As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Monoclonal, Humanized ; immunology ; Antigens ; Epitopes ; metabolism ; Protein Domains ; immunology ; Receptors, Antigen ; chemistry ; immunology ; Sharks

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.
Epitopes, B-Lymphocyte/*chemistry/genetics/*immunology ; Female ; Humans ; Immunodominant Epitopes/chemistry/genetics/*immunology ; Malaria, Vivax/immunology/*parasitology ; Middle Aged ; Plasmodium vivax/chemistry/genetics/*immunology ; Protein Structure, Tertiary ; Protozoan Proteins/chemistry/genetics/*immunology ; Reticulocytes/*parasitology

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

The secondary structures of fusion protein pp150/MDBP, including alpha-helix, beta-sheet, turn regions, were analyzed by Garnier-Robson's and Chou-Fasman's methods; the antigenic epitopes of B cells were analysed by using hydrophilicity plot. The results showed that the fusion protein pp150/MDBP might have less alpha-helix, but be rich in beta-sheet and turn regions. The epitopes recognized by B cells may be at 7-56 amino acid residues or adjacent to 137-192 amino acid residues.
Amino Acid Sequence ; Binding Sites ; Cytomegalovirus ; chemistry ; Epitopes ; Humans ; Molecular Sequence Data ; Phosphoproteins ; chemistry ; immunology ; Protein Structure, Secondary ; Viral Fusion Proteins ; chemistry ; immunology ; Viral Matrix Proteins ; chemistry ; immunology

To establish a relation between an protein amino acid sequence and its tendencies to generate antibody response, and to investigate an improved in silico method for linear B-cell epitope (LBE) prediction. We present a sequence-based LBE predictor developed using deep maxout network (DMN) with dropout training techniques. A graphics processing unit (GPU) was used to reduce the training time of the model. A 10-fold cross-validation test on a large, non-redundant and experimentally verified dataset (Lbtope_Fixed_ non_redundant) was performed to evaluate the performance. DMN-LBE achieved an accuracy of 68.33% and an area under the receiver operating characteristic curve (AUC) of 0.743, outperforming other prediction methods in the field. A web server, DMN-LBE, of the improved prediction model has been provided for public free use. We anticipate that DMN-LBE will be beneficial to vaccine development, antibody production, disease diagnosis, and therapy.
Amino Acid Sequence ; Computational Biology ; methods ; Epitopes, B-Lymphocyte ; chemistry ; immunology ; ROC Curve

CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.
Amino Acid Sequence ; Antibodies, Monoclonal/*immunology ; Antigens, CD/*chemistry/*immunology ; Blotting, Western ; Cell Adhesion Molecules/*chemistry/*immunology ; *Epitope Mapping ; Epitopes/*chemistry/*immunology ; Glutathione Transferase ; Human ; Molecular Sequence Data ; Peptide Fragments/chemistry/immunology ; Recombinant Fusion Proteins/chemistry/immunology

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.
Binding Sites ; Blood Group Antigens ; chemistry ; immunology ; Caliciviridae Infections ; immunology ; virology ; Crystallography, X-Ray ; Epitopes ; chemistry ; immunology ; Evolution, Molecular ; Humans ; Lewis Blood-Group System ; chemistry ; immunology ; Norovirus ; chemistry ; immunology ; pathogenicity ; Protein Binding ; Viral Proteins ; chemistry ; immunology

The abnormal glycans expressing on the surface of tumor cells are good targets to develop carbohydrate-based anti-cancer vaccines. However, one of the major problems is that carbohydrate antigens possess weak immunogenicity. This review summarizes the recent efforts to overcome this problem: glycoconjugates produced by coupling the carbohydrate antigens and proper carrier proteins improve their immunogenicity, many glycoconjugates have entered clinical trials; the vaccines become chemically well-defined when coupling the carbohydrate antigens with a T-cell peptide epitope and an immunostimulant to form fully synthetic multi-component glycoconjugate vaccines; the modification of carbohydrate antigens in combination with the technology of metabolic oligosaccharide engineering of tumor cells induces a strong immune response; and the fact that the antibodies elicited against the unnatural carbohydrate antigens can recognize the native carbohydrate antigens on tumor cells provides a new promising strategy for the development of anti-cancer vaccines.
Animals ; Antigens, Tumor-Associated, Carbohydrate ; chemistry ; immunology ; Cancer Vaccines ; chemical synthesis ; chemistry ; immunology ; therapeutic use ; Carbohydrates ; chemistry ; immunology ; Epitopes, T-Lymphocyte ; chemistry ; immunology ; Glycoconjugates ; chemistry ; immunology ; Humans ; Immune Tolerance ; Metabolic Engineering ; methods ; Neoplasms ; prevention & control ; therapy ; Oligosaccharides ; chemistry

BACKGROUNDTo characterize the intertype epitopes on human adenovirus (HAdV) hexon.

METHODSBased on computerized analysis on adenoviruses sequence of genomic alignment, antigenicity prediction and 3-D structure characteristics of hexon subunit, several peptides of hexon of adenoviruses were chosen to be synthesized or recombinant proteins of the hexon were expressed in E. coli by use of PGEX-5X. To identify the existence of intertype epitopes, the antisera raised with synthetic peptides or purified recombinant proteins were analyzed with Western blot and immunofluorescent assay.

RESULTSThe results of Western blot indicated that both peptide and recombinant antibodies showed specific reactivities with hexons of HADv-3, 4, 7 individually. Meanwhile, typical stain of immunofluorescence was found on HeLa cells infected with these HAdV by incubation with peptide as well as recombinant antibodies. Also, antibodies raised against peptide recognized the recombinant hexon protein in which a corresponding region of peptides was covered.

CONCLUSIONSMost of the predicted intertype epitopes of HAdV hexon wer e exclusively found in synthetic peptides and recombinant proteins. These intertype epitopes showed to be continuous and sequential which could be employed for development of antibodies of diagnostic use.

Adenoviruses, Human ; immunology ; Amino Acid Sequence ; Animals ; Capsid ; chemistry ; immunology ; Capsid Proteins ; immunology ; Epitopes ; immunology ; HeLa Cells ; Humans ; Mice ; Peptide Fragments ; immunology