Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-ulsed dendritic cells and cytokine-induced killer cells

AIM： To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells （DCs） and cytokine-induced killer cells. METHODS： Freshly collected hepatocellular carcinoma （HCC） tumor tissues were incubated with a mixture of neuraminidase and recombinant αl,3-galactosyltrans- ferase （αI,3GT） to synthesize α-Gal epitopes on car- bohydrate chains of the glycoproteins of tumor mem- branes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage 111 primary HCC were randomly chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS： The evaluation demonstrated that the pro- cedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly pro- longed the survival of treated patients as compared with the controls （17.1 ± 2.01 mo vs 10.1 ±4.5 mo, P = 0.00121）. After treatment, all patients in the study group had positive delayed hypersensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-y-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO- and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the se- rum. CONCLUSION： This new tumor-specific immunotherapy is safe, effective and has a great potential for the treat- ment of tumors.

Antigen epitopes of animal coronaviruses:a mini-review

Coronaviruses are widespread in nature and can infect mammals and poultry,making them a public health concern.Globally,prevention and control of emerging and re-emerging animal coronaviruses is a great challenge.The mecha-nisms of virus-mediated immune responses have important implications for research on virus prevention and control.The antigenic epitope is a chemical group capable of stimulating the production of antibodies or sensitized lympho-cytes,playing an important role in antiviral immune responses.Thus,it can shed light on the development of diagnos-tic methods and novel vaccines.Here,we have reviewed advances in animal coronavirus antigenic epitope research,aiming to provide a reference for the prevention and control of animal and human coronaviruses.

Recent advances in the study of epitopes,allergens and immunologic cross-reactivity of edible mango

Mango(Mangifera indica L.)is a tropical fruit that is widely consumed as both fresh fruits and processed products around the world.The high incidence of mango allergy,on the other hand,has sparked widespread concern.Therefore,a summary and analysis of the current status and issues in mango allergen research can guide in-depth study on the mechanism of mango allergy and reveal effective desensitization methods.We described the incidence of fruit allergy,as well as the mechanism and clinical symptoms of mango allergy,in this review.We also looked into the structural properties of mango allergens,the effect of processing methods on mango allergens,prediction methods for mango allergen epitopes,and the current state of research on mango cross-reactive allergens and preventive measures.Finally,the research directions and ideas for the future are proposed and discussed.

鸭坦布苏病毒E蛋白特异性抗原表位鉴定及Epitope-ELISA血清学诊断方法建立

旨在建立鸭坦布苏病毒(DTMUV)的特异性血清学诊断方法。本研究以纯化的单克隆抗体1A5为固相筛选分子,利用噬菌体展示技术鉴定DTMUV囊膜(E)蛋白的特异性抗原表位,并利用DNAStar分析抗原表位序列在DTMUV中保守性和与其他黄病毒的E蛋白相应位点的氨基酸序列相似性。结果成功筛选到DTMUV E蛋白的一个线性抗原表位_(87)YAEYI_(91),该抗原表位在DTMUV中具有高度的保守性,无氨基酸位点差异,而与其他黄病毒E蛋白相应位点的氨基酸序列不具有相似性。Dot-Blotting结果表明表位_(87)YAEYI_(91)与DTMUV阳性血清具有良好的亲和性,而与JEV、DENV、WNV等黄病毒阳性血清间不出现交叉反应,可见抗原表位_(87)YAEYI_(91)为DTMUV所特有的抗原表位。利用DTMUV特异性抗原表位87YAEYI_(91)建立Epitope-ELISA血清学诊断方法并对126份鸭血清样品进行检测,以中和试验作为标准进行比较,结果显示Epitope-ELISA检测方法的相对特异性为100%,相对敏感度为96%,并且Epitope-ELISA与其他黄病毒血清间不出现交叉反应性,表明本研究建立的Epitope-ELISA检测方法是一种特异的、灵敏的ELISA血清学诊断方法。

Prediction of B Cell Epitope of DMRT Protein in Oreochromis niloticus

[Objective] The research aimed to predict the B cell epitope of DMRT protein in Oreochromis niloticus. [Method] The secondary structure of amino acid sequence of DMRT protein was revealed by Garnier-Robson, Chou-Fasman and Karplus-Schulz methods. The hydrophilicity plot, surface probability and antigenic index were obtained by Kyte-Doolittle, Emini and Jameson-Wolf methods, respectively. Based on the above results, the B cell epitopes for DMRT were predicted. [Result] Both the prediction results from Garnier-Robson, Chou-Fasman methods indicated that the α-helix centers of DMRT protein in O. niloticus were in the N terminal No. 31-56, 68-75, 110-116, 209-211 and 239-243; the β-sheet centers of DMRT protein in O. niloticus were in the N terminal No. 95-99, 177-183, 225-234 and 251-254. With the assistant of Kyte-Doolittle, Emini and Jameson-Wolf methods, the B cell epitopes for DMRT were located in or nearby the N terminal No. 13-16, 35-38, 47-54,84-93, 101-109, 127-156, 166-177 and 198-201. [Conclusion] These results are helpful for preparing the antibody of DMRT protein and revealing the sex determination mechanism of O. niloticus.

Prediction of Secondary Structure and B Cell Epitope of GH Protein from Acipenser sinensis

[ Objective] The aim was to predict the secondary structure and B cell epitope of growth hormone （GH） protein from Acipenser sinensis. [Method] With the amino acid sequence of GH protein from A. sinensis as the base, the secondary structure of GH protein from A. sinensis was predicted by the method of Gamier-Robson, Chou-Fasman and Karpius-Schulz, and its cell epitope was predicted by the method of Kyte- Doolittle, Emini and Jameson-Wolf. [Result] The sections of 18 -23, 55 -55, 67 -73, 83 -86,112 -114,151 -157 and 209 -211 in the N terminal of GH protein molecule had softer structure and these sections could sway or fold to produce more complex tertiary structure. The sections of 19 -23, 51 -71,84 -95, 128 -139, 164 -176 and 189 -195 in the N terminal of GH protein could be the epitope of B cell and there were flexible regions in these sections or near these sections of GH protein molecule. So the dominant regions could be in these sections or near these sections. [ Conclusion] The research provided the basis for the preparation of monoctonal antibody of GH protein from A. sinensis and provided the reference for the discussion for the molecular regulation mechanism of A. sinensis.

The Prediction of B Cell Epitopes for VP73 Protein of African Fever Virus

[Objective] The B cell epitopes for VP73 protein of African swine fever virus was predicted. [ Method] Based on the analysis of the amino acid sequence and the flexible regions of VP73 protein, the B cell epitopes for VP73 protein of African swine fever virus were predicted by method of Kyte-Doolittie, Emini and Jameson-Wolf. [Result] The B cell epitopes were located at or adjacent to the N-terminal No. 11 - 18,26 -48,73 -82,136 - 150,159 - 174,181 - 189,191 - 210,247 - 276,279 - 295,313 - 323 and 382 - 392. [Conclusion] The multi-parameters analytic method was adopted to predict the B cell epitopes for VP73 protein of African swine fever virus, which laid solid foundation for further characterizing the protein of VP73 and researching epitope vaccine.

Analysis of BAC_5 mcAb-Related Epitope Using Random Peptide library

Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

Bioinformatics Analysis on B cell Epitopes of Rice Allergen RAG1

[Objective] To predict the secondary structure and B cell epitopes of the rice major allergen RAG1. [Method] The amino acid sequence of rice allergen RAG1 was acquired from Expasy protein database. The secondary structure of RAG1 was predicted by DNAStar Protean software with Gamier-Robson program, Chou-Fasman program and Karplus-Schulz program; the B cell epitopes of RAG1 was predicted with the Kyte Doolittle hydrophilic program, Emini surface accessibility program and Jameson-Wolf antigenic index program. [Result] The predictions on secondary structure and B cell epitopes showed that the regions of 33-44, 119-129, 155-163 were the dominant B cell epitopes. [Conclusion] This study predicted the potential dominant B cell epitopes in rice allergen RAG1 by comprehensive use of multi-methods and multi-parameters, and provided a theoretical basis for further researches on identification, antigen modification and epitope vaccine design of RAG1 B cell epitopes.

Prediction on Antigenic Epitope Characteristics of Bt Cry2Ab Protein in Transgenic Crops

[Objective] This study aimed to predict the structural characteristics of Bt Cry2Ab protein in transgenic crops with bioinformatic analysis to provide the theoretical clues for design of antibody Cry2Ab. [Method] The amino acid sequence of Cry2Ab protein was searched from NCBI database. The B cell epitopes were predicted with DNAStar. The binding affinity between Cry2Ab protein and MHC-II molecules was analyzed with NetMHCII 2.2 Server to predict the T cell epitopes. [Result] Prediction result suggested the potential B cell epitope of Cry2Ab locating in the region of 208-215. Analysis of the binding affinity between Cry2Ab and MHC-II molecules suggested the regions of 177-185, 299-307 and 255-263 were the potential T cell epitopes. Human with HLA-DRB10101 alleles and HLA-DRB10701 alleles were more sensitive to Cry2Ab protein. [Conclusion] This study facilitates to understand the structural characteristics of Cry2Ab protein and provides a new clue to improve the assessment method for potential allergenicity of genetically modified food.

Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum

Objective:To analyze the structure of aquaporins-3(AQP-3) from Schistosoma japonicum(SJAQP-3) using bioinformalical methods,and to provid of references for vaccine targets research.Methods:Protparam,BepiPred,TMHMM Server,MLRC,Geno3d,DNA star software packages were used to predict the physical and chemical properties,hydrophilicity plot, flexibility regions,antigenic index,surface probability plot,secondary structure,and tertiary structure of amino acid sequence of SJAQP-3.Results:SJAQP-3 had six transmembrane regions and two half-spanning helices that form a central channel.The half-spanning helices fold into the centre of the channel.Either of the half-spanning helix had a conserved motif of NPA common to all aquaporins.Predicted linear B-Cell epitopes were most likely at the N-terminal amino acid residues of Saa-7aa,59aa- 62aa,225aa-230aa,282aa -288aa,294aa -29Saa and 305aa -307aa area.59aa- 62aa,22Saa-230aa located outside the membrane,the others located inside the cell.Conclusions:SJAQP-3 is a integral membrane protein in Schistosoma japonicum tegument.There are six potential epitopes in SJ AQP-3.It might be a potential molecular target for the development of vaccines.

Preparation and Initial Application of a Monoclonal Antibody Specific for a Newly Discovered Conserved Linear Epitope of Rabies Virus Nucleoprotein

Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate （FITC） after purification and used in fluorescent antibody test （FAT）. Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line （BSR）. Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies

Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

AIM:To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). METHODS:Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore,the inner lipoyl peptide 167-184 was used in an unlip oylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA posit ive PBC patients,63 patients with other liver disorders and 22 healthy blood donors served as controls.RESULTS:Of the 95 PBC-sera,74% reacted with the peptide 475-499 and 58% with the pept ide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylatedand lip oylated pept ide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera,respectively; using ovalbumin-coupled peptides,the incidence increased up to 57% (unlipoylated form). CONCLUSION:Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodomin ant epitopes recognized by AMA in PBC.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction

In recent years,the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein,a general overview of different tools currently available,including T cell and B cell epitopes prediction tools,is presented. And the principles of different prediction algorithms are reviewed briefly. Finally,several examples are present to illustrate the application of the prediction tools.

