Objective To prepare mouse anti-human lymphocyte activation gene 3 (LAG3) monoclonal antibody (mAb) and perform immunological identification of the antibody. Methods BALB/c mice were immunized with LAG3-mLumin-3T3 cells, which stably express the extracellular and transmembrane regions of human LAG3 in mouse 3T3 cells. The secretion of anti-human LAG3 antibodies in mouse serum was assessed using flow cytometry and immunofluorescence. SP2/0 cells were injected subcutaneously into the mice to elicit solid myelomas, and mouse myeloma cells were subsequently isolated. Spleen cells from the immunized mice were fused with the myeloma cells to establish hybridomas, which were then separated using the limiting dilution method. Flow cytometry was used to detect LAG3 mAbs in the hybridoma culture medium. To map the epitopes recognized by these mAbs, 3T3 cells expressing individual extracellular domains of LAG3(LAG3 domains 1/-2/-3/-4-3T3) were used. Flow cytometry was also applied to analyze LAG3 expression on activated human peripheral blood mononuclear cells (PBMC) before and after co-culture with the LAG3 mAbs. Results Mice immunized with the recombinant eukaryotic cell antigen produced anti-LAG3 antibodies. The generated hybridomas secreted mouse anti-human LAG3 mAbs, with each hybridoma line recognizing different LAG3 antigenic domains. Conclusion Mouse anti-human LAG3 mAbs were successfully generated, with different hybridoma clones secreting antibodies that recognize distinct LAG3 epitopes. These findings lay the groundwork for further studies into the biological properties of LAG3 and the development of diagnostic reagents and therapeutic blocking antibodies for cancer treatment.
Animals ; Humans ; Mice ; Lymphocyte Activation Gene 3 Protein ; Antibodies, Monoclonal/immunology* ; Mice, Inbred BALB C ; Hybridomas/immunology* ; Antigens, CD/genetics* ; Immunization ; Recombinant Proteins/immunology* ; Female ; Eukaryotic Cells/immunology* ; Flow Cytometry ; Epitopes/immunology*

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.
Amino Acid Sequence ; Capsid Proteins ; genetics ; immunology ; Computational Biology ; Enterovirus ; genetics ; pathogenicity ; Epitopes, B-Lymphocyte ; genetics ; immunology ; Humans

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

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

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

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

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

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.
Adenoviridae/genetics/immunology ; Administration, Intranasal ; Animals ; Capsid Proteins/*genetics/immunology ; Circoviridae Infections/*immunology ; Circovirus/*genetics/immunology ; Epitopes/genetics/immunology ; Female ; Immunity, Mucosal/immunology ; Immunoglobulin A/blood/immunology ; Immunoglobulin G/blood/immunology ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides/genetics ; Vaccines, Synthetic/genetics/immunology ; Viral Vaccines/administration & dosage/*genetics/immunology

OBJECTIVETo understand the genotypic characteristics and the neutralizing antibody levels of Japanese encephalitis virus (JEV) and Japanese encephalitis (JE) in both vector mosquitoes and in healthy people of Zhejiang province.

METHODSVirus was isolated from mosquitos sampled from the Monitoring Stations located in Xianju county during 2012 to 2013. Phylogenetic and homological studies were carried out on the E gene. A total of 1 263 blood specimens from 642 healthy people were collected before and after the seasons of JE epidemics. JEV neutralizing antibody was detected by the micro-neutralization test.

RESULTSTwenty-five JEV strains were isolated from a total of 11 650 mosquitoes. The identity of nucleotide appeared as 87.8%-99.7% both from 2012 to 2013 and from 1982 to 2010 while as 87.7%-88.0% with vaccine strain SA14-14-2, in Zhejiang. The phylogeny tree of E gene indicated that the newly isolated virus belonged to genotype I but no mutation of amino acid sequence coding conformational epitope was identified in the envelop protein. Both positive rates and the geometric mean titer (GMT) of neutralizing antibody in healthy people were 31.5%-42.0% and 1 : 2.56-1 : 3.53 in Xianju county, during 2012 and 2013, respectively. Both of the two positive rates (χ(2)≤1.76, P > 0.05) and the two GMTs (u≤0.64, P > 0.5) for antibodies pre or post the epidemic season did not show significant differences.

CONCLUSIONJEV isolated in Xianju during 2012 and 2013 belonged to genotype I. The positive rates of JEV neutralizing antibody from healthy people in Xianju were less than 42.0%, which showed no significant differendes pre or post JE epidemic season.

Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; China ; Culicidae ; virology ; Disease Vectors ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; virology ; Epitopes ; Genotype ; Humans ; Neutralization Tests ; Phylogeny