific methods have been used to make a graphical...
Garza-Cuartero, Laura; Geurden, Thomas; Mahan, Suman M; Hardham, John M; Dalton, John P; Mulcahy, Grace
Fasciola hepatica infection causes important economic losses in livestock and food industries around the world. In the Republic of Ireland F. hepatica infection has an 76% prevalence in cattle. Due to the increase of anti-helminthic resistance, a vaccine-based approach to control of Fasciolosis is urgently needed. A recombinant version of the cysteine protease cathepsin L1 (rmFhCL1) from F. hepatica has been a vaccine candidate for many years. We have found that vaccination of cattle with this immunodominant antigen has provided protection against infection in some experimental trials, but not in others. Differential epitope recognition between animals could be a source of variable levels of vaccine protection. Therefore, we have characterised for first time linear B-cell epitopes recognised within the FhCL1 protein using sera from F. hepatica-infected and/or vaccinated cattle from two independent trials. Results showed that all F. hepatica infected animals recognised the region 19-31 of FhCL1, which is situated in the N-terminal part of the pro-peptide. Vaccinated animals that showed fluke burden reduction elicited antibodies that bound to the regions 120-137, 145-155, 161-171 of FhCL1, which were not recognised by non-protected animals. This data, together with the high production of specific IgG2 in animals showing vaccine efficacy, suggest important targets for vaccine development. Copyright © 2018 Elsevier Ltd. All rights reserved.
Williams, Kristina M.; Bigley, Elmer C.; Raybourne, Richard B.
The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on
Rune A. Høglund
Full Text Available Memory B cells acting as antigen-presenting cells are believed to be important in multiple sclerosis (MS, but the antigen they present remains unknown. We hypothesized that B cells may activate CD4+ T cells in the central nervous system of MS patients by presenting idiotopes from their own immunoglobulin variable regions on human leukocyte antigen (HLA class II molecules. Here, we use bioinformatics prediction analysis of B cell immunoglobulin variable regions from 11 MS patients and 6 controls with other inflammatory neurological disorders (OINDs, to assess whether the prerequisites for such idiotope-driven T–B cell collaboration are present. Our findings indicate that idiotopes from the complementarity determining region (CDR 3 of MS patients on average have high predicted affinities for disease associated HLA-DRB1*15:01 molecules and are predicted to be endosomally processed by cathepsin S and L in positions that allows such HLA binding to occur. Additionally, complementarity determining region 3 sequences from cerebrospinal fluid (CSF B cells from MS patients contain on average more rare T cell-exposed motifs that could potentially escape tolerance and stimulate CD4+ T cells than CSF B cells from OIND patients. Many of these features were associated with preferential use of the IGHV4 gene family by CSF B cells from MS patients. This is the first study to combine high-throughput sequencing of patient immune repertoires with large-scale prediction analysis and provides key indicators for future in vitro and in vivo analyses.
Full Text Available Dengue fever of tropics is a mosquito transmitted devastating disease caused by dengue virus (DENV. There is no effective vaccine available, so far, against any of its four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4. There is a need for the development of preventive and therapeutic vaccines against DENV to decrease the prevalence of dengue fever, especially in Pakistan. In this research, linear and conformational B-cell epitopes of envelope glycoprotein of DENV-2 and DENV-3 (the most prevalent serotypes in Pakistan were predicted. We used Kolaskar and Tongaonkar method for linear epitope prediction, Emini's method for surface accessibility prediction and Karplus and Schulz's algorithm for flexibility determination. To propose three dimensional epitopes, the E proteins for both serotypes were homology modeled by using Phyre2 V 2.0 server, and ElliPro was used for the prediction of surface epitopes on their globular structure. Total 21 and 19 linear epitopes were predicted for DENV-2 and DENV-3 Pakistani isolates respectively. Whereas, 5 and 4 discontinuous epitopes were proposed for DENV-2 and DENV-3 Pakistani isolates respectively. Moreover, the values of surface accessibility, flexibility and solvent-accessibility can be helpful in analyzing vaccines against DENV-2 and DENV-3. In conclusion, the proposed continuous and discontinuous antigenic peptides can be valuable candidates for diagnostic and therapeutics of DENV.
Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang
A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.
Zhang, Wen; Niu, Yanqing; Zou, Hua; Luo, Longqiang; Liu, Qianchao; Wu, Weijian
T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system. In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set. Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant. The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.
Full Text Available T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system.In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve on the training set.Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant.The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.
Dec 1, 2009 ... learning techniques such as Support Vector Machine (SVM) and Artificial Neural Network (ANN). This method has been trained and tested on non-redundant dataset of T cell epitopes and non-epitopes that includes 1137 experimen- tally proven MHC class 1 restricted T cell epitopes. (Bhasin and Raghava ...
Khoshhali, Mehri; Mahjub, Hossein; Saidijam, Massoud; Poorolajal, Jalal; Soltanian, Ali Reza
The present study was conducted to predict survival time in patients with diffuse large B-cell lymphoma, DLBCL, based on microarray data using Cox regression model combined with seven dimension reduction methods. This historical cohort included 2042 gene expression measurements from 40 patients with DLBCL. In order to predict survival, a combination of Cox regression model was used with seven methods for dimension reduction or shrinkage including univariate selection, forward stepwise selecti...
Xu-Monette, Zijun Y; Li, Ling; Byrd, John C
, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37(-), including TP53 mutation, NF-κB(high), Myc(high), phosphorylated STAT3(high), survivin......(high), p63(-), and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37(-) status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R...
Full Text Available Abstract Background The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP to the endoplasmic reticulum (ER, where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM, EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.
Guo, Chunyan; Xie, Xin; Li, Huijin; Zhao, Penghua; Zhao, Xiangrong; Sun, Jingying; Wang, Haifang; Liu, Yang; Li, Yan; Hu, Qiaoxia; Hu, Jun; Li, Yuan
Influenza A virus infection is a persistent threat to public health worldwide due to hemagglutinin (HA) variation. Current vaccines against influenza A virus provide immunity to viral isolates similar to vaccine strains. Antibodies against common epitopes provide immunity to diverse influenza virus strains and protect against future pandemic influenza. Therefore, it is vital to analyze common HA antigenic epitopes of influenza virus. In this study, 14 strains of monoclonal antibodies with high sensitivity to common epitopes of influenza virus antigens identified in our previous study were selected as the tool to predict common HA epitopes. The common HA antigenic epitopes were divided into four categories by ELISA blocking experiments, and separately, into three categories according to the preliminary results of computer simulation. Comparison between the results of computer simulations and ELISA blocking experiments indicated that at least two classes of common epitopes are present in influenza virus HA. This study provides experimental data for improving the prediction of HA epitopes of influenza virus (H1 subtype) and the development of a potential universal vaccine as well as a novel approach for the prediction of epitopes on other pathogenic microorganisms. Copyright © 2014 Elsevier Inc. All rights reserved.
Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K.
BACKGROUND: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein....... of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL.All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php....
Muthusamy, Karthikeyan; Gopinath, Krishnasamy; Nandhini, Dharmalingam
Epitope-based vaccines (EVs) are specific, safe and easy to produce. However, vaccine failure has been frequently reported due to variation within epitopic regions. Therefore, development of vaccines based on conserved epitopes may prevent such vaccine failure. This study was undertaken to identify highly conserved antigenic regions in the four dengue serotypes to produce an epitope-based dengue vaccine. Polyprotein sequences of all four dengue serotypes were collected and aligned using MAFFT multiple sequence alignment plugin with Geneious Pro v6.1. Consensus sequences of the polyproteins for all four dengue serotypes were designed and screened against experimentally proven epitopes to predict potential antigenic regions that are conserved among all four dengue serotypes. The antigenic region VDRGWGNGCGLFGKG was 100 per cent conserved in the consensus polyprotein sequences of all four dengue serotypes. Fifteen experimentally proven epitopes were identical to the immunodominant antigenic region. Computationally predicted antigenic regions may be considered for use in the development of EVs for protection against dengue virus. Such vaccines would be expected to provide protection against dengue infections caused by all dengue serotypes because these would contain antigenic regions highly conserved across those serotypes. Therefore, the immunodominant antigenic region (VDRGWGNGCGLFGKG) and 15 potential epitopes may be considered for use in dengue vaccines.
Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections
Full Text Available Abstract Background Differential diagnose of Japanese encephalitis virus (JEV infection from other flavivirus especially West Nile virus (WNV and Dengue virus (DV infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. Results To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120 was detected by enzyme-linked immunosorbent assay (ELISA. By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118. This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. Conclusion Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.
Identification of a novel linear B-cell epitope using a monoclonal antibody against the carboxy terminus of the canine distemper virus nucleoprotein and sequence analysis of the identified epitope in different CDV isolates.
Yi, Li; Cao, Zhigang; Tong, Mingwei; Cheng, Yuening; Yang, Yong; Li, Shuang; Wang, Jianke; Lin, Peng; Sun, Yaru; Zhang, Miao; Cheng, Shipeng
The Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly. In this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401-523). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis.The results indicated that 444 GDKYPIHFNDER 455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence. This collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.
S K Rosendahl Huber
Full Text Available Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP, polymerase basic protein 1 (PB1 and matrix protein 1 (M1. C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.
Xu-Monette, Z.Y.; Li, L; Byrd, J.C.; Jabbar, K.J.; Manyam, G.C.; Winde, C. Maria de; Brand, M. van den; Tzankov, A.; Visco, C.; Wang, J; Dybkaer, K.; Chiu, A.; Orazi, A.; Zu, Y.; Bhagat, G.; Richards, K.L.; Hsi, E.D.; Choi, W.W.; Huh, J.; Ponzoni, M.; Ferreri, A.J.; Moller, M.B.; Parsons, B.M.; Winter, J.N.; Wang, M.; Hagemeister, F.B.; Piris, M.A.; Krieken, J.H. van; Medeiros, L.J.; Li, Y.; Spriel, A.B. van; Young, K.H.
CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its
Lundegaard, Claus; Nielsen, Morten; Lamberth, K.
The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding...... for predicting peptides binding to specific MHC class I alleles. The method combines advanced automatic scoring matrix generation with empirical position specific differential anchor weighting. The method leads to predictions with a comparable or higher accuracy than other established prediction servers, even...
de Lalla, C; Sturniolo, T; Abbruzzese, L; Hammer, J; Sidoli, A; Sinigaglia, F; Panina-Bordignon, P
Although atopic allergy affects Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.
Full Text Available Cancer subtype classification and survival prediction both relate directly to patients' specific treatment plans, making them fundamental medical issues. Although the two factors are interrelated learning problems, most studies tackle each separately. In this paper, expression levels of genes are used for both cancer subtype classification and survival prediction. We considered 350 diffuse large B-cell lymphoma (DLBCL subjects, taken from four groups of patients (activated B-cell-like subtype dead, activated B-cell-like subtype alive, germinal center B-cell-like subtype dead, and germinal center B-cell-like subtype alive. As classification features, we used 11,271 gene expression levels of each subject. The features were first ranked by mRMR (Maximum Relevance Minimum Redundancy principle and further selected by IFS (Incremental Feature Selection procedure. Thirty-five gene signatures were selected after the IFS procedure, and the patients were divided into the above mentioned four groups. These four groups were combined in different ways for subtype prediction and survival prediction, specifically, the activated versus the germinal center and the alive versus the dead. Subtype prediction accuracy of the 35-gene signature was 98.6%. We calculated cumulative survival time of high-risk group and low-risk groups by the Kaplan-Meier method. The log-rank test p-value was 5.98e-08. Our methodology provides a way to study subtype classification and survival prediction simultaneously. Our results suggest that for some diseases, especially cancer, subtype classification may be used to predict survival, and, conversely, survival prediction features may shed light on subtype features.
Jenniffer M. Mabuka
Full Text Available Immunological events in acute HIV-1 infection before peak viremia (hyperacute phase may contribute to the development of broadly cross-neutralizing antibodies. Here, we used pre-infection and acute-infection peripheral blood mononuclear cells and plasma samples from 22 women, including 10 who initiated antiretroviral treatment in Fiebig stages I–V of acute infection to study B cell subsets and B-cell associated cytokines (BAFF and CXCL13 kinetics for up to ~90 days post detection of plasma viremia. Frequencies of B cell subsets were defined by flow cytometry while plasma cytokine levels were measured by ELISA. We observed a rapid but transient increase in exhausted tissue-like memory, activated memory, and plasmablast B cells accompanied by decline in resting memory cells in untreated, but not treated women. B cell subset frequencies in untreated women positively correlated with viral loads but did not predict emergence of cross-neutralizing antibodies measured 12 months post detection of plasma viremia. Plasma BAFF and CXCL13 levels increased only in untreated women, but their levels did not correlate with viral loads. Importantly, early CXCL13 but not BAFF levels predicted the later emergence of detectable cross-neutralizing antibodies at 12 months post detection of plasma viremia. Thus, hyperacute HIV-1 infection is associated with B cell subset changes, which do not predict emergence of cross-neutralizing antibodies. However, plasma CXCL13 levels during hyperacute infection predicted the subsequent emergence of cross-neutralizing antibodies, providing a potential biomarker for the evaluation of vaccines designed to elicit cross-neutralizing activity or for natural infection studies to explore mechanisms underlying development of neutralizing antibodies.
Lundegaard, Claus; Hoof, Ilka; Lund, Ole; Nielsen, Morten
Sequence based T-cell epitope predictions have improved immensely in the last decade. From predictions of peptide binding to major histocompatibility complex molecules with moderate accuracy, limited allele coverage, and no good estimates of the other events in the antigen-processing pathway, the field has evolved significantly. Methods have now been developed that produce highly accurate binding predictions for many alleles and integrate both proteasomal cleavage and transport events. Moreov...
Larsen, Mette Voldby
: The bacteria Mycobacterium tuberculosis, Influenza A virus, HIV, Yellow fever virus, and West Nile virus. For each of the above-mentioned viruses, a number of predicted CTL epitopes was subsequently selected in such a way that they together constitute a broad coverage of the available viral strains. Part IV...
Welner, Simon; Nielsen, Morten; Lund, Ole
an effective CTL response against PRRSV, we have taken a bioinformatics approach to identify common PRRSV epitopes predicted to react broadly with predominant swine MHC (SLA) alleles. First, the genomic integrity and sequencing method was examined for 334 available complete PRRSV type 2 genomes leaving 104...... by the PopCover algorithm, providing a final list of 54 epitopes prioritized according to maximum coverage of PRRSV strains and SLA alleles. This bioinformatics approach provides a rational strategy for selecting peptides for a CTL-activating vaccine with broad coverage of both virus and swine diversity...
Full Text Available Abstract Background In our recent study, we determined the cut-off value of CD20 expression at the level of 25 000 molecules of equivalent soluble fluorochrome (MESF to be the predictor of response to rituximab containing treatment in patients with B-cell lymphomas. In 17.5% of patients, who had the level of CD20 expression below the cut-off value, the response to rituximab containing treatment was significantly worse than in the rest of the patients with the level of CD20 expression above the cut-off value. The proportion of patients with low CD20 expression who might not benefit from rituximab containing treatment was not necessarily representative. Therefore the aim of this study was to quantify the CD20 expression in a larger series of patients with B-cell lymphomas which might allow us to determine more reliably the proportion of patients with the CD20 expression below the cut-off. Methods Cytological samples of 64 diffuse large B-cell lymphomas (DLBCL, 56 follicular lymphomas (FL, 31 chronic lymphocytic leukemias (CLL, 34 mantle cell lymphomas (MCL, 18 marginal zone lymphomas (MZL and 15 B-cell lymphomas unclassified were analyzed for CD20 expression by quantitative four-color flow cytometric measurements using FACSCalibur flow cytometer (BD Biosciences. Results The range of CD20 expression in different B-cell lymphomas was very broad, varying from 2 737 to 115 623 MESF in CLL and 3 549 to 679 577 MESF in DLBCL. However, when we compared the CD20 expression in the groups of patients with DLBCL, FL, MCL, MZL, CLL and B-cell lymphomas unclassified, it was found to be significantly lower (p = 0.002 only in CLL but did not significantly differ in other lymphoma types (p = NS. Fifty-three out of 218 (24.3% patients with B-cell lymphomas had the CD20 expression below the cut-off value. Conclusions The CD20 expression in CLL is significantly lower than in most histological types of mature B-cell lymphomas in which it appears to be comparable
Lundegaard, Claus; Hoof, Ilka; Lund, Ole
Sequence based T-cell epitope predictions have improved immensely in the last decade. From predictions of peptide binding to major histocompatibility complex molecules with moderate accuracy, limited allele coverage, and no good estimates of the other events in the antigen-processing pathway......, the field has evolved significantly. Methods have now been developed that produce highly accurate binding predictions for many alleles and integrate both proteasomal cleavage and transport events. Moreover have so-called pan-specific methods been developed, which allow for prediction of peptide binding...... to MHC alleles characterized by limited or no peptide binding data. Most of the developed methods are publicly available, and have proven to be very useful as a shortcut in epitope discovery. Here, we will go through some of the history of sequence-based predictions of helper as well as cytotoxic T cell...
Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J
T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.
Stranzl, Thomas; Larsen, Mette Voldby; Lundegaard, Claus
Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates...... predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I...... molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I...
Challa, A; Pound, J D; Armitage, R J; Gordon, J
The induction of IgE synthesis in naive B cells requires two T-cell-derived signals: one delivered through CD40 and the other via interleukin-4 (IL-4). The natural counterstructure to CD40 is the CD40 ligand (CD40L). We have asked about the interplay between CD40L and CD40 mAb that recognize distinct epitopes in delivering signals for regulating IL-4-dependent IgE synthesis and the expression of CD23, the low-affinity IgE receptor, in resting B cells. After culture of purified human tonsillar B cells with CD40 agonists and IL-4, surface CD23 was determined by flow cytometric analysis. CD23 levels in cell lysates and supernatants were quantified by ELISA, as were those of secreted IgE. With regard to both induction of CD23 and IgE production, soluble CD40L trimer (sCD40LT) showed synergistic interaction with two mAb to CD40 which bind to epitopes located outside the ligand binding site (EA5 and 5C3), but not with a mAb (G28-5) which effectively competes for CD40L binding to CD40. Each of the two noncompeting mAb to CD40 was able to cooperate strongly with sCD40LT in promoting high-level induction of CD23 even in the absence of IL-4, an effect mirrored in the promotion of strong homotypic clustering and high-rate DNA synthesis. G28-5, uniquely, induced a down-regulation in IL-4-induced CD23 expression with time, a change that was accompanied by an increase in the amount of soluble CD23 detected. While the two noncompeting mAb consistently synergized with sCD40LT for the promotion of IL-4-dependent IgE synthesis, sCD40LT and G28-5 (which, by itself, was the most potent of the CD40 mAb at inducing IL-4-dependent IgE production) exhibited mutual antagonism in this regard, the level of which could be quite profound. This study demonstrates that appropriate targeting of CD40 can modulate IgE synthesis either positively or negatively.
Full Text Available Abstract Background Ebola viruses (EBOVs cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs. Results Computer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV, GPCAGDFAF and LYDRLASTV (Zaire EBOV could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma. Conclusion Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.
Full Text Available BACKGROUND: Recent reports indicate that in vitro drug screens combined with gene expression profiles (GEP of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information. MATERIALS AND METHODS: Microarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA, sparse partial least squares (SPLS and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis. PRINCIPAL FINDINGS: Both cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value. CONCLUSION: The present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to
Faisal, Ar-Rafi Md; Imtiaz, Syed Hassan; Zerin, Tasnim; Rahman, Tania; Shekhar, Hossain Uddin
Lassa virus (LASV) is an arena virus causing hemorrhagic fever and it is endemic in several regions of West Africa. The disease-causing virus records high mortality rate in endemic regions due to lack of appropriate treatment and prevention strategies. Therefore, it is of interest to design and develop viable vaccine components against the virus. We used the Lassa virus envelope glyco-proteins as a vaccine target to identify linear peptides as potential epitopes with immunogenic properties by computer aided epitope prediction tools. We report a T-cell epitope 'LLGTFTWTL' and a B-cell epitope 'AELKCFGNTAVAKCNE' with predicted potential immunogenicity for further in vivo and in vitro consideration.
Lundegaard, Claus; Lund, Ole; Nielsen, Morten
In this paper, we describe the methodologies behind three different aspects of the NetMHC family for prediction of MHC class I binding, mainly to HLAs. We have updated the prediction servers, NetMHC-3.2, NetMHCpan-2.2, and a new consensus method, NetMHCcons, which, in their previous versions, hav...
Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K
Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have al.......The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php....
Larsen, Malene Erup; Kloverpris, H.; Stryhn, A.
HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal......Traditionally, T cell epitope discovery requires considerable amounts of tedious, slow, and costly experimental work. During the last decade, prediction tools have emerged as essential tools allowing researchers to select a manageable list of epitope candidates to test from a larger peptide...
The potassium channel inhibitor alpha-KTx 3.8, a 38-residue peptide was isolated from the venom of Mesobuthus tamulus sindicus. In this assay we have predicted the binding affinity of alpha-KTx 3.8 having 38 amino acids, which shows 30 nonamers. Peptide fragments of the neurotoxin can be used to select nonamers for ...
Biccler, Jorne; Eloranta, Sandra; de Nully Brown, Peter
The international prognostic index (IPI) and similar models form the cornerstone of clinical assessment in newly diagnosed diffuse large B-cell lymphoma (DLBCL). While being simple and convenient to use, their inadequate use of the available clinical data is a major weakness. In this study, we...... compared performance of the International Prognostic Index (IPI) and its variations (R-IPI and NCCN-IPI) to a Cox proportional hazards (CPH) model using the same covariates in nondichotomized form. All models were tested in 4863 newly diagnosed DLBCL patients from population-based Nordic registers. The CPH...
Møller, Michael B; Pedersen, Niels T; Christensen, Bjarne E
The International Prognostic Index (IPI) is widely used for risk stratification of patients with diffuse large B-cell lymphoma (DLBCL). However, even among patients with low-risk disease, according to the IPI a substantial proportion of patients ultimately succumb to their disease. Using mature...... population-based data from the Danish Lymphoma Group, we analyzed if prognostic clinical pretreatment factors could be identified in patients with low-risk DLBCL. One hundred seventy-seven patients, all with a prognostic profile as favorable as possible according to the IPI and treated with anthracycline...... prognosis, with a survival at 5 and 15 years of 90% and 80%, respectively. In contrast, patients with both adverse factors had poor outcome, with survival at 5 and 15 years of 70% and 29%, respectively (PIPI score...
Full Text Available Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.
Dong, Wei-Feng; Canil, Sarah; Lai, Raymond; Morel, Didier; Swanson, Paul E.; Izevbaye, Iyare
A new automated MYC IHC classifier based on bivariate logistic regression is presented. The predictor relies on image analysis developed with the open-source ImageJ platform. From a histologic section immunostained for MYC protein, 2 dimensionless quantitative variables are extracted: (a) relative distance between nuclei positive for MYC IHC based on euclidean minimum spanning tree graph and (b) coefficient of variation of the MYC IHC stain intensity among MYC IHC-positive nuclei. Distance between positive nuclei is suggested to inversely correlate MYC gene rearrangement status, whereas coefficient of variation is suggested to inversely correlate physiological regulation of MYC protein expression. The bivariate classifier was compared with 2 other MYC IHC classifiers (based on percentage of MYC IHC positive nuclei), all tested on 113 lymphomas including mostly diffuse large B-cell lymphomas with known MYC fluorescent in situ hybridization (FISH) status. The bivariate classifier strongly outperformed the “percentage of MYC IHC-positive nuclei” methods to predict MYC+ FISH status with 100% sensitivity (95% confidence interval, 94-100) associated with 80% specificity. The test is rapidly performed and might at a minimum provide primary IHC screening for MYC gene rearrangement status in diffuse large B-cell lymphomas. Furthermore, as this bivariate classifier actually predicts “permanent overexpressed MYC protein status,” it might identify nontranslocation-related chromosomal anomalies missed by FISH. PMID:27093450
Heydarian, Mohammad; Luperchio, Teresa Romeo; Cutler, Jevon; Mitchell, Christopher J; Kim, Min-Sik; Pandey, Akhilesh; Sollner-Webb, Barbara; Reddy, Karen
An increasingly common method for predicting gene activity is genome-wide chromatin immuno-precipitation of 'active' chromatin modifications followed by massively parallel sequencing (ChIP-seq). In order to understand better the relationship between developmentally regulated chromatin landscapes and regulation of early B cell development, we determined how differentially active promoter regions were able to predict relative RNA and protein levels at the pre-pro-B and pro-B stages. Herein, we describe a novel ChIP-seq quantification method (cRPKM) to identify active promoters and a multi-omics approach that compares promoter chromatin status with ongoing active transcription (GRO-seq), steady state mRNA (RNA-seq), inferred mRNA stability, and relative proteome abundance measurements (iTRAQ). We demonstrate that active chromatin modifications at promoters are good indicators of transcription and steady state mRNA levels. Moreover, we found that promoters with active chromatin modifications exclusively in one of these cell states frequently predicted the differential abundance of proteins. However, we found that many genes whose promoters have non-differential but active chromatin modifications also displayed changes in abundance of their cognate proteins. As expected, this large class of developmentally and differentially regulated proteins that was uncoupled from chromatin status used mostly post-transcriptional mechanisms. Strikingly, the most differentially abundant protein in our B-cell development system, 2410004B18Rik, was regulated by a post-transcriptional mechanism, which further analyses indicated was mediated by a micro-RNA. These data highlight how this integrated multi-omics data set can be a useful resource in uncovering regulatory mechanisms. This data can be accessed at: https://usegalaxy.org/u/thereddylab/p/prediction-of-gene-activity-based-on-an-integrative-multi-omics-analysis.
De Puysseleyr, K; Kieckens, E; De Puysseleyr, L; Van den Wyngaert, H; Ahmed, B; Van Lent, S; Creasy, H H; Myers, G S A; Vanrompay, D
Chlamydia suis infections lead to economic loss in the pork industry. Chlamydia suis infections could be successfully treated with tetracyclines until the appearance of a tetracycline resistant phenotype, which was acquired via horizontal gene transfer of the tet(C) gene. Given the importance of C. suis as a swine pathogen and as a recently emerged tetracycline resistant pathogen with zoonotic potential, our aim was to develop a sensitive C. suis-specific antibody ELISA based on the polymorphic membrane proteins (Pmps). Chlamydia Pmps are important virulence factors and candidate antigens for serodiagnosis. We identified nine Pmps (PmpA to I) in C. suis strain MD56 using a recently developed Hidden-Markov model. PmpC was the most promising candidate for the development of a C. suis-specific antibody ELISA as the protein was absent in C. abortus, C. pecorum and C. psittaci which also infect pigs and as the protein contained C. suis-specific amino acid regions, absent in C. trachomatis PmpC. We identified an immunodominant B-cell epitope in C. suis PmpC using experimental porcine sera. The sensitivity and specificity of the PmpC ELISA was compared to the complement fixation test (CFT) and to a recombinant MOMP ELISA using experimental sera. The PmpC ELISA detected all positive control sera and was in contrast to CFT and the rMOMP ELISA 100% C. suis specific as positive control sera against other Chlamydia species did not react in the PmpC ELISA. The test was successfully validated using slaughterhouse sera and sera from clinically affected pigs. The PmpC ELISA could assist in diminishing the spread of C. suis infections in the pork industry. © 2018 Blackwell Verlag GmbH.
Nielsen, Morten; Lundegaard, Claus; Worning, Peder
to the MHC class II complex HLA-DR4(B1*0401). Prior identification of information-rich (anchor) positions in the binding motif is shown to improve the predictive performance of the Gibbs sampler. Similarly, a consensus solution obtained from an ensemble average over suboptimal solutions is shown......Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus......, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates...
Tipu, Hamid Nawaz
To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.
Zheng, Juzeng; Lin, Xianfan; Wang, Xiuyan; Zheng, Liyu; Lan, Songsong; Jin, Sisi; Ou, Zhanfan; Wu, Jinming
Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.
Nielsen, Morten; Lundegaard, Claus; Worning, Peder
calculations we show that peptides that bind to the HLA A*0204 complex display signal of higher order sequence correlations. Neural networks are ideally suited to integrate such higher order correlations when predicting the binding affinity. It is this feature combined with the use of several neural networks......In this paper we describe an improved neural network method to predict T-cell class I epitopes. A novel input representation has been developed consisting of a combination of sparse encoding, Blosum encoding, and input derived from hidden Markov models. We demonstrate that the combination...... of several neural networks derived using different sequence-encoding schemes has a performance superior to neural networks derived using a single sequence-encoding scheme. The new method is shown to have a performance that is substantially higher than that of other methods. By use of mutual information...
Zhang, Yan-Huan; Lu, Ai-Dong; Yang, Lu; Li, Ling-Di; Chen, Wen-Min; Long, Ling-Yu; Zhang, Le-Ping; Qin, Ya-Zhen
To investigate the prognostic value of PRAME expression in pediatric acute lymphoblastic leukemia(ALL), we measured PRAME transcript levels at diagnosis in 191 patients(146 B-ALL; 45T-ALL)receiving chemotherapy only. PRAME overexpression was defined as transcript levels higher than 0.30%, which is the upper limit of normal bone marrow and the optimal cutoff value derived from ROC curve analysis. PRAME overexpression was identified in 45.5% of patients. In B-ALL, PRAME overexpression was significantly associated with lower CIR(cumulative incidence of relapse), higher DFS (disease-freesurvival), and OS(overall survival) rates at 3 years, respectively (5.8% vs. 14.9%, P=0.014; 94.2% vs. 85.1%, P=0.014; 96.0% vs. 87.4%, P=0.039). PRAME overexpression had no impact on outcome in T-ALL patients. Among B-ALL patients with non-poor cytogenetic risk, those with PRAME overexpression showed significantly lower CIR, higher DFS and OS rates at 3 years, respectively (8.47% vs. 14.5%, P=0.009; 96.5% vs. 85.5%, P=0.009; 98.4% vs. 88.0%, P=0.023). Furthermore, PRAME overexpression was an independent good prognostic factor for relapse in all B-ALL patients and B-ALL patients with non-poor cytogenetic risk. Therefore, the prognostic significance of PRAME overexpression differed by ALL subtype; It predicted good outcome in pediatric B-ALL receiving chemotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.
Cotugno, Nicola; De Armas, Lesley; Pallikkuth, Suresh; Rinaldi, Stefano; Issac, Biju; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita
Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27 + CD21 - ) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27 + CD21 + ) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses ( APRIL, BTK, BLIMP1 ) and
Full Text Available Despite effective antiretroviral therapy (ART, HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27+CD21− from HIV when compared to HC despite long-term viral control (>24 months. Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM B cells (CD27+CD21+ was able to distinguish vaccine responders from non-responders (NR. In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses (APRIL, BTK, BLIMP1 and
Full Text Available Meningitis infection is one of the major threats during Hajj season in Mecca. Meningitis vaccines are available, but their uses are limited in some countries due to religious reasons. Furthermore, they only give protection to certain serogroups, not to all types of meningitis-inducing bacteria. Recently, research on epitope-based vaccines has been developed intensively. Such vaccines have potential advantages over conventional vaccines in that they are safer to use and well responded to the antibody. In this study, we developed epitope-based vaccine candidates against various meningitis-inducing bacteria, including Streptococcus pneumoniae, Neisseria meningitidis , and Haemophilus influenzae type b. The epitopes were selected from their protein of polysaccharide capsule. B-cell epitopes were predicted by using BCPred, while T-cell epitope for major histocompatibility complex (MHC class I was predicted using PAProC, TAPPred, and Immune Epitope Database. Immune Epitope Database was also used to predict T-cell epitope for MHC class II. Population coverage and molecular docking simulation were predicted against previously generated epitope vaccine candidates. The best candidates for MHC class I- and class II-restricted T-cell epitopes were MQYGDKTTF, MKEQNTLEI, ECTEGEPDY, DLSIVVPIY, YPMAMMWRNASNRAI, TLQMTLLGIVPNLNK, ETSLHHIPGISNYFI, and SLLYILEKNAEMEFD, which showed 80% population coverage. The complexes of class I T-cell epitopes-HLA-C * 03:03 and class II T-cell epitopes-HLA-DRB1 * 11:01 showed better affinity than standards as evaluated from their δ G binding value and the binding interaction between epitopes and HLA molecules. These peptide constructs may further be undergone in vitro and in vivo testings for the development of targeted vaccine against meningitis infection.
Full Text Available Abstract Background HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An in silico approach was applied for the analysis of entire HCV proteome of Pakistani origin, aimed to identify the viral epitopes and their conservancy in HCV genotypes 1, 2 and 3 of diverse origin. Results Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide. Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100% conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2. Conclusions The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was
Stranzl, Thomas; Larsen, Mette V; Lund, Ole; Nielsen, Morten; Brunak, Søren
Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I supertype representatives consistently found to contain fewer predicted epitopes compared to normal tissue. We observed a significant difference in amino acid composition between protein sequences associated with normal versus cancer tissue, as transcripts found in cancer are enriched with hydrophilic amino acids. This variation contributes to the observed significant lower likelihood of cancer-specific peptides to be predicted epitopes compared to peptides found in normal tissue.
Full Text Available Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I supertype representatives consistently found to contain fewer predicted epitopes compared to normal tissue. We observed a significant difference in amino acid composition between protein sequences associated with normal versus cancer tissue, as transcripts found in cancer are enriched with hydrophilic amino acids. This variation contributes to the observed significant lower likelihood of cancer-specific peptides to be predicted epitopes compared to peptides found in normal tissue.
Denisova, Galina F; Denisov, Dimitri A; Yeung, Jeffrey; Loeb, Mark B; Diamond, Michael S; Bramson, Jonathan L
Understanding antibody function is often enhanced by knowledge of the specific binding epitope. Here, we describe a computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification. We applied this methodology to the prediction of epitopes for five monoclonal antibodies against the West Nile virus (WNV) E protein, two of which exhibit therapeutic activity in vivo. This strategy was validated by comparison of our results with existing F(ab)-E protein crystal structures and mutational analysis by yeast surface display. We demonstrate that by combining the results of the mimotope method with our data from mutational analysis, epitopes could be predicted with greater certainty. The two methods displayed great complementarity as the mutational analysis facilitated epitope prediction when the results with the mimotope method were equivocal and the mimotope method revealed a broader number of residues within the epitope than the mutational analysis. Our results demonstrate that the combination of these two prediction strategies provides a robust platform for epitope characterization.
Lazarovici, Julien; Petrovanu, Cynthia; Danu, Alina; Ferme, Christophe; Ribrag, Vincent; Ghez, David [Gustave Roussy, Villejuif (France). Dept. of Hematology; Paris Saclay Univ., Saint-Aubin (France); Terroir, Marie [Gustave Roussy, Villejuif (France). Dept. of Nuclear Medicine and Endocrine Oncology; Paris Saclay Univ., Saint-Aubin (France); Arfi-Rouche, Julia [Gustave Roussy, Villejuif (France). Dept. of Radiology; Paris Saclay Univ., Saint-Aubin (France); Michot, Jean-Marie [Gustave Roussy, Villejuif (France). Dept. of Drug Development (DITEP); Paris Saclay Univ., Saint-Aubin (France); Mussot, Sacha; Florea, Valentina [Marie Lannelongue Hospital, Le Plessis Robinson (France). Dept. of Thoracic Surgery; Ghigna, Maria-Rosa [Marie Lannelongue Hospital, Le Plessis Robinson (France). Dept. of Pathology; Dartigues, Peggy [Gustave Roussy, Villejuif (France). Dept. of Pathology; Paris Saclay Univ., Saint-Aubin (France)
Though commonly used to assess response to therapy, the prognostic value of interim FDG-PET/CT in Primary Mediastinal Large B-cell Lymphoma (PMBCL) is unclear. We conducted a retrospective study on 36 consecutive patients treated at our institution for a PMBCL between 2006 and 2014. All patients with a positive interim FDG-PET/CT had undergone histological restaging consisting either in a surgical debulking of the residual lesion (15 patients) or a CT-guided core needle biopsy (two patients). All FDG-PET/CT were secondarily reviewed according to the more recent Deauville criteria. Interim FDG-PET/CT was considered positive in 17/36 patients using visual evaluation. Among these patients, 14 had a Deauville score of 4. Histological restaging was negative in all but one case, showing inflammation and/or fibrosis. After a median follow-up of 48.5 months, a total of five patients have relapsed, two patients in the positive FDG-PET/CT group, and three patients in the negative FDG-PET/CT group, respectively. These data indicate that a positive interim FDG-PET/CT does not reflect persistence of active disease in the vast majority of PMBCL cases. The relapse rate appears similar regardless of interim FDG-PET/CT results and interpretation criteria. This suggests that interim FDG-PET/CT has a poor positive predictive value, thus kt should be used with caution in PMBCL. (orig.)
Tipu, H. N.
Objective: To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Study Design: Cross-sectional study. Place and Duration of Study: Combined Military Hospital, Khuzdar Cantt, in May 2015. Methodology: Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLA class I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. Results: HLA A*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. A total of 35 nanomers from GP1, and 3 from GP2 were identified. HLA B*0702 bound maximum number of peptides (6), while HLA B*4001 showed strongest binding affinity. Conclusion: HLA specific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates. (author)
Lundegaard, Claus; Nielsen, Morten; Lamberth, K.
The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding pre...... in situations where only very limited data are available for training....
Stranzl, Thomas; Larsen, Mette Voldby; Lund, Ole
Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes...... is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based...... on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I...
Hou, Minbo; Zhou, Defang; Li, Gen; Guo, Huijun; Liu, Jianzhu; Wang, Guihua; Zheng, Qiankun; Cheng, Ziqiang
Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry. Copyright © 2016 Elsevier Ltd. All rights reserved.
Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus... vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then...constructed two DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to
Calis, Jorg J A; Reinink, Peter; Keller, Christin; Kloetzel, Peter M; Keşmir, Can
Proteolysis is the general term to describe the process of protein degradation into peptides. Proteasomes are the main actors in cellular proteolysis, and their activity can be measured in in vitro digestion experiments. However, in vivo proteolysis can be different than what is measured in these experiments if other proteases participate or if proteasomal activity is different in vivo. The in vivo proteolysis can be measured only indirectly, by the analysis of peptides presented on MHC-I molecules. MHC-I presented peptides are protected from further degradation, thus enabling an indirect view on the underlying in vivo proteolysis. The ligands presented on different MHC-I molecules enable different views on this process; in combination, they might give a complete picture. Based on in vitro proteasome-only digestions and MHC-I ligand data, different proteolysis predictors have been developed. With new in vitro digestion and MHC-I ligand data sets, we benchmarked how well these predictors capture in vitro proteasome-only activity and in vivo whole-cell proteolysis, respectively. Even though the in vitro proteasome digestion patterns were best captured by methods trained on such data (ProteaSMM and NetChop 20S), the in vivo whole-cell proteolysis was best predicted by a method trained on MHC-I ligand data (NetChop Cterm). Follow-up analysis showed that the likely source of this difference is the activity from proteases other than the proteasome, such as TPPII. This non-proteasomal in vivo activity is captured by NetChop Cterm and should be taken into account in MHC-I ligand predictions.
Tanu, Arifur Rahman; Ashraf, Mohammad Arif; Hossain, Md Faruk; Ismail, Md; Shekhar, Hossain Uddin
This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development.
Long Zheng; Peisheng Hu; Brandon Wolfe; Caryn Gonsalves; Luqing Ren; Leslie A. Khawli; Harvey R. Kaslow; Alan L. Epstein
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas...
Arefa Jafarzadeh Kohneloo
Full Text Available Background: Recent studies have shown that effective genes on survival time of cancer patients play an important role as a risk factor or preventive factor. Present study was designed to determine effective genes on survival time for diffuse large B-cell lymphoma patients and predict the survival time using these selected genes. Materials & Methods: Present study is a cohort study was conducted on 40 patients with diffuse large B-cell lymphoma. For these patients, 2042 gene expression was measured. In order to predict the survival time, the composition of the semi-parametric additive survival model with two gene selection methods elastic net and lasso were used. Two methods were evaluated by plotting area under the ROC curve over time and calculating the integral of this curve. Results: Based on our findings, the elastic net method identified 10 genes, and Lasso-Cox method identified 7 genes. GENE3325X increased the survival time (P=0.006, Whereas GENE3980X and GENE377X reduced the survival time (P=0.004. These three genes were selected as important genes in both methods. Conclusion: This study showed that the elastic net method outperformed the common Lasso method in terms of predictive power. Moreover, apply the additive model instead Cox regression and using microarray data is usable way for predict the survival time of patients.
Green, Tina Marie; Nielsen, Ole; de Stricker, Karin
Determining the presence of MYC gene rearrangements is becoming an increasingly important part of the diagnostic workup in aggressive lymphoma. Cytogenetic MYC alterations aid in differentiating diffuse large B-cell lymphoma (DLBCL) from Burkitt lymphoma. In addition, MYC aberrations are associated...... with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry......, and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found...
Sommer Kristensen, Lasse; Hansen, Jakob Werner; Kristensen, Søren Sommer
III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after......BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients...... treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter. CONCLUSIONS: Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating...
Pedersen, Lasse Eggers; Rasmussen, Michael; Harndahl, Mikkel
The affinity for and stability of peptides bound by major histocompatibility complex (MHC) class I molecules are instrumental factors in presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). In swine, such peptide presentations by swine leukocyte antigens (SLA) are crucial for swine i...
Pedersen, Lasse Eggers; Rasmussen, Michael; Harndah, Mikkel
The affinity with which major histocompatibility complex (MHC) class I molecules bind peptides is instrumental to presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). We analyzed three swine leukocyte antigen (SLA) molecules for complete nonamer peptide-based binding matrices in orde...
Young, Ken H; Leroy, Karen; Møller, Michael Boe
The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients and the overall survival (OS......) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (p=0.0006). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA binding domains were the strongest predictors of poor OS (p=0.0003). Mutations in the Loop......-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (p=0.002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His(158), His(175), Ser(245), Gln(248), His(273), Arg(280) and Arg(282)) in the DNA binding domains had the worst prognosis. Multivariate...
Saha, Chayan Kumar; Mahbub Hasan, Md; Saddam Hossain, Md; Asraful Jahan, Md; Azad, Abul Kalam
To explore a common B- and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV). Membrane proteins F, G and M of HeV and NiV were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B- and T-cell epitopes that shared maximum identity with HeV and NiV were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope (VDPLRVQWRNNSVIS) showed at least 66% identity with all NiV and HeV G protein sequences, while the 15-mer M-epitope (GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all NiV and HeV M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II (MHC II) and class-I (MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against HeV and NiV. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction. Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.
Avalos, Ana M.; Bilate, Angelina M.; Witte, Martin D.; Tai, Albert K.; He, Jiang; Frushicheva, Maria P.; Thill, Peter D.; Meyer-Wentrup, Friederike; Theile, Christopher S.; Chakraborty, Arup K.; Zhuang, Xiaowei; Ploegh, Hidde L.
Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR.
Lucas Y. H. Goh
Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.
Kristensen, Lasse Sommer; Asmar, Fazila; Dimopoulos, Konstantinos
found that DAPK1 promoter methylation was associated with shorter overall survival (p=0.017) and disease-specific survival (p=0.023). In multivariate analyses DAPK1 methylation remained as an independent prognostic factor predicting disease-specific survival (p=0.038). When only considering individuals...
Lu, J; Celis, E
Tumor cells can be effectively recognized and eliminated by CTLs. One approach for the development of CTL-based cancer immunotherapy for solid tumors requires the use of the appropriate immunogenic peptide epitopes that are derived from defined tumor-associated antigens. Because CTL peptide epitopes are restricted to specific MHC alleles, to design immune therapies for the general population it is necessary to identify epitopes for the most commonly found human MHC alleles. The identification of such epitopes has been based on MHC-peptide-binding assays that are costly and labor-intensive. We report here the use of two computer-based prediction algorithms, which are readily available in the public domain (Internet), to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). These algorithms identified three candidate peptides that we studied for their capacity to induce CTL responses in vitro using lymphocytes from HLA-B7+ normal blood donors. The results show that one of these peptides, CEA9(632) (IPQQHTQVL) was efficient in the induction of primary CTL responses when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that express HLA-B7 and produce CEA. The identification of this HLA-B7-restricted CTL epitope will be useful for the design of ethnically unbiased, widely applicable immunotherapies for common solid epithelial tumors expressing CEA. Moreover, our strategy of identifying MHC class I-restricted CTL epitopes without the need of peptide/HLA-binding assays provides a convenient and cost-saving alternative approach to previous methods.
Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza
Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All
Full Text Available Recent studies provide convincing evidence that a combined immunohistochemical or fluorescence in situ hybridization (FISH score of MYC, BCL2, BCL6 proteins and MYC translocations predicted outcome in diffuse large B-cell lymphoma (DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP. However, by far, all these researches are based on Western populations. Therefore, we investigate the prognostic relevance of MYC-, BCL2- and BCL6-rearrangements and protein expression by immunohistochemistry and FISH from 336 de novo DLBCL, NOS treated with CHOP or R-CHOP. Breaks in MYC and BCL6, and fusion in IGH/BCL2 were detected in 9.7%, 20.0%, and 11.1% of the cases, respectively, and were not significantly associated with clinical outcomes. Protein overexpression of MYC (≥40%, BCL2 (≥70% and BCL6 (≥50% was encountered in 51%, 51% and 36% of the tumors, respectively. On the basis of MYC, BCL2 and BCL6 expression, double-hit scores (DHSs and triple-hit score (THS were assigned to all patients with DLBCL. Patients with high MYC/BCL2 DHS, high MYC/BCL6 DHS and high THS had multiple adverse prognostic factors including high LDH level, poor performance status, advanced clinical stage, high International Prognostic Index (IPI score, and non-germinal center B-cell. In univariate analysis, high MYC/BCL2 DHS, high MYC/BCL6 DHS and high THS were associated with inferior OS and PFS in both CHOP and R-CHOP cohorts (P0.05. These data together suggest that the immunohistochemical DHSs and THS defined a large subset of DLBCLs with double-hit biology and was strongly associated with poor outcome in patients treated with R-CHOP or CHOP.
Tayo Alex Adekiya
Full Text Available Major histocompatibility complex class 1 chain-related gene sequence A is a polymorphic gene found at about 46.6 kb centromeric to HLA-B. It encodes a transmembrane protein, which is a non-classical human leukocyte antigen whose expression is normally induced by stress conditions like cancer and viral infections. The expression of MIC-A leads to the activation of NKG2D receptors of natural killer and T cells, leading to the generation of innate immune response that can easily eliminate/cleanse tumour cells and other cells that express the protein. Several bioinformatics and immunoinformatics tools were used to analyse the sequence and structure of the MIC-A protein. These tools were used in building and evaluating modelled structure of MIC-A, and to predict several antigenic determinant sites on the protein. The MIC-A protein structure generated an average antigenic propensity of 1.0289. Additionally, the hydrophilic regions on the surface of the MIC-A protein where antibodies can be attached were revealed. A total of fourteen antigenic epitopes were predicted, with six found in the transmembrane protein topology, and are predicted to play a role in the development of vaccines that can reactivate the functionalities of the MIC-A protein on the surface of cancer cells in order to elicit a desired immune response.
Sutton, Rosemary; Venn, Nicola C; Law, Tamara; Boer, Judith M; Trahair, Toby N; Ng, Anthea; Den Boer, Monique L; Dissanayake, Anuruddhika; Giles, Jodie E; Dalzell, Pauline; Mayoh, Chelsea; Barbaric, Draga; Revesz, Tamas; Alvaro, Frank; Pieters, Rob; Haber, Michelle; Norris, Murray D; Schrappe, Martin; Dalla Pozza, Luciano; Marshall, Glenn M
To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P 5 × 10 -5 (P < 0·0001) and High National Cancer Institute (NCI) risk (P < 0·0001). We created a predictive model based on a risk score (RS) for deletions, MRD and NCI risk, extending from an RS of 0 (RS0) for patients with no unfavourable factors to RS2 + for patients with 2 or 3 high risk factors. RS0, RS1, and RS2 + groups had RFS of 93%, 78% and 49%, respectively, and overall survival (OS) of 99%, 91% and 71%. The RS provided greater discrimination than MRD-based risk stratification into standard (89% RFS, 96% OS) and medium risk groups (79% RFS, 91% OS). We conclude that this RS may enable better early therapeutic stratification and thus improve cure rates for childhood ALL. © 2017 John Wiley & Sons Ltd.
Kasper Winther Jørgensen
Full Text Available Major Histocompatibility class II (MHC-II molecules sample peptides from the extracellular space allowing the immune system to detect the presence of foreign microbes from this compartment. Prediction of MHC class II ligands is complicated by the open binding cleft of the MHC class II molecule, allowing binding of peptides extending out of the binding groove. Furthermore, only a few HLA-DR alleles have been characterized with a sufficient number of peptides (100-200 peptides per allele to derive accurate description of their binding motif. Little work has been performed characterizing structural properties of MHC class II ligands. Here, we perform one such large-scale analysis. A large set of SYFPEITHI MHC class II ligands covering more than 20 different HLA-DR molecules was analyzed in terms of their secondary structure and surface exposure characteristics in the context of the native structure of the corresponding source protein. We demonstrated that MHC class II ligands are significantly more exposed and have significantly more coil content than other peptides in the same protein with similar predicted binding affinity. We next exploited this observation to derive an improved prediction method for MHC class II ligands by integrating prediction of MHC- peptide binding with prediction of surface exposure and protein secondary structure. This combined prediction method was shown to significantly outperform the state-of-the-art MHC class II peptide binding prediction method when used to identify MHC class II ligands. We also tried to integrate N- and O-glycosylation in our prediction methods but this additional information was found not to improve prediction performance. In summary, these findings strongly suggest that local structural properties influence antigen processing and/or the accessibility of peptides to the MHC class II molecule.
Biccler, Jorne; Eloranta, Sandra; de Nully Brown, Peter; Frederiksen, Henrik; Jerkeman, Mats; Smedby, Karin E; Bøgsted, Martin; El-Galaly, Tarec C
The international prognostic index (IPI) and similar models form the cornerstone of clinical assessment in newly diagnosed diffuse large B-cell lymphoma (DLBCL). While being simple and convenient to use, their inadequate use of the available clinical data is a major weakness. In this study, we compared performance of the International Prognostic Index (IPI) and its variations (R-IPI and NCCN-IPI) to a Cox proportional hazards (CPH) model using the same covariates in nondichotomized form. All models were tested in 4863 newly diagnosed DLBCL patients from population-based Nordic registers. The CPH model led to a substantial increase in predictive accuracy as compared to conventional prognostic scores when evaluated by the area under the curve and other relevant tests. Furthermore, the generation of patient-specific survival curves rather than assigning patients to one of few predefined risk groups is a relevant step toward personalized management and treatment. A test-version is available on lymphomapredictor.org. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
Chesneau, M; Danger, R; Soulillou, J-P; Brouard, S
Transplantation is currently the therapy of choice for endstage organ failure even though it requires long-term immunosuppresive therapy, with its numerous side effects, for acceptance of the transplanted organ. In rare cases however, patients develop operational tolerance, that is, graft survival without immunosuppression. Studies conducted on these patients reveal genetic, phenotypic, and functional signatures. They provide a better understanding of the immunological mechanisms involved in operational tolerance and define biomarkers that could be used to adapt immunosuppressive treatment to the individual, safely reduce immunosuppression doses, and ideally and safely guide immunosuppression withdrawal. This review summarizes studies that suggest a role for B cells as biomarkers of operational tolerance and discusses the use of B cells as a predictive tool for immunologic risk. Copyright © 2018. Published by Elsevier Inc.
Jørgensen, Kasper Winther; Buus, Søren; Nielsen, Morten
Major Histocompatibility class II (MHC-II) molecules sample peptides from the extracellular space allowing the immune system to detect the presence of foreign microbes from this compartment. Prediction of MHC class II ligands is complicated by the open binding cleft of the MHC class II molecule...... properties of MHC class II ligands. Here, we perform one such large-scale analysis. A large set of SYFPEITHI MHC class II ligands covering more than 20 different HLA-DR molecules was analyzed in terms of their secondary structure and surface exposure characteristics in the context of the native structure...... of the corresponding source protein. We demonstrated that MHC class II ligands are significantly more exposed and have significantly more coil content than other peptides in the same protein with similar predicted binding affinity. We next exploited this observation to derive an improved prediction method for MHC...
Song, Yoo Sung; Lee, Won Woo; Lee, Jong Seok; Kim, Sang Eun
Central nervous system (CNS) relapse of diffuse large B-cell lymphoma (DLBCL) is a rare complication, but has a poor prognosis with unknown pathophysiology. Recent trials of CNS prophylaxis have shown to be ineffective, despite patient's selection using several known clinical risk factors. In this study, the authors evaluated the value of pretreatment [F]2-Fluoro-2-deoxyglucose positron emission tomography in predicting CNS relapse in DLBCL patients.The authors analyzed 180 pathologically confirmed DLBCL patients, retrospectively. Patients underwent [F]2-Fluoro-2-deoxyglucose positron emission tomography/computed tomography before first line rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone therapy. Clinical characteristics were evaluated and total lesion glycolysis (TLG) with a threshold margin of 50% was calculated.Among age, sex, Ann Arbor stage, International Prognostic Index, revised International Prognostic Index, high serum lactate dehydrogenase level, presence of B symptoms, bulky disease (≥10 cm), extranodal lesion involvement, bone marrow involvement, high metabolic tumor volume ( >450 mL), and high TLG50 (>2000), the high TLG50 was the only significant prognostic factor for predicting CNS relapse in a multivariate analysis (P = 0.04). Kaplan-Meir survival analysis between high TLG50 (>2000) and low TLG50 (≤2000) groups revealed significantly different mean progression free survival (PFS) of 1317.2 ± 134.3 days and 1968.6 ± 18.3 days, respectively (P positron emission tomography/computed tomography is the most significant predictor of CNS relapse in un-treated DLBCL patients.
Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. Results We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A, lysine (K or aspartic acid (D, respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. Conclusions We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.
Lundegaard, Claus; Lund, Ole; Buus, Søren
Over the last decade, in silico models of the major histocompatibility complex (MHC) class I pathway have developed significantly. Before, peptide binding could only be reliably modelled for a few major human or mouse histocompatibility molecules; now, high-accuracy predictions are available...
Bjerregaard, Anne-Mette; Nielsen, Morten; Hadrup, Sine Reker
, including HLA binding and similarity to normal peptides. The peptides are sorted according to a priority score which is intended to roughly predict immunogenicity. We applied MuPeXI to three tumors for which predicted MHC-binding peptides had been screened for T cell reactivity, and found that Mu......PeXI was able to prioritize immunogenic peptides with an area under the curve of 0.63. Compared to other available tools, MuPeXI provides more information and is easier to use. MuPeXI is available as stand-alone software and as a web server at http://www.cbs.dtu.dk/services/MuPeXI ....
Full Text Available Abstract Background Schistosomiasis remains a major public health problem in endemic countries and is caused by infections with any one of three primary schistosome species. Although there are no vaccines available to date, this strategy appears feasible since natural immunity develops in individuals suffering from repeated infection during a lifetime. Since vaccinations resulting in both Th1- and Th2-type responses have been shown to contribute to protective immunity, a vaccine formulation with the capacity for stimulating multiple arms of the immune response will likely be the most effective. Previously we developed partially protective, single Th- and B cell-epitope-based peptide-DNA dual vaccines (PDDV (T3-PDDV and B3-PDDV, respectively capable of eliciting immune responses against the Schistosoma japonicum 22.6 kDa tegument antigen (Sj22.6 and a 62 kDa fragment of myosin (Sj62, respectively. Results In this study, we developed PDDV cocktails containing multiple epitopes of S. japonicum from Sj22.6, Sj62 and Sj97 antigens by predicting cytotoxic, helper, and B-cell epitopes, and evaluated vaccine potential in vivo. Results showed that mice immunized with a single-epitope PDDV elicited either Tc, Th, or B cell responses, respectively, and mice immunized with either the T3- or B3- single-epitope PDDV formulation were partially protected against infection. However, mice immunized with a multicomponent (3 PDDV components formulation elicited variable immune responses that were less immunoprotective than single-epitope PDDV formulations. Conclusions Our data show that combining these different antigens did not result in a more effective vaccine formulation when compared to each component administered individually, and further suggest that immune interference resulting from immunizations with antigenically distinct vaccine targets may be an important consideration in the development of multicomponent vaccine preparations.
Takahashi, Yoshimasa; Kelsoe, Garnett
Virus-specific memory B cells (B mem ) play a crucial role in protecting against variant viruses. The ability to recognize these variant viruses, defined as antibody breadth, is achieved in B mem populations by two very different pathways, germline-encoded cross-reactivity and affinity-driven, somatic evolution in germinal centers (GCs) for conserved viral epitopes. The latter class of broadly-reactive B mem cells are not cross-reactive per se, but bind epitopes crucial for viral fitness. Although these conserved epitopes are often weakly immunogenic, the GC reaction is surprisingly permissive for the continued survival/proliferation of B cells that bind with low affinity or react to cryptic epitopes, increasing their chance of memory recruitment. In this review, we discuss the adaptive strategies of B-cell memory to viral antigenic variations. Copyright © 2017 Elsevier Ltd. All rights reserved.
Mirza, Osman Asghar; Henriksen, A; Ipsen, H
The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces...
Gazi, Md Amran; Kibria, Mohammad Golam; Mahfuz, Mustafa; Islam, Md Rezaul; Ghosh, Prakash; Afsar, Md Nure Alam; Khan, Md Arif; Ahmed, Tahmeed
The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In
Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel
Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Dommaraju, Kalpana; Kijak, Gustavo; Carlson, Jonathan M; Larsen, Brendan B; Tovanabutra, Sodsai; Geraghty, Dan E; Deng, Wenjie; Maust, Brandon S; Edlefsen, Paul T; Sanders-Buell, Eric; Ratto-Kim, Silvia; deSouza, Mark S; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttihum, Punnee; Kaewkungwal, Jaranit; O'Connell, Robert J; Robb, Merlin L; Michael, Nelson L; Mullins, James I; Kim, Jerome H; Rolland, Morgane
The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84-91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.
Kalina, Jennifer L; Neilson, David S; Lin, Yen-Yi; Hamilton, Phineas T; Comber, Alexandra P; Loy, Emma M H; Sahinalp, S Cenk; Collins, Colin C; Hach, Faraz; Lum, Julian J
Purpose: Gene fusions are frequently found in prostate cancer and may result in the formation of unique chimeric amino acid sequences (CASQ) that span the breakpoint of two fused gene products. This study evaluated the potential for fusion-derived CASQs to be a source of tumor neoepitopes, and determined their relationship to patterns of immune signatures in prostate cancer patients. Experimental Design: A computational strategy was used to identify CASQs and their corresponding predicted MHC class I epitopes using RNA-Seq data from The Cancer Genome Atlas of prostate tumors. In vitro peptide-specific T-cell expansion was performed to identify CASQ-reactive T cells. A multivariate analysis was used to relate patterns of in silico -predicted tumor-infiltrating immune cells with prostate tumors harboring these mutational events. Results: Eighty-seven percent of tumors contained gene fusions with a mean of 12 per tumor. In total, 41% of fusion-positive tumors were found to encode CASQs. Within these tumors, 87% gave rise to predicted MHC class I-binding epitopes. This observation was more prominent when patients were stratified into low- and intermediate/high-risk categories. One of the identified CASQ from the recurrent TMPRSS2:ERG type VI fusion contained several high-affinity HLA-restricted epitopes. These peptides bound HLA-A*02:01 in vitro and were recognized by CD8 + T cells. Finally, the presence of fusions and CASQs were associated with expression of immune cell infiltration. Conclusions: Mutanome analysis of gene fusion-derived CASQs can give rise to patient-specific predicted neoepitopes. Moreover, these fusions predicted patterns of immune cell infiltration within a subgroup of prostate cancer patients. Clin Cancer Res; 23(24); 7596-607. ©2017 AACR . ©2017 American Association for Cancer Research.
Full Text Available Twenty B candidate epitopes of glycoproteins B (gB2, C (gC2, E (gE2, G (gG2, and I (gI2 of herpes simplex virus type 2 (HSV-2 were predicted using DNAstar, Biosun, and Antheprot methods combined with the polynomial method. Subsequently, the biological functions of the peptides were tested via experiments in vitro. Among the 20 epitope peptides, 17 could react with the antisera to the corresponding parent proteins in the EIA tests. In particular, five peptides, namely, gB2466–473 (EQDRKPRN, gC2216–223 (GRTDRPSA, gE2483–491 (DPPERPDSP, gG2572–579 (EPPDDDDS, and gI2286-295 (CRRRYRRPRG had strong reaction with the antisera. All conjugates of the five peptides with the carrier protein BSA could stimulate mice into producing antibodies. The antisera to these peptides reacted strongly with the corresponding parent glycoproteins during the Western Blot tests, and the peptides reacted strongly with the antibodies against the parent glycoproteins during the EIA tests. The antisera against the five peptides could neutralize HSV-2 infection in vitro, which has not been reported until now. These results suggest that the immunodominant epitopes screened using software algorithms may be used for virus diagnosis and vaccine design against HSV-2.
Conclusion: Our study results show that for patients with relapsed/refractory B-cell NHL, rituximab-containing salvage treatment is feasible and generally tolerable. A high ALC-R value was significantly associated with a better response to this treatment.
Wong, K K; Gascoyne, D M; Brown, P J
We previously identified autoantibodies to the endocytic-associated protein Huntingtin-interacting protein 1-related (HIP1R) in diffuse large B-cell lymphoma (DLBCL) patients. HIP1R regulates internalization of cell surface receptors via endocytosis, a process relevant to many therapeutic strateg...
Kristensen, Lasse Sommer; Asmar, Fazila; Dimopoulos, Konstantinos
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Improvements in overall survival have been observed with the introduction of rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), however, prognostic markers...
Loo, Suet Kee; Ch'ng, Ewe Seng; Lawrie, Charles H
DNMT1 is a target of approved anti-cancer drugs including decitabine. However, the prognostic value of DNMT1 protein expression in R-CHOP-treated diffuse large B-cell lymphomas (DLBCLs) remains unexplored. Here we showed that DNMT1 was expressed in the majority of DLBCL cases (n = 209/230, 90.9%)...
Ghaffar, Ammarah; Tariq, Aamira
Pasteurella multocida represents a highly diverse group of bacteria infecting various hosts like the fowl, goat and buffalo leading to huge economic loss to the poultry and cattle industry. Previous reports indicated that the outer membrane proteins contribute significantly to the pathogenesis of Pasteurella multocida. The comparative in-silico genome wide analysis of four pathogenic Pasteurella multocida strains (Anand1-poultry, Anand1-goat, PMTB and VTCCBAA264) with their respective hosts was performed. A pipeline was developed to identify the list of non-homologous proteins of Pasteurella multocida strains and their hosts. The list was further analyzed for the identification of the essential outer membrane proteins responsible for the pathogenicity. Outer membrane proteins were further selected from these antigenic proteins on the basis of their pathogenic potential. A common B-cell epitope (TDYRNRDRS, ARRSVTSKEN, and KINDQWRW) determined via sequential and structural approach from the lipopolysaccharide (LPS) assembly outer membrane complex protein was predicted from fowl, goat and buffalo. Furthermore, we identified T-cell epitopes based on the lipopolysaccharide (LPS) assembly outer membrane complex protein via docking studies which were either similar to the B-cell epitopes or were occurring in the same patch except for MHC class II M fowl. We propose that this difference in epitope sequence is due to different interacting MHC class II protein predicted from the fowl. Hence, in the current study we found that a unique epitope based on the common antigenic lipopolysaccharide (LPS) outer membrane complex protein present in fowl, goat and buffalo can be a suitable target for vaccine development against the two economic devastating diseases; fowl cholera (FC) and hemorrhagic septicemia (HS). Copyright © 2016 Elsevier B.V. All rights reserved.
Geneugelijk, K; Hönger, G; van Deutekom, H W M|info:eu-repo/dai/nl/341356816; Thus, K A; Kesmir, C.|info:eu-repo/dai/nl/304843393; Hösli, I; Schaub, S; Spierings, E
Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child-specific HLA antibodies. We previously demonstrated that donor-specific HLA antibody formation after kidney transplantation is associated with donor-derived HLA epitopes
Geneugelijk, K.; Hönger, G.; Van Deutekom, H. W M; Thus, K. A.; Keşmir, C.; Hösli, I.; Schaub, S.; Spierings, E.
Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child-specific HLA antibodies. We previously demonstrated that donor-specific HLA antibody formation after kidney transplantation is associated with donor-derived HLA epitopes
Full Text Available Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154 linked to a T-cell epitope (3A 21 to 35 of FMD virus (FMDV elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T or four (B4T copies of the B-cell epitope from type O FMDV (a widespread circulating serotype were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFNγ. Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.
Valerie A Walshe
Full Text Available Predictive models of peptide-Major Histocompatibility Complex (MHC binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.Using an in-house, flow cytometry-based MHC stabilization assay we generated novel peptide binding data, from which we derived a precise two-dimensional quantitative structure-activity relationship (2D-QSAR binding model. This allowed us to explore the peptide specificity of HLA-Cw*0102 molecule in detail. We used this model to design peptides optimized for HLA-Cw*0102-binding. Experimental analysis showed these peptides to have high binding affinities for the HLA-Cw*0102 molecule. As a functional validation of our approach, we also predicted HLA-Cw*0102-binding peptides within the HIV-1 genome, identifying a set of potent binding peptides. The most affine of these binding peptides was subsequently determined to be an epitope recognized in a subset of HLA-Cw*0102-positive individuals chronically infected with HIV-1.A functionally-validated in silico-in vitro approach to the reliable and efficient prediction of peptide binding to a previously uncharacterized human MHC allele HLA-Cw*0102 was developed. This technique is generally applicable to all T cell epitope identification problems in immunology and vaccinology.
Madhumathi, J; Prince, P R; Rao, D N; Karande, A A; Reddy, M V R; Kaliraj, P
Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.
Stranzl, Thomas; Larsen, Mette V.; Lund, Ole; Nielsen, Morten; Brunak, Søren
Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the c...
M, Autio; Jørgensen, Judit Meszaros; SK, Leivonen
treatment-specific roles in diffuse large B-cell lymphoma. For the high risk DLBCL patients treated with dose-dense immunochemotherapy, high expression of type 1/2 immune response signature genes predicts a poor outcome. A detailed characterization of immune cell composition in the tumor microenvironment......Introduction: Despite better therapeutic options and improved survival of diffuse large B-cell lymphoma (DLBCL), 30-40% of the patients still relapse and have dismal prognosis. Recently, the impact of genomic aberrations, allowing lymphoma cells to escape immune recognition on DLBCL pathogenesis...... has been recognized. However, whether immune related signatures could be used as determinants for treatment outcome has not been rigorously evaluated. Here, our aim was to elucidate the immunologic characteristics of the tumor microenvironment, and associate the findings with outcome in patients...
Perry, Heather M.; Bender, Timothy P.; McNamara, Coleen A.
Atherosclerosis, the underlying cause of heart attacks and strokes, is a chronic inflammatory disease of the artery wall. Immune cells, including lymphocytes modulate atherosclerotic lesion development through interconnected mechanisms. Elegant studies over the past decades have begun to unravel a role for B cells in atherosclerosis. Recent findings provide evidence that B cell effects on atherosclerosis may be subset-dependent. B-1a B cells have been reported to protect from atherosclerosis ...
Sebastian Carrasco Pro
Full Text Available Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.
Chu, Zhulang; Zou, Weilong; Xu, Yanan; Sun, Qiquan; Zhao, Yong
B cells mediate allograft rejection through antigen presentation, and production of cytokines and antibodies. More and more immunosuppressive agents specifically targeting B cells and plasma cells have been applied in clinical transplantation. However, recent studies have indicated the regulatory roles of B cells. Therefore, it is vital to clarify the different effects of B cell subsets in organ transplantation so that we can completely understand the diverse functions of B cells in transplantation. Areas covered: This review focuses on the regulatory roles of B cells in transplantation. B cell subsets with immune modulation and factors mediating immunosuppressive functions of regulatory B (Breg) cells were analyzed. Therapies targeting B cells and the application of B cells for transplant tolerance induction were discussed. Expert commentary: Besides involving rejection, B cells could also play regulatory roles in transplantation. Breg cells and the related markers may be used to predict the immune tolerant state in transplant recipients. New therapeutic strategies targeting B cells should be explored to promote tolerance induction with less impact on the host's protective immunity in organ transplanted patients.
N Lance Hepler
Full Text Available Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab, determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license, documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.
Hepler, N Lance; Scheffler, Konrad; Weaver, Steven; Murrell, Ben; Richman, Douglas D; Burton, Dennis R; Poignard, Pascal; Smith, Davey M; Kosakovsky Pond, Sergei L
Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.
Azhar R Hussain
Full Text Available Activated B-cell lymphoma (ABC, one of the three subtypes of Diffuse Large B-cell Lymphoma (DLBCL has the worst survival rate after upfront chemotherapy and is characterized by constitutively activated NFκB. We therefore studied the role of NFκB In a cohort of clinical DLBCL samples and ABC cell lines. In our clinical tissue microarray cohort of DLBCL samples, p-IκBα was detected in 38.3% of ABC DLBCL and was an independent prognostic marker for poor survival. In vitro, we found that Thymoquinone (TQ, a natural compound isolated from Nigella sativa caused release of ROS in ABC cells. TQ-mediated release of ROS in turn inhibited NFκB activity by dephosphorylating IκBα and decreased translocation of p65 subunit of NFκB in the nuclear compartment in ABC cell lines. This led to inhibition of cell viability and induction of mitochondrial dependent apoptosis in ABC-DLBCL cell lines. Additionally, TQ treatment also caused up-regulation of death receptor 5 (DR5, however, up-regulation of DR5 did not play a role in TQ-induced apoptosis. Finally, combination of sub-optimal doses of TQ and TRAIL induced efficient apoptosis in ABC-DLBCL cell lines. These data show that p-IκBα can be used as a prognostic marker and target for therapy in this aggressive sub-type of DLBCL and TQ may play an important role in the management of DLBCL in the future.
Zheng, Long; Hu, Peisheng; Wolfe, Brandon; Gonsalves, Caryn; Ren, Luqing; Khawli, Leslie A; Kaslow, Harvey R; Epstein, Alan L
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgamma null (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.
Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.
Meesters, T M; Hoet, M; van den Hoogen, F H; Verheijen, R; Habets, W J; van Venrooij, W J
In this paper an immunodominant epitope of Topoisomerase I is described. An epitope expression sublibrary was constructed from Topoisomerase I cDNA. The subclones were screened with an antiserum from a patient with systemic sclerosis (SSc). The positive clones defined one immunodominant B cell epitope (epitope III), which was located at the carboxyterminal part of the protein. The epitope, 52 amino acids in length, neither contains the p30gag sequence nor the suggested active site Tyr-723, both presumed antibody recognition sites. More than 70% of our anti-TopoI sera recognize this epitope III, indicating that it is a major recognition site of the anti-TopoI autoantibodies in SSc sera. DNA relaxation experiments show that all sera that recognize epitope III and most sera with antibodies to other epitopes inhibit Topoisomerase I activity.
Cao, Xi-Xi; Fan, Jian; Chen, Jiang; Li, Yu-Hong; Fan, Ming-Wen
The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.
Kirsten E Wiens
Full Text Available The HLA (human leukocyte antigen molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure.We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines.We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes.Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.
Wiens, Kirsten E; Swaminathan, Harish; Copin, Richard; Lun, Desmond S; Ernst, Joel D
The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.
Vega, Gabriel G.; Avilés-Salas, Alejandro; Chalapud, J. Ramón; Martinez-Paniagua, Melisa; Pelayo, Rosana; Mayani, Héctor; Hernandez-Pando, Rogelio; Martinez-Maza, Otoniel; Huerta-Yepez, Sara; Bonavida, Benjamin; Vega, Mario I.
The p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients. Tissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed. Tumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38 − and 34 % for p38 + and for OS was 83 % for p38 − and 47 % for p38 + . The p-p38 + tissues expressed Bcl-2 and 90 % of p-p38 − where Bcl-2 − . The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB. The findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors. The online version of this article (doi:10.1186/s12885-015-1778-8) contains supplementary material, which is available to authorized users
Hampe, Christiane S.
The role of B cells in autoimmune diseases involves different cellular functions, including the well-established secretion of autoantibodies, autoantigen presentation and ensuing reciprocal interactions with T cells, secretion of inflammatory cytokines, and the generation of ectopic germinal centers. Through these mechanisms B cells are involved both in autoimmune diseases that are traditionally viewed as antibody mediated and also in autoimmune diseases that are commonly classified as T cell...
Zhong, Weijie; Xu, Xin; Zhu, Zhigang; Du, Qinghua; Du, Hong; Yang, Li; Ling, Yanying; Xiong, Huabao; Li, Qingshan
The immunological pathogenesis of diffuse large B cell lymphoma (DLBCL) remains elusive. Searching for new prognostic markers of DLBCL is a crucial focal point for clinical scientists. The aim of the present study was to examine the prognostic value of interferon regulatory factor 8 (IRF8) expression and its effect on the development of Th17 cells in the tumor microenvironment of DLBCL patients. Flow cytometry, immunohistochemistry, and quantitative real-time PCR were used to detect the distribution of Th17 cells and related cytokines and IRF8 in tumor tissues from DLBCL patients. Two DLBCL cell lines (OCI-LY10 and OCI-LY1) with IRF8 knockdown or overexpression and two human B lymphoblast cell lines were co-cultured with peripheral blood mononuclear cells (PBMCs) in vitro to determine the effect of IRF8 on the generation of Th17 cells. Quantitative real-time PCR and Western blotting were used to investigate the involvement of retinoic acid receptor-related orphan receptor gamma t (RORγt) in the effect of IRF8 on Th17 cell generation. The survival of 67 DLBCL patients was estimated using the Kaplan-Meier method and log-rank analysis. The percentage of Th17 cells was lower in DLBCL tumor tissues than in PBMCs and corresponding adjacent benign tissues. Relative expression of interleukin (IL)-17A was lower, whereas that of interferon (IFN)-γ was higher in tumor tissues than in benign tissues. Co-culture with DLBCL cell lines inhibited the generation of Th17 cells in vitro. IRF8 upregulation was detected in DLBCL tumor tissues, and it was associated with decreased DLBCL patient survival. Investigation of the underlying mechanism suggested that IRF8 upregulation in DLBCL, through an unknown mechanism, inhibited Th17 cell generation by suppressing RORγt in neighboring CD4+ T cells. Tumor cells may express soluble or membrane-bound factors that inhibit the expression of RORγt in T cells within the tumor microenvironment. Our findings suggest that IRF8 expression could
Wang, De-Wang; Ni, Wei-Wei; Zhou, Yan-Jun; Huang, Wen; Cao, Meng-Da; Meng, Ling; Wei, Ji-Fu
Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ‑3.0 and ‑2.2. In total, eight B cell epitopes (15‑24, 60‑66, 78‑86, 109‑124, 232‑240, 260‑269, 298‑306 and 315‑322) and five T cell epitopes (62‑67, 86‑91, 125‑132, 217‑222 and 343‑350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen‑associated allergic reactions.
Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation-selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.
Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman
Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.
Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C
The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.
Hnida, Kathrin; Stamnaes, Jorunn; du Pré, M Fleur
Transglutaminase 2 (TG2) is a Ca(2+)-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2...... of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients....
Tegar A. P. Siregar
Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify
Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.
Riley, Richard L; Khomtchouk, Kelly; Blomberg, Bonnie B
With old age (∼2y old), mice show substantial differences in B cell composition within the lymphoid tissues. In particular, a novel subset of IgM + CD21/35 lo/- CD23 - mature B cells, the age-associated B cells or ABC, increases numerically and proportionately. This occurs at the expense of other B cell subsets, including B2 follicular B cells in spleen and recirculating primary B cells in bone marrow. Our studies suggest that ABC have a distinctive antibody repertoire, as evidenced by relatively high reactivity to the self-antigens phosphorylcholine (PC) and malondialdehyde (MDA). While PC and MDA are found on apoptotic cells and oxidized lipoproteins, antibodies to these antigens are also cross-reactive with epitopes on bacterial species. In old mice, ABC express TNFα and are pro-inflammatory. ABC can inhibit growth and/or survival in pro-B cells as well as common lymphoid progenitors (CLP). In particular, ABC cause apoptosis in pro-B cells with relatively high levels of the surrogate light chain (SLC) and, consequently, promote an "SLC low" pathway of B cell differentiation in old mice. SLC together with μ heavy chain comprises the pre-B cell receptor (preBCR) critical for pre-B cell expansion and selection of the μ heavy chain Vh repertoire. The low level of SLC likely impairs normal preBCR driven proliferation and alters μ heavy chain Vh selection thereby affecting the antibody specificities of new B cells. In this manner, ABC may contribute to both qualitative and quantitative disruptions of normal B lymphopoiesis in old age. Copyright © 2017. Published by Elsevier Inc.
Chentoufi, Aziz Alami; Zhang, Xiuli; Lamberth, Kasper
epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10...
Troppan, K T; Schlick, K; Deutsch, A; Melchardt, T; Egle, A; Stojakovic, T; Beham-Schmid, C; Weiss, L; Neureiter, D; Wenzl, K; Greil, R; Neumeister, P; Pichler, M
High levels of C-reactive protein (CRP), an acute phase protein, proofed being associated with decreased clinical outcome in small-scale studies in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to evaluate the prognostic impact of pretreatment CRP levels on overall survival (OS) and disease-free survival (DFS) in a large bicentre study of DLBCL patients. Data from 477 DLBCL patients, diagnosed and treated between 2004 and 2013 at two Austrian centres, were evaluated retrospectively. The prognostic influence of CRP and other factors, including age, tumour stage, and revised International Prognostic Index (R-IPI) on 5-year OS and 5-year DFS, were studied by Kaplan-Meier curves as well as univariate and multivariate Cox regression models. Influence of CRP on the predictive accuracy of the R-IPI score was determined by the Harrell concordance index. Kaplan-Meier curves revealed elevated CRP as a factor for decreased 5-year OS and DFS in DLBCL patients (PIPI score and 0.79 when CRP was added. In the present study, we demonstrated high CRP levels at diagnosis of DLBCL as an independent poor prognostic factor for clinical outcome. Adding CRP to the well-established prognostic models such as the R-IPI score might improve their predictive ability.
Scholtalbers, Jelle; Boegel, Sebastian; Bukur, Thomas; Byl, Marius; Goerges, Sebastian; Sorn, Patrick; Loewer, Martin; Sahin, Ugur; Castle, John C
Human cancer cell lines are an important resource for research and drug development. However, the available annotations of cell lines are sparse, incomplete, and distributed in multiple repositories. Re-analyzing publicly available raw RNA-Seq data, we determined the human leukocyte antigen (HLA) type and abundance, identified expressed viruses and calculated gene expression of 1,082 cancer cell lines. Using the determined HLA types, public databases of cell line mutations, and existing HLA binding prediction algorithms, we predicted antigenic mutations in each cell line. We integrated the results into a comprehensive knowledgebase. Using the Django web framework, we provide an interactive user interface with advanced search capabilities to find and explore cell lines and an application programming interface to extract cell line information. The portal is available at http://celllines.tron-mainz.de.
Vaughan, Kerrie; Peters, Bjoern; Mallone, Roberto; von Herrath, Matthias; Roep, Bart O.; Sette, Alessandro
Background The Immune Epitope Database (IEDB), originally focused on infectious diseases, was recently expanded to allergy, transplantation and autoimmunity diseases. Here we focus on diabetes, chosen as a prototype autoimmune disease. We utilize a combined tutorial and meta-analysis format, which demonstrates how common questions, related to diabetes epitopes can be answered. Results A total of 409 references are captured in the IEDB describing >2,500 epitopes from diabetes associated antigens. The vast majority of data were derived from GAD, insulin, IA-2/PTPRN, IGRP, ZnT8, HSP, and ICA-1, and the experiments related to T cell epitopes and MHC binding far outnumbers B cell assays. We illustrate how to search by specific antigens, epitopes or host. Other examples include searching for tetramers or epitopes restricted by specific alleles or assays of interest, or searching based on the clinical status of the host. Conclusions The inventory of all published diabetes epitope data facilitates its access for the scientific community. While the global collection of primary data from the literature reflects potential investigational biases present in the literature, the flexible search approach allows users to perform queries tailored to their preferences, including or excluding data as appropriate. Moreover, the analysis highlights knowledge gaps and identifies areas for future investigation. PMID:25140192
Kim, Dongbum; Lee, Younghee; Kwon, Hyung-Joo
Antibody production using synthetic peptides has been investigated extensively to develop therapeutic antibodies and prophylactic vaccines. Previously, we reported that a complex of CpG-DNA and synthetic peptides corresponding to B cell epitopes, encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex, significantly enhanced the synthetic peptide-specific IgG production. Here, we describe synthetic peptide-based epitope screening and antibody production without conventional carriers.
Full Text Available Abstract Background Human herpes simplex virus (HSV 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2. Methods The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN from envelope glycoprotein B, 216-223 (GRTDRPSA from C, 6-18 (DPSLKMADPNRFR from D, 483-491 (DPPERPDSP from E, 572-579 (EPPDDDDS from G and 286-295 (CRRRYRRPRG from I glycoprotein of HSV-2, four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL from D, 162-177 (KDVTVSQVWFGHRYSQ from B, 205-224 (KAYQQGVTVDSIGMLPRFIP from D and 245-259 (KPPYTSTLLPPELSD from D and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK from D and 268-276 (ALLEDPAGT from D, are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290 of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3, and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. Results The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell
Christine N. Vuong
Full Text Available In vivo targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, Clostridium perfringens alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected in silico using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for in vitro toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin’s hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and de facto identification of neutralizing epitopes in microbial
Ma, Ning; Zhang, Yu; Liu, Qilin; Wang, Zhiding; Liu, Xiaoling; Zhu, Gaizhi; Yu, Dandan; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Wang, Tianxiao; Ma, Yuanfang; Shen, Beifen; Li, Yan; Xiao, He; Wang, Renxi
B cell activating factor (BAFF) regulates B cell maturation, survival, function, and plays a critical pathogenic role in autoimmune diseases. It remains unclear how BAFF affects IL-10 - B cells versus regulatory B cells (Bregs) in inflammatory responses. In this study, we found that IL-10-expressing Bregs decreased in lupus-prone MRL/lpr mice and experimental allergic encephalomyelitis (EAE) mice. On blockade of the effects of BAFF with TACI-IgG, IL-10 + Bregs were upregulated in MRL/lpr and EAE mice. In addition, BAFF expanded IL-10 + B cells over IL-10 - B cells under noninflammatory conditions in vitro, whereas it expanded IL-10 - B cells over IL-10 + B cells during inflammatory responses, such as stimulation with autoantigen and LPS. Finally, the selection of IL-10 - B cells over IL-10 + B cells by BAFF was dependent on BAFF receptors (BAFFR, TACI, and BCMA) that were upregulated by inflammatory responses. This study suggests that BAFF selects IL-10 - B cells over IL-10 + regulatory B cells via BAFF receptors in inflammatory responses. Copyright © 2017 Elsevier Ltd. All rights reserved.
Zhang, Qing; Wang, Peng; Kim, Yohan
We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total...
Sher, Gene; Zhi, Degui; Zhang, Shaojie
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). 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. 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.
van den Berg, T. K.; Hasbold, J.; Renardel de Lavalette, C.; Döpp, E. A.; Dijkstra, C. D.; Klaus, G. G.
In this study we describe the tissue distribution of mouse CD40 using two monoclonal antibodies (mAb) against different epitopes of the molecule. In lymphoid tissues CD40 was expressed by B lymphocytes. Most B cells in typical B-cell compartments were CD40-positive, including germinal centre B
El-Galaly, Tarec Christoffer; Villa, Diego; Alzahrani, Musa; Hansen, Jakob Werner; Sehn, Laurie H; Wilson, Don; de Nully Brown, Peter; Loft, Annika; Iyer, Victor; Johnsen, Hans Erik; Savage, Kerry J; Connors, Joseph M; Hutchings, Martin
18F-fluorodeoxyglucose PET/CT (PET/CT) is the current state-of-the-art in the staging of diffuse large B-cell lymphoma (DLBCL) and has a high sensitivity for extranodal involvement. Therefore, reassessment of extranodal involvement and the current prognostic indices in the PET/CT era is warranted. We screened patients with newly diagnosed DLBCL seen at the academic centers of Aalborg, Copenhagen, and British Columbia for eligibility. Patients that had been staged with PET/CT and treated with R-CHOP(-like) 1(st) line treatment were retrospectively included. In total 443 patients met the inclusion criteria. With a median follow-up of 2.4 years, the 3-year overall (OS) and progression-free survival (PFS) were 73% and 69%, respectively. The Ann Arbor classification had no prognostic impact in itself with the exception of stage IV disease (HR 2.14 for PFS, P2 extranodal sites, including HR 7.81 (P 3 sites. Bone/bone marrow involvement was the most commonly involved extranodal site identified by PET/CT (29%) and was associated with an inferior PFS and OS. The IPI, R-IPI, and NCCN-IPI were predictive of PFS and OS, and the two latter could identify a very good prognostic subgroup with 3-year PFS and OS of 100%. PET/CT-ascertained extranodal involvement in DLBCL is common and involvement of >2 extranodal sites is associated with a dismal outcome. The IPI, R-IPI, and NCCN-IPI predict outcome with high accuracy. © 2015 Wiley Periodicals, Inc.
Hemstreet, George P; Rossi, Gabriela R; Pisarev, Vladimir M; Enke, Charles A; Helfner, Laura; Hauke, Ralph J; Tennant, Lucinda; Ramsey, William Jay; Vahanian, Nicholas N; Link, Charles J
The immunogenicity of a cellular immunotherapy using genetically modified vaccines to express α(1,3)galactosyl epitopes (αGal) was evaluated in advanced prostate cancer (PC) patients. In this dose escalation phase I study, we report safety, feasibility, and immunologic data of an immunotherapy composed of 2 human PC cell lines engineered to express αGal epitopes (HyperAcute-Prostate, HAP, NewLink Genetics). Eight patients received up to 12 biweekly vaccinations with HAP. Enrolled patients (aged range, 53-85 y) had American Joint Committee on Cancer stage IV, any T, any N, M1, Eastern Cooperative Oncology Group PS≤2, at least 1 prior hormonal treatment and HAP immunotherapy induced humoral immune responses to autoantigens in 2 of 8 patients. These patients developed IgG antibody to multiple epitopes overexpressed in PC after immunization. These responding patients received higher doses of the immunotherapy suggesting a dose response. Two immunogenic proteins (prostate-specific membrane antigen, hepsin) belong to the extracellular molecules family participating in malignant cell invasion. Median overall survival for patients was 25.1 months with 1 patient surviving over 70 months with stable PSA and bone metastasis before expiring of other causes. Three of 8 patients showed PSA stabilization (>100 d). In conclusion, HAP immunotherapy induces IgG responses to epitopes from autoantigens overexpressed in PC suggesting dose-dependent effect. HAP represents a viable immunotherapy approach to induce immune responses against tumor cells and may provide clinical benefit with minimal toxicity.
Keller, H.J.H.G.; Pomp, H.; Bakker, J.; Schots, A.
A phage library containing 2.7 × 10(9) randomly expressed peptides was used to determine the epitopes of three monoclonal antibodies that bind to the coat protein of Potato Virus Y. Construction of the consensus sequences for the peptides obtained after three selection rounds indicated that each
Li, Yan; Chen, Zhongbiao; Ye, Jianbin; Ning, Lijun; Luo, Jun; Zhang, Lili; Jiang, Yin; Xi, Yue; Ning, Yunshan
The epitope vaccine is an attractive potential for prophylactic and therapeutic vaccination against Helicobacter pylori (H. pylori) infection. Lpp20 is one of major protective antigens which trigger immune response after H. pylori invades host and has been considered as an excellent vaccine candidate for the control of H. pylori infection. In our previous study, one B-cell epitope and two CD4(+) T-cell epitopes of Lpp20 were identified. In this study, an epitope vaccine composed of mucosal adjuvant cholera toxin B subunit (CTB) and these three identified Lpp20 epitopes were constructed to investigate the efficacy of this epitope vaccine in mice. The epitope vaccine including CTB, one B-cell, and two CD4(+) T-cell epitopes of Lpp20 was constructed and named CTB-Lpp20, which was then expressed in Escherichia coli and used for intraperitoneal immunization in BALB/c mice. The immunogenicity, specificity, and ability to induce antibodies against Lpp20 and cytokine secretion were evaluated. After that, CTB-Lpp20 was intragastrically immunized to investigate the prophylactic and therapeutic efficacy in infected mice. The results indicated that the epitope vaccine CTB-Lpp20 possessed good immunogenicity and immunoreactivity and could elicit specific high level of antibodies against Lpp20 and the cytokine of IFN-γ and IL-17. Additionally, CTB-Lpp20 significantly decreased H. pylori colonization in H. pylori challenging mice, and the protection was correlated with IgG, IgA, and sIgA antibody and Th1-type cytokines. This study will be better for understanding the protective immunity of epitope vaccine, and CTB-Lpp20 may be an alternative strategy for combating H. pylori invasion. © 2015 John Wiley & Sons Ltd.
Moise, Leonard; McMurry, Julie A; Buus, Søren
Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...
van Geffen Wouter H
Full Text Available Abstract There is increasing evidence that a specific immune response contributes to the pathogenesis of COPD. B-cell follicles are present in lung tissue and increased anti-elastin titers have been found in plasma of COPD patients. Additionally, regulatory T cells (Tregs have been implicated in its pathogenesis as they control immunological reactions. We hypothesize that the specific immune response in COPD is smoke induced, either by a direct effect of smoking or as a result of smoke-induced lung tissue destruction (i.e. formation of neo-epitopes or auto antigens. Furthermore, we propose that Tregs are involved in the suppression of this smoke-induced specific immune response. The presence of B cells, memory B cells and Tregs was assessed by flow cytometry in peripheral blood of 20 COPD patients and 29 healthy individuals and related to their current smoking status. COPD patients had lower (memory B-cell percentages and higher Treg percentages in peripheral blood than healthy individuals, with a significant negative correlation between these cells. Interestingly, current smokers had higher percentages of (class-switched memory B cells than ex-smokers and never smokers, irrespective of COPD. This increase in (class-switched memory B cells in current smokers is intriguing and suggests that smoke-induced neo-antigens may be constantly induced in the lung. The negative correlation between B cells and Tregs in blood is in line with previously published observations that Tregs can suppress B cells. Future studies focusing on the presence of these (class switched memory B cells in the lung, their antigen specificity and their interaction with Tregs are necessary to further elucidate the specific B-cell response in COPD.
Hicar, Mark D; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U; Kalams, Spyros A; Doranz, Benjamin J; Spearman, Paul; Crowe, James E
Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.
Full Text Available Hepatitis C virus (HCV has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL. Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.
Full Text Available Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4+ and CD8+ T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania (L. infantum proteins, CPA, histone H1, KMP-11 and LeIF were analysed for the prediction of binding epitopes to H2d MHC class I and class II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and class II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in CFA/IFA. CPA_p2, CPA_p3, H1_p1 and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3 and LeIF_p6 induced IFN-γ-producing CD4+ T cells indicating a TH1 type response. In addition, CPA_p2, CPA_p3 and H1_p1 induced also the induction of CD8+ T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3 and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.
Bergh, Ann-Charlotte; Evaldsson, Chamilly; Pedersen, Lone Bredo
Chronic lymphocytic leukemia B cells express auto/xeno antigen-reactive antibodies that bind to self-epitopes and resemble natural IgM antibodies in their repertoire. One of the antigenic structures recognized is oxidation-induced malonedialdehyde that is present on low-density lipoprotein......, apoptotic blebs, and on certain microbes. The poor-prognostic stereotyped subset #1 (Clan I IGHV genes-IGKV1(D)-39) express IgM B-cell receptors that bind oxidized low-density lipoprotein. In this study, we have used for the first time this authentic cognate antigen for analysis of downstream B......-cell receptor-signal transduction events, since it is more faithful to B-cell physiology than anti-IgM. Multivalent oxidized low-density lipoprotein showed specific binding to subset #1 IgM/IgD B-cell receptors, whereas native low-density lipoprotein did not. The antigen binding induced prompt receptor...
