Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.
Adolescent ; Adult ; Allergens/*chemistry/*immunology ; Amino Acid Sequence ; Animals ; Bombyx/*chemistry/genetics/growth & development/*immunology ; Epitopes/immunology ; Female ; Food Hypersensitivity/etiology ; Glycoproteins/*chemistry/genetics/*immunology ; Hot Temperature ; Humans ; Immunoglobulin E/immunology ; Male ; Molecular Sequence Data ; Molecular Weight ; Proteomics ; Pupa/chemistry/immunology ; Recombinant Proteins/biosynthesis/chemistry/immunology ; Sequence Alignment

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

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.
Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; immunology ; Dengue ; virology ; Dengue Virus ; chemistry ; genetics ; immunology ; Epitope Mapping ; Epitopes, B-Lymphocyte ; chemistry ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Viral Envelope Proteins ; chemistry ; genetics ; immunology

To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.
Amino Acid Sequence ; Animals ; Antibodies, Viral ; immunology ; Epitope Mapping ; Epitopes, B-Lymphocyte ; chemistry ; genetics ; immunology ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Nucleoproteins ; chemistry ; genetics ; immunology ; Rabies ; immunology ; virology ; Rabies virus ; chemistry ; genetics ; immunology

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
Amino Acid Sequence ; Animals ; Antibodies/*immunology ; Antigen-Antibody Reactions ; Epitope Mapping ; Epitopes/chemistry/genetics/*immunology ; Glycosylation ; HEK293 Cells ; Heart Failure/immunology ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Natriuretic Peptide, Brain/chemistry/genetics/*immunology ; Peptide Fragments/chemistry/genetics/*immunology ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/immunology

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
Adult ; Amino Acid Sequence ; Animals ; Binding Sites ; Epitopes ; chemistry ; immunology ; metabolism ; Female ; Genotype ; HEK293 Cells ; HLA-A2 Antigen ; metabolism ; Hepatitis B Core Antigens ; chemistry ; immunology ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Hydrogen Bonding ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Middle Aged ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

The molecular characterization and phylogenetic analysis of hemagglutinin (HA) genes of human influenza H3N2 viruses in Guangdong, China from 2007 to 2010 were studied in this study. By space-time sampling of strains, the HA genes of H3N2 strains from Guangdong were sequenced and searched from Internet, and then the variation and evolution of HA genes were conducted by Lasergene 7.1 and Mega 5.05 and evolutionary rates were analyzed by epidemiological data. The phylogenetic tree was established by alignment of 17 Guangdong strains and 26 global reference strains. Ks rates and Ka rates of HA genes were 2.06 x 10(-3)-2.23 x 10(-3) Nt/Year and 1.05 x 10(-3)-1.21 x 10(3) Nt/Year during 2007-2010, while the velocity of HA1 evolution of Ka was 3. 13 times than that of HA2 evolution. Compared with HA of vaccine strain A/Perth/16/2009, the genetic homologies of Guangdong strains in 2009 reached to 98.8%-99.7% and of Guangdong strains in 2010 reached to 98.0%-98.4%. There were some amino acid substitutions in five epitope regions of HA1 during 2007-2010, especially in B region (N160K) and D region (K174R/N); the K189E/N/Q and T228A in RBS (receptor-binding site) occurred in 2010 as two glycoproteins sites substituted impacted on the HA1 antigenicity. The antigenicity of epidemic H3N2 strains in 2010 was to some degree different that of the vaccine strain A/ Perth/16/2009. According to that there were variations of B and D epitopes and two sites of RBS and two glycoprotein in Guangdong H3N2 HA1 genes, WHO/ CDC should recommend new representative strains during 2011-2012 influenza seasons if H3N2 HA genes further evolve in the near future.
Amino Acid Substitution ; China ; Disulfides ; chemistry ; Epitopes ; genetics ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Mutation ; Phylogeny

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

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA