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1.
J Agric Food Chem ; 67(46): 12918-12926, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31668066

RESUMO

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.


Assuntos
Braquiúros/enzimologia , Epitopos/química , Triose-Fosfato Isomerase/imunologia , Sequência de Aminoácidos , Animais , Braquiúros/química , Braquiúros/genética , Braquiúros/imunologia , Reações Cruzadas , Cristalização , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoglobulina E/imunologia , Conformação Molecular , Conformação Proteica , Alinhamento de Sequência , Frutos do Mar/análise , Hipersensibilidade a Frutos do Mar/imunologia , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética
2.
Acta Virol ; 63(3): 301-308, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507196

RESUMO

Transmissible gastroenteritis virus (TGEV) causes great economic loss to swine industry worldwide. Vaccination is an important method to control the TGEV infection. In this study, a TGEV oral vaccine was generated by transferring a eukaryotic expression recombinant plasmid carrying the SAD (A and D antigenic sites of the S protein) epitope of TGEV into a swine-origin Lactobacillus acidophilus (L. acidophilus). In orally immunized BALB/c mice, the TGEV L. acidophilus oral vaccine induced significantly higher level of SIgA antibodies specific to TGEV compared with the mice immunized with a commercial inactivated TGEV vaccine and similar levels of IgG specific to TGEV as the inactivated vaccine. Furthermore, the TGEV L. acidophilus oral vaccine induced higher levels of IFN-γ, which suggested that the vaccine was able to induce immune response. In brief, this novel TGEV L. acidophilus oral vaccine could induce high levels of both mucosal and humoral immune responses, which has a potential to be used in the pig industries in the future. Keywords: transmissible gastroenteritis virus (TGEV); live L. acidophilus oral vaccine; SIgA antibody; IgG antibody; IFN-γ; IL-4.


Assuntos
Anticorpos Antivirais , Epitopos , Gastroenterite Suína Transmissível , Lactobacillus acidophilus , Vírus da Gastroenterite Transmissível , Vacinas Virais , Administração Oral , Animais , Anticorpos Antivirais/sangue , Epitopos/genética , Epitopos/imunologia , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/patologia , Imunogenicidade da Vacina/imunologia , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/virologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Suínos , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
3.
Nat Biotechnol ; 37(10): 1137-1144, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31427818

RESUMO

The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.


Assuntos
DNA Mitocondrial/genética , Epitopos/genética , Epitopos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Pluripotentes Induzidas , Animais , Antígenos , Transplante de Células/métodos , Células-Tronco Embrionárias , Rejeição de Enxerto/imunologia , Humanos , Transplante de Rim/efeitos adversos , Transplante de Fígado/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Transplante Autólogo
4.
J Agric Food Chem ; 67(37): 10458-10469, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31469568

RESUMO

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Braquiúros/genética , Clonagem Molecular , Epitopos/imunologia , Cadeias Leves de Miosina/imunologia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Braquiúros/química , Braquiúros/imunologia , Degranulação Celular , Epitopos/química , Epitopos/genética , Humanos , Mastócitos/imunologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Fases de Leitura Aberta , Alinhamento de Sequência
5.
Nat Commun ; 10(1): 3017, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289267

RESUMO

Differences among hosts, resulting from genetic variation in the immune system or heterogeneity in drug treatment, can impact within-host pathogen evolution. Genetic association studies can potentially identify such interactions. However, extensive and correlated genetic population structure in hosts and pathogens presents a substantial risk of confounding analyses. Moreover, the multiple testing burden of interaction scanning can potentially limit power. We present a Bayesian approach for detecting host influences on pathogen evolution that exploits vast existing data sets of pathogen diversity to improve power and control for stratification. The approach models key processes, including recombination and selection, and identifies regions of the pathogen genome affected by host factors. Our simulations and empirical analysis of drug-induced selection on the HIV-1 genome show that the method recovers known associations and has superior precision-recall characteristics compared to other approaches. We build a high-resolution map of HLA-induced selection in the HIV-1 genome, identifying novel epitope-allele combinations.


Assuntos
Evolução Molecular , HIV-1/genética , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Modelos Genéticos , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Teorema de Bayes , Conjuntos de Dados como Assunto , Epitopos/efeitos dos fármacos , Epitopos/genética , Epitopos/imunologia , Genoma Viral/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/imunologia , Seleção Genética/efeitos dos fármacos , Seleção Genética/imunologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-31300131

RESUMO

Small peptides require large carriers to stimulate the humoral immune system. The filamentous phages, such as M13, have been proposed as vectors for expressing and carrying these peptides on their capsid surface. M2e 2-9 (SLLTEVET) residues of the transmembrane protein M2 of Influenza A virus are conserved and considered as a suitable target for immunization against a wide range of Influenza A virus strains. Here, M2e (2-9) sequence of Influenza A virus was fused to the N-terminus of the major coat protein gpVIII of M13 phage and was used to immunize broiler chickens. The results showed that the SLLTEVET peptide on the surface of M13 phage was expressed, the hybrid phage was immunogenic and produced specific antibodies against M2e (2-9) in broiler chickens.


Assuntos
Bacteriófago M13/genética , Epitopos/imunologia , Vírus da Influenza A , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/veterinária , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/genética , Técnicas de Visualização da Superfície Celular , Galinhas/imunologia , Epitopos/genética , Imunogenicidade da Vacina , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/genética
7.
Immunogenetics ; 71(7): 479-487, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270568

RESUMO

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.


Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Antígenos Heterófilos/genética , Arginina/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Suínos
8.
PLoS Pathog ; 15(6): e1007836, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242272

RESUMO

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linhagem Celular , Cricetinae , Dengue/genética , Dengue/imunologia , Vírus da Dengue/genética , Epitopos/genética , Humanos , Proteínas do Envelope Viral/genética
9.
PLoS Pathog ; 15(6): e1007716, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170257

RESUMO

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the ß-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra Dengue , Epitopos , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Epitopos/genética , Epitopos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Macaca mulatta
10.
PLoS Pathog ; 15(6): e1007819, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194843

RESUMO

Recently identified broadly neutralizing antibodies (bnAbs) show great potential for clinical interventions against HIV-1 infection. However, resistant strains may impose substantial challenges. Here, we report on the identification and characterization of a panel of HIV-1 strains with broad and potent resistance against a large number of bnAbs, particularly those targeting the CD4-binding site (CD4bs). Site-directed mutagenesis revealed that several key epitope mutations facilitate resistance and are located in the inner domain, loop D, and ß23/loop V5/ß24 of HIV-1 gp120. The resistance is largely correlated with binding affinity of antibodies to the envelope trimers expressed on the cell surface. Our results therefore demonstrate the existence of broadly resistant HIV-1 strains against CD4bs neutralizing antibodies. Treatment strategies based on the CD4bs bnAbs must overcome such resistance to achieve optimal clinical outcomes.


Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/genética , Antígenos CD4/genética , Linhagem Celular , Epitopos/genética , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos
11.
PLoS Comput Biol ; 15(6): e1007056, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170145

RESUMO

Developing HIV-1 vaccines that trigger broadly neutralizing antibodies (bnAbs) is a priority as bnAbs are considered key to elicitation of a protective immune response. To investigate whether the breadth of a neutralizing antibody (nAb) depended on the conservation of its epitope among circulating viruses, we examined Antibody:Envelope (Ab:Env) interactions and worldwide Env diversity. We found that sites corresponding to bnAb epitopes were as variable as other accessible, non-hypervariable Env sites (p = 0.50, Mann-Whitney U-test) with no significant relationship between epitope conservation and neutralization breadth (Spearman's ρ = -0.44, adjusted p = 0.079). However, when accounting for key sites in the Ab:Env interaction, we showed that the broadest bnAbs targeted more conserved epitopes (Spearman's ρ = -0.70, adjusted p = 5.0e-5). Neutralization breadth did not stem from the overall conservation of Ab epitopes but depended instead on the conservation of key sites of the Ab:Env interaction, revealing a mechanistic basis for neutralization breadth that could be exploited for vaccine design.


Assuntos
Anticorpos Neutralizantes , Sequência Conservada , Anticorpos Anti-HIV , Vacinas contra a AIDS , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Biologia Computacional , Sequência Conservada/genética , Sequência Conservada/imunologia , Epitopos/genética , Epitopos/imunologia , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Simulação de Dinâmica Molecular , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
12.
mSphere ; 4(3)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092602

RESUMO

The Plasmodium vivax Duffy binding protein region II (DBPII) is a vital ligand for the parasite's invasion of reticulocytes, thereby making this molecule an attractive vaccine candidate against vivax malaria. However, strain-specific immunity due to DBPII allelic variation in Bc epitopes may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target conserved epitopes that are potential targets of strain-transcending neutralizing immunity. The minimal epitopes reactive with functionally inhibitory anti-DBPII monoclonal antibody (MAb) 3C9 and noninhibitory anti-DBPII MAb 3D10 were mapped using phage display expression libraries, since previous attempts to deduce the 3C9 epitope by cocrystallographic methods failed. Inhibitory MAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3, while noninhibitory MAb 3D10 binds to a linear epitope in subdomain 1 of DBPII, consistent with previous studies. Immunogenicity studies using synthetic linear peptides of the minimal epitopes determined that the 3C9 epitope, but not the 3D10 epitope, could induce functionally inhibitory anti-DBPII antibodies. Therefore, the highly conserved binding-inhibitory 3C9 epitope offers the potential as a component in a broadly inhibitory, strain-transcending DBP subunit vaccine.IMPORTANCE Vivax malaria is the second leading cause of malaria worldwide and the major cause of non-African malaria. Unfortunately, efforts to develop antimalarial vaccines specifically targeting Plasmodium vivax have been largely neglected, and few candidates have progressed into clinical trials. The Duffy binding protein is considered a leading blood-stage vaccine candidate because this ligand's recognition of the Duffy blood group reticulocyte surface receptor is considered essential for infection. This study identifies a new target epitope on the ligand's surface that may serve as the target of vaccine-induced binding-inhibitory antibody (BIAb). Understanding the potential targets of vaccine protection will be important for development of an effective vaccine.


Assuntos
Antígenos de Protozoários/imunologia , Epitopos/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Ligantes , Vacinas Antimaláricas , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Plasmodium vivax/química , Proteínas de Protozoários/genética , Receptores de Superfície Celular/genética
13.
Can J Vet Res ; 83(2): 97-103, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31097871

RESUMO

In Korea, for the past 30 years (1987-present), porcine epidemic diarrhea (PED) has been established as an endemic situation in which multiple genogroups of classical G1 and G2b, and the recently introduced pandemic G2a, coexisted. Because of the dynamic nature of the virus, continuous field monitoring for PEDV strains is required. This study is the first to reveal prevalence of PEDV in 9 sampling provinces, with an overall detection rate of 6.70%. Porcine endemic diarrhea virus (PEDV) was present in pigs of all ages, especially in the non-PED vaccinated groups. The highest detection rate was in the finisher group (2.34%), followed by that in the newborn group (1.56%). Secondly, using Sanger sequencing, this study recovered a complete genome (28 005 nucleotides long) of NB1 strain from a farm severely affected by PED. Analyses of nucleotide and deduced amino acid sequences showed that NB1 differed from 18 other Korean PEDV mostly in 4 protein coding genes: ORF1a, ORF1b, S, and N. Two amino acid substitutions (V635E and Y681Q) in the COE and S1D neutralizing epitopes of NB1 resulted in antigenic index alteration of the adjacent sites, one of which contributed to a mutation that escaped neutralizing antibodies.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/virologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Epitopos/genética , Fezes/virologia , Variação Genética , Genoma Viral , Vírus da Diarreia Epidêmica Suína/classificação , República da Coreia/epidemiologia , Suínos , Doenças dos Suínos/epidemiologia
14.
Virol J ; 16(1): 72, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138240

RESUMO

BACKGROUND: Human papillomavirus (HPV) E6 and E7 oncoproteins play a crucial role in HPV-related diseases, such as cervical cancer, and can be used as ideal targets for therapeutic vaccines. Human leukocyte antigen (HLA) participates in the immune response to block HPV infection and invasion by its target/recognition function. HPV-33 and HPV-58 are highly prevalent among Chinese women. Therefore, it is of great significance to study the E6 and E7 region-specific gene polymorphisms of HPV-33 and HPV-58 in Southwest China and to identify ideal epitopes for vaccine design. Both HPV-33 and HPV-58 belong to α-9 genus HPV and are highly homologous, so their correlations are included in our research. METHODS: To study the E6 and E7 variations and polymorphisms of HPV-33 and HPV-58 in Southwest China, we collected samples, extracted and sequenced DNA, and identified variants. Nucleotide sequences were translated into amino acids by Mega 6.0 software. The physical/chemical properties, amino acid-conserved sequences and secondary structure of protein sequences were analysed by the Protparam server, ConSurf server and PSIPRED software. The T and B cell epitopes of the E6/E7 reference and variant sequences in HPV-33 and HPV-58 were predicted by the Immune Epitope Database (IEDB) analysis server and the ABCpred server, respectively. RESULTS: Five and seven optimal HLA-I restricted T cell epitopes were selected from HPV-33 and HPV-58 E6, respectively, and these optimal epitopes are mainly located in 41-58EVYDFAFADLTVVYREGN of HPV-33 E6 and 40-60SEVYDFVFADLRIVYRDGNPF of HPV-58 E6. Six optimal HLA-I-restricted T cell epitopes were selected from HPV-33 and HPV-58 E7, and these epitopes are mainly located in 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7. CONCLUSIONS: HPV-33/HPV-58 E6/E7 gene polymorphisms and T/B cell epitopes of their reference and variant sequences were studied, and candidate epitopes were selected by bioinformatics techniques for therapeutic vaccine design for people in Southwest China. This study was the first to investigate the correlation of epitopes between HPV-33 and HPV-58. After experimental validation, these selected epitopes will be employed to induce a wide range of immune responses in heterogeneous HLA populations.


Assuntos
Epitopos/imunologia , Variação Genética , Papillomaviridae/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Epitopos/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Infecções por Papillomavirus/epidemiologia , Filogenia , Neoplasias do Colo do Útero/virologia
15.
Nat Commun ; 10(1): 2073, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061402

RESUMO

Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Simulação por Computador , Desenho de Drogas , Mapeamento de Epitopos/métodos , Epitopos/genética , Epitopos/imunologia , Evolução Molecular , Hepacivirus/genética , Hepatite C/prevenção & controle , Hepatite C/virologia , Humanos , Modelos Biológicos , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/imunologia
16.
Nat Protoc ; 14(6): 1926-1943, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101906

RESUMO

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Eletroporação/métodos , Epitopos/genética , Humanos , Imunoterapia/métodos , Neoplasias/terapia , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção/métodos
17.
Emerg Microbes Infect ; 8(1): 516-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30938227

RESUMO

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética
18.
Microb Cell Fact ; 18(1): 70, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971255

RESUMO

BACKGROUND: Bacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on the Escherichia coli cell surface and then compared it with plasmid-induced expression of OmpF and OmpC. RESULTS: Enhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, onto OmpF of E. coli by genome editing. The resultant fused OmpF proteins were constitutively expressed in the edited E. coli and purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome. CONCLUSIONS: Enhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications.


Assuntos
Técnicas de Visualização da Superfície Celular , Epitopos/genética , Porinas/genética , Anticorpos Antibacterianos , Proteínas do Capsídeo/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Edição de Genes , Proteínas Oncogênicas Virais/genética , Plasmídeos/genética , Mutação Puntual , Proteínas do Envelope Viral/genética
19.
J Agric Food Chem ; 67(17): 4958-4966, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30966750

RESUMO

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.


Assuntos
Aptâmeros de Peptídeos/genética , Arginina Quinase/genética , Arginina Quinase/imunologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Braquiúros/imunologia , Hipersensibilidade a Frutos do Mar/imunologia , Adolescente , Adulto , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Aptâmeros de Peptídeos/imunologia , Arginina Quinase/química , Proteínas de Artrópodes/química , Braquiúros/enzimologia , Braquiúros/genética , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Adulto Jovem
20.
Dokl Biochem Biophys ; 484(1): 52-54, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012013

RESUMO

To enhance the synthesis of antigenic envelope proteins L1 of high-grade papillomavirus types HPV16, HPV18, HPV31, and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This made it possible to increase the production of these antigenic proteins to 25-27 µg/mg total soluble protein.


Assuntos
Cucumovirus , Epitopos , Lycopersicon esculentum , Papillomaviridae/genética , Vacinas contra Papillomavirus , Proteínas Virais , Cucumovirus/genética , Cucumovirus/metabolismo , Epitopos/biossíntese , Epitopos/química , Epitopos/genética , Lycopersicon esculentum/química , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/virologia , Vacinas contra Papillomavirus/biossíntese , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/genética , Proteínas Virais/biossíntese , Proteínas Virais/química , Proteínas Virais/genética
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