Banerjee, Shyamashree; Sen Gupta, Parth Sarthi; Bandyopadhyay, Amal Kumar
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese Encephalitis (JE) and Acute Encephalitis Syndrome (AES) in humans. Genotype-I (as co-circulating cases with Genotype-III) was isolated in 2010 (JEV28, JEV21) and then in 2011 (JEV45) from Midnapur district, West Bengal (WB) for the first time from clinical patients who were previously been vaccinated with live attenuated SA14-14-2 strain. We apply bioinformatics and immunoinformatics on sequence and structure of E protein for analysis of crucial substitutions that might cause the genotypic transition, affecting protein-function and altering specificity of epitopes. Although frequency of substitutions in E glycoprotein of JEV28, JEV21 and JEV45 isolates vary, its homologous patterns remain exactly similar as earlier Japan isolate (Ishikawa). Sequence and 3D model-structure based analyses of E protein show that only four of all substitutions are critical for genotype-I specific effect of which N103K is common among all isolates indicating its role in the transition of genotype-III to genotype-I. Predicted B-cell and T-cell epitopes are seen to harbor these critical substitutions that affect overall conformational stability of the protein. These epitopes were subjected to conservation analyses using a large set of the protein from Asian continent. The study identifies crucial substitutions that contribute to the emergence of genotype-I. Predicted epitopes harboring these substitutions may alter specificity which might be the reason of reported failure of vaccine. Conservation analysis of these epitopes would be useful for design of genotype-I specific vaccine.
Bitencourt Chaves, Lana; Perce-da-Silva, Daiana de Souza; Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Cassiano, Gustavo Capatti; Machado, Ricardo Luiz Dantas; Pratt-Riccio, Lilian Rose; Banic, Dalma Maria; Lima-Junior, Josué da Costa
The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (pisolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate.
Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Waldenstrom Macroglobulinemia; Lymphocytosis; Lymphoma, Non-Hodgkin; B-Cell Chronic Lymphocytic Leukemia; Hematological Malignancies
Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.
Overlapping CD8+ and CD4+ T-cell epitopes identification for the progression of epitope-based peptide vaccine from nucleocapsid and glycoprotein of emerging Rift Valley fever virus using immunoinformatics approach.
Adhikari, Utpal Kumar; Rahman, M Mizanur
Rift Valley fever virus (RVFV) is an emergent arthropod-borne zoonotic infectious viral pathogen which causes fatal diseases in the humans and ruminants. Currently, no effective and licensed vaccine is available for the prevention of RVFV infection in endemic as well as in non-endemic regions. So, an immunoinformatics-driven genome-wide screening approach was performed for the identification of overlapping CD8+ and CD4+ T-cell epitopes and also linear B-cell epitopes from the conserved sequences of the nucleocapsid (N) and glycoprotein (G) of RVFV. We identified overlapping 99.39% conserved 1 CD8+ T-cell epitope (MMHPSFAGM) from N protein and 100% conserved 7 epitopes (AVFALAPVV, LAVFALAPV, FALAPVVFA, VFALAPVVF, IAMTVLPAL, FFDWFSGLM, and FLLIYLGRT) from G protein and also identified IL-4 and IFN-γ induced (99.39% conserved) 1 N protein CD4+ T-cell epitope (HMMHPSFAGMVDPSL) and 100% conserved 5 G protein CD4+ T-cell epitopes (LPALAVFALAPVVFA, PALAVFALAPVVFAE, GIAMTVLPALAVFAL, GSWNFFDWFSGLMSW, and FFLLIYLGRTGLSKM). The overlapping CD8+ and CD4+ T-cell epitopes were bound with most conserved HLA-C*12:03 and HLA-DRB1*01:01, respectively with the high binding affinity (kcal/mol). The combined population coverage analysis revealed that the allele frequencies of these epitopes are high in endemic and non-endemic regions. Besides, we found 100% conserved and non-allergenic 2 decamer B-cell epitopes, GVCEVGVQAL and RVFNCIDWVH of G protein had the sequence similarity with the nonamer CD8+ T-cell epitopes, VCEVGVQAL and RVFNCIDWV, respectively. Consequently, these epitopes may be used for the development of epitope-based peptide vaccine against emerging RVFV. However, in vivo and in vitro experiments are required for their efficient use as a vaccine. Copyright © 2017 Elsevier B.V. All rights reserved.
ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.
Guo, Le; Yang, Hua; Tang, Feng; Yin, Runting; Liu, Hongpeng; Gong, Xiaojuan; Wei, Jun; Zhang, Ying; Xu, Guangxian; Liu, Kunmei
that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.
Full Text Available Japanese encephalitis virus (JEV non-structural protein 1 (NS1 contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA, five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5AIDITRK(11, (72RDELNVL(78, (251KSKHNRREGY(260, (269DENGIVLD(276, and (341DETTLVRS(348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.
Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard
Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families.......Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individual...
Full Text Available B lymphocyte receptors are generated randomly during the bone marrow developmental phase of B cells. Hence, the B cell repertoire consists of both self and foreign antigen specificities necessitating specific tolerance mechanisms to eliminate self-reactive B cells. This review summarizes the major mechanisms of B cell tolerance, which include clonal deletion, anergy and receptor editing. In the bone marrow presentation of antigen in membrane bound form is more effective than soluble form and the role of dendritic cells in this process is discussed. Toll like receptor derived signals affect activation of B cells by certain ligands such as nucleic acids and have been shown to play crucial roles in the development of autoimmunity in several animal models. In the periphery availability of BAFF, a B cell survival factor plays a critical role in the survival of self-reactive B cells. Antibodies against BAFF have been found to be effective therapeutic agents in lupus like autoimmune diseases. Recent developments are targeting anergy to control the growth of chronic lymphocytic leukemia cells.
Hu, Yeguang; Yoshida, Toshimi; Georgopoulos, Katia
Loss of IKAROS in committed B cell precursors causes a block in differentiation while at the same time augments aberrant cellular properties, such as bone marrow stromal adhesion, self-renewal and resistance to glucocorticoid-mediated cell death. B cell acute lymphoblastic leukaemias originating from these early stages of B cell differentiation and associated with IKAROS mutations share a high-risk cellular phenotype suggesting that deregulation of IKAROS-based mechanisms cause a highly malignant disease process. Recent studies show that IKAROS is critical for the activity of super-enhancers at genes required for pre-B cell receptor (BCR) signalling and differentiation, working either downstream of or in parallel with B cell master regulators such as EBF1 and PAX5. IKAROS also directly represses a cryptic regulatory network of transcription factors prevalent in mesenchymal and epithelial precursors that includes YAP1, TEAD1/2, LHX2 and LMO2, and their targets, which are not normally expressed in lymphocytes. IKAROS prevents not only expression of these 'extra-lineage' transcription factors but also their cooperation with endogenous B cell master regulators, such as EBF1 and PAX5, leading to the formation of a de novo for lymphocytes super-enhancer network. IKAROS coordinates with the Polycomb repression complex (PRC2) to provide stable repression of associated genes during B cell development. However, induction of regulatory factors normally repressed by IKAROS starts a feed-forward loop that activates de-novo enhancers and elevates them to super-enhancer status, thereby diminishing PRC2 repression and awakening aberrant epithelial-like cell properties in B cell precursors. Insight into IKAROS-based transcriptional circuits not only sets new paradigms for cell differentiation but also provides new approaches for classifying and treating high-risk human B-ALL that originates from these early stages of B cell differentiation.
Heizmann, Beate [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Sellars, MacLean [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Macias-Garcia, Alejandra [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Institute for Medical Engineering and Science at MIT, Cambridge, MA 02139 (United States); Chan, Susan, E-mail: firstname.lastname@example.org [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Kastner, Philippe, E-mail: email@example.com [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Faculté de Médecine, Université de Strasbourg, Strasbourg (France)
The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.
Heizmann, Beate; Sellars, MacLean; Macias-Garcia, Alejandra; Chan, Susan; Kastner, Philippe
The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.
Gupta, Neetu; DeFranco, Anthony L
B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secrete pathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process them for presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasma membrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation including B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we review the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.
This report documents the methods used to collect samples and analyze data necessary to verify and/or determine the radionuclide content of the 324 Facility B-Cell decontamination and decommissioning waste stream.
Cooper, Max D
The separate development of functionally intertwined lineages of lymphocytes known as B cells and T cells is now recognized as a fundamental organizing principle of the adaptive immune system in all vertebrates. Immunologists strive to define the different sublineages of the clonally diverse B cells and T cells, how they interact with each other and how they interact with innate lymphoid cells and other elements of the innate immune system to counter infections, cancer and the development of autoimmune and inflammatory diseases. On the 50th anniversary of the recognition of B cells as a discrete cell lineage, this Timeline article recounts some of the milestones marking the development of the concept that B cells are a functionally and developmentally distinct arm of the adaptive immune system.
Pozsgay, Judit; Szarka, Eszter; Huber, Krisztina; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Sarmay, Gabriella
Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes.
LUCCHESE, A.; GUIDA, A.; CAPONE, G.; DONNARUMMA, G.; LAINO, L.; PETRUZZI, M.; SERPICO, R.; SILVESTRE, F.; GARGARI, M.
SUMMARY Purpose To identify potential antigenic targets for Porphyromonas gingivalis vaccine development. Materials and methods In the present study, we analyzed the Porphyromonas gingivalis, fimA type II primary amino acid sequence and characterized the similarity to the human proteome at the pentapeptide level. Results We found that exact peptide-peptide profiling of the fimbrial antigen versus the human proteome shows that only 19 out of 344 fimA type II pentapeptides are uniquely owned by the bacterial protein. Conclusions The concept that protein immunogenicity is allocated in rare peptide sequences and the search the Porphyromonas gingivalis fimA type II sequence for peptides unique to the bacterial protein and absent in the human host, might be used in new therapeutical approaches as a significant adjunct to current periodontal therapies. PMID:28042435
Simmons, Daimon P; Kafetzi, Maria L; Wood, Isabelle; Macaskill, Peter C; Milford, Edgar L; Guleria, Indira
HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies. Published by Elsevier Inc.
Fan, Shuhua; Wu, Yanan; Wang, Song; Wang, Zhenbao; Jiang, Bo; Liu, Yanjie; Liang, Ruiying; Zhou, Wenzhong; Zhang, Nianzhi; Xia, Chun
The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. SLA-3*hs0202 binding with sβ2m and a KMNTQFTAV (hemagglutinin [HA]-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune responses. Notably, we found that different HA-KMN9 conformations are caused, not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG), but also by the skewing of α1 and α2 helixes forming the PBG. In addition, alanine scanning and circular-dichroism (CD) spectra confirmed that the B, D, and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC-I) structures, the fundamental motif for SLA-3*hs0202 was determined to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine. We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previous findings showing that variable peptide conformations are caused only by the flexibility of the side chains in the groove
Sánchez-Burgos, Gilma; Ramos-Castañeda, José; Cedillo-Rivera, Roberto; Dumonteil, Eric
We used T cell epitope prediction tools to identify epitopes from Dengue virus polyprotein sequences, and evaluated in vivo and in vitro the immunogenicity and antigenicity of the corresponding synthetic vaccine candidates. Twenty-two epitopes were predicted to have a high affinity for MHC class I (H-2Kd, H-2Dd, H-2Ld alleles) or class II (IAd alleles). These epitopes were conserved between the four virus serotypes, but with no similarity to human and mouse sequences. Thirteen synthetic peptides induced specific antibodies production with or without T cells activation in mice. Three synthetic peptides induced mostly IgG antibodies, and one of these from the E gene induced a neutralizing response. Ten peptides induced a combination of humoral and cellular responses by CD4+ and CD8+ T cells. Twelve peptides were novel B and T cell epitopes. These results indicate that our bioinformatics strategy is a powerful tool for the identification of novel antigens and its application to human HLA may lead to a potent epitope-based vaccine against Dengue virus and many other pathogens. (c) 2010 Elsevier B.V. All rights reserved.
William S DeWitt
Full Text Available The vast diversity of B-cell receptors (BCR and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.
B lymphocyte differentiation is dependent on an intricate interplay of transcription factors and signaling pathways to establish a lineage-specific program of gene expression. Functional perturbations of several transcription factors by gain- or loss-of-function experiments indicated that E2A, EBF1, and FoxO1 are required for the specification of the B cell lineage, whereas Pax5 antagonizes alternative cell fates by repressing genes that allow for responsiveness to T lymphoid- and myeloid-promoting signals. However, genome-wide analysis of EBF1-binding sites and their functional interrogation indicated that EBF1 is involved in both activation of the B cell program and repression of alternative cell fates. Recent studies indicate that EBF1 function is required throughout the B cell lineage until the onset of plasma cell differentiation and includes a role in the maintenance of B cell identity. Thus, early B cell differentiation requires intertwined networks of transcription factors in which EBF1 collaborates with E2A and FoxO1 to activate the B lineage program and acts together with Pax5 to antagonize alternative cell fates. Copyright © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.
Becerra, E; De La Torre, I; Leandro, M J; Cambridge, G
Serum levels of B cell-activating factor (BAFF) rise following rituximab (RTX) therapy in patients with rheumatoid arthritis (RA). Initiation of naive B cell return to the periphery and autoreactive B cell expansion leading to relapse after RTX may therefore be linked to interactions between BAFF and BAFF-binding receptors (BBR). Relationships between serum BAFF and BBR expression [(BAFFR, calcium signal modulating cyclophilic ligand interactor (TACI) and B cell maturation antigen (BCMA)] were determined on B cell subsets, defined using immunoglobulin (Ig)D/CD38. Twenty pre-RTX and 18 RA patients relapsing after B cell depletion were included. Results were analysed with respect to timing of relapse up to 7 months after peripheral B cell return (≥ 5 B cells/μl) and to serum BAFF levels. After B cell return, B cell populations from relapsing patients had significantly lower BAFFR + expression compared to HC and pre-RTX patients. The percentage of BAFFR + B cells increased with time after B cell return and was correlated inversely with serum BAFF levels. BAFFR expression remained reduced. The percentage of TACI + memory B cells were lower in RA patients after RTX compared with healthy controls (HC). BCMA expression (% and expression) did not differ between patients and HC. Relapse following B cell return appeared largely independent of the percentage of BAFFR + or percentage of BCMA + B cells or serum BAFF levels. The lower percentage of TACI + memory B cells may reduce inhibitory signalling for B cell differentiation. In patients relapsing at longer periods after B cell return, recovery of the B cell pool was more complete, suggesting that selection or expansion of autoreactive B cells may be needed to precipitate relapse. © 2017 British Society for Immunology.
Borhis, Gwenoline; Trovato, Maria; Chaoul, Nada; Ibrahim, Hany M.; Richard, Yolande
With the goal to design effective HIV vaccines, intensive studies focused on broadly neutralizing antibodies, which arise in a fraction of HIV-infected people. Apart from identifying new vulnerability sites in the viral envelope proteins, these studies have shown that a fraction of these antibodies are produced by self/poly-reactive B-cells. These findings prompted us to revisit the B-cell differentiation and selection process during HIV/SIV infection and to consider B-cells as active players possibly shaping the helper T-cell program within germinal centers (GCs). In this context, we paid a particular attention to B-cell-activating factor (BAFF), a key cytokine in B-cell development and immune response that is overproduced during HIV/SIV infection. As it does in autoimmune diseases, BAFF excess might contribute to the abnormal rescue of self-reactive B-cells at several checkpoints of the B-cell development and impair memory B-cell generation and functions. In this review, we first point out what is known about the functions of BAFF/a proliferation-inducing ligand and their receptors [B-cell maturation, transmembrane activator and CAML interactor (TACI), and BAFF-R], in physiological and pathophysiological settings, in mice and humans. In particular, we highlight recent results on the previously underappreciated regulatory functions of TACI and on the highly regulated production of soluble TACI and BAFF-R that act as decoy receptors. In light of recent data on BAFF, TACI, and BAFF-R, we then revisit the altered phenotypes and functions of B-cell subsets during the acute and chronic phase of HIV/SIV infection. Given the atypical phenotype and reduced functions of memory B-cells in HIV/SIV infection, we particularly discuss the GC reaction, a key checkpoint where self-reactive B-cells are eliminated and pathogen-specific memory B-cells and plasmablasts/cells are generated in physiological settings. Through its capacity to differentially bind and process BAFF-R and
Full Text Available Abstract Background Untreated human immunodeficiency virus (HIV disease disrupts B cell populations causing reduced memory and reduced naïve resting B cells leading to increases in specific co-infections and impaired responses to vaccines. To what extent antiretroviral treatment reverses these changes in an African population is uncertain. Methods A cross-sectional study was performed. We recruited HIV-uninfected and HIV-infected Malawian adults both on and off antiretroviral therapy attending the Queen Elizabeth Central hospital in Malawi. Using flow cytometry, we enumerated B cells and characterized memory B cells and compared these measurements by the different recruitment groups. Results Overall 64 participants were recruited - 20 HIV uninfected (HIV-, 30 HIV infected ART naïve (HIV+N and 14 HIV-infected ART treated (HIV+T. ART treatment had been taken for a median of 33 months (Range 12-60 months. Compared to HIV- the HIV+N adults had low absolute number of naïve resting B cells (111 vs. 180 cells/μl p = 0.008; reduced memory B cells (27 vs. 51 cells/μl p = 0.0008. The HIV+T adults had B-cell numbers similar to HIV- except for memory B cells that remained significantly lower (30 vs. 51 cells/μl p = 0.02. In the HIV+N group we did not find an association between CD4 count and B cell numbers. Conclusions HIV infected Malawian adults have abnormal B-cell numbers. Individuals treated with ART show a return to normal in B-cell numbers but a persistent deficit in the memory subset is noted. This has important implications for long term susceptibility to co-infections and should be evaluated further in a larger cohort study.
Weisel, Florian; Shlomchik, Mark
We comprehensively review memory B cells (MBCs), covering the definition of MBCs and their identities and subsets, how MBCs are generated, where they are localized, how they are maintained, and how they are reactivated. Whereas naive B cells adopt multiple fates upon stimulation, MBCs are more restricted in their responses. Evolving work reveals that the MBC compartment in mice and humans consists of distinct subpopulations with differing effector functions. We discuss the various approaches to define subsets and subset-specific roles. A major theme is the need to both deliver faster effector function upon reexposure and readapt to antigenically variant pathogens while avoiding burnout, which would be the result if all MBCs generated only terminal effector function. We discuss cell-intrinsic differences in gene expression and signaling that underlie differences in function between MBCs and naive B cells and among MBC subsets and how this leads to memory responses.
Jackson, Shaun W; Scharping, Nicole E; Jacobs, Holly M; Wang, Shari; Chait, Alan; Rawlings, David J
Patients with systemic lupus erythematosus exhibit accelerated atherosclerosis, a chronic inflammatory disease of the arterial wall. The impact of B cells in atherosclerosis is controversial, with both protective and pathogenic roles described. For example, natural IgM binding conserved oxidized lipid epitopes protect against atherosclerosis, whereas anti-oxidized low-density lipoprotein (oxLDL) IgG likely promotes disease. Because BAFF promotes B cell class-switch recombination and humoral autoimmunity, we hypothesized that excess BAFF would accelerate atherosclerosis. In contrast, BAFF overexpression markedly reduced hypercholesterolemia and atherosclerosis in hyperlipidemic mice. BAFF-mediated atheroprotection required B cells and was associated with increased protective anti-oxLDL IgM. Surprisingly, high-titer anti-oxLDL IgM production and reduced atherosclerosis was dependent on the BAFF family receptor transmembrane activator and CAML interactor. In summary, we identified a novel role for B cell-specific, BAFF-dependent transmembrane activator and CAML interactor signals in atherosclerosis pathogenesis, of particular relevance to the use of BAFF-targeted therapies in systemic lupus erythematosus. Copyright © 2016 by The American Association of Immunologists, Inc.
Saulep-Easton, Damien; Vincent, Fabien B.; Quah, Pin Shie; Wei, Andrew; Ting, Stephen B.; Croce, Carlo M.; Tam, Constantine; Mackay, Fabienne
Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory players with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B cell-activating factor of the tumour necrosis factor (TNF) family (BAFF) is critical for B cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing B regulatory cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor TACI. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL. PMID:26139429
Full Text Available Persistent polyclonal B cell lymphocytosis (PPBL is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19+CD27+IgM+ memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.
Full Text Available Objective: Multiple sclerosis (MS is a central nervous system (CNS disease characterized by autoimmune inflammation and neurodegeneration. Damage to the CNS is thought to be mediated predominantly by activated pro-inflammatory T cells and antibody secreting B cells. Strong evidence of B cell functions in MS pathogenesis has come from trials of B cell- depleting treatment. In this study, the peripheral blood frequencies of B cell subsets were measured using flow cytometry in patients to determine the disease-specific B cell differences that might be associated with the evolution to progressive forms of MS. Materials and Methods: Peripheral blood mononuclear cells were separated from patients and healthy controls [relapsing-remitting MS (RRMS and secondary progressive MS (SPMS]. Cells were stained with anti-human monoclonal antibodies (CD19-APC, CD27-FITC, IgD-APC/Cy7, CD138-PE, CD24-PerCP and CD38-Alexa fluor 700, and analyzed using flow cytometry Results: There were no significant differences between the MS group and healthy controls by means of peripheral blood frequencies of B cells, immature, naïve, classic memory, plasma, plasmablasts, and regulatory B cells. Only higher naïve B cell frequency tendency was determined in patients with RRMS as compared with patients with SPMS and healthy controls. Conclusion: Peripheral blood B cell subset measurements are not likely to be used as a biomarker for prediction of disease progression. Although B cells have a well-known pathogenic significance, B cell population alterations do not occur during the progression of the disease
Babakhin, A A; Gushchin, I S; Andreev, S M; Petrukhina, A I; Viler, A V; Stokinger, B; Nolte, G; Dubuske, L M; Khaitov, R M; Petrpv, R V
Modification of a model allergen ovalbumin (OA) with succinylation led to a decrease of its allergenicity measured by passive cutaneous anaphylaxis reaction, RAST inhibition assay and basophil histamine release. Modified OA stimulated OA-specific T-cell hybrid 3DO-548 to produce IL-2 at the same level as in case of non-modified OA. Modified OA did not induce anti-OA IgE, but did induce anti-OA IgG antibodies. This approach to chemical modification of allergen-selective blockade of B-cell epitopes while not affecting T-cell epitopes suggests new opportunities in creation of safe and effective allergovaccines.
Unizony, Sebastian; Lim, Noha; Phippard, Deborah J.; Carey, Vincent J.; Miloslavsky, Eli M.; Tchao, Nadia K.; Iklé, David; Asare, Adam L.; Merkel, Peter A.; Monach, Paul A.; Seo, Philip; St Clair, E. William; Langford, Carol A.; Spiera, Robert; Hoffman, Gary S.; Kallenberg, Cees G. M.; Specks, Ulrich; Stone, John H.
Objective CD5+ B cells have been conceptualized as a possible surrogate for Breg cells. The aim of the present study was to determine the utility of CD5+ B cells as biomarkers in antineutrophil cytoplasmic antibody–associated vasculitis (AAV). Methods The absolute and relative numbers (percentages) of CD5+ B cells (explanatory variables) were measured longitudinally during 18 months in 197 patients randomized to receive either rituximab (RTX) or cyclophosphamide (CYC) followed by azathioprine (AZA) for the treatment of AAV (Rituximab in ANCA-Associated Vasculitis [RAVE] trial). Outcome variables included disease activity (status of active disease versus complete remission), responsiveness to induction therapy, disease relapse, disease severity, and, in RTX-treated patients, relapse-free survival according to the percentage of CD5+ B cells detected upon B cell repopulation. Results CD5+ B cell numbers were comparable between the treatment groups at baseline. After an initial decline, absolute CD5+ B cell numbers progressively increased in patients in the RTX treatment arm, but remained low in CYC/AZA-treated patients. In both groups, the percentage of CD5+ B cells increased during remission induction and slowly declined thereafter. During relapse, the percentage of CD5+ B cells correlated inversely with disease activity in RTX-treated patients, but not in patients who received CYC/AZA. No significant association was observed between the numbers of CD5+ B cells and induction treatment failure or disease severity. The dynamics of the CD5+ B cell compartment did not anticipate disease relapse. Following B cell repopulation, the percentage of CD5+ B cells was not predictive of time to flare in RTX-treated patients. Conclusion The percentage of peripheral CD5+ B cells might reflect disease activity in RTX-treated patients. However, sole staining for CD5 as a putative surrogate marker for Breg cells did not identify a subpopulation of B cells with clear potential for
Tyler K Nygaard
Full Text Available Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen produces numerous molecules that can interfere with recognition and binding by host innate immune cells, an initial step required for the ingestion and subsequent destruction of microbes by phagocytes. To better understand the interaction of this pathogen with human immune cells, we compared the association of S. aureus and S. epidermidis with leukocytes in human blood. We found that a significantly greater proportion of B cells associated with S. epidermidis relative to S. aureus. Complement components and complement receptors were important for the binding of B cells with S. epidermidis. Experiments using staphylococci inactivated by ultraviolet radiation and S. aureus isogenic deletion mutants indicated that S. aureus secretes molecules regulated by the SaeR/S two-component system that interfere with the ability of human B cells to bind this bacterium. We hypothesize that the relative inability of B cells to bind S. aureus contributes to the microbe's success as a human pathogen.
Butler, J. E.; Zhao, Y.; Šinkora, Marek; Wertz, N.; Kacskovics, I.
Roč. 33, č. 3 (2009), s. 321-333 ISSN 0145-305X R&D Projects: GA ČR GA523/07/0088 Institutional research plan: CEZ:AV0Z50200510 Keywords : swine * immunoglobulin * b cell Subject RIV: EC - Immunology Impact factor: 3.290, year: 2009
Nygaard, Tyler K.; Kobayashi, Scott D.; Freedman, Brett; Porter, Adeline R.; Voyich, Jovanka M.; Otto, Michael; Schneewind, Olaf; DeLeo, Frank R.
Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen produces numerous molecules that can interfere with recognition and binding by host innate immune cells, an initial step required for the ingestion and subsequent destruction of microbes by phagocytes. To better understand the interaction of this pathogen with human immune cells, we compared the association of S. aureus and S. epidermidis with leukocytes in human blood. We found that a significantly greater proportion of B cells associated with S. epidermidis relative to S. aureus. Complement components and complement receptors were important for the binding of B cells with S. epidermidis. Experiments using staphylococci inactivated by ultraviolet radiation and S. aureus isogenic deletion mutants indicated that S. aureus secretes molecules regulated by the SaeR/S two-component system that interfere with the ability of human B cells to bind this bacterium. We hypothesize that the relative inability of B cells to bind S. aureus contributes to the microbe’s success as a human pathogen. PMID:27711145
Pan, Keyao; Deem, Michael W.
The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high-affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a 'microscopic' random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principle calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment.
Jill M Brooks
Full Text Available Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501, as well as subdominant responses through common class I alleles (e.g. B7 and C*0304. Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.
Lee, Ho-Bin; Piao, Da-Chuan; Lee, Jun-Yeong; Choi, Jae-Yun; Bok, Jin-Duck; Cho, Chong-Su; Kang, Sang-Kee; Choi, Yun-Jaie
Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.
Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.
Vijayakumar, Subramaniyan; Ramesh, Venkatachalam; Prabhu, Srinivasan; Manogar, Palani
Reverse vaccinology method was used to predict the monovalent peptide vaccine candidate to produce antibodies for therapeutic purpose and to predict tetravalent vaccine candidate to act as a common vaccine to cover all the fever dengue virus serotypes. Envelope (E)-proteins of DENV-1-4 serotypes were used for vaccine prediction using NCBI, Uniprot/Swissprot, Swiss-prot viewer, VaxiJen V2.0, TMHMM, BCPREDS, Propred-1, Propred and MHC Pred,. E-proteins of DENV-1-4 serotypes were identified as antigen from which T cell epitopes, through B cell epitopes, were predicted to act as peptide vaccine candidates. Each selected T cell epitope of E-protein was confirmed to act as vaccine and to induce complementary antibody against particular serotype of dengue virus. Chimeric tetravalent vaccine was formed by the conjugation of four vaccines, each from four dengue serotypes to act as a common vaccine candidate for all the four dengue serotypes. It can be justifiably concluded that the monovalent 9-mer T cell epitope for each DENV serotypes can be used to produce specific antibody agaomst dengue virus and a chimeric common tetravalent vaccine candidate to yield a comparative vaccine to cover any of the four dengue virus serotype. This vaccine is expected to act as highly immunogenic against preventing dengue fever.
Kam Leng Aw-Yong
Full Text Available Enterovirus A71 (EV-A71 is one of the main causative agents of hand, foot and mouth disease (HFMD. Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4, and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41-55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.
Full Text Available Adipose tissue surrounding major arteries (Perivascular adipose tissue or PVAT has long been thought to exist to provide vessel support and insulation. Emerging evidence suggests that PVAT regulates artery physiology and pathology, such as, promoting atherosclerosis development through local production of inflammatory cytokines. Yet the immune subtypes in PVAT that regulate inflammation are poorly characterized. B cells have emerged as important immune cells in the regulation of visceral adipose tissue inflammation and atherosclerosis. B cell-mediated effects on atherosclerosis are subset-dependent with B-1 cells attenuating and B-2 cells aggravating atherosclerosis. While mechanisms whereby B-2 cells aggravate atherosclerosis are less clear, production of immunoglobulin type M (IgM antibodies is thought to be a major mechanism whereby B-1 cells limit atherosclerosis development. B-1 cell-derived IgM to oxidation specific epitopes (OSE on low density lipoproteins (LDL blocks oxidized LDL-induced inflammatory cytokine production and foam cell formation. However, whether PVAT contains B-1 cells and whether atheroprotective IgM is produced in PVAT is unknown. Results of the present study provide clear evidence that the majority of B cells in and around the aorta are derived from PVAT. Interestingly, a large proportion of these B cells belong to the B-1 subset with the B-1/B-2 ratio being 10-fold higher in PVAT relative to spleen and bone marrow. Moreover, PVAT contains significantly greater numbers of IgM secreting cells than the aorta. ApoE−/− mice with B cell-specific knockout of the gene encoding the helix-loop-helix factor Id3, known to have attenuated diet-induced atherosclerosis, have increased numbers of B-1b cells and increased IgM secreting cells in PVAT relative to littermate controls. Immunostaining of PVAT on human coronary arteries identified fat associated lymphoid clusters (FALCs harboring high numbers of B cells, and flow
da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S
Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.
Angeletti, Davide; Gibbs, James S; Angel, Matthew; Kosik, Ivan; Hickman, Heather D; Frank, Gregory M; Das, Suman R; Wheatley, Adam K; Prabhakaran, Madhu; Leggat, David J; McDermott, Adrian B; Yewdell, Jonathan W
Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.
Tafalla, Carolina; González, Lucia; Castro, Rosario; Granja, Aitor G.
In mammals, B cell functionality is greatly influenced by cytokines released by innate cells, such as macrophages or dendritic cells, upon the early recognition of common pathogen patterns through invariant receptors. B cell-activating factor (BAFF) is one of these innate B cell-helper signals and plays a key role in the survival and differentiation of B cells. Although, evolutionarily, teleost fish constitute the first animal group in which adaptive immunity based on Ig receptors is present, fish still rely greatly on innate responses. In this context, we hypothesized that BAFF would play a key role in the control of B cell responses in fish. Supporting this, our results show that teleost BAFF recapitulates mammalian BAFF stimulating actions on B cells, upregulating the expression of membrane MHC II, improving the survival of fish naïve B cells and antibody-secreting cells, and increasing the secretion of IgM. Surprisingly, we also demonstrate that BAFF is not only produced in fish by myeloid cells but is also produced by a subset of splenic B cells. Thus, if this B cell-produced BAFF proves to be actively regulating this same B cell subset, our findings point to an ancient mechanism to control B cell differentiation and survival in lower vertebrates, which has been silenced in mammals in physiological conditions, but reemerges under pathological conditions, such as B cell lymphomas and autoimmune diseases. PMID:28360916
Full Text Available Diffuse large B-cell lymphoma is the most common form of lymphoma. It usually begins in the lymph nodes; up to 40% may have an extranodal presentation. According to a definition of primary extranodal lymphoma with presentation only in extranodal sites, there are reports of large B-cell lymphomas limited to liver or spleen as separate entities, and to date there have been only three documented cases of primary hepatosplenic presentation. This paper reports a fourth case. Due to a review of the literature and the clinical course of the case reported, we conclude that primary hepatosplenic large B-cell lymphoma has been found predominantly in females older than 60 years. The patients reported had <2 months of evolution prior to diagnosis, prominent B symptoms, splenomegaly in three and hepatomegaly in two, none with lymph node involvement. All had thrombocytopenia and abnormal liver function tests; three had anemia and elevated serum lactic dehydrogenase levels, two with hemophagocytosis in bone marrow. Because of the previously mentioned data, it can be stated that primary hepatosplenic lymphoma is an uncommon and aggressive form of disease that requires immediate recognition and treatment.
Xie, Li-Jun; Cui, Zhao; Chen, Fang-Jin; Pei, Zhi-Yong; Hu, Shui-Yi; Gu, Qiu-Hua; Jia, Xiao-Yu; Zhu, Li; Zhou, Xu-Jie; Zhang, Hong; Liao, Yun-Hua; Lai, Lu-Hua; Hudson, Billy G; Zhao, Ming-Hui
Goodpasture's disease is closely associated with HLA, particularly DRB1*1501. Other susceptible or protective HLA alleles are not clearly elucidated. The presentation models of epitopes by susceptible HLA alleles are also unclear. We genotyped 140 Chinese patients and 599 controls for four-digit HLA II genes, and extracted the encoding sequences from the IMGT/HLA database. T-cell epitopes of α3(IV)NC1 were predicted and the structures of DR molecule-peptide-T-cell receptor were constructed. We confirmed DRB1*1501 (OR = 4·6, P = 5·7 × 10 -28 ) to be a risk allele for Goodpasture's disease. Arginine at position 13 (ARG13) (OR = 4·0, P = 1·0 × 10 -17 ) and proline at position 11 (PRO11) (OR = 4·0, P = 2·0 × 10 -17 ) on DRβ1, encoded by DRB1*1501, were associated with disease susceptibility. α 134-148 (HGWISLWKGFSFIMF) was predicted as a T-cell epitope presented by DRB1*1501. Isoleucine 137 , tryptophan 140 , glycine 142 , phenylalanine 143 and phenylalanine 145 , were presented in peptide-binding pockets 1, 4, 6, 7 and 9 of DR2b, respectively. ARG13 in pocket 4 interacts with tryptophan 140 and forms a hydrogen bond. In conclusion, we propose a mechanism for DRB1*1501 susceptibility for Goodpasture's disease through encoding ARG13 and PRO11 on MHC-DRβ1 chain and presenting T-cell epitope, α 134-148 , with five critical residues. © 2017 John Wiley & Sons Ltd.
Engmark, Mikael; Andersen, Mikael Rørdam; Laustsen, Andreas Hougaard
Insight into the molecular details of polyclonal antivenom antibody specificity is a prerequisite for accurate prediction of cross-reactivity and can provide a basis for design of novel antivenoms. In this work, a highthroughput approach was applied to characterize linear elements in epitopes in 82...... toxins from four African mamba and three neurotoxic cobra snakes obtained from public databases....
Oyarzun, P; Kobe, B
Epitope-based vaccines (EVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant - immunogenic and conserved - antigen regions. Experimental screening of large sets of peptides is time-consuming and costly; therefore, in silico methods that facilitate T-cell epitope mapping of protein antigens are paramount for EV development. The prediction of T-cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T-cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T-cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state-of-the-art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens. © 2015 John Wiley & Sons Ltd.
Giuseppina Li Pira
Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.
Full Text Available BAFF is a potent B-cell survival factor, and it plays an essential role in B-cell homeostasis and B-cell function in the periphery. Both normal and autoreactive B cells are BAFF dependent; however, excess BAFF promotes the survival, growth, and maturation of autoreactive B cells. When overexpressed, BAFF protects B cells from apoptosis, thereby contributing to autoimmunity. Three independent studies have shown higher BAFF levels in the circulation of MG patients. BAFF may play an important role in the pathogenesis of MG. BAFF antagonists may well provide new treatment options for MG patients, particularly those patients with thymic lymphoid follicular hyperplasia.
kill the infected cells. The focus of my PhD project has been on improving a method for CTL epitope pathway prediction, on analyzing the epitope density in the alternative cancer exome, and on a study investigating minor histocompatibility antigens (mHags) associated with leukemia. Part I......The human immune system is a highly adaptable system, defending our bodies against pathogens and tumor cells. Cytotoxic T cells (CTL) are cells of the adaptive immune system, capable of inducing a programmed cell death and thus able to eliminate infected or tumor cells. CTLs discriminate between...... healthy and infected cells based on peptide fragments presented on the cells surface. All nucleated cells present these peptide fragments in complex with Major Histocompatibility Complex (MHC) class I molecules. Peptides that are recognized by CTLs are called epitopes and induce the CTLs to subsequently...
Full Text Available The germinal center (GC is a dynamic microenvironment where antigen (Ag-activated B cells rapidly expand and differentiate, generating plasma cells (PC that produce high-affinity antibodies. Precise regulation of survival and proliferation of Ag-activated B cells within the GC is crucial for humoral immune responses. The follicular dendritic cells (FDC are the specialized stromal cells in the GC that prevent apoptosis of GC-B cells. Recently, we reported that human GC-B cells consist of CD9+ and CD9− populations and that it is the CD9+ cells that are committed to the PC lineage. In this study, we investigated the functional role of CD9 on GC-B cells. Tonsillar tissue section staining revealed that in vivo CD9+ GC-B cells localized in the light zone FDC area. Consistent this, in vitro CD9+ GC-B cells survived better than CD9− GC-B cells in the presence of HK cells, an FDC line, in a cell–cell contact-dependent manner. The frozen tonsillar tissue section binding assay showed that CD9+ GC-B cells bound to the GC area of tonsillar tissues significantly more than the CD9− GC-B cells did and that the binding was significantly inhibited by neutralizing anti-integrin β1 antibody. Furthermore, CD9+ cells bound to soluble VCAM-1 more than CD9− cells did, resulting in activation and stabilization of the active epitope of integrin β1. All together, our data suggest that CD9 on GC-B cells contributes to survival by strengthening their binding to FDC through the VLA4/VCAM-1 axis.
Li, Junxin; Sun, Wenji; Subrahmanyam, Priyanka B; Page, Carly; Younger, Kenisha M; Tiper, Irina V; Frieman, Matthew; Kimball, Amy S; Webb, Tonya J
Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.
Al Kindi, M; Mundy, J; Sullivan, T; Smith, W; Kette, F; Smith, A; Heddle, R; Hissaria, P
Abnormalities in peripheral blood B cell subsets have been identified in common variable immunodeficiency (CVID) patients and classification systems based upon their numbers have been proposed to predict the clinical features. We analysed B lymphocyte subsets by multi-colour flow cytometry (MFC) in a cohort of well-characterized CVID patients to look at their clinical relevance and validate the published association of different classification criteria (Freiburg, Paris and Euroclass) with clinical manifestations. CVID patients had a reduced proportion of total and switched memory B cells (MBC, swMBC) compared to normal controls (P < 0·0006). Patients classified in Freiburg Ia had a higher prevalence of granulomatous diseases (P = 0·0034). The previously published associations with autoimmune diseases could not be confirmed. The Euroclass classification was not predictive of clinical phenotypes. The absolute numbers of all B cell subsets were reduced in CVID patients compared to controls. There was a significant linear correlation between low absolute total B cells and MBC with granulomatous disease (P < 0·05) and a trend towards lower B cells in patients with autoimmune diseases (P = 0·07). Absolute number of different B cell subsets may be more meaningful than their relative percentages in assessing the risk of granulomatous diseases and possibly autoimmunity. PMID:22236004
Padmaja, Radhakrishnan Jayasree; Halami, Prakash Motiram
Leukotoxin M/F'-PV (LukM/F'-PV) produced by bovine mastitis causing Staphylococcus aureus structurally comprises three domains, the β-sandwich, rim and stem domain. The rim and stem domains interacting with target cell membrane lipid rafts contributes to the virulent trait of the toxin. In the present study, two facts were hypothesized that neutralization of these domains will ebb LukM/F'-PV leukotoxicity. Secondly, the neutralizing antibodies can improve the leukotoxin detection sensitivity in bovine mastitis milk samples. The in silico mapping of S. aureus LukM C-termini comprising these domains predicted seven linear B-cell antigenic epitopes. The immune response of C-terminal truncated recombinant peptides rCtM19 (19 kDa; near carboxy-terminal) having four epitopes and rCtM15 (15 kDa; C-terminal) with three epitopes were evaluated for their diagnostic and neutralization potential. Anti-rCtM19 and anti-rCtM15 antibodies with enhanced immunogenicity had the most striking outcome in IgG-ELISA for detecting native determinants of leukotoxin. For the obtained ELISA values, ROC curve inferred a cut-off score of >0.102 OD405. The assay sensitivity in the range of 90-96% along with 100% specificity and AUC of 0.93-0.98 categorized subclinical and clinical from healthy bovine milk samples. As observed through in vitro neutralization and LDH assays, C-terminus specific antibodies (1:42 titer) deactivating leukotoxicity abolished LukM from interacting with lipid bilayer and LukF for forming pores on bovine neutrophil membrane. As a proof of concept, it was proved that peptide antibodies can be a more specific serodiagnostic and passive therapeutic molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.
Full Text Available Clinical studies of B cell depletion in Multiple Sclerosis (MS have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.Activation-Induced Deaminase (AID is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID(-/- mice with the extracellular domain (amino acids 1-120 of recombinant human MOG protein (rhMOG and examined the incidence and severity of disease in AID(-/- versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab.AID(-/- mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.
Martelli, Maurizio; Ferreri, Andrés; Di Rocco, Alice; Ansuinelli, Michela; Johnson, Peter W M
Primary mediastinal large B-cell lymphoma (PMLBCL) is a distinct clinical and biological disease from other types of DLBCL. It is more frequent in young female and constitutes 6%-10% of all DLBCL. PMLBCL is characterized by a diffuse proliferation of medium to large B-cells associated with sclerosis. Molecular analysis shows it to be a distinct entity from other DLBCL. Rituximab CHOP/MACOP-B-like regimens followed by mediastinal radiotherapy (RT) were associated with a 5-years PFS of 75%-85%. More intensive regimens, as DA-EPOCH-R without mediastinal RT, have shown very promising results, but this therapeutic advance needs to be confirmed in further prospective trials. The role of consolidative mediastinal RT should be still better assess in prospective comparative studies. PET-CT scan is a powerful tool to define the real quality of response and it is hoped that future prospective trials may allow its role in the de-escalation of mediastinal RT. Copyright © 2017 Elsevier B.V. All rights reserved.
Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes
Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics
Tridandapani, S; Kelley, T; Cooney, D; Pradhan, M; Coggeshall, K M
Negative signaling in B cells is initiated by co-crosslinking of the antigen receptor and the Fcy receptor, resulting in cessation of B-cell signaling events and, in turn, inhibiting B-cell proliferation and antibody secretion. Here, a competitive role is proposed for SHIP in blocking the interaction of Shc with the Grb2-Sos complex of proteins that lead to Ras activation in B cells.
In celebration of the 50th anniversary of the discovery of B cells, I take a look back at the history of T cell help to B cells, which was discovered 47 years ago. In addition, I summarize and categorize the distinct molecules that are expressed by CD4(+) T cells that constitute 'help' to B cells, and particularly the molecules expressed by T follicular helper (TFH) cells, which are the specialized providers of help to B cells.
Full Text Available The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.
Taylor, G M; Wade, R; Hussain, A; Thompson, P; Hann, I; Gibson, B; Eden, T; Richards, S
We previously reported that children in the UKALL XI ALL trial with HLA-DP 1 and -DP 3 supertypes had significantly worse event-free survival (EFS) than children with other DP supertypes. As DP 1 and DP 3 share two of four key antigen-binding amino-acid polymorphisms (aspartic acid84-lysine69), we asked whether Asp84-Lys69 or Asp84 alone were independent prognostic indicators in childhood acute lymphoblastic leukemia (ALL). We analysed EFS in 798 UKALL XI patients, stratified by Asp84-Lys69 vs non-Asp84-Lys69, for a median follow-up of 12.5 years. Asp84-Lys69 was associated with a significantly worse EFS than non-Asp84-Lys69 (5-year EFS: Asp84-Lys69: 58.8% (95% CI (confidence of interval): 52.7-64.9%); non-Asp84-Lys69: 67.3% (63.4-71.2%); 2P=0.007). Post-relapse EFS was 10% less in Asp84-Lys69 than non-Asp84-Lys69 patients. EFS was significantly worse (P=0.03) and post-relapse EFS marginally worse (P=0.06) in patients with Asp84 compared with Gly84. These results suggest that Asp84-Lys69 predicted adverse EFS in the context of UKALL XI because of Asp84, and may have influenced post-relapse EFS. We speculate that this may be due to the recruitment of Asp84-Lys69-restricted regulatory T cells in the context of this regimen, leading to the re-emergence of residual disease. However, functional and molecular studies of the prognostic value of this and other HLA molecular signatures in other childhood ALL trials are needed.
Maria S. Khan MD, FACP
Full Text Available Case Presentation . A 69-year-old Hispanic male, with a past history of diabetes and coronary disease, was admitted for fever, diarrhea, and confusion of 4 weeks duration. Physical examination showed a disoriented patient with multiple ecchymoses, possible ascites, and bilateral scrotal swelling. Hemoglobin was 6.7, prothrombin time (PT 21.4 seconds with international normalized ratio 2.1, partial thromboplastin time (PTT 55.6 seconds, fibrin split 10 µg/L, and lactate dehydrogenase (LDH 1231 IU/L. Except for a positive DNA test for Epstein–Barr virus (EBV infection, extensive diagnostic workup for infections, malignancy, or a neurological cause was negative. Mixing studies revealed a nonspecific inhibitor of PT and PTT but Factor VIII levels were normal. The patient was empirically treated with antibiotics but developed hypotension and died on day 27 of admission. At autopsy, patient was found to have intravascular diffuse large B-cell lymphoma involving skin, testes, lung, and muscles. The malignant cells were positive for CD20, CD791, Mum-1, and Pax-5 and negative for CD3, CD5, CD10, CD30, and Bcl-6. The malignant cells were 100% positive for Ki-67. Discussion . Intravascular large cell B-cell lymphoma (IVLBCL is rare form of diffuse large B-cell lymphoma and tends to proliferate within small blood vessels, particularly capillaries and postcapillary venules. The cause of its affinity for vascular bed remains unknown. In many reports, IVLBCL was associated with HIV, HHV8, and EBV infections. The fact that our case showed evidence of EBV infection lends support to the association of this diagnosis to viral illness. The available literature on this subject is scant, and in many cases, the diagnosis was made only at autopsy. The typical presentation of this disorder is with B symptoms, progressive neurologic deficits, and skin findings. Bone marrow, spleen, and liver are involved in a minority of patients. Nearly all patients have elevated LDH
Veronika I Zarnitsyna
Full Text Available The development of next-generation influenza vaccines that elicit strain-transcendent immunity against both seasonal and pandemic viruses is a key public health goal. Targeting the evolutionarily conserved epitopes on the stem of influenza's major surface molecule, hemagglutinin, is an appealing prospect, and novel vaccine formulations show promising results in animal model systems. However, studies in humans indicate that natural infection and vaccination result in limited boosting of antibodies to the stem of HA, and the level of stem-specific antibody elicited is insufficient to provide broad strain-transcendent immunity. Here, we use mathematical models of the humoral immune response to explore how pre-existing immunity affects the ability of vaccines to boost antibodies to the head and stem of HA in humans, and, in particular, how it leads to the apparent lack of boosting of broadly cross-reactive antibodies to the stem epitopes. We consider hypotheses where binding of antibody to an epitope: (i results in more rapid clearance of the antigen; (ii leads to the formation of antigen-antibody complexes which inhibit B cell activation through Fcγ receptor-mediated mechanism; and (iii masks the epitope and prevents the stimulation and proliferation of specific B cells. We find that only epitope masking but not the former two mechanisms to be key in recapitulating patterns in data. We discuss the ramifications of our findings for the development of vaccines against both seasonal and pandemic influenza.
Mirza, Osman Asghar; Henriksen, A; Ipsen, H
leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16......, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2...... as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order...
Pelzek, Adam J; Shopsin, Bo; Radke, Emily E; Tam, Kayan; Ueberheide, Beatrix M; Fenyö, David; Brown, Stuart M; Li, Qianhao; Rubin, Ada; Fulmer, Yi; Chiang, William K; Hernandez, David N; El Bannoudi, Hanane; Sause, William E; Sommerfield, Alexis; Thomsen, Isaac P; Miller, Andy O; Torres, Victor J; Silverman, Gregg J
Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureus possesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureus isolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureus antigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureus antigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti- S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses. IMPORTANCE The contribution of B-cell memory and long-term antibody responses to host defenses against S. aureus exotoxins remains poorly understood. Our studies
Souwer, Yuri; Griekspoor, Alexander; Jorritsma, Tineke; de Wit, Jelle; Janssen, Hans; Neefjes, Jacques; van Ham, S. Marieke
The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and
Kwakkenbos, Mark J.; Diehl, Sean A.; Yasuda, Etsuko; Bakker, Arjen Q.; van Geelen, Caroline M. M.; Lukens, Michaël; van Bleek, Grada M.; Widjojoatmodjo, Myra N.; Bogers, Willy M. J. M.; Mei, Henrik; Radbruch, Andreas; Scheeren, Ferenc A.; Spits, Hergen; Beaumont, Tim
The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced
Kwakkenbos, Mark J.; Diehl, Sean A.; Yasuda, Etsuko; Bakker, Arjen Q.; van Geelen, Caroline M. M.; Lukens, Michaël V.; van Bleek, Grada M.; Widjojoatmodjo, Myra N.; Bogers, Willy M. J. M.; Mei, Henrik; Radbruch, Andreas; Scheeren, Ferenc A.; Spits, Hergen; Beaumont, Tim
The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced
Hodson, Daniel J; Shaffer, Arthur L; Xiao, Wenming; Wright, George W; Schmitz, Roland; Phelan, James D; Yang, Yandan; Webster, Daniel E; Rui, Lixin; Kohlhammer, Holger; Nakagawa, Masao; Waldmann, Thomas A; Staudt, Louis M
The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.
Larsen, Mette Voldby; Lelic, A.; Parsons, R.
bioinformatics methods to predict WNV-specific CD8(+) T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8(+) T cell...... identified CD8(+) T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8(+) T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine....
Diffuse Large B-Cell Lymphoma; Mediastinal (Thymic) Large B-Cell Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mediastinal (Thymic) Large B-Cell Cell Lymphoma
Thais Melo Mendes
Full Text Available Leishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.Seventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2 protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1-28 and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67-81 and 122-135 were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL. The assay proved to be highly sensitive (98% and specific (99%.Our work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.
Lakshmi Kiran C
Full Text Available Complement-mediated cytotoxic antibodies in conventional cross match, often result in misappropriation of true positives and borderline positives which are detrimental to allograft survival. Flow cross matches (FCXM are sensitive to capture even non comple-ment fixing cytotoxic antibodies. This retrospective study evaluates the utility of FCXM in effectively predicting acute allograft rejection. A total of 17 cases were processed for FCXM (T and B cell of whom seven had no rejection episodes, while the remaining 11 had acute rejection despite negative cross match and panel reacting antibodies being ne-gative (less than 20%. The sensitivity and specificity of the FCXM outcome demons-trated that positive B-cell FCXM has potential to be a good tool in pre-transplant scree-ning. The current analysis proposes the possible utility of B-cell positive FCXM as a more sensitive parameter in predicting acute allograft rejection prior to transplantation.
Patzelt, Thomas; Keppler, Selina J.; Gorka, Oliver; Thoene, Silvia; Wartewig, Tim; Reth, Michael; Förster, Irmgard; Lang, Roland; Buchner, Maike; Ruland, Jürgen
The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1. Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma. PMID:29507226
Nguyen, Phuong-Cac T.; Lewis, Kenneth B.; Ettinger, Ruth A.; Schuman, Jason T.; Lin, Jasper C.; Healey, John F.; Meeks, Shannon L.; Lollar, Pete; Pratt, Kathleen P.
Amino acid residues comprising B-cell epitopes recognized by neutralizing anti-factor VIII antibodies (inhibitors) have been identified.Amino acids contributing significant antigen–antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.
Full Text Available Primary cardiac lymphoma (PCL is a rare and fatal disorder. It may often mimic other common cardiac tumors like cardiac myxoma because of similarities in the clinical presentation. We report a case of PCL of diffuse large B-cell type, in a 38-year-old, immunocompetent male who presented with superior vena cava syndrome that was excised as a myxoma. Histology revealed a large cell population diffusely and strongly expressing CD45, CD20, MUM1/IRF4 and FOXP1 hinting at an activated B-cell (ABC-like phenotype. After four cycles of Rituximab with CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisolone the tumor regressed completely but the patient had a relapse and subsequently succumbed to the disease confirming the aggressive nature. The aggressive behavior of PCL may be possibly linked to its ABC-like origin.
Full Text Available Aging is the greatest risk factor for developing chronic diseases. Inflamm-aging, the age-related increase in low-grade chronic inflammation, may be a common link in age-related diseases. This review summarizes recent published data on potential cellular and molecular mechanisms of the age-related increase in inflammation, and how these contribute to decreased humoral immune responses in aged mice and humans. Briefly, we cover how aging and related inflammation decrease antibody responses in mice and humans, and how obesity contributes to the mechanisms for aging through increased inflammation. We also report data in the literature showing adipose tissue infiltration with immune cells and how these cells are recruited and contribute to local and systemic inflammation. We show that several types of immune cells infiltrate the adipose tissue and these include macrophages, neutrophils, NK cells, innate lymphoid cells, eosinophils, T cells, B1, and B2 cells. Our main focus is how the adipose tissue affects immune responses, in particular B cell responses and antibody production. The role of leptin in generating inflammation and decreased B cell responses is also discussed. We report data published by us and by other groups showing that the adipose tissue generates pro-inflammatory B cell subsets which induce pro-inflammatory T cells, promote insulin resistance, and secrete pathogenic autoimmune antibodies.
Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria
Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Faurschou, Mikkel; Jayne, David R W
Several monoclonal antibodies targeting B cells have been tested as therapeutics for inflammatory rheumatic diseases. We review important observations from randomized clinical trials regarding the efficacy and safety of anti-B cell antibody-based therapies for rheumatoid arthritis, systemic lupus...... and functions in rheumatic disorders. Future studies should also evaluate how to maintain disease control by means of conventional and/or biologic immunosuppressants after remission-induction with anti-B cell antibodies....
Zhang, JunMei; Jia, Ge; Liu, Qun; Hu, Jue; Yan, Mei; Yang, BaiFeng; Yang, Huan; Zhou, WenBin; Li, Jing
MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR-146a was up-regulated in activated B cells in response to rat acetylcholine receptor (AChR) α-subunit 97-116 peptide, and this up-regulation was significantly attenuated by AntagomiR-146a. Our data also demonstrated that silencing miR-146a with its inhibitor AntagomiR-146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR-146a was knocked down in B cells, including decreased anti-R97-116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B-1 cells, and weakened activation of B cells. Previously, miR-146a has been identified as a nuclear factor-κB-dependent gene and predicted to base pair with the tumour necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR-146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR-146a in B cells does not involve these two target molecules. We conclude that silencing miR-146a exerts its therapeutic effects by influencing the B-cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis. © 2014 John Wiley & Sons Ltd.
Feldhahn, Magdalena; Dönnes, Pierre; Thiel, Philipp; Kohlbacher, Oliver
Over the last decade, immunoinformatics has made significant progress. Computational approaches, in particular the prediction of T-cell epitopes using machine learning methods, are at the core of modern vaccine design. Large-scale analyses and the integration or comparison of different methods become increasingly important. We have developed FRED, an extendable, open source software framework for key tasks in immunoinformatics. In this, its first version, FRED offers easily accessible prediction methods for MHC binding and antigen processing as well as general infrastructure for the handling of antigen sequence data and epitopes. FRED is implemented in Python in a modular way and allows the integration of external methods. FRED is freely available for download at http://www-bs.informatik.uni-tuebingen.de/Software/FRED.
Rahmanzadeh, R; Weber, M S; Brück, W; Navardi, S; Sahraian, M A
For decades, B cells were ignored in multiple sclerosis (MS) pathogenesis, and the disease was always regarded as a T cell-mediated disorder. Recent evidence shows that there is an antigen-driven B-cell response in the central nervous system of patients with MS, and memory B cells/plasma cells are detectable in MS lesions. The striking efficacy of B cell-depleting therapies in reducing the inflammatory activity of the disease highlights that B cells may play more pathogenetic roles than expected. B cells express several unique characteristic markers on their surface, for example, CD19, CD20 molecules, that provide selective targets for monoclonal antibodies. In this respect, several B cell-targeted therapies emerged, including anti-CD20 antibodies (rituximab, ocrelizumab, and ofatumumab), anti-CD19 antibody (inebilizumab), and agents targeting the BAFF/APRIL signaling pathway (atacicept, belimumab, and LY2127399). In this review, we discuss, in detail, the immunobiology of B cells and their protective and destructive roles in MS pathogenesis. In the second part, we list the completed and ongoing clinical trials investigating the safety and efficacy of B cell-related monoclonal antibodies in MS. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Wang, Ping; Zheng, Song Guo
The regulatory T (Treg) cells play an important role in the maintenance of homeostasis and the prevention of autoimmune diseases. Although most studies are focusing on the role of Treg cells in T cells and T cells-mediated diseases, these cells also directly affect B cells and other non-T cells. This manuscript updates the role of Treg cells on the B cells and B cell-mediated diseases. In addition, the mechanisms whereby Treg cells suppress B cell responses have been discussed.
The B-cell receptor (BCR) signaling pathway plays an essential role in the survival, proliferation, differentiation and trafficking of lymphocytic. Recent findings associate aberrant BCR signaling with specific disease pathologies, including B-cell malignancies and autoimmune disorders. Inhibition of the BCR signaling pathway may therefore provide promising new strategies for the treatment of B-cell diseases. This special issue of International Reviews of Immunology focuses on atypical B-cell receptor signaling, its role in immune diseases and cancer, and its implications for potential therapeutic intervention.
Hoof, Ilka; Vita, R; Zarebski, L
The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive...... immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course...
Miyazaki, Yusei; Niino, Masaaki; Takahashi, Eri; Suzuki, Masako; Mizuno, Masanori; Hisahara, Shin; Fukazawa, Toshiyuki; Amino, Itaru; Nakano, Fumihito; Nakamura, Masakazu; Akimoto, Sachiko; Minami, Naoya; Fujiki, Naoto; Doi, Shizuki; Shimohama, Shun; Terayama, Yasuo; Kikuchi, Seiji
Patients with multiple sclerosis (MS) who are treated with fingolimod have an increased proportion of transitional B cells in the circulation, but the underlying mechanism is not known. We hypothesized that B cell-activating factor of the tumor necrosis factor family (BAFF) is involved in the process. Compared with healthy controls and untreated MS patients, fingolimod-treated MS patients had significantly higher serum concentrations of BAFF, which positively correlated with the proportions and the absolute numbers of transitional B cells in blood. Despite the elevated concentrations of BAFF in fingolimod-treated MS patients, serum levels of soluble transmembrane activator and calcium-modulating cyclophilin ligand interactor, and B cell maturation antigen were not elevated. Our results show that fingolimod induces BAFF in the circulation and expands transitional B cells, but does not activate memory B cells or plasma cells in MS, which is favorable for the treatment of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.
Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou; Ardito, Matthew T; Boyle, Christine M; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D; De Groot, Anne S
HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.
Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo
of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data......-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope....
Ali H Dalloul
Full Text Available Solid organs have been transplanted for decades. Since the improvement in graft selection and in medical and surgical procedures, the likelihood of graft function after one year is now close to 90%. Nonetheless even well-matched recipients continue to need medications for the rest of their lives hence adverse side effects and enhanced morbidity. Understanding Immune rejection mechanisms, is of increasing importance since the greater use of living-unrelated donors and genetically unmatched individuals. Chronic rejection is devoted to T-cells, however the role of B-cells in rejection has been appreciated recently by the observation that B-cell depletion improve graft survival. By contrast however, B-cells can be beneficial to the grafted tissue. This protective effect is secondary to either the secretion of protective antibodies or the induction of B-cells that restrain excessive inflammatory responses, chiefly by local provision of IL-10, or inhibit effector T-cells by direct cellular interactions. As a proof of concept B-cell-mediated infectious transplantation tolerance could be achieved in animal models, and evidence emerged that the presence of such B-cells in transplanted patients correlate with a favorable outcome. Among these populations, regulatory B-cells constitute a recently described population. These cells may develop as a feedback mechanism to prevent uncontrolled reactivity to antigens and inflammatory stimuli. The difficult task for the clinician, is to quantify the respective ratios and functions of tolerant vs effector B-cells within a transplanted organ, at a given time point in order to modulate B-cell-directed therapy. Several receptors at the B-cell membrane as well as signaling molecules, can now be targeted for this purpose. Understanding the temporal expansion of regulatory B-cells in grafted patients and the stimuli that activate them will help in the future to implement specific strategies aimed at fighting chronic
Horowitz, Mark C; Fretz, Jackie A; Lorenzo, Joseph A
It is now well established that important regulatory interactions occur between the cells in the hematopoietic, immune and skeletal systems (osteoimmunology). B lymphocytes (B cells) are responsible for the generation and production of antibodies or immunoglobulins in the body. Together with T cells these lymphocytes comprise the adaptive immune system, which allows an individual to develop specific responses to an infection and retain memory of that infection, allowing for a faster and more robust response if that same infection occurs again. In addition to this immune function, B cells have a close and multifaceted relationship with bone cells. B cells differentiate from hematopoietic stem cells (HSCs) in supportive niches found on endosteal bone surfaces. Cells in the osteoblast lineage support HSC and B cell differentiation in these niches. B cell differentiation is regulated, at least in part, by a series of transcription factors that function in a temporal manner. While these transcription factors are required for B cell differentiation, their loss causes profound changes in the bone phenotype. This is due, in part, to the close relationship between macrophage/osteoclast and B cell differentiation. Cross talk between B cells and bone cells is reciprocal with defects in the RANKL-RANK, OPG signaling axis resulting in altered bone phenotypes. While the role of B cells during normal bone remodeling appears minimal, activated B cells play an important role in many inflammatory diseases with associated bony changes. This review examines the relationship between B cells and bone cells and how that relationship affects the skeleton and hematopoiesis during health and disease. Copyright 2010 Elsevier Inc. All rights reserved.
B cells were often thought of as simple precursors of end-stage effector cells that are merely in charge of antibody production. Research in the last decades has shown that B cells possess important other roles as well, including their involvement in the regulation and functioning of T cell-mediated
Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.
T follicular helper (Tfh) cells help B cells to generate affinity-matured antibodies. Three papers in this issue of Immunity (Choi et al., 2011; Kerfoot et al., 2011; Kitano et al., 2011) provide information about the reciprocal relationship between B cells and Tfh cells. Copyright © 2011 Elsevier Inc. All rights reserved.
Boer, R.J. de; Freitas, A.A. (António); Perelson, A.S. (Alan)
Previous experiments with mouse chimeras demonstrated that cellular competition for antigen- specific survival signals plays a crucial role in the maintenance of the naive B cell repertoire. Transgenic (Tg) B cell populations in these chimeras have a shortened lifespan and poor competitive
Full Text Available Objective: To construct an immune alpaca phage display library, in order to obtain a single domain anti-BAFF (B cell-activating factor antibody. Methods: Using phage display technology, we constructed an immune alpaca phage display library, selected anti-BAFF single domain antibodies (sdAbs, cloned three anti-BAFF single-domain antibody genes into expression vector pSJF2, and expressed them efficiently in Escherichia coli. The affinity of different anti-BAFF sdAbs were measured by Bio layer interferometry. The in vitro biological function of three sdAbs was investigated by cell counting kit-8 (CCK-8 assay and a competitive enzyme-linked immunosorbent assay (ELISA. Results: We obtained three anti-BAFF single domain antibodies (anti-BAFF64, anti-BAFF52 and anti-BAFFG3, which were produced in high yield in Escherichia coli and inhibited tumor cell proliferation in vitro. Conclusion: The selected anti-BAFF antibodies could be candidates for B-cell lymphoma therapies.
Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun
Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.
Full Text Available Waterfowl parvovirus (WPV infection causes high mortality and morbidity in both geese (Anser anser and Muscovy ducks (Cairina moschata, resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized.To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA, we localized the VP3 epitope between amino acids (aa 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity.We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.
Peters, B.; Sidney, J.; Bourne, P.
is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide...... a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, "The Immune Epitope Database and Analysis Resource." We describe how we plan to capture, structure......, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide....
Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard
Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.
Full Text Available B cells, key cells in allergic inflammation, differentiate in the bone marrow and their precursors include pro-B, pre-B and immature B cells. Eosinophil progenitor cells increase in the lung after allergen exposure. However, the existence and possible role of B cell precursors in the lung during allergic inflammation remains elusive.A BALB/c mouse model of allergic airway inflammation was utilized to perform phenotypic and quantification analyses of pro-B and pre-B cells in the lung by flow cytometry. B cell maturation factors IL-7 and B cell-activating factor (BAFF and their receptors (CD127 and BAFFR, BCMA, TACI, respectively were also evaluated in the lung and serum. The effect of anti-BAFF treatment was investigated both in vivo (i.p. administration of BAFF-R-Ig fusion protein and in vitro (colony forming cell assay. Finally, BAFF levels were examined in the bronchoalveolar lavage (BAL of asthmatic patients and healthy controls.Precursor pro and pre-B cells increase in the lung after allergen exposure, proliferate in the lung tissue in vivo, express markers of chemotaxis (CCR10 and CXCR4 and co-stimulation (CD40, CD86 and are resistant to apoptosis (Bax. Precursor B cells express receptors for BAFF at baseline, while after allergen challenge both their ligand BAFF and the BCMA receptor expression increases in B cell precursors. Blocking BAFFR in the lung in vivo decreases eosinophils and proliferating precursor B cells. Blocking BAFFR in bone marrow cultures in vitro reduces pre-B colony formation units. BAFF is increased in the BAL of severe asthmatics.Our data support the concept of a BAFF-mediated role for B cell precursors in allergic airway inflammation.
Liu, Xingjun; Li, Yue-Sheng; Shinton, Susan A; Rhodes, Jennifer; Tang, Lingjuan; Feng, Hui; Jette, Cicely A; Look, A Thomas; Hayakawa, Kyoko; Hardy, Richard R
CD79a and CD79b proteins associate with Ig receptors as integral signaling components of the B cell Ag receptor complex. To study B cell development in zebrafish, we isolated orthologs of these genes and performed in situ hybridization, finding that their expression colocalized with IgH-μ in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to enhanced GFP to identify B cells, as demonstrated by PCR analysis of IgH-μ expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene (Rag)2:mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry + cells and the timing of Ig H and L chain expression revealed simultaneous expression of both IgH-μ- and IgL-κ-chains, without progressing through the stage of IgH-μ-chain alone. Rag2:mCherry + cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared with CD79a and CD79b:GFP + B cells, which showed strongly reduced Rag mRNAs. Thus, B cell development in zebrafish does not go through a Rag hi CD79 + IgH-μ + pre-B cell stage, different from mammals. After the generation of CD79:GFP + B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-μ exon usage, similar to in mammals. This confirmed a conserved role for CD79 in B cell development and differentiation, without the requirement of a pre-B cell stage in zebrafish. Copyright © 2017 by The American Association of Immunologists, Inc.
Boller, Sören; Li, Rui; Grosschedl, Rudolf
Hematopoiesis is regulated by signals from the microenvironment, transcription factor networks, and changes of the epigenetic landscape. Transcription factors interact with and shape chromatin to allow for lineage- and cell type-specific changes in gene expression. During B lymphopoiesis, epigenetic regulation is observed in multilineage progenitors in which a specific chromatin context is established, at the onset of the B cell differentiation when early B cell factor 1 (EBF1) induces lineage-specific changes in chromatin, during V(D)J recombination and after antigen-driven activation of B cells and terminal differentiation. In this review, we discuss the epigenetic changes underlying B cell differentiation, focusing on the role of transcription factor EBF1 in B cell lineage priming. Copyright © 2018 Elsevier Ltd. All rights reserved.
Obmolova, Galina; Teplyakov, Alexey; Malia, Thomas J.; Wunderler, Nicole; Kwok, Deborah; Barone, Linda; Sweet, Raymond; Ort, Tatiana; Scully, Michael; Gilliland, Gary L. (Janssen)
CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.
Full Text Available Hepatitis C virus (HCV infections affect more than 170 million people worldwide. Most of these individuals are chronically infected, but some clear the infection rapidly. Host factors seem to play a key role in HCV clearance, among them the human leukocyte antigen (HLA class I molecules. Certain HLA molecules, e.g. B*27 and B*57, are associated with viral clearance. To identify potential mechanisms for these associations, we assess epitope distribution differences between HLA molecules using experimentally verified and in silico predicted HCV epitopes. Specifically, we show that the NS5B protein harbors the largest fraction of conserved regions among all HCV proteins, which could be good targets for cytotoxic T cell (CTL responses. We find that the protective HLA-B*27 molecule preferentially presents cytotoxic T cell (CTL epitopes from NS5B, and in general presents the most strongly conserved epitopes among the 23 HLA molecules analyzed. In contrast, HLA molecules known to be associated with HCV persistence do not have similar preferences, and appear to target the variable P7 protein. Overall, our analysis suggests that by targeting highly constrained - and thereby conserved - regions of HCV, the protective HLA molecule HLA-B*27 reduces the ability of HCV to escape the cytotoxic T cell response of the host. For visualizing the distribution of both experimentally verified and predicted epitopes across the HCV genome, we created the HCV epitope browser, which is available at theory.bio.uu.nl/ucqi/hcv.
Full Text Available To maintain the integrity of an organism constantly challenged by pathogens, the immune system is endowed with a variety of cell types. B-lymphocytes were initially thought to only play a role in the adaptative branch of immunity. However, a number of converging observations revealed that two B-cell subsets, marginal zone (MZ and B1 cells, exhibit unique developmental and functional characteristics, and can contribute to innate immune responses. In addition to their capacity to mount local antibody response against type 2 T-independent (TI-2 antigens, MZ B-cells can participate to T-dependent (TD immune response through the capture and import of blood-borne antigens to follicular areas of the spleen. Here, we discuss the multiple roles of MZ B-cells in rodents and primates. We also summarize studies —performed in transgenic mice expressing fully human antibodies on their B-cells and macaques whose infection with Simian Immunodeficiency Virus (SIV represents a suitable model for HIV-1 infection in humans— showing that infectious agents have developed strategies to subvert MZ B-cell functions. In these two experimental models, we observed that two microbial superantigens for B-cells (protein A from Staphylococcus aureus and protein L from Peptostreptococcus magnus as well as inactivated AT-2 virions of HIV-1 and infectious SIV preferentially deplete innate-like B-cells —MZ B-cells and/or B1 B-cells— with different consequences on TI and TD antibody responses. These data revealed that viruses and bacteria have developed strategies to deplete innate-like B-cells during the acute phase of infection and to impair the antibody response. Unraveling the intimate mechanisms responsible for targeting MZ B-cells in humans will be important for understanding disease pathogenesis and for designing novel vaccine strategies.
Nivarthi, Usha K; Kose, Nurgun; Sapparapu, Gopal; Widman, Douglas; Gallichotte, Emily; Pfaff, Jennifer M; Doranz, Benjamin J; Weiskopf, Daniela; Sette, Alessandro; Durbin, Anna P; Whitehead, Steve S; Baric, Ralph; Crowe, James E; de Silva, Aravinda M
The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination. IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses
Full Text Available Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE. In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease.
Flores-Fernández, Rocio; Blanco-Favela, Francisco; Fuentes-Pananá, Ezequiel M.; Chávez-Sánchez, Luis; Gorocica-Rosete, Patricia; Pizaña-Venegas, Alberto; Chávez-Rueda, Adriana Karina
Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. PMID:27314053
Full Text Available Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.
Gondre-Lewis, Timothy A; Hartmann, Constance B; Caffrey, Rebecca E; McCoy, Kathleen L
Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4(+) T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.
Barfod, L.; Bernasconi, N. L.; Dahlback, M.
-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-epsilon domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates...
Mahanonda, Rangsini; Champaiboon, Chantrakorn; Subbalekha, Keskanya; Sa-Ard-Iam, Noppadol; Rattanathammatada, Warattaya; Thawanaphong, Saranya; Rerkyen, Pimprapa; Yoshimura, Fuminobu; Nagano, Keiji; Lang, Niklaus P; Pichyangkul, Sathit
The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19(+)CD27(+)CD38(-) memory B cells, the major B cell component in periodontitis was CD19(+)CD27(+)CD38(+)CD138(+)HLA-DR(low) plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell-activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis. Copyright © 2016 by The American Association of Immunologists, Inc.
Lundgren, E; Carballeira, N; Vazquez, R; Dubinina, E; Bränden, H; Persson, H; Wolf-Watz, H
The Yersinia pseudotuberculosis cell surface-located protein invasin was found to promote binding between the pathogen and resting peripheral B cells via beta 1 integrin receptors (CD29). B cells responded by expressing several activation markers and by growing, In contrast, T cells did not react, although these cells express CD29. An isogenic invA mutant failed to activate B cells. The mutation could be complemented by providing the invA+ gene in trans. Purified invasin alone did not activat...
Surendra S Negi
Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web
Lauren Folgosa Cooley
Full Text Available ADAM10, as the sheddase of the low affinity IgE receptor (CD23, promotes IgE production and thus is a unique target for attenuating allergic disease. Herein, we describe that B cell levels of ADAM10, specifically, are increased in allergic patients and Th2 prone WT mouse strains (Balb/c and A/J. While T cell help augments ADAM10 expression, Balb WT B cells exhibit increased ADAM10 in the naïve state and even more dramatically increased ADAM10 after anti-CD40/IL4 stimulation compared C57 (Th1 prone WT B cells. Furthermore, ADAM17 and TNF are reduced in allergic patients and Th2 prone mouse strains (Balb/c and A/J compared to Th1 prone controls. To further understand this regulation, ADAM17 and TNF were studied in C57Bl/6 and Balb/c mice deficient in ADAM10. C57-ADAM10B-/- were more adept at increasing ADAM17 levels and thus TNF cleavage resulting in excess follicular TNF levels and abnormal secondary lymphoid tissue architecture not noted in Balb-ADAM10B-/-. Moreover, the level of B cell ADAM10 as well as Th context is critical for determining IgE production potential. Using a murine house dust mite airway hypersensitivity model, we describe that high B cell ADAM10 level in a Th2 context (Balb/c WT is optimal for disease induction including bronchoconstriction, goblet cell metaplasia, mucus, inflammatory cellular infiltration, and IgE production. Balb/c mice deficient in B cell ADAM10 have attenuated lung and airway symptoms compared to Balb WT and are actually most similar to C57 WT (Th1 prone. C57-ADAM10B-/- have even further reduced symptomology. Taken together, it is critical to consider both innate B cell levels of ADAM10 and ADAM17 as well as Th context when determining host susceptibility to allergic disease. High B cell ADAM10 and low ADAM17 levels would help diagnostically in predicting Th2 disease susceptibility; and, we provide support for the use ADAM10 inhibitors in treating Th2 disease.
Busch, Clara J; Binder, Christoph J
Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder. Copyright © 2016 Elsevier B.V. All rights reserved.
Full Text Available Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow (BM is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse-anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin-V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment.
Xiao, Gang; Chan, Lai N; Klemm, Lars; Braas, Daniel; Chen, Zhengshan; Geng, Huimin; Zhang, Qiuyi Chen; Aghajanirefah, Ali; Cosgun, Kadriye Nehir; Sadras, Teresa; Lee, Jaewoong; Mirzapoiazova, Tamara; Salgia, Ravi; Ernst, Thomas; Hochhaus, Andreas; Jumaa, Hassan; Jiang, Xiaoyan; Weinstock, David M; Graeber, Thomas G; Müschen, Markus
B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD. Copyright © 2018 Elsevier Inc. All rights reserved.
Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi
Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.
Full Text Available Trichinellosis is a worldwide zoonosis and remains a serious public health problem. Interrupting parasite transmission via vaccination of livestocks with a potent vaccine is a practical approach to prevent human Trichinellosis. Our previous studies have identified that paramyosin of Trichinella spiralis (Ts-Pmy is a good vaccine candidate against Trichinellosis. In this study, a novel multi-epitope vaccine (MEP was constructed by using four CD4+ T cell epitopes (P2, P3, P4, and P5 and one B cell epitope (YX1 from Ts-Pmy and expressed as a soluble recombinant protein (rMEP in Escherichia coli. Mice immunized with rMEP vaccine produced significant higher muscle larval reduction (55.4% than that induced by immunization of parental rTs-Pmy (34.4% against T. spiralis infection. The better protection is associated with rMEP induced high levels of anti-rMEP specific IgG and subclass IgG1/IgG2a, elevated T cell proliferation of splenocytes and secretion of IFN-γ, IL-4 and IL-5. The cellular response to individual T cell epitope also showed that splenocytes from mice immunized with rMEP strongly response to the stimulation of synthetic epitope peptide P2, P3, and P4, but not to P5, suggesting that most of T cell epitopes are exposed and processed well during immunization that may contribute to the high protection induced by the immunization of rMEP. This study implies that epitope vaccine is a promising approach for the development of vaccines against Trichinellosis.
In this study, we established a computational model describing the molecular circuit underlying B cell terminal differentiation and how TCDD may affect this process by impinging upon various molecular targets.
This report documents the methods used to collect and analyze samples to obtain data necessary to verify and/or determine the radionuclide content of the 324 Facility B-Cell decontamination and decommissioning waste stream
Tarlinton, David; Good-Jacobson, Kim
Immunological memory is the residuum of a successful immune response that in the B cell lineage comprises long-lived plasma cells and long-lived memory B cells. It is apparent that distinct classes of memory B cells exist, distinguishable by, among other things, immunoglobulin isotype, location, and passage through the germinal center. Some of this variation is due to the nature of the antigen, and some appears to be inherent to the process of forming memory. Here, we consider the heterogeneity in development and phenotype of memory B cells and whether particular functions are partitioned into distinct subsets. We consider also how understanding the details of generating memory may provide opportunities to develop better, functionally targeted vaccines.
Griffin, T A; Hostoffer, R W; Tserng, K Y; Lebovitz, D J; Hoppel, C L; Mosser, J L; Kaplan, D; Kerr, D S
The mechanisms of hypocalcemia, recurrent infections and hypogammaglobulinemia associated with metabolic decompensation of propionic acidemia due to propionyl-CoA carboxylase deficiency have not been defined. A 7-week-old infant with this disorder presented with severe hypocalcemia and B cell lymphopenia during an episode of metabolic acidosis and hyperammonemia. Hypocalcemia (1.1 mmol l-1) was associated with elevated serum intact parathyroid hormone (122 ng l-1), hyperphosphatemia, hypophosphaturia and hypercalcuria, indicating parathyroid hormone resistance. B cell lymphopenia (20 cells microliters-1) was associated with transient neutropenia, anemia and subsequent hypogamma-globulinemia (IgG < 294 mg dl-1, IgM < 8 mg dl-1, IgA < 8 mg dl-1), while T cells were normal. Parathyroid hormone resistance and B cell lymphopenia resolved following treatment with hemodialysis, diet and carnitine. These complications may be due to interference with parathyroid hormone renal tubular action and B cell maturation/proliferation by accumulated organic acids.
Sandra M. McLachlan
Full Text Available Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg, thyroid peroxidase (TPO, and the thyrotropin receptor (TSHR A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of spontaneously arising pathogenic TSHR antibodies in transgenic human thyrotropin receptor/NOD.H2h4 mice by injecting a non-pathogenic form of TSHR A-subunit protein also provides evidence for original antigenic sin. From other studies, antigen presentation by B cells, not dendritic cells, is likely responsible for original antigenic sin. Recognition of restricted epitopes on the large glycosylated thyroid autoantigens (60-kDa A-subunit, 100-kDa TPO, and 600-kDa Tg facilitates exploring the amino acid locations in the immunodominant regions. Epitope spreading has also been revealed by autoantibodies in thyroid autoimmunity. In humans, and in mice that spontaneously develop autoimmunity to all three thyroid autoantigens, autoantibodies develop first to Tg and later to TPO and the TSHR A-subunit. The pattern of intermolecular epitope spreading is related in part to the thyroidal content of Tg, TPO and TSHR A-subunit and to the molecular sizes of these proteins. Importantly, the epitope spreading pattern provides a rationale for future antigen-specific manipulation to block the development of all thyroid autoantibodies by inducing tolerance to Tg, first in the autoantigen cascade. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment, preventing the development of pathogenic TSHR antibodies.
Taylor, J J; Laudenbach, M; Tucker, A M; Jenkins, M K; Pravetoni, M
Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address this obstacle, we developed a fluorescent antigen-based enrichment method paired with flow cytometry to characterize hapten-specific B cells. Using this approach, we studied naïve and activated B cells specific for structurally-related model haptens based on derivatization of the morphinan structure at the C6 position on oxycodone or at the C8 position on hydrocodone, and showing different pre-clinical efficacy against the prescription opioid oxycodone. Prior to vaccination, naïve B cells exhibited relatively higher affinity for the more effective C6-derivatized oxycodone-based hapten (6OXY) and the 6OXY-specific naïve B cell population contained a higher number of B cells with greater affinity for free oxycodone. Higher affinity of naïve B cells for hapten or oxycodone reflected greater efficacy of vaccination in blocking oxycodone distribution to brain in mice. Shortly after immunization, activated hapten-specific B cells were detected prior to oxycodone-specific serum antibodies and provided earlier evidence of vaccine failure or success. Analysis of hapten-specific naïve and activated B cells may aid rational vaccine design and provide screening tools to predict vaccine clinical efficacy against drugs of abuse or other small molecules. Copyright © 2014 Elsevier B.V. All rights reserved.
Tang, Sheila Tuyet; Wang, M.; Lamberth, K.
lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-gamma release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity......It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T...
Earlier studies have identified transcription factors that specify B-cell fate, but the underlying mechanisms remain to be revealed. Two new studies by Miyai and colleagues (pp. 112-126) and Li and colleagues (pp. 96-111) in this issue of Genes & Development provide new and unprecedented insights into the genetic and epigenetic mechanisms that establish B-cell identity. © 2018 Murre; Published by Cold Spring Harbor Laboratory Press.
Full Text Available Long noncoding RNAs (lncRNAs have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n = 7, and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n = 6, respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as "guilt by association". By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations.
Full Text Available MicroRNAs have the potential to be useful biomarkers in the development of individualized treatment since they are easy to detect, are relatively stable during sample handling, and are important determinants of cellular processes controlling pathogenesis, progression, and response to treatment of several types of cancers including B-cell malignancies. miR-155 is an oncomiR with a crucial role in tumor initiation and development of several B-cell malignancies. The present review elucidates the potential of miR-155 as a diagnostic, prognostic, or predictive biomarker in B-cell malignancies using a systematic search strategy to identify relevant literature. miR-155 was upregulated in several malignancies compared to nonmalignant controls and overexpression of miR-155 was further associated with poor prognosis. Elevated expression of miR-155 shows potential as a diagnostic and prognostic biomarker in diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Additionally, in vitro and in vivo studies suggest miR-155 as an efficient therapeutic target, supporting its oncogenic function. The use of inhibiting anti-miR structures indicates promising potential as novel anticancer therapeutics. Reports from 53 studies prove that miR-155 has the potential to be a molecular tool in personalized medicine.
De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T; Skowron, Gail; Mayer, Kenneth H; Buus, Soren; Boyle, Christine M; Martin, William D
Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs.
Kowalczyk-Quintas, Christine; Schuepbach-Mallepell, Sonia; Vigolo, Michele; Willen, Laure; Tardivel, Aubry; Smulski, Cristian R.; Zheng, Timothy S.; Gommerman, Jennifer; Hess, Henry; Gottenberg, Jacques-Eric; Mackay, Fabienne; Donzé, Olivier
B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro. A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo. These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice. PMID:27451394
Holen, E; Elsayed, S
Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.
Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas
meningitis patients, CSF antibodies are focussed to only one single glycoform peptide of a major viral glycoprotein. Thus, dependent on the viral disease, the serological response may be variable or constant with respect to the number of targeted peptide glycoforms. Mapping of these epitopes relies......Viral envelope glycoproteins are major targets for antibodies that bind to and inactivate viral particles. The capacity of a viral vaccine to induce virus-neutralizing antibodies is often used as a marker for vaccine efficacy. Yet the number of known neutralization target epitopes is restricted...... owing to various viral escape mechanisms. We expand the range of possible viral glycoprotein targets, by presenting a previously unknown type of viral glycoprotein epitope based on a short peptide stretch modified with small O-linked glycans. Besides being immunologically active, these epitopes have...
Cai, Ruikun; Liu, Zexian; Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu
As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-A(g7) in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-A(g7) and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-A(g7) and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-A(g7) and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org.
Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan
Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.
Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe
Follicular helper T (T FH ) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of T FH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on T FH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate T FH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing T FH cell maturation. In cocultures they differentiated B cells into CD138 + plasma and IgD - CD27 + memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented T FH cell development. Added to T FH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3 + CXCR5 + PD-1 + follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on T FH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control T FH cell maturation, expand follicular regulatory T cells, and inhibit the T FH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the T FH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
McGuire, Andrew T.; Gray, Matthew D.; Dosenovic, Pia; Gitlin, Alexander D.; Freund, Natalia T.; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B.; Glenn, Jolene; Seaman, Michael S.; Schief, William R.; Strong, Roland K.; Nussenzweig, Michel C.; Stamatatos, Leonidas
VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype. PMID:26907590
McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas
VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.
Ye, Qing; Xu-Monette, Zijun Y; Tzankov, Alexandar
Double-hit B-cell lymphoma is a common designation for a group of tumors characterized by concurrent translocations of MYC and BCL2, BCL6, or other genes. The prognosis of concurrent MYC and BCL6 translocations is not well known. In this study, we assessed rearrangements and expression of MYC, BCL2...... with MYC translocation (observed in 2.0% of diffuse large B-cell lymphoma) predicted poorer survival in diffuse large B-cell lymphoma patients. Diffuse large B-cell lymphoma patients with MYC/BCL6 co-expression did have significantly poorer survival, however, MYC/BCL6 co-expression had no effect...... on prognosis in the absence of MYC/BCL2 co-expression, and had no additive impact in MYC+/BCL2+ cases. The isolated MYC+/BCL6+/BCL2- subset, more frequent in germinal center B-cell like diffuse large B-cell lymphoma, had significantly better survival compared with the isolated MYC+/BCL2+/BCL6- subset (more...
Fu, Fang; Li, Lin; Shan, Lingling; Yang, Beibei; Shi, Hongyan; Zhang, Jiaoer; Wang, Hongfeng; Feng, Li; Liu, Pinghuang
Porcine epidemic diarrhea (PED), caused by an alpha coronavirus, is a highly contagious disease and causes high morbidity and mortality in suckling piglets. Isolating PEDV neutralizing antibodies from porcine B cells is critical to elucidate the development of PEDV neutralizing antibodies and the protective mechanism of PEDV infection. Here, we described the isolation of a PEDV-neutralizing antibody from the B cell of a vaccinated pig. The antibody, named PC10, was demonstrated to target the conformational epitope of PEDV spike protein, specifically bind to the infected cells of PEDV genogroup 1 and 2 strains, and potently neutralize PEDV infection. PC10 neutralized PEDV infection through interfering with the viral life stages after cellular attachment instead of blocking the attachment of PEDV to cells. These results suggest that PC10 could be a promising candidate for passive protection and inform PEDV vaccine design because of its specificity and substantial neutralization potency. Copyright © 2017 Elsevier B.V. All rights reserved.
Priyamvada, Lalita; Cho, Alice; Onlamoon, Nattawat; Zheng, Nai-Ying; Huang, Min; Kovalenkov, Yevgeniy; Chokephaibulkit, Kulkanya; Angkasekwinai, Nasikarn; Pattanapanyasat, Kovit; Ahmed, Rafi; Wilson, Patrick C; Wrammert, Jens
Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis. In addition to their role in protection, antibody responses have
Antigen stimulation may be important for splenic marginal zone lymphoma pathogenesis. To address this hypothesis, the occurrence of stereotyped B-cell receptors was investigated in 133 SMZL (26 HCV+) compared with 4,414 HCDR3 sequences from public databases. Sixteen SMZL (12%) showed stereotyped BCR; 7 of 86 (8%) SMZL sequences retrieved from public databases also belonged to stereotyped HCDR3 subsets. Three categories of subsets were identified: i) SMZL-specific subsets (n=5), composed only of 12 SMZL (9 HCV- from our series); ii) Non-Hodgkin\\'s lymphoma-like subsets (n=5), comprising 5 SMZL (4 from our series) clustering with other indolent lymphomas; iii) "CLL-like subsets" (n=6), comprising 6 SMZL (3 from our series) that belonged to known CLL subsets (n=4) or clustered with public CLL sequences. Immunoglobulin 3D modeling of 3 subsets revealed similarities in antigen binding regions not limited to HCDR3. Overall, data suggest that the pathogenesis of splenic marginal zone lymphoma may involve also HCV unrelated epitopes or an antigenic trigger common to other indolent lymphomas. ©2010 Ferrata Storti Foundation.
Wang, M.J.; Lamberth, K.; Harndahl, M.
The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation......, the latter encompassing 12 different HLA class I supertypes with > 99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-1 interactions were synthesized, tested for peptide-HLA-1 interactions...... in a biochemical assay and for influenza-specific, HLA-1-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-1 binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes...
Full Text Available The use of serotype 5 adenovirus (Ad-derived vectors in vaccination is confronted to preexisting anti-Ad immunity. Epitope display on Ad capsid is currently being investigated as an alternative approach of vaccination. The present study seeks to better understand virus- and host-related factors controlling the efficacy of this new vaccination approach. In contrast to an Ad vector expressing ovalbumin as a transgene, Ad displaying an ovalbumin-derived B-cell epitope inserted into the fiber protein was able to elicit antibody responses in both Ad-naive and Ad-immune mice. Moreover, introduction of a set of mutations abrogating Ad interaction with its receptors did not modify the virus capacity to elicit a humoral response against the inserted epitope while reducing its capacity to mount antibody responses against the transgene product. Taken as a whole these data indicate that the efficacy of Ad displaying epitopes requires neither Ad binding to its receptors nor the infection process. In addition, the use of genetically deficient mice demonstrated that both toll-like receptor (TLR/MyD88 and RIG-I/mitochondrial antiviral-signaling (MAVS innate immunity pathways were dispensable to mount anti-epitope antibody responses. However, they also revealed that TLR/MyD88 pathway but not RIG-I/MAVS pathway controls the nature of antibodies directed against the displayed epitope.
Full Text Available Characterization of the pollen allergens and identification of the main T-cell and B-cell epitopes is of crucial importance, since the clarification of the cellular and molecular mechanisms will allow long-term control of this type allergic diseases and development of safe immunotherapeutic medication. The objective of this study was to identify which pollen allergens are responsible for the T-cell activation in atopic patients with pollinosis and to determine whether the same allergens are responsible for the IgE-mediated reactions. Thirty-seven patients with pollinosis and thirteen non-allergic subjects were recruited. Peripheral blood samples were collected out of the pollen season. T-cell responses (IFN-γ production towards different pollen allergens and levels of pollen specific IgE and IgG in the sera were measured by ELISA. The T-cell reactivity in most patients was directed towards the grass pollen B1, tree pollen I and autumn pollen B5. 70% of the allergic individuals responded to allergens from Lolium perenne, 84% to Dactylis glomerata, 11% to Phleum pratense, 65% to Betula pendula and 70% to Taxus baccata. Elevated serum levels of specific IgE in the allergic patients (p<0.01 were measured against the tree pollen I and autumn pollen, which include widespread deciduous trees (birch, willow, poplar and yew and Artemisia absinthium, respectively. Our results show that the T-cell reactivity and antibody responses may be directed towards different or the same allergens. In addition, we propose that short synthetic peptides, which contain overlapping T- and B-cell epitopes can be used for specific immunotherapy treatment if they lack antibody recognition domains.
Full Text Available Sensitization to human leukocyte antigens (HLA in organ transplant patients causes graft rejection, according to the humoral theory of transplantation. Sensitization is almost ubiquitous as anti-HLA antibodies are found in almost all sera of transplant recipients. Advances in testing assays and amino acid sequencing of HLA along with computer software contributed further to the understanding of antibody-antigen reactivity. It is commonly understood that antibodies bind to HLA antigens. With current knowledge of epitopes, it is more accurate to describe that antibodies bind to their target epitopes on the surface of HLA molecular chains. Epitopes are present on a single HLA (private epitope or shared by multiple antigens (public epitope. The phenomenon of cross-reactivity in HLA testing, often explained as cross-reactive groups (CREGs of antigens with antibody, can be clearly explained now by public epitopes. Since 2006, we defined and reported 194 HLA class I unique epitopes, including 56 cryptic epitopes on dissociated HLA class I heavy chains, 83 HLA class II epitopes, 60 epitopes on HLA-DRB1, 15 epitopes on HLA-DQB1, 3 epitopes on HLA-DQA1, 5 epitopes on HLA-DPB1, and 7 MICA epitopes. In this paper, we provide a summary of our findings.
Song, Jae-Min; Lee, Kwang-Hee; Seong, Baik-Lin
Neuraminidase (NA) of influenza virus contains stalk region that shows a great deal of variability in both amino acid sequence and length. In this paper, we investigated generation of recombinant influenza viruses that had hepatitis B virus (HBV) B cell epitopes in the NA stalk region as a dual vaccine candidate. We used the WSH-HK reassortant helper virus for rescue of recombinant influenza virus containing HBV epitopes and reverse genetic protocol based on the use of micrococcal nuclease-treated virus cores for reconstitution of ribonucleoproteins. We successfully generated a chimeric influenza viruses which contained 22 amino acid peptides in the stalk region derived from the surface and pre-surface protein HBV. The growth kinetics of the recombinant viruses was investigated after infection of Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney (MDBK) cells and the rIV-BVPreS virus showed higher titer than other viruses in MDCK cells. We also confirmed the presence of HBV epitopes in the chimeric viruses by enzyme-linked immunosorbent assay (ELISA) using anti-HBV polyclonal antibody. When the ratio of recombinant virus verse wild type virus was calculated by ELISA, recombinant viruses exhibited 2 fold higher values than the wild type virus. These results suggest that chimeric influenza virus which contained foreign antigens can be used as dual vaccine against both HBV and influenza viruses.
Guo, J; Li, G; Tang, J; Cao, X-B; Zhou, Q-Y; Fan, Z-J; Zhu, B; Pan, X-H
Hepsin is a type II transmembrane serine protease that is overexpressed in prostate cancer, and it is associated with prostate cancer cellular migration and invasion. Therefore, HPN is a biomarker for prostate cancer. CD8(+) T cells play an important role in tumour immunity. This study predicted and identified HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in human hepsin protein. HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: (1) a computer program generated predicted epitopes from the amino acid sequence of human hepsin; (2) an HLA-A2-binding assay detected the affinity of the predicted epitopes to the HLA-A2 molecule; (3) the primary T cell response against the predicted epitopes was stimulated in vitro; and (4) the induced CTLs towards different types of hepsin- or HLA-A2-expressing prostate cancer cells were detected. Five candidate peptides were identified. The effectors that were induced by human hepsin epitopes containing residues 229 to 237 (Hpn229; GLQLGVQAV), 268 to 276 (Hpn268; PLTEYIQPV) and 191 to 199 (Hpn199; SLLSGDWVL) effectively lysed LNCaP prostate cancer cells that were hepsin-positive and HLA-A2 matched. These peptide-specific CTLs did not lyse normal liver cells with low hepsin levels. Hpn229, Hpn268 and Hpn199 increased the frequency of IFN-γ-producing T cells compared with the negative peptide. These results suggest that the Hpn229, Hpn268 and Hpn199 epitopes are novel HLA-A2-restricted CTL epitopes that are capable of inducing hepsin-specific CTLs in vitro. Hpn229, Hpn268 and Hpn199 peptide-based vaccines may be useful for immunotherapy in patients with prostate cancer. © 2013 John Wiley & Sons Ltd.
McLaughlin, Kerry A; Richardson, Carolyn C; Williams, Stefan; Bonifacio, Ezio; Morgan, Diana; Feltbower, Richard G; Powell, Michael; Rees Smith, Bernard; Furmaniak, Jadwiga; Christie, Michael R
Diversification of autoimmunity to islet autoantigens is critical for progression to Type 1 diabetes. B-cells participate in diversification by modifying antigen processing, thereby influencing which peptides are presented to T-cells. In Type 1 diabetes, JM antibodies are associated with T-cell responses to PTP domain peptides. We investigated whether this is the consequence of close structural alignment of JM and PTP domain determinants on IA-2. Fab fragments of IA-2 antibodies with epitopes mapped to the JM domain blocked IA-2 binding of antibodies that recognise epitopes in the IA-2 PTP domain. Peptides from both the JM and PTP domains were protected from degradation during proteolysis of JM antibody:IA-2 complexes and included those representing major T-cell determinants in Type 1 diabetes. The results demonstrate close structural relationships between JM and PTP domain epitopes on IA-2. Stabilisation of PTP domain peptides during proteolysis in JM-specific B-cells may explain determinant spreading in IA-2 autoimmunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Kowalczyk-Quintas, Christine; Schuepbach-Mallepell, Sonia; Vigolo, Michele; Willen, Laure; Tardivel, Aubry; Smulski, Cristian R; Zheng, Timothy S; Gommerman, Jennifer; Hess, Henry; Gottenberg, Jacques-Eric; Mackay, Fabienne; Donzé, Olivier; Schneider, Pascal
B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Alexander S. Garruss
Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.
Owens, T; Zeine, R
The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...
Full Text Available Aggressive B-cell lymphomas share high proliferative and invasive attitudes and dismal prognosis despite heterogeneous biological features. In the interchained sequence of events leading to cancer progression, neoplastic clone-intrinsic molecular events play a major role. Nevertheless, microenvironment-related cues have progressively come into focus as true determinants for this process. The cancer-associated microenvironment is a complex network of nonneoplastic immune and stromal cells embedded in extracellular components, giving rise to a multifarious crosstalk with neoplastic cells towards the induction of a supportive milieu. The immunological and stromal microenvironments have been classically regarded as essential partners of indolent lymphomas, while considered mainly negligible in the setting of aggressive B-cell lymphomas that, by their nature, are less reliant on external stimuli. By this paper we try to delineate the cardinal microenvironment-centred dynamics exerting an influence over lymphoid clone progression in aggressive B-cell lymphomas.
Schreterova, Eva; Bhide, Mangesh; Potocnakova, Lenka; Borszekova Pulzova, Lucia
Introduction and objective. Lyme disease (LD) is the most common vector-borne disease in the temperate zone of the Northern Hemisphere. Diagnosis of LD is mainly based on clinical symptoms supported with serology (detection of anti-Borrelia antibodies) and is often misdiagnosed in areas of endemicity. In this study, the chimeric proteins (A/C-2, A/C-4 and A/C-7.1) consisting of B-cell epitopes of outer surface proteins OspA and OspC from Borrelia genospecies prevalent in Eastern Slovakia, were designed, over-expressed in E. coli, and used to detect specific anti-Borrelia antibodies in serologically characterized sera from patients with Lyme-like symptoms to evaluate their diagnostic potential. Results showed that chimeras vary in their immuno-reactivity when tested with human sera. Compared with the results obtained from a two-tier test, the application of recombinant multi-epitope chimeric proteins as diagnosis antigens, produced fair agreement in the case of A/C-2 (0.20<κ<0.40) and good agreement (0.60<κ<0.80) when A/C-7.1 was used as capture antigen. Chimera A/C-4 were excluded from further study due to loss of reactivity with OspA-specific antibodies. The combination of specific B-cell epitopes from OspA and OspC proteins may improve the diagnostic accuracy of serologic assays, but further studies are required to address this hypothesis.
Mandrup, Charlotte; Petersen, Anders; Højfeldt, Anne Dirks
MicroRNA expression in nodal and extranodal Diffuse Large B-cell Lymphoma C. Mandrup1, A. Petersen1, A. D. Hoejfeldt1, H. F. Thomsen1, J. Madsen1, J. Dahlgaard1, P. Johansen2, A. Bukh1, K. Dybkaer1 and H. E Johnsen1. 1Department of Hematology, 2Pathological Institute, Aalborg Hospital, Aarhus...... University Hospital, Aalborg, Denmark Introduction: The aim of this project was to analyse microRNA (miRNA) expression in nodal and extranodal diffuse large B-cell lymphoma (DLBCL). Manifestation at diagnosis may be nodal and/or extranodal. At present, there are no known determinants for none...... of the manifestations, and no way to predict the potential progression from nodal to extranodal disease. miRNA are small regulatory RNA molecules with core function to repress/cleave sequence complementary mRNA targets. Abnormalities in miRNA genetics and expression are known to affect initiation and development...
Ok, Chi Y; Chen, Jiayu; Xu-Monette, Ziju
PURPOSE: Activated signal transducer and activator of transcription 3 (STAT3) regulates tumor growth, invasion, cell proliferation, angiogenesis, immune response, and survival. Data regarding expression of phosphorylated (activated) STAT3 in diffuse large B-cell lymphoma (DLBCL) and the impact...... of phosphorylated STAT3 (pSTAT3) on prognosis are limited. EXPERIMENTAL DESIGN: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma...... biomarkers, and genetic translocations. RESULTS: pSTAT3 expression was observed in 16% of DLBCL and was associated with advanced stage, multiple extranodal sites of involvement, activated B-cell-like (ABC) subtype, MYC expression, and MYC/BCL2 expression. Expression of pSTAT3 predicted inferior overall...
Marina D Kraaij
Full Text Available Marina D Kraaij, Jacob M van LaarMusculoskeletal Research Group, Institute of Cellular Medicine, School of Clinical Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United KingdomAbstract: Systemic sclerosis (SSc is a connective disease characterized by features of autoimmunity, vasculopathy, inflammation, and fibrosis. The disease typically starts with Raynaud’s phenomenon, followed by skin thickening in the extremities due to inflammation and fibrosis. Fibrosis results from excessive collagen production by fibroblasts, which constitutes the final common pathway of complex cellular interactions including B cells. Several studies have indicated that B cells may play a role in SSc. Lesional skin infiltrates from SSc patients consist of a variety of cells, including eosinophils, neutrophils, lymphocytes, plasma cells, and macrophages. Autoantibodies of several specificities are present in the serum of SSc patients of which antitopoisomerase 1 is the most common, and evidence has been gathered for a potential pathogenic role of some autoantibodies, eg, anti-PDGF antibodies. The blood of SSc patients contains an increased proportion of naïve B cells but a decreased proportion of memory B cells. Furthermore, serum levels of interleukin-6, an important pro-inflammatory cytokine, have been shown to correlate with skin fibrosis. Animal models of SSc have provided more in-depth information on the role of B lymphocytes, eg, through disruption of B cell function. In this review we will discuss the evidence that B cells are involved in the pathogenesis of SSc.Keywords: B lymphocyte, systemic sclerosis, fibrosis
Full Text Available Immunization with myelin components can elicit experimental autoimmune encephalomyelitis (EAE. EAE susceptibility varies between mouse strains, depending on the antigen employed. BL/6 mice are largely resistant to EAE induction with proteolipid protein (PLP, probably a reflection of antigen-specific tolerance. However, the extent and mechanism(s of tolerance to PLP remain unclear. Here, we identified three PLP epitopes in PLP-deficient BL/6 mice. PLP-sufficient mice did not respond against two of these, whereas tolerance was “leaky” for an epitope with weak predicted MHCII binding, and only this epitope was encephalitogenic. In TCR transgenic mice, the “EAE-susceptibility-associated” epitope was “ignored” by specific CD4 T cells, whereas the “resistance-associated” epitope induced clonal deletion and Treg induction in the thymus. Central tolerance was autoimmune regulator dependent and required expression and presentation of PLP by thymic epithelial cells (TECs. TEC-specific ablation of PLP revealed that peripheral tolerance, mediated by dendritic cells through recessive tolerance mechanisms (deletion and anergy, could largely compensate for a lack of central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self.
Sheikh, Qamar M; Gatherer, Derek; Reche, Pedro A; Flower, Darren R
Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. To exemplify our approach we designed two epitope ensemble vaccines comprising highly conserved and experimentally verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96 and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97 and 88% coverage of observed subtypes. http://imed.med.ucm.es/Tools/episopt.html CONTACT: firstname.lastname@example.org. © The Author 2016. Published by Oxford University Press.
Vanshylla, Kanika; Bartsch, Caren; Hitzing, Christoffer; Krümpelmann, Laura; Wienands, Jürgen; Engels, Niklas
The B cell antigen receptor (BCR) employs enzymatically inactive adaptor proteins to facilitate activation of intracellular signaling pathways. In animal model systems, adaptor proteins of the growth factor receptor-bound 2 (Grb2) family have been shown to serve critical functions in lymphocytes. However, the roles of Grb2 and the Grb2-related adaptor protein (GRAP) in human B lymphocytes remain unclear. Using TALEN-mediated gene targeting, we show that in human B cells Grb2 and GRAP amplify signaling by the immunoglobulin tail tyrosine (ITT) motif of mIgE-containing BCRs and furthermore connect immunoreceptor tyrosine-based activation motif (ITAM) signaling to activation of the Ras-controlled Erk MAP kinase pathway. In contrast to mouse B cells, BCR-induced activation of Erk in human B cells is largely independent of phospholipase C-ɣ activity and diacylglycerol-responsive members of Ras guanine nucleotide releasing proteins. Together, our results demonstrate that Grb2 family adaptors are critical regulators of ITAM and ITT signaling in naïve and IgE-switched human B cells.
Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank
Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626
Shimada, Midori; Fukuda, Minoru; Horio, Kensuke; Suyama, Takayuki; Kitazaki, Takeshi; Hashiguchi, Kohji; Fukuda, Masaaki; Shigematsu, Kazuto; Nakamura, Yoichi; Honda, Takuya; Ashizawa, Kazuto; Mukae, Hiroshi
Primary mediastinal large B-cell lymphoma (PMLBCL) is one of the subtypes of diffuse large B-cell lymphoma. We experienced a rare case of PMLBCL that exhibited endobronchial involvement. A 33-year-old Japanese female with the chief complaints of epigastralgia, back pain, and nausea visited a primary care hospital. Computed tomography of the chest and abdomen demonstrated a bulky mass in the left anterior mediastinum, multiple pulmonary nodules, axillary lymph node swelling, and a pancreatic tumor. Fiberoptic bronchoscopy showed a white-tinged irregularly shaped endobronchial tumor accompanied by capillary vessel dilation in the left upper lobar bronchus. Taken together, these findings resulted in a diagnosis of PMLBCL.
Selva, R La; Violetti, S Alberti; Delfino, C; Grandi, V; Cicchelli, S; Tomasini, C; Fierro, M T; Berti, E; Pimpinelli, N; Quaglino, P
The term "Primary Cutaneous B-Cell Lymphoma" (PCBCL) comprehends a variety of lymphoproliferative disorders characterized by a clonal proliferation of B-cells primarily involving the skin. The absence of evident extra-cutaneous disease must be confirmed after six-month follow-up in order to exclude a nodal non-Hodgkin's lymphoma (NHL) with secondary cutaneous involvement, which may have a completely different clinical behavior and prognosis. In this article, we have summarized the clinico-pathological features of main types of PCBCL and we outline the guidelines for management based on a review of the available literature.
Di Tullio, Alessandro, 1982-
Our earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBPα at very high frequencies and also that a clonal pre-B cell line with an inducible form of C/EBPα can be converted into macrophage-like cells. Using these systems we have performed a systematic analysis of the questions whether during transdifferentiation the cells retrodifferentiate to a precursor cell state and whether cell cycle is required for reprogramming. As for the first ...
Vilagos, Bojan; Hoffmann, Mareike; Souabni, Abdallah; Sun, Qiong; Werner, Barbara; Medvedovic, Jasna; Bilic, Ivan; Minnich, Martina; Axelsson, Elin; Jaritz, Markus; Busslinger, Meinrad
The transcription factor EBF1 is essential for lineage specification in early B cell development. In this study, we demonstrate by conditional mutagenesis that EBF1 is required for B cell commitment, pro–B cell development, and subsequent transition to the pre–B cell stage. Later in B cell development, EBF1 was essential for the generation and maintenance of several mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular (FO) and germinal center (GC) B cells were redu...
Full Text Available West Nile virus (WNV is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1 of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24 were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV, Newcastle Disease Virus (NDV, Duck Plague Virus (DPV and Goose Parvovirus (GPV antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and
Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A; Drake, James R
Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.
Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent
The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent ex...... as compared to mice vaccinated with wild type epitopes. Most of the modifications did not adversely affect the ability of the plasmids to induce complete protection of mice against homologous challenge....... example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively...
Lund, Ole; Nascimento, Eduardo J. M.; Maciel, Milton, Jr
Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV...
L. C. Pontes de Carvalho
Full Text Available Despite the existence of erythrocyte-autoreactive B cells in normal animals, erythrocyte-autoantibodies could not be detected during polyclonal B-cell activation (PBA both in patients with visceral leishmaniasis and in bacterial lipopolysacharide (LPS - injected mice. The failure to detect these autoantibodies in mice with PBA di not seem to be due to suppressor-cell activity, since (1 transfer of spleen cells from LPS-treated mice to naive recipients did not affect the erythrocyte-autoantibody response elicited by subsequent injections of rat erythrocytes and (2 low doses of X-radiation did no lead to erythrocyte-autoantibody detection in LPS-treated mice. The possibility that the detection of erytrocyte-autoantibodies could be affected by autoantibodies with idiotopes mimicring erythrocyte epitopes, the synthesis of which would also be triggerred in PBA, is discussed. Indirect evidence for the existence in normal animal of an expanded lymphocyte population with DNP-binding. Ia-mimicring antigen receptors is presented.
Luxenburger, Hendrik; Graß, Franziska; Baermann, Janina; Boettler, Tobias; Marget, Matthias; Emmerich, Florian; Panning, Marcus; Thimme, Robert; Nitschke, Katja; Neumann-Haefelin, Christoph
Virus-specific CD8 + T-cell responses play an important role in the outcome of hepatitis C virus (HCV) infection. To date, most HCV-specific CD8 + T-cell epitopes have been defined in HCV genotype 1 infection. In contrast, the HCV genotype 4-specific CD8 + T-cell response is poorly defined. Here, we analysed whether known HCV-specific CD8 + T-cell epitopes are also recognized in HCV genotype 4-infected patients and set out to identify the first HCV genotype 4-specific CD8 + T-cell epitopes. We studied patients chronically infected with HCV genotype 1 (n = 20) or 4 (n = 21) using 91 well-described HCV-specific epitope peptides. In addition, we analysed 24 genotype 4-infected patients using 40 epitope candidates predicted using an in silico approach. HCV-specific CD8 + T-cell responses targeting previously described epitopes were detectable in the majority of genotype 1-infected patients (11 of 20). In contrast, patients infected with HCV genotype 4 rarely targeted these epitopes (4 of 21; P = .0247). Importantly, we were able to identify eight novel HCV genotype 4-specific CD8 + T-cell epitopes. Only one of these epitopes was shared between genotype 1 and genotype 4. These results indicate that there is little overlap between CD8 + T-cell repertoires targeting HCV genotype 1 and 4. Prophylactic vaccination studies based on HCV genotype 1 are currently underway. However, in countries with the highest prevalence of HCV infection, such as Egypt, most patients are infected with HCV genotype 4. Thus, prophylactic vaccination strategies need to be adapted to HCV genotype 4 before their application to regions where HCV genotype 4 is endemic. © 2018 John Wiley & Sons Ltd.
Cytotoxic T cell epitopes are the targets for a T cell mediated immunotherapy of cancer. The thesis reports on their identification in the tumor associated proteins BCR-ABL and PRAME by the reverse immunology (prediction) strategy. An extended strategy is used, including the analysis of the
Soysal, S. D.; Muenst, S.; Kan-Mitchell, J.; Huarte, E.; Zhang, X.; Wilkinson-Ryan, I.; Fleming, T.; Tiriveedhi, V.; Mohanakumar, T.; Li, L.; Herndon, J.; Oertli, D.; Goedegebuure, S. P.; Gillanders, W. E.
Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80 % of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for a breast cancer prevention vaccine, and we recently initiated a phase 1 clinical trial of a MAM-A DNA vaccine. Previously, we have identified multiple MAM-A CD8 T cell epitopes using a reverse immunology candidate epitope approach based on predicted binding, but to date n...
Åhsberg, Josefine; Ungerbäck, Jonas; Strid, Tobias; Welinder, Eva; Stjernberg, Jenny; Larsson, Malin; Qian, Hong; Sigvardsson, Mikael
Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.
Åhsberg, Josefine; Ungerbäck, Jonas; Strid, Tobias; Welinder, Eva; Stjernberg, Jenny; Larsson, Malin; Qian, Hong; Sigvardsson, Mikael
Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1+/− pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development. PMID:24078629
In silico analysis of MHC-I restricted epitopes of Chikungunya virus proteins: Implication in understanding anti-CHIKV CD8(+) T cell response and advancement of epitope based immunotherapy for CHIKV infection.
Pratheek, B M; Suryawanshi, Amol R; Chattopadhyay, Soma; Chattopadhyay, Subhasis
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus, responsible for acute febrile infection. The high morbidity and socio-economic loss associated with the recent CHIKV epidemics worldwide have raised a great public health concern and emphasize the need to study the immunological basis of CHIKV infection to control the disease. MHC-I restricted CD8(+) T cell response represent one of the major anti-viral immune responses. Accordingly, it is essential to have a detailed understanding towards CHIKV specific MHC-I restricted immunogenic epitopes for anti-viral CD8(+) CTL immunogenicity. In the present study, a computational approach was used to predict the conserved MHC-I epitopes for mouse haplotypes (H2-Db and H2-Dd) and some alleles of the major HLA-I supertypes (HLA-A2, -A3, -A24, -B7, -B15) of all CHIKV proteins. Further, an in-depth computational analysis was carried out to validate the selected epitopes for their nature of conservation in different global CHIKV isolates to assess their binding affinities to the appropriate site of respective MHC-I molecules and to predict anti-CHIKV CD8(+) CTL immunogenicity. Our analyses resulted in fifteen highly conserved epitopes for H2-Db and H2-Dd and fifty epitopes for different HLA-I supertypes. Out of these, the MHC-I epitopes VLLPNVHTL and MTPERVTRL were found to have highest predictable CTL immunogenicities and least binding energies for H2-Db and H2-Dd, whereas, for HLA-I, the epitope FLTLFVNTL was with the highest population coverage, CTL immunogenicity and least binding energy. Hence, our study has identified MHC-I restricted epitopes that may help in the advancement of MHC-I restricted epitope based anti-CHIKV immune responses against this infection and this will be useful towards the development of epitope based anti-CHIKV immunotherapy in the future. However, further experimental investigations for cross validation and evaluation are warranted to establish the ability of epitopes to induce CD8(+) T cell
Full Text Available The chimeric antibodies anti-CD20 rituximab (Rtx and anti-TNFα infliximab (Ifx induce antidrug antibodies (ADAs in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.
Salvat, Regina S; Parker, Andrew S; Guilliams, Andrew; Choi, Yoonjoo; Bailey-Kellogg, Chris; Griswold, Karl E
Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed "T cell epitopes." Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure-function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4-5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.
Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou
HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinfor......HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using...... immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity...... time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine....
de Rooij, M.F.M.
Chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM) are B-cell malignancies which are still incurable. In these lymphomas, the cells proliferate in specialized niches in lymph nodes and bone marrow, in which they are provided by stromal-derived
Duncan K Ralph
Full Text Available The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called "rearrangement" forming progenitor B cells, then a Darwinian process of lineage diversification and selection called "affinity maturation." The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem "clonal family inference." In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets.
Poudrier, J; Owens, T
Sustained interaction with Th1 cells has been shown to induce IL-2 responsiveness by murine B cells. This is equivalently dependent on CD40, CD54/ICAM-1 and MHC II ligation, and co-cross-linking of CD54 and MHC II in the presence of IL-5 up-regulates a functional IL-2R on B cells. We now show...... that IL-5 (125 U/ml) synergizes with Th1 cells to induce B cell responses to IL-2, that are maintained following T-cell removal, e.g. autonomous. Th1 help in the absence of IL-5 resulted in weak or undetectable responses following T cell removal. The mechanism of IL-5 synergy involved persistence of IL-2R...... anti-Ig did not circumvent the need for IL-5 for autonomous IL-2 responses. Consistent with the above, interaction with an IL-5-producing Th2 clone induced strong autonomous B cell responses to IL-2. Qualitative differences of Th2 help over that of Th1 may thus be attributable to their differential...
Pal Singh, S. (Simar); F. Dammeijer (Floris); R.W. Hendriks (Rudi)
textabstractBruton's tyrosine kinase (BTK) is a non-receptor kinase that plays a crucial role in oncogenic signaling that is critical for proliferation and survival of leukemic cells in many B cell malignancies. BTK was initially shown to be defective in the primary immunodeficiency X-linked
textabstractSignalling from the BCR or its immature form, the pre-BCR, was shown to be crucial for B cell development. Gene-targeted mice have defined differential roles of components of the (pre-) BCR complex or its downstream signalling pathways. One of the proteins involved in (pre-) BCR
Jiang, Manli; Bennani, N Nora; Feldman, Andrew L
Lymphomas are classified based on the normal counterpart, or cell of origin, from which they arise. Because lymphocytes have physiologic immune functions that vary both by lineage and by stage of differentiation, the classification of lymphomas arising from these normal lymphoid populations is complex. Recent genomic data have contributed additional complexity. Areas covered: Lymphoma classification follows the World Health Organization (WHO) system, which reflects international consensus and is based on pathological, genetic, and clinical factors. A 2016 revision to the WHO classification of lymphoid neoplasms recently was reported. The present review focuses on B-cell non-Hodgkin lymphomas, the most common group of lymphomas, and summarizes recent changes most relevant to hematologists and other clinicians who care for lymphoma patients. Expert commentary: Lymphoma classification is a continually evolving field that needs to be responsive to new clinical, pathological, and molecular understanding of lymphoid neoplasia. Among the entities covered in this review, the 2016 revision of the WHO classification particularly impact the subclassification and genetic stratification of diffuse large B-cell lymphoma and high-grade B-cell lymphomas, and reflect evolving criteria and nomenclature for indolent B-cell lymphomas and lymphoproliferative disorders.
Full Text Available The research on T cell immunosuppression therapies has attracted most of the attention in clinical transplantation. However, B cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. Indeed, humoral immune responses can lead to renal allograft rejection even in patients whose cell-mediated immune responses are well controlled. On the other hand, newly studied B cell subsets with regulatory effects have been linked to tolerance achievement in transplantation. Better understanding of the regulatory and effector B cell responses may therefore lead to new therapeutic approaches.Mesenchymal Stem Cells (MSC are arising as a potent therapeutic tool in transplantation due to their regenerative and immunomodulatory properties. The research on MSCs has mainly focused on their effects on T cells and although data regarding the modulatory effects of MSCs on alloantigen-specific humoral response in humans is scarce, it has been demonstrated that MSCs significantly affect B cell functioning. In the present review we will analyze and discuss the results in this field.
Poulsen, Christian Bjørn; Borup, Rehannah; Borregaard, Niels
We have investigated metallothionein (MT) I and II mRNA and protein in B-cell lymphomas with particular reference to diffuse large B-cell lymphoma (DLBCL). The mRNA profiling was performed on Affymetrix arrays and showed up-regulated MT mRNA in 15 of 48 DLBCLs, including 12 of 23 activated B......-cell (ABC) and 3 of 9 type-3 lesions. In contrast, MT mRNA was low to undetectable in 16 germinal center B-cell (GCB)-type DLBCLs. Only 1 of 15 patients with up-regulated MT mRNA achieved a sustained remission, suggesting that up-regulated MT mRNA constitutes a significant risk factor for treatment failure......, in 115 DLBCLs, MT labeling of more than 20% lymphoma cells was associated with a significantly poorer 5-year survival, independent of the age, stage, or International Prognostic Index. Taken together, it is suggested that both increased MT mRNA and MT protein expression by more than 20% lymphoma cells...
Bendtsen, Mette Dahl; Munksgaard, Peter Svenssen; Severinsen, Marianne Tang
Objective: The impact of body mass index (BMI) and body surface area (BSA) on survival in diffuse large B-cell lymphoma (DLBCL) is controversial. Recent studies show superior outcomes for overweight and obese patients. Patients and methods: A total of 653 R-CHOP(-like)-treated DLBCL patients were...
Lee, Donald W.; Khavrutskii, Ilja V.; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L.; Chaudhury, Sidhartha
The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate
Ahmetspahic, Diana; Schwarte, Kathrin; Ambrée, Oliver; Bürger, Christian; Falcone, Vladislava; Seiler, Katharina; Kooybaran, Mehrdad Rahbar; Grosse, Laura; Roos, Fernand; Scheffer, Julia; Jörgens, Silke; Koelkebeck, Katja; Dannlowski, Udo; Arolt, Volker; Scheu, Stefanie; Alferink, Judith
Pro-inflammatory activity and cell-mediated immune responses have been widely observed in patients with major depressive disorder (MDD). Besides their well-known function as antibody-producers, B cells play a key role in inflammatory responses by secreting pro- and anti-inflammatory factors. However, homeostasis of specific B cell subsets has not been comprehensively investigated in MDD. In this study, we characterized circulating B cells of distinct developmental steps including transitional, naïve-mature, antigen-experienced switched, and non-switched memory cells, plasmablasts and regulatory B cells by multi-parameter flow cytometry. In a 6-weeks follow-up, circulating B cells were monitored in a small group of therapy responders and non-responders. Frequencies of naïve lgD + CD27 - B cells, but not lgD + CD27 + memory B cells, were reduced in severely depressed patients as compared to healthy donors (HD) or mildly to moderately depressed patients. Specifically, B cells with immune-regulatory capacities such as CD1d + CD5 + B cells and CD24 + CD38 hi transitional B cells were reduced in MDD. Also Bm1-Bm5 classification in MDD revealed reduced Bm2' cells comprising germinal center founder cells as well as transitional B cells. We further found that reduced CD5 surface expression on transitional B cells was associated with severe depression and normalized exclusively in clinical responders. This study demonstrates a compromised peripheral B cell compartment in MDD with a reduction in B cells exhibiting a regulatory phenotype. Recovery of CD5 surface expression on transitional B cells in clinical response, a molecule involved in activation and down-regulation of B cell responses, further points towards a B cell-dependent process in the pathogenesis of MDD.
Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø
The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted....... Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...
Rita R Barbosa
Full Text Available IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17 share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID, defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21(lowCD38(low. Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies.
Engmark, Mikael; Lomonte, Bruno; Gutiérrez, José María
Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations...... for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high......-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs...
Primary mediastinal large B-cell lymphoma has been recognised as a distinct entity with unique clinical, pathologic, and genetic features. According to WHO 2008 classification it is marked as a variant of diffuse large B-cell lymphoma but shares characteristics with classic Hodgkin lymphoma. Genetic analysis has shown that amplification of the 9p24.1 region is the disease's specific structural alteration. Aggressive behaviour and a tendency to invade surrounding tissues of the thoracic cavity, often causing superior vena cava syndrome, or pleural or pericardial effusions, are the clinical hallmarks of this disease. For a long period of time it has been considered as a disease with poor prognosis, which responds poorly to the conventional treatment created for diffuse large B-cell lymphoma. An elective treatment has not yet been established, but recently the situation has became much more favourable. After the introduction of rituximab the cure rates have risen to over 80%, and the most recent results have demonstrated a new insight with dose-adjusted intensified continuous treatments, in which the cure rates have exceeded 90%. Current trends have led to the introduction of dose-adjusted intensified protocols becoming a standard of care, whereas the use of radiotherapy remains controversial because of the questionable predictive value of post-treatment PET/CT validity. The relapse rate is very low after two years of sustained complete remission. If the disease relapses or is resistant the outcome is very poor regardless of the applied treatment modality. PMID:26843837
Twa, David D W; Steidl, Christian
Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive non-Hodgkin lymphoma that displays phenotypic and genotypic similarity to Hodgkin lymphoma and diffuse large B-cell lymphoma. Studies using genome-wide discovery tools have revealed specific, recurrent structural aberrations as critical somatic events in the pathogenesis of PMBCL. These structural alterations prominently include transcript and protein altering rearrangements and copy number variations of the programmed death ligands 1 (CD274) and 2 (PDCD1LG2), CIITA, JAK2 and REL. Importantly, evidence is emerging that these acquired structural genomic changes, in synergy with other somatic alterations, contribute to PMBCL pathogenesis by influencing tumor microenvironment interactions that favor malignant B-cell growth. The means by which these rearrangements arise are not well understood. However, analysis of breakpoint junctions at base-pair resolution provides preliminary insight into putative rearrangement mechanisms. As the field also anticipates predictive value and therapeutic targeting of structural changes involving programmed death ligands and JAK2, a review of therapies that will likely shape future lymphoma treatment is needed.
Engelberts, Patrick J.; Voorhorst, Marleen; Schuurman, Janine; van Meerten, Tom; Bakker, Joost M.; Vink, Tom; Mackus, Wendy J. M.; Breij, Esther C. W.; Derer, Stefanie; Valerius, Thomas; van de Winkel, Jan G. J.; Parren, Paul W. H. I.; Beurskens, Frank J.
Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20(+) B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab ')2 fragments, as well as C1q-binding-deficient IgG mutants,
Full Text Available BACKGROUND: The envelope (E protein of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217 at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. CONCLUSIONS/SIGNIFICANCE: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.
Full Text Available A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL, whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL. We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.
Fu, Linglin; Wang, Jinbao; Ni, Saiqiao; Wang, Chong; Wang, Yanbo
Chinese shrimp ( Penaeus chinensis) is widely cultured and consumed in Asia but is also a major food allergen locally. Although they may be the foundation for preventing and treating allergies, the allergenic epitopes of the major allergens tropomyosin (TM) and arginine kinase (AK) in Penaeus chinensis have not been identified. Here, we applied Immunoinfo-CB (immunoinformatics coupled with competitive-binding strategy) to address the point. Potential allergenic epitopes of TM and AK were predicted by multiple immunoinformatics tools, followed by validating with inhibitory dot-blot assay, indirect competition ELISA, and mast cell degranulation assay. Furthermore, critical amino acids in allergenic epitopes were also identified by Immunoinfo-CB. Our findings provide new insight into allergenic epitopes and critical amino acids of TM and AK responsible for the anaphylactic response. The Immunoinfo-CB therefore offers promises for characterization of IgE-binding epitopes that might be used as new targets for immunotherapy of food allergy.
Defendenti, Caterina; Grosso, Silvia; Atzeni, Fabiola; Croce, Annamaria; Senesi, Olivia; Saibeni, Simone; Bollani, Simona; Almasio, Piero Luigi; Bruno, Savino; Sarzi-Puttini, Piercarlo
B lymphocytes express various different types of surface immunoglobulins that are largely unrelated to other hematological lines, although some reports have described a relationship between malignant B cells and other cells such as macrophages. Multiple genes of hematopoietic lineage, including transcription factors, are co-expressed in hematopoietic stem cells and progenitors, a phenomenon referred to as "lineage priming". Changes in the expression levels and timing of transcription factors can induce the lineage conversion of committed cells, which indicates that the regulation of transcription factors might be particularly critical for maintaining hierarchical hematopoietic development. The aim of this study was to evaluate the surface markers of particular IgM-positive and irregularly nucleated cells detected in patients with inflammatory bowel disease (IBD), and to assess their association with diagnosis and inflammatory cell recruitment. Small intestine, colon and rectal biopsy specimens of 96 IBD patients were studied. Immunoglobulin-producing cells (IPCs) were analyzed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The specimens positive for B cells with irregular nuclei were assessed using monoclonal antibodies specific for CD79, and λ and κ chains in order to confirm their B cell nature. CD15+ cells, an important marker of inflammatory cell recruitment, were also evaluated. Statistical correlations were sought between the histological findings and clinical expression. 34 (35.4%) of the 96 patients (64 with ulcerative colitis and 32 with Crohn's disease) presented a periglandial localization of IPCs with irregular nuclei, which showed surface markers specific for the B cell subset, such as IgM and CD79, but quantitative differences in λ and κ chains. These specimens also contained CD15-positive cells, which are usually absent in healthy controls. The quantitative aspects and localization of the CD15
Barfod, Lea; Dobrilovic, Tina; Magistrado, Pamela
Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes...... monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas...
Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Tongtawe, Pongsri; Glab-Ampai, Kittirat; Malik, Aijaz Ahmad; Supasorn, Oratai; Chiawwit, Phatcharaporn; Poovorawan, Yong; Chaicumpa, Wanpen
Enterovirus-71 (EV71) and coxsackievirus-A16 (CA16) frequently cause hand-foot-mouth disease (HFMD) epidemics among infants and young children. CA16 infections are usually mild, while EV71 disease may be fatal due to neurologic complications. As such, the ability to rapidly and specifically recognize EV71 is needed to facilitate proper case management and epidemic control. Accordingly, the aim of this study was to generate antibodies to EV71-virion protein-2 (VP2) by phage display technology for further use in specific detection of EV71. A recombinant peptide sequence of EV71-VP2, carrying a predicted conserved B cell epitope fused to glutathione-S-transferase (GST) (designated GST-EV71-VP2/131-160), was produced. The fusion protein was used as bait in in-solution biopanning to separate protein-bound phages from a murine scFv (MuscFv) phage display library constructed from an immunoglobulin gene repertoire from naïve ICR mice. Three phage-transformed E. coli clones (clones 63, 82, and 83) produced MuscFvs that bound to the GST-EV71-VP2/131-160 peptide. The MuscFv of clone 83 (MuscFv83), which produced the highest ELISA signal to the target antigen, was further tested. MuscFv83 also bound to full-length EV71-VP2 and EV71 particles, but did not bind to GST, full-length EV71-VP1, or the antigenically related CA16. MuscFv83 could be a suitable reagent for rapid antigen-based immunoassay, such as immunochromatography (ICT), for the specific detection and/or diagnosis of EV71 infection as well as epidemic surveillance.
Sonet, Anne; Bosly, André
Rituximab is an anti-CD20 chimeric monoclonal antibody with activity in nearly all subtypes of B-cell lymphomas. Association of rituximab with chemotherapy (mostly the cyclophosphamide, doxorubicin, vincristine and prednisolone [CHOP] regimen) in diffuse large B-cell lymphoma (DLBCL) represents an extraordinary revolution in the prognosis of DLBCL, and is the new standard of therapy in elderly and young, low-risk patients. Despite the lack of randomized, clinical trials in younger patients with high risk, rituximab is also a standard of care in these patients in clinical practice, at least in North America. The practice is based on observational trials (e.g., the British Columbia Registry) and the missing logic in classifying patients as 'younger' or 'older': 60 years old or 65 years old. In Europe, trials are ongoing to establish the best treatment for young, high-risk patients. Association of rituximab and chemotherapy deeply modifies prognostic factors defined before the rituximab era.
Tonti, Elena; Jiménez de Oya, Nereida; Galliverti, Gabriele; Moseman, E. Ashley; Di Lucia, Pietro; Amabile, Angelo; Sammicheli, Stefano; De Giovanni, Marco; Sironi, Laura; Chevrier, Nicolas; Sitia, Giovanni; Gennari, Luigi; Guidotti, Luca G.; von Andrian, Ulrich H.; Iannacone, Matteo
Summary Bisphosphonates are a class of drugs that are widely used to inhibit loss of bone mass in patients. We show here that the administration of clinically relevant doses of bisphosphonates in mice increases antibody responses to live and inactive viruses, proteins, haptens and existing commercial vaccine formulations. Bisphosphonates exert this adjuvant-like activity in the absence of CD4+ and γδ T cells, neutrophils or dendritic cells and their effect does not rely on local macrophage depletion nor does it depend upon Toll-like receptor signaling or the inflammasome. Rather, bisphosphonates target directly B cells and enhance B cell expansion and antibody production upon antigen encounter. These data establish bisphosphonates as a novel class of adjuvants that boost humoral immune responses. PMID:24120862
Full Text Available Bisphosphonates are a class of drugs that are widely used to inhibit loss of bone mass in patients. We show here that the administration of clinically relevant doses of bisphosphonates in mice increases antibody responses to live and inactive viruses, proteins, haptens, and existing commercial vaccine formulations. Bisphosphonates exert this adjuvant-like activity in the absence of CD4+ and γδ T cells, neutrophils, or dendritic cells, and their effect does not rely on local macrophage depletion, Toll-like receptor signaling, or the inflammasome. Rather, bisphosphonates target directly B cells and enhance B cell expansion and antibody production upon antigen encounter. These data establish bisphosphonates as an additional class of adjuvants that boost humoral immune responses.
In rare cases, B cells can supply HIV-1-infected individuals with unconventional antibodies equipped to neutralize the wide diversity of viral variants. Innovations in single-cell cloning, high-throughput sequencing, and structural biology methods have enabled the capture and thorough characterization of these exceptionally potent broadly neutralizing antibodies (bNAbs). Here, I review the recent findings in humoral responses to HIV-1, focusing on the interplay between naturally occurring bNAbs and the virus both at systemic and mucosal levels. In this context, I discuss how an improved understanding of bNAb generation may provide invaluable insight into the fundamental mechanisms governing adaptive B cell responses to viruses, and how this knowledge is currently contributing to the development of vaccine and therapeutic strategies against HIV-1. Copyright © 2014 Elsevier Ltd. All rights reserved.
Spangfort, Michael D; Mirza, Osman; Ipsen, Henrik
approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular...... surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid...... substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45...
Edgar E. Lara-Ramírez
Full Text Available The past pandemic strain H1N1 (A (H1N1pdm09 has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI database were used for the analysis. The epitope compositions in A (H1N1pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB, were compared at genomic level. The result showed that A (H1N1 pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA, which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1pdm09 virus. This study may be helpful for better understanding of A (H1N1pdm09 and the production of new influenza vaccines.
Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.
Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648
Gonzalez, Santiago F.; Lukacs-Kornek, Veronika; Kuligowski, Michael P.
an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of Ag to the B cell compartment. In this review, we discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine, such as mannose-binding lectin...... opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes....
Neuberger, M S; Ehrenstein, M R; Rada, C; Sale, J; Batista, F D; Williams, G; Milstein, C
In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene...
Schmitz, Roland; Wright, George W; Huang, Da Wei; Johnson, Calvin A; Phelan, James D; Wang, James Q; Roulland, Sandrine; Kasbekar, Monica; Young, Ryan M; Shaffer, Arthur L; Hodson, Daniel J; Xiao, Wenming; Yu, Xin; Yang, Yandan; Zhao, Hong; Xu, Weihong; Liu, Xuelu; Zhou, Bin; Du, Wei; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Connors, Joseph M; Campo, Elias; Lopez-Guillermo, Armando; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M; Tay Kuang Wei, Kevin; Zelenetz, Andrew D; Leonard, John P; Bartlett, Nancy L; Tran, Bao; Shetty, Jyoti; Zhao, Yongmei; Soppet, Dan R; Pittaluga, Stefania; Wilson, Wyndham H; Staudt, Louis M
Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88 L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).
Full Text Available A 4-year-old child with B-cell acute lymphoblastic leukemia presented with vitreous hemorrhage due to proliferative retinopathy in both eyes. Pars plana vitrectomy was performed in both eyes to clear nonresolving vitreous hemorrhage after systemic stabilization. Visual recovery was limited by the disc drag in the right eye and subfoveal exudation in the left eye. Etiopathogenesis and management of proliferative retinopathy in acute leukemias are discussed.
Mizote, Yu; Taniguchi, Taku; Tanaka, Kei; Isobe, Midori; Wada, Hisashi; Saika, Takashi; Kita, Shoichi; Koide, Yukari; Uenaka, Akiko; Nakayama, Eiichi
Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination. (c) 2010 Elsevier Ltd. All rights reserved.
Altered B cell homeostasis and Toll-like receptor 9-driven response in patients affected by autoimmune polyglandular syndrome Type 1: Altered B cell phenotype and dysregulation of the B cell function in APECED patients.
Perri, Valentina; Gianchecchi, Elena; Scarpa, Riccardo; Valenzise, Mariella; Rosado, Maria Manuela; Giorda, Ezio; Crinò, Antonino; Cappa, Marco; Barollo, Susi; Garelli, Silvia; Betterle, Corrado; Fierabracci, Alessandra
APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation. Total B and switched memory (SM) B cells were reduced while IgM memory were increased in patients. In those having more than 15 years from the first clinical manifestation the defect included also mature and transitional B cells; total memory B cells were increased, while SM were unaffected. In patients with shorter disease duration, total B cells were unaltered while SM and IgM memory behaved as in the total group. A defective B cell proliferation was detected after 4day-stimulation. In conclusion APECED patients show, in addition to a significant alteration of the B cell phenotype, a dysregulation of the B cell function involving peripheral innate immune mechanisms particularly those with longer disease duration. Copyright © 2016 Elsevier GmbH. All rights reserved.
Magomedova, A U; Fastova, E A; Kovrigina, A M; Obukhova, T N; Skidan, N I; Mangasarova, Ya K; Vorobyev, A I; Kravchenko, S K
Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct type of large B-cell lymphoma. In this type of the disease, the neoplastic process is located in the anterior and superior mediastinum, frequently with compression of the superior vena cava and with tumor invasion into the adjacent organs and tissues: the pericardium, lung, pleura, etc. Despite the fact that in PMBCL progression, there may be involvement of extranodal organs, such as the kidney, adrenal glands, liver, and central nervous system, bone marrow (BM) injury is generally absent. Since BM injury in patients with diffuse large B-cell lymphoma is an independent poor prognostic indicator, there is reason to believe that BM involvement in PMBCL affects the prognosis. These cases may need intensified induction therapy followed by autologous hematopoietic stem cell transplantation; and BM injury should be monitored during the therapy. The paper gives reports of clinical cases of bone marrow involvement in 2 PMBCL patients treated at the National Research Center for Hematology, Ministry of Health of the Russian Federation.
Poudrier, J; Owens, T
The mechanism whereby small resting (high buoyant density) murine B cells are induced to express interleukin-2 receptors (IL-2R) and to respond to IL-2 was addressed by staining with anti-IL-2R alpha and -IL-2R beta monoclonal antibodies (mAb), and using receptor-specific cDNA probes. Resting B...... cells expressed undetectable levels of both IL-2R alpha and beta chains on their surface and did not respond to IL-2, even at supra-physiological concentrations. Sepharose-coupled, but not streptavidin-cross-linked, plastic-adsorbed or soluble, anti-mu up-regulated the expression of IL-2R alpha and beta...... chains and mRNA to levels comparable to those seen in activated T cells. Anti-mu-stimulated B cells responded to IL-2 by incorporation of [3H]thymidine and high rate immunoglobulin (Ig) secretion. Both IL-5 (at optimal concentration) and suboptimal lipopolysaccharide (LPS; 20 ng/ml) induced surface...
Turner, Marian L; Hawkins, Edwin D; Hodgkin, Philip D
Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.
Flatland, Bente; Fry, Michael M; Newman, Shelley J; Moore, Peter F; Smith, Joanne R; Thomas, William B; Casimir, Roslyn H
A 5-year-old female spayed domestic shorthair cat was presented for evaluation of tetraparesis. The neurologic lesion was localized to the cervical spinal segment (C1-C6). A left axillary mass was identified, and the results of fine needle aspiration cytology indicated malignant round cell neoplasia of possible histiocytic origin. The cells were large, had marked anisocytosis and anisokaryosis, occasional bi- and multinucleation, and cytoplasmic vacuolation. Euthanasia was performed due to the poor prognosis associated with severe, progressive neurologic signs and a malignant neoplasm. Postmortem examination revealed spinal cord compression and an extradural mass at the C1-C2 spinal segment, with neoplastic cells in the adjacent vertebral bodies, surrounding skeletal muscle, left axillary lymph node, and bone marrow from the right femur. The initial histologic diagnosis was anaplastic sarcoma, but immunohistochemical results indicated the cells were CD20+ and CD45R+ and CD3-, compatible with a diagnosis of B-cell lymphoma. CD79a staining was nonspecific and uninterpretable. Weak to moderate CD18 positivity and E-cadherin positivity were also observed. Clonality of the B-cell population could not be demonstrated using PCR testing for antigen receptor gene rearrangement. To the authors' knowledge, this is the first reported case of a feline spinal anaplastic B-cell lymphoma exhibiting bi- and multinucleated cells. The prognostic significance of this cell morphology and immunophenotype is unknown.
Full Text Available Diffuse Large B-cell Lymphomas (DLBCL are the most frequent Non-Hodgkin Lymphomas (NHL. The addition of Rituximab to the standard chemotherapy CHOP improved the outcome in this patients, but so far 40% of patients experienced relapse or progressive disease. Lenalidomide, an immunomodulatory agent, had direct tumoricidal and antiangiogenetic actions on tumor cells and was able to modulate tumor-cell microenvironment, with the restoration of impaired T-cell activity and the formation of immuno-synapsis. Based on these actions, lenalidomide represented an active drug on aggressive relapsed NHL. In this review, the most relevant clinical trials for the use of lenalidomide in DLBCL were reported. Monotherapy with lenalidomide showed an activity in term of overall response rate, with acceptable hematological and extrahematological toxicities in relapsed/refractory aggressive NHL. The role of lenalidomide as salvage therapy in both cell of origin patterns in DLBCL (germinal center B-cell/activated B-cell was reported in preliminary data. Preliminary data regarding the role of lenalidomide in addition to chemoimmunotherapy (R-CHOP in first line clinical trials were discussed; data of safety, feasibility and efficacy were promising.
The decommissioning of the 324 Facility B Cell includes the onsite transport of grouted remote-handled radioactive waste from the 324 Facility to the 200 Areas for disposal. The grouted waste has been transported in the leased ATG Nuclear Services 3-82B Radioactive Waste Shipping Cask (3-82B cask). Because the 3-82B cask is a U.S. Nuclear Regulatory Commission (NRC)-certified Type B shipping cask, the lease cost is high, and the cask operations in the onsite environment may not be optimal. An alternatives study has been performed to develop cost and schedule information on alternative waste transportation systems to assist in determining which system should be used in the future. Five alternatives were identified for evaluation. These included continued lease of the 3-82B cask, fabrication of a new 3-82B cask, development and fabrication of an onsite cask, modification of the existing U.S. Department of Energy-owned cask (OH-142), and the lease of a different commercially available cask. Each alternative was compared to acceptance criteria for use in the B Cell as an initial screening. Only continued leasing of the 3-82B cask, fabrication of a new 3-82B cask, and the development and fabrication of an onsite cask were found to meet all of the B Cell acceptance criteria
Full Text Available The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.
van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.
ALK-Positive Large B-Cell Lymphoma; Atypical Burkitt/Burkitt-Like Lymphoma; Burkitt-Like Lymphoma With 11q Aberration; Diffuse Large B-Cell Lymphoma Activated B-Cell Type; Diffuse Large B-Cell Lymphoma Associated With Chronic Inflammation; Diffuse Large B-Cell Lymphoma Germinal Center B-Cell Type; Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; EBV-Positive Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; EBV-Positive Mucocutaneous Ulcer; High-Grade B-Cell Lymphoma With MYC, BCL2, and BCL6 Rearrangements; Human Herpesvirus 8-Positive Neoplastic Cells Present; Intravascular Large B-Cell Lymphoma; Large B-Cell Lymphoma With IRF4 Rearrangement; Plasmablastic Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type; Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System; Primary Effusion Lymphoma; Recurrent B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classical Hodgkin Lymphoma; Recurrent Burkitt Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Lymphomatoid Granulomatosis; Recurrent Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classical Hodgkin Lymphoma; Refractory Burkitt Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Small Intestinal High Grade B-Cell Lymphoma, Not Otherwise Specified; T-Cell/Histiocyte-Rich Large B-Cell Lymphoma
CD20 Positive; Recurrent B-Cell Non-Hodgkin Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory B-Cell Non-Hodgkin Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Transformed Recurrent Non-Hodgkin Lymphoma
Wiebe, C; Nevins, T E; Robiner, W N; Thomas, W; Matas, A J; Nickerson, P W
Predicting long-term outcomes in renal transplant recipients is essential to optimize medical therapy and determine the frequency of posttransplant histologic and serologic monitoring. Nonadherence and human leukocyte antigen (HLA) mismatch are risk factors that have been associated with poor long-term outcomes and may help individualize care. In the present study, class II HLA mismatches were determined at the HLA epitope level in 195 renal transplant recipients in whom medication adherence was prospectively measured using electronic monitors in medication vial caps. Recipients were grouped by medication adherence and high (≥10 HLA-DR, ≥17 HLA-DQ) or low epitope-mismatch load. We found that the combination of higher epitope mismatch and poor adherence acted synergistically to determine the risk of rejection or graft loss. Nonadherent recipients with HLA-DR epitope mismatch ≥10 had increased graft loss (35% vs. 8%, p mismatch. At the HLA-DQ locus nonadherent recipients with HLA-DQ epitope mismatch ≥17 had increased graft loss (33% vs. 10%, p mismatch. Subclinical nonadherence early posttransplant combined with HLA class II epitope mismatch may help identify recipients that could benefit from increased clinical, histologic, and serologic monitoring. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Yuan, Bangqing; Zhao, Lin; Xian, Ronghua; Zhao, Gang
Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors. Copyright © 2012 Elsevier Inc. All rights reserved.
Full Text Available Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV, we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1 along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.
Longo, Diane M; Louie, Brent; Mathi, Kavita; Pos, Zoltan; Wang, Ena; Hawtin, Rachael E; Marincola, Francesco M; Cesano, Alessandra
Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males]. Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune
Longo Diane M
Full Text Available Abstract Background Single-cell network profiling (SCNP is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. Methods The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs, 5 European Americans (36-56 yrs, all males]. Results Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt, the MAPK pathways (Erk and p38, and the NF-κB pathway (NF-κB. In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. Conclusions SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from
Escalona, Emilia; Sáez, Darwin; Oñate, Angel
Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus. PMID:28232837
Valentino, Michael D; Abdul-Alim, C Siddiq; Maben, Zachary J; Skrombolas, Denise; Hensley, Lucinda L; Kawula, Thomas H; Dziejman, Michelle; Lord, Edith M; Frelinger, Jeffrey A; Frelinger, John G
Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens. Copyright © 2011 Elsevier B.V. All rights reserved.
Engelberts, P. J.; Voorhorst, M.; Schuurman, J.
. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship...... between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells...... isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC...
Treiber, Thomas; Mandel, Elizabeth M; Pott, Sebastian; Györy, Ildiko; Firner, Sonja; Liu, Edison T; Grosschedl, Rudolf
The transcription factor early B cell factor-1 (Ebf1) is a key determinant of B lineage specification and differentiation. To gain insight into the molecular basis of Ebf1 function in early-stage B cells, we combined a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Among 565 genes that are occupied and transcriptionally regulated by Ebf1, we identified large sets involved in (pre)-B cell receptor and Akt signaling, cell adhesion, and migration. Interestingly, a third of previously described Pax5 targets was found to be occupied by Ebf1. In addition to Ebf1-activated and -repressed genes, we identified targets at which Ebf1 induces chromatin changes that poise the genes for expression at subsequent stages of differentiation. Poised chromatin states on specific targets could also be established by Ebf1 expression in T cells but not in NIH 3T3 cells, suggesting that Ebf1 acts as a "pioneer" factor in a hematopoietic chromatin context. Copyright 2010 Elsevier Inc. All rights reserved.
Kristensen, Birte; Hegedüs, Laszlo; Lundy, Steven K
A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT...... patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors......, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups...
Vilagos, Bojan; Hoffmann, Mareike; Souabni, Abdallah; Sun, Qiong; Werner, Barbara; Medvedovic, Jasna; Bilic, Ivan; Minnich, Martina; Axelsson, Elin; Jaritz, Markus; Busslinger, Meinrad
The transcription factor EBF1 is essential for lineage specification in early B cell development. In this study, we demonstrate by conditional mutagenesis that EBF1 is required for B cell commitment, pro-B cell development, and subsequent transition to the pre-B cell stage. Later in B cell development, EBF1 was essential for the generation and maintenance of several mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular (FO) and germinal center (GC) B cells were reduced in the absence of EBF1. Activation of the B cell receptor resulted in impaired intracellular signaling, proliferation and survival of EBF1-deficient FO B cells. Immune responses were severely reduced upon Ebf1 inactivation, as GCs were formed but not maintained. ChIP- and RNA-sequencing of FO B cells identified EBF1-activated genes that encode receptors, signal transducers, and transcriptional regulators implicated in B cell signaling. Notably, ectopic expression of EBF1 efficiently induced the development of B-1 cells at the expense of conventional B cells. These gain- and loss-of-function analyses uncovered novel important functions of EBF1 in controlling B cell immunity.
Ia-restricted B-B cell interaction. I. The MHC haplotype of bone marrow cells present during B cell ontogeny dictates the self-recognition specificity of B cells in the polyclonal B cell activation by a B cell differentiation factor, B151-TRF2
Ono, S.; Takahama, Y.; Hamaoka, T.
We have demonstrated that B cell recognition of Ia molecules is involved in polyclonal B cell differentiation by B151-TRF2. The present study was undertaken to examine the Ia recognition specificity of B151-TRF2-responsive B cells in fully major histocompatibility complex (MHC)-allogeneic P1----P2, semiallogeneic P1----(P1 x P2)F1, and double donor (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 radiation bone marrow chimeras. The B cells from both P1----P2 and P1----(P1 x P2)F1 chimeras could give rise to in vitro immunoglobulin M-producing cells upon stimulation with B151-TRF2 comparable in magnitude to that of normal P1 B cells, and their responses were inhibited by anti-I-AP1 but not by anti-I-AP2 monoclonal antibody even in the presence of mitomycin C-treated T cell-depleted P2 spleen cells as auxiliary cells. In contrast, the B151-TRF2 responses of P1 B cells isolated from both (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 double bone marrow chimeras became sensitive to the inhibition of not only anti-I-AP1 but also anti-I-AP2 monoclonal antibody only when the culture was conducted in the presence of P2 auxiliary cells, demonstrating that they adaptively differentiate to recognize as self-structures allogeneic as well as syngeneic Ia molecules. Moreover, the experiments utilizing B cells from H-2-congenic mice and B cell hybridoma clones as auxiliary cells revealed that B151-TRF2-responsive B cells recognize Ia molecules expressed on B cells. Taken together, these results demonstrate that B151-TRF2-responsive B cells recognize Ia molecules expressed by B cells as self-structures and that their self-recognition specificity is dictated by the MHC haplotype of bone marrow cells present during the B cell ontogeny but not by the MHC haplotype of a radiation-resistant host environment
Levy, C; Fusil, F; Amirache, F; Costa, C; Girard-Gagnepain, A; Negre, D; Bernadin, O; Garaulet, G; Rodriguez, A; Nair, N; Vandendriessche, T; Chuah, M; Cosset, F-L; Verhoeyen, E
Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc -/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc -/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein
Full Text Available B cell Expansion with NF-κB and T cell Anergy (BENTA disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound in vitro survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.
Zinzani, Pier Luigi; Broccoli, Alessandro
Primary mediastinal B-cell lymphoma is characterized by a high chance of cure, and cured patients have a long disease-free life-expectancy; however, prognosis is severe in the case of relapsed or refractory disease. The initial use of the most effective chemoimmunotherapy regimen is therefore crucial. Understanding who will benefit from postinduction radiotherapy is also of paramount importance; positron emission tomography may be a reliable guide for physicians in determining which patients will require consolidation. New drugs with mechanisms of action including the most relevant biologic features of the tumor may allow better disease control. Copyright Â© 2016 Elsevier Inc. All rights reserved.
Full Text Available Although diffuse large B-cell lymphoma (DLBCL usually occurs in the lymph nodes, approximately 30–40% of the time it can have an extranodal site of involvement and it can arise in nearly every body site such as intestine, bone, breast, liver, skin, lung, and central nervous system. Muscle involvement of DLBCL is especially uncommon, comprising 0.5% of extranodal NHL. We report a case of a 72-year-old man with extranodal DLBCL of a unique manifestation in the calf muscle, involving predominantly the gastrocnemius muscle. The patient achieved complete response and remained free of local recurrence or metastasis following diagnosis.
Carlos Bortoluzzi, Marcelo
The authors report a case of diffuse large B-cell lymphoma (DLBL) of the oral cavity. The patient was a 73-year-old white man who first presented at the Division of Stomatology with a large nodular mass in the hard palate and a nodular lesion in the upper lip, which were diagnosed as DLBL. The patient was treated with eight cycles of CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisone), but the disease recurred 22 months after the end of the therapy. Both primary sites hard palate and upper lip were involved again and the patient was resubmitted to chemotherapy. (author)
Gies, Vincent; Schickel, Jean-Nicolas; Jung, Sophie; Joublin, Aurélie; Glauzy, Salomé; Knapp, Anne-Marie; Soley, Anne; Poindron, Vincent; Guffroy, Aurélien; Choi, Jin-Young; Gottenberg, Jacques-Eric; Anolik, Jennifer H; Martin, Thierry; Soulas-Sprauel, Pauline; Meffre, Eric; Korganow, Anne-Sophie
B cells play a central role in systemic lupus erythematosus (SLE) pathophysiology but dysregulated pathways leading to a break in B cell tolerance remain unclear. Since Toll-like receptor 9 (TLR9) favors the elimination of autoreactive B cells in the periphery, we assessed TLR9 function in SLE by analyzing the responses of B cells and plasmacytoid dendritic cells (pDCs) isolated from healthy donors and patients after stimulation with CpG, a TLR9 agonist. We found that SLE B cells from patients without hydroxychloroquine treatment displayed defective in vitro TLR9 responses, as illustrated by the impaired upregulation of B cell activation molecules and the diminished production of various cytokines including antiinflammatory IL-10. In agreement with CD19 controlling TLR9 responses in B cells, decreased expression of the CD19/CD21 complex on SLE B cells was detected as early as the transitional B cell stage. In contrast, TLR7 function was preserved in SLE B cells, whereas pDCs from SLE patients properly responded to TLR9 stimulation, thereby revealing that impaired TLR9 function in SLE was restricted to B cells. We conclude that abnormal CD19 expression and TLR9 tolerogenic function in SLE B cells may contribute to the break of B cell tolerance in these patients.
Gallagher, Sandra; Turman, Sean; Lekstrom, Kristen; Wilson, Susan; Herbst, Ronald; Wang, Yue
Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistical