Sequence characteristics of Rhipicephalus microplus Enolase gene and prediction of structure and antigenic epitopes of its encoding protein / 中国血吸虫病防治杂志

Objective To analyze the sequence characteristics of Rhipicephalus microplus Enolase gene, and to predict the secondary and tertiary structure and antigenic epitopes of the Enolase protein. Methods Sixty-two engorged female R. microplus were sampled from a yellow cattle breeding farm in Zhijiang County, Huaihua City, Hunan Province in June 25, 2022. Genomic DNA was isolated from R. microplus, and the Enolase gene was amplified using PCR assay, followed by cloning, sequencing and expression of the amplification product. The sequence characteristics of the Enolase gene were analyzed using the software Clustal X, and the gene sequence was translated into amino acid sequences. The secondary and tertiary structures of the Enolase protein were deduced using the software PRABI, and the physicochemical properties of the Enolase protein were analyzed using the software PRABI. In addition, the B- and T-cell epitopes of the Enolase protein were predicted using the software ABCpred Prediction, Scratch, IEDB and NetCTL. Results The R. microplus Enolase gene sequence was 1 323 bp in size, and the contents of A, T, G and C bases were 24.5%, 22.5%, 27.0% and 26.0%,with 47.0% of A + T content and 53.0% of G + C content. The R. microplus Enolase gene encoded 434 amino acids, and the Enolase protein had a molecular weight of 47.12 kDa. The secondary structure of the Enolase protein contained 186 α-helixes (42.86%), 32 β-turns (7.37%), 144 random coils (33.18%) and 72 extended strands (16.59%). The Enolase protein was most probably present in cytoplasm (76.7%), followed by in mitochondrion (39.1%) and nucleus (21.7%), and the Enolase protein had no signal peptide or transmembrane domain. In addition, the Enolase protein had 14 B-cell dominant epitopes and 8 T-cell dominant epitopes. Conclusions The R. microplus Enolase gene sequence exhibits a GC preference, and its encoding Enolase protein is an acidic and hydrophilic protein, with α-helixes and random coils as its primary structure, and presenting B- and T-cell dominant epitopes, which is a potential target for development of vaccines against R. microplus.

Bioinformatics analysis of VP1 of Coxsackievirus A10 / 公共卫生与预防医学

Objective To predict and analyze the physicochemical properties, structural characteristics, and antigenic epitopes of viral protein (VP) VP1 of Coxsackievirus A10 (CV-A10) by bioinformatics methods. Methods The physicochemical properties and structural characteristics of CV-A10 VP1 were predicted by ProtParam, SOPMA, SWISS-MODEL, PDBsum, and ProSA-web. The antigenic epitopes of CV-A10 VP1 were predicted and analyzed by DNAstar, ABCpred, Bepipred 2.0, ElliPro, DiscoTope-2.0, NetMHCpan-4.1, NetMHCIIpan-4.0, Consurf, VaxiJen v.2.0, AllerTOP v.2.0, ToxinPred2, and IEDB immunogenicity. Results Bioinformatics analysis showed that CV-A10 VP1 was a basic, unstable, and hydrophilic protein, of which the secondary structure mainly consisted of random coil. The analysis revealed that CV-A10 VP1 had multiple potential B and T cell antigenic epitopes as well as a dominant antigenic epitope based on the potential epitope. Conclusion CV-A10 VP1 has multiple potential sites that induce specific humoral and cellular immunity, providing important support for its experimental identification, molecular epidemiological studies, and vaccine development.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus / 生物工程学报

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

Genetic evolution analysis of SARS-CoV-2 ORF 1ab/S/M protein and screening of epitopes of mRNA vaccine / 解放军医学杂志

Objective To analyze the genetic and evolutionary properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF 1ab/S/M proteins and select antigen epitope sequences of mRNA vaccines. Methods: We analyzed the worldwide SARS-CoV-2 genome sequences in this study and have focused on the protein and nucleic acid sequences of the ORF 1ab/S/M. The neighbor-joining tree was employed to map the global distribution of genetic differences. Based on current research on SARS-CoV-2 and SARS-CoV-2 genetic differences, we predicted candidate mRNA vaccines for SARS-CoV-2. Results: The SARS-CoV-2 ORF 1ab nucleic acid sequence similarity is 100.0%, while the homology is 99.3% in the global hot region; the S-protein nucleic acid sequence similarity is 100.0%, while the homology is 97.5%; the M-protein nucleic acid sequence similarity is 100.0%, while the homology is 99.9%. Global distribution of ORF 1ab/S/M proteins indicates that there is a significant genetic difference between the Americas and Eurasia. Potential vaccine antigen epitope mRNA sequences (11 B cell responses and 13 T cell responses) were selected for SARS-CoV-2 ORF 1ab protein; 6 B cell responses and 4 T cell responses antigen epitope mRNA sequences were selected for the Spike protein; 3 B cell responses and 7 T cell responses antigen epitope mRNA sequences were selected for the membrane protein. Conclusion: There are significant genetic differences in the global hot spot of SARS-CoV-2 in the Americas and Eurasia. Through our new antigen design strategy to screen linear epitopes, we predicted many sequences in ORF 1ab/S/M coding region that potentially raising an immune response. Our study will benefit the discovery of the mRNA vaccine (tandem antigen epitope sequence), antibody discovery, and potentially understanding related immune mechanisms.

Construction of recombinant influenza virus carrying human metapneumovirus epitopes / 中华微生物学和免疫学杂志

Objective To construct and rescue recombinant influenza virus strains expressing hu-man metapneumovirus ( hMPV) epitopes. -ethods B cell, CTL and Th epitopes predicted by bioinformat-ics software were coupled together in different combinations. These different array genes were inserted into the NS1 gene of influenza virus strain A/PR/8/34 ( PR8 ) , respectively. Recombinant PR8 influenza virus vectors expressing different hMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system. Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric hMPV epitopes. Hemagglutination ( HA) titers, half tissue culture infection dose ( TCID50 ) and growth curves were detected. Results Interval sequences GPGPG and KK were introduced into hMPV epitope combinations to construct multi-epitope antigens (MEA). These MEA were inserted into the PR8 NS gene, respectively. Using 8 plasmid system, three recombinant influenza virus strains were rescued successfully. After cultured for three passages in Madin-Darby canine kidney ( MDCK) cells and one in eggs, these three recombinant strains could proliferate steadily. Whole genome sequencing verified that the three recombinant strains car-ried the chimeric MEA sequences, named as rFLU/hMPV/B, rFLU/hMPV/CTL-Th and rFLU/hMPV/B-Th. HA titers of the recombinant strains were 128, 128 and 256 using turkey erythrocyte, respectively. Their TCID50 were 107. 0/ml, 106. 8/ml and 107. 0/ml, respectively. Growth curve tests also verified that the recombinant strains could proliferate steadily in MDCK cells. Conclusions Three recombinant influenza vi-rus vector strains carrying the B cell, CTL and Th epitopes of hMPV were rescued successfully. This study lays the foundation for further evaluation of the immune effects of these recombinant viruses and their poten-tial application value in vaccine development.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Predominant antigenic epitopes on Hap adhesin of nontypeable Haemophilus influenzae and their immunogenicity / 中华微生物学和免疫学杂志

Objective To investigate the distribution and sequence conservation of Hap adhensin encoding gene (hap) in clinical isolates of nontypeable Haemophilus influenzae (NTHi), to screen out and identify the predominant T-and B-cell (T-B) combined antigenic epitopes on Hap protein and to analyze their immunogenicity.Methods Sequence conservation of hap genes in NTHi strains and T-B combined antigenic epitopes were predicted using bioinformatic softwares.PCR was used to amplify the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap gene (hap-5′-156 and hap-3′-855) and the amplified products were sequenced.Phage display systems of seven T-B combined antigenic epitopes located on the 55 aa segment at N-terminal and the 285 aa segment at C-terminal of Hap protein (Hap-N52 and Hap-C285) were constructed.Western blot assay and ELISA were performed to detect the antigenicity and immunoreactivity of different T-B combined epitopes displayed by recombinant phage PⅢ protein (rPⅢ).Results Hap protein encoded by the hap gene in NTHi was located on membrane surface.Sequences of the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap genes extracted from different NTHi strains were relatively conservative, but many mutations were found in sequences at the middle regions of these hap genes.All of the 56 NTHi strains carried hap-5′-156 and hap-3′-855 segments and shared 92.3％-100％ identities in nucleotide and amino acid sequences of these segements.Hap-N5-24 in the Hap-N52 segment as well as Hap-C4-27, Hap-C28-47, Hap-C114-129, Hap-C150-173, Hap-C200-227 and Hap-C241-267 in the Hap-C285 segment was predicted as the T-B combined antigenic epitope with a higher score and less mutations.Results of Western blot assay and ELISA confirmed that the rPⅢ-displayed Hap-C4-27 and Hap-C150-173 epitopes presented clear hybridization bands with NTHi antisera, and 96.9％ (63/65) and 92.3％ (60/65) of serum samples from children with NTHi infection were positive for antibodies against Hap-C4-27 and Hap-C150-173 epitopes, respectively.Conclusion The gene of hap is widely distributed in clinical isolates of NTHi.Moreover, sequences of the 156 pb segment at 5′-end and the 855 bp segment at 3′-end of hap gene are conservative.Hap-C4-27 and Hap-C150-173 are the predominant T-B combined antigenic epitopes on Hap protein, suggesting that they can be used as epitope candidates for developing multiple antigenic peptide vaccines against NTHi.

Recent advances of neoantigen in tumor therapy / 中国生化药物杂志

As the research work went further and more detailed, a variety of new treatments compete to come out.However, it remains unclear that how the antigen works to distinguish cancer cells and normal cells.Neoantigen, which is located in the tumor cell surface of a specific antigen, its presence makes human immunotherapy into new areas which may make personalized treatment possible in the near future.Emerging data suggest that the identification of such newantigens is a major factor in clinical immunotherapy.They can form a biomarker in cancer immunotherapy to provide targets for a variety of therapeutic approaches to attack, which allows T cells to selectively enhance the immune response against this class of antigens.

Identification and immunogenicity analysis of predominant T-B combined antigenic epitopes on the ;outer membrane protein Loa22 of Leptospira interrogans strains / 中华微生物学和免疫学杂志

Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

Bioinformatics prediction on glutathione S-transferase antigen epitopes of Echinoccocus granulosus / 国际检验医学杂志

Objective To analyse the secondary structure of glutathions S-transferase of Echinoccocus granulosus(EgGST)and predict the B cell and T cell epitopes with informatics tools,in order to provide basic data and references for the following design of epitope vaccine.Methods The B cell and T cell epitopes of EgGST were predicted by DNAstar,Biosun software and Propred MHC class-Ⅱ Binding Peptide Prediction Server software.Results Many distinct antigenic epitopes of EgGST were identified by comput-er,there existed 8 B cell epitopes and 7 T cell epitopes.Conclusion Analysis of predicted epitopes of EgGST antigenic might be sig-nificant for future research of epitope vaccine.

Identification of epitope recognized by mAb 15 A11 sepecific against cartilage oli-gomeric matrix protein / 中国免疫学杂志

Objective:To identify the epitope of mAb15A11 which is specific against RA associated autoantigen cartilage oligomeric matrix protein ( COMP ).Methods: A filamentous phage library displaying random linear dodecapeptides was used to mapping the epitope of mAb15A11.After three rounds of screenings,40 phage clones were selected at random and sequenced.The specificity of phages was confirmed by enzyme immunoassays.Homology search by ClustalW2 and structure analysis by PyMol to identified the epitope amino acid sequence.Western blot analysis of COMP and ELISA analysis of COMP-derived peptides were used to confirm epitope′s characterization.Results: After repeated screenings using bio-panning method, 2 clones were identified, which interacted specifically with mAb 15A11.Homology search did not find succession consensus sequence within COMP molecular,which indicated that the epitope was not linear.PyMol Structure analysis identified the rationality of conformational epitope.Western blot analysis and ELISA of EDTA-treated COMP further prove an conformational structure of the epitope recognized by mAb 15A11.ELISA analysis of COMP-derived peptides demonstrated both disulfide bonds between 229 C-243 C and 237 C-253 C and every epitope amino acid of 232 G,238 H,240 H,241 A,244 V,247 R and 251 R were essential to the binding of mAb 15A11 with COMP.Conclusion: In this study, the potential B cell antigentic epitopes of mAb 15A11 was identified by phage display library.The epitope amino acids sequence and char-acterization were also recognized.It may have important theoretical value for the study of reaction mechanism of COMP antibody and antigen and may also show application significance in the detection of rheumatoid arthritis.

Prediction of antigenic epitopes on HA, NA amino acid sequences of novel influenza A (H7N9) virus and analysis association between susceptibility and HLA-Ⅱalleles / 中国免疫学杂志

Objective:To compare the amino acid sequences difference of HA,NA novel influenza virus A/H7N9 isolates, decipher possible B cell epitopes and T cell epitopes of HA,NA protein,and analyze the association between susceptibility and HLA polymorphisms.Methods:The amino acid sequences of novel influenza A ( H7N9) virus were downloaded from Genbank.Phylogenetic trees were constructed based on the amino acid sequences of HA and NA by using software Clustal X and MEGA 4.0.B cell and T cell epitopes were respectively predicted with Protean software and NetMHCⅡ2.2 Server online server.Results:The homology of HA and NA proteins of H7N9 virus was high.10 B cell epitopes and 15 T cell epitopes were randomly distributed throughout HA sequence and 12 B cell epitopes and 9 T cell epitopes were randomly distributed throughout NA sequence.HLA-DRB1*0701 allele which was commonly observed in Northern Chinese population have a high binding affinity for 9-mer peptides of HA and NA proteins.Conclusion:The prediction of B and T cell epitopes of HA and NA proteins with multiple methods benefits the research and development of vaccine against human infection with avian influenza A H7N9 virus.HLA-DRB1*0701 allele may contribute to susceptibility to novel influenza A (H7N9) virus.H7N9 influenza virus is more easily spread in Urumqi,Harbin,Shandong Province,Liaoning Province,Beijing, Shijiazhuang and Tianjin of China.

Identification of predominant T-and B-combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori / 中华微生物学和免疫学杂志

Objective To screen and identify the predominant T-and B-cell (T-B) combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori (H.pylori).Methods The entire groEL gene of H.pylori strain NCTC11637 was amplified by PCR,and then a prokaryotic expression system for groEL gene was constructed.The expressed target recombinant protein rGroEL was analyzed by SDSPAGE and purified by Ni-NTA affinity chromatography.Laser confocal microscopy was applied to determine the distribution of GroEL in H.pyloristrains of NCTC11637 and SS1.The outer membrane protein (OMP) samples were extracted from the two strains using Triton X-114 method.GroEL in OMP samples was detected by Western blot assay.Special bioinformatic softwares were used to predict T-B combined antigenic epitopes on GroEL molecule,and phage display systems for every T-B combined antigenic epitope were established.Western blot assay and ELISA were used to detect the immunoreactivity of recombinant T-B combined antigenic epitope-containing phage PⅢ proteins and artificially synthesized T-B combined antigenic epitope peptides,respectively.Results The groEL genes from H.pylori strains of NCTC11637 and SS1 showed 97.68％ ～99.63％ identities in their nucleotide and amino acid sequences.The established prokaryotic expression system could efficiently express soluble rGroEL.GroEL located on the outer membrane of H.pylori strains of NCTC11637 and SS1.Among the six major predicted T-B combined epitopes (GroEL168,GroEL258,GroEL288,GroEL365,GroEL396 and GroEL438),GroEL168 showed the strongest positive immunoblotting signal.The results of ELISA showed that the immunoreactivity of GroEL168 was also the strongest (P＜0.01),followed by GroEL258 and GroEL288 (P＜0.05).Conclusion GroEL is an outer membrane protein of H.pylori.GroEL168 being the predominant T-B combined antigenic epitope of GroEL can be used to develop multiple antigenic peptide vaccines of H.pylori.

Preparation and identification of monoclonal antibodies against human IgE / 中华微生物学和免疫学杂志

Objective To obtain monoclonal antibodies(McAb) against human IgE by recombinant IgE antigen.Methods BLAB/c mice were immunized with recombinant IgE.The spleen cells of immunized mice were used to prepare the McAb by hybridoma techniques.IgE-McAb was selected by indirect ELISA with culture supernatant of hybridoma cells.ELISA blocking assay were employed to identify the function of McAb.Alanine scanning mutagenesis were used to confirm the antigenic epitopes of McAb.Results A monoclonal antibody against IgE(178) was produced.The antigenic epitope of 178 was found to be spatially close to the receptor-binding site.Functional assay revealed that 178 could markedly inhibit IgE binding to receptors.Conclusion A hybridoma cell line secreting IgE-McAb stably was obtained by recombinant IgE antigen,and the IgE-McAb might block IgE binding to receptors via steric hindrance.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli / 中国病毒学·英文版

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both ≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Expression and Antigenic Analysis of the Recombinant Epitope of Herpes Simplex Virus Type 2 Glycoprotein G / 中国生物工程杂志

A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.

A preliminary study on targeting antigenic epitopes of monoclonal antibody against human TNF-? / 中国免疫学杂志

Objective:To locate the region of antigenic epitopes recognized by monoclonal antibody(Z8) against human tumor necrosis factor ?(hTNF ?).Methods:Several recombinant fusion expression vectors were constructed which contained hTNF ? mutant genes deleted different parts respectively. After induction with IPTG, the fusion proteins were expressed and analyzed by SDS PAGE and Western blot.Results:Z8 specially recognized the fusion products which contained amino acids from 92 to 157 of hTNF ?, but not GST and its fusion protein with amino acids from 1 to 91 of hTNF ?.Conclusion:The antigenic epitopes recognized by Z8 were located in the sequence region 92 157 of hTNF ?.