Identification of an epitope of SARS-coronavirus nucleocapsid protein

The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SAR,S-CoV. Furthermore, it was confirmed that Nl peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective:To predict B cell and T cell epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins.Methods:The sequences of 22-kDa,47-kDa,56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA,DNAstar,Bcepred,ABCpred,NetMHC,NetMHCⅡand IEDB.The 58-kDa tertiary structure model was built by MODELLER9.17.Results:The 22-kDa B-cell epitopes were located at positions 194-200,20-26 and 143-154,whereas the T-cell epitopes were located at positions 154-174,95-107,17-25 and 57-65.The 47-kD a protein B-cell epitopes were at positions 413-434,150-161 and 283-322,whereas the T-cell epitopes were located at positions 129-147,259-267,412-420 and 80-88.The 56-kDa protein B-cell epitopes were at positions 167-173,410-419 and 101-108,whereas the T-cell epitopes were located at positions 88-104,429-439,232-240 and 194-202.The 58-kDa protein B-cell epitopes were at positions 312-317,540-548 and 35-55,whereas the T-cell epitopes were located at positions 415-434,66-84 and 214-230.Conclusions:We identified candidate epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins from Orientia tsutsugamushi.In the case of 58-kDa,the dominant antigen is displayed on tertiary structure by homology modeling.Our findings will help target additional recombinant antigens with strong specificity,high sensitivity,and stable expression and will aid in their isolation and purification.

Immune Potential of a Novel Multiple-epitope Vaccine to FMDV Type Asia 1 in Guinea Pigs and Sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized. The biologically functional molecule, the ovine IgG heavy constant region (oIgG) as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed. The recombinant proteins, RE and RE-oIgG, were expressed as a formation of inclusion bodies in E. coli. The immune potential of this vaccine regime in guinea pigs and sheep was evaluated. The results showed that IgG could significantly enhance the immune potential of antigenic epitopes. The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs, but confer complete protection in guinea pigs against virus challenge. Although the recombinant protein RE could not confer protection in the vaccinated animals, it could delay the appearance of the clinical signs and reduce the severity of disease. Inspiringly, the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination. Therefore, we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.

Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

AIM:To investigate the variability of the main immunodominant motifs of hepatitis B virus(HBV) core gene by ultra-deep-pyrosequencing(UDPS).METHODS:Four samples(2 genotype A and 2 genotype D) from 4 treatment-na ve patients were assessed for baseline variability.Two additional samples from one patient(patient 4,genotype D) were selected for analysis:one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment.The HBV region analyzed covered amino acids 40 to 95 of the core gene,and included the two main epitopic regions,Th50-69 and B74-84.UDPS was carried out in the Genome Sequencer FLX system(454 Life Sciences,Roche).After computer filtering of UDPS data based on a Poisson statistical model,122 813 sequences were analyzed.The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture.RESULTS:Positions with highest variability rates were mainly located in the main core epitopes,confirming their role as immune-stimulating regions.In addition,the distribution of variability showed a relationship with HBV genotype.Patient 1(genotype A) presented the lowest variability rates and patient 2(genotype A) had 3 codons with variability higher than 1%.Patient 3 and 4(both genotype D) presented 5 and 8 codons with variability higher than 1%,respectively.The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed,approaching significance(P = 0.07,sample 1 and P = 0.05,sample 2).In contrast,there were no significant differences in variability between the epitopic and other positions in genotype D cases.Interestingly,patient 1 presented a completely mutated motif from amino acid 64 to 67(E 64 LMT 67),which is commonly recognized by T helper cells.Additionally,the variability observed in all 4 patients was particularly associated with the E 64 LMT 67 motif.Codons 78 and 79 were highly conserved in all samples,in keeping with their involvement in the interaction between the HBV virion capsid and the surface antigens(HBsAg).Of note,codon 76 was even more conserved than codons 78 and 79,suggesting a possible role in HBsAg interactions or even in hepatitis B e antigen conformation.Sequential analysis of samples from patient 4(genotype D) illustrated the dynamism of the HBV quasispecies,with strong selection of one minor baseline variant coinciding with a decrease in core variability during the treatment-free and lamivudinetreated period.The drop in variability seemed to result from a "steady state" situation of the HBV quasispecies after selection of the variant with greatest fitness.CONCLUSION:Host immune pressure seems to be the main cause of HBV core evolution.UDPS analysis is a useful technique for studying viral quasispecies.