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1.
BMC Vet Res ; 15(1): 274, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370852

RESUMO

BACKGROUND: In Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy's disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies. RESULTS: Two recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4-6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4-6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4-6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity. CONCLUSION: In conclusion, the VP3ep4-6 ELISA method described here can be used for the detection of antibodies to GPV in serum.


Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática/normas , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/diagnóstico , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
2.
Protein Pept Lett ; 26(12): 904-909, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31429685

RESUMO

BACKGROUND: Rotavirus is the most common cause of infectious diarrhea in infants and young children around the world. The inner capsid protein VP6 has been discussed as alternative vaccine as it can induce cross-protective immune responses against different RV strai. The use of ferritin nanoparticle may enhance the immunogenicity of the subunit vaccine. OBJECTIVE: In this article, our motivation is to design and obtain a self-assemble rotavirus nanoparticle vaccine which can induce efficiency immune response. METHODS: The VP6 protein was fused with ferritin and expressed in the Escherichia coli expression system. The recombinant VP6-ferritin (rVP6-ferritin) protein was purified by His-tag affinity chromatography and fast protein liquid chromatography. Transmission electron micrographic analysis was used to detect the nanostructure of the self-assembled protein. Mice were gavage with the protein and ELISA was used to detect the titer of the VP6 specific antibody. RESULTS: The recombined VP6-ferritin was expressed in the Escherichia coli as an inclusion body form and the purified protein has similar antigenicity to rotavirus VP6. Transmission electron micrographic analysis of rVP6-ferritin exhibited spherical architecture with a uniform size distribution, which is similar to the ferritin nanocage. Immune response analysis showed that mice immunized by rVP6-ferritin protein induced 8000 (8000±1093) anti-VP6 IgG titers or 1152 (1152±248.8) anti-VP6 IgA titers. CONCLUSION: According to the above research, the rotavirus VP6-ferritin protein can be easily express and self-assemble to the nano-vaccine and induce efficiency humoral and mucosal immunity. Our research makes a foundation for the development of oral rotavirus vaccine.


Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Escherichia coli/metabolismo , Imunidade nas Mucosas/imunologia , Nanopartículas , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Rotavirus/imunologia , Rotavirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Escherichia coli/genética , Feminino , Ferritinas/genética , Humanos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Vacinas contra Rotavirus/genética
3.
Talanta ; 205: 120110, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450419

RESUMO

Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DµFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DµFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95-10.0 × 103 fg mL-1 and ultralow detection limit of 0.3 fg mL-1. The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.


Assuntos
Closterovirus/isolamento & purificação , Dispositivos Lab-On-A-Chip , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/imunologia , Citrus/virologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Imunoensaio/métodos , Separação Imunomagnética/métodos , Limite de Detecção , Nanopartículas Metálicas/química , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Doenças das Plantas/virologia , Folhas de Planta/virologia , Reprodutibilidade dos Testes
4.
J Vet Sci ; 20(4): e35, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31364320

RESUMO

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21-42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.


Assuntos
Vacinas contra Adenovirus/administração & dosagem , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Imunização/veterinária , Doenças dos Suínos/prevenção & controle , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Feminino , Proteínas Recombinantes/imunologia , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Vacinas Sintéticas/administração & dosagem , Yersinia pseudotuberculosis/genética
5.
Mol Immunol ; 114: 41-48, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336248

RESUMO

Viral myocarditis, mainly caused by enteroviruses specially coxsackievirus B3 (CVB3) infection, is a common clinical cardiovascular disease and characterized by cardiac massive inflammation. Our previous study showed that CVB3-induced myocardial NLRP3 contributed to the development of viral myocarditis. In this study, we found that beside of being up-regulated in myocardiocytes, NLPR3 was also obviously increased in the cardiac infiltrating macrophages. While whether this accumulated NLRP3 influences, macrophage inflammatory responses remains unknown. By adoptive transfer assays, we found that mice receiving NLRP3 up-regulated macrophages showed much more abundant cardiac IL-1ß production and more severe myocardial inflammation, while those receiving NLRP3 down-regulated macrophages showed much less IL-1ß production and milder myocarditis, indicating that NLRP3 up-regulated macrophages played a pathological role in CVB3-induced myocarditis. In addition, we further found that it was CVB3 capsid proteins VP1 (predominant) and VP2, but not viral RNAs, robustly triggered macrophage NLRP3 up-regulation and activation. Our study demonstrated macrophage NLRP3 inflammasome could be efficiently be activated by CVB3 capsid proteins, and contributed to the pathogenesis of viral myocarditis. It might provide some clues to the development of new therapeutic strategies based on macrophage NLRP3 modulation.


Assuntos
Proteínas do Capsídeo/imunologia , Infecções por Coxsackievirus/imunologia , Enterovirus/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Miocardite/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Transferência Adotiva/métodos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo/imunologia , Células HeLa , Coração/virologia , Humanos , Inflamação/imunologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/virologia , Miocárdio/imunologia , Células RAW 264.7 , Regulação para Cima/imunologia , Viroses/imunologia , Viroses/virologia
6.
BMC Vet Res ; 15(1): 232, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286975

RESUMO

BACKGROUND: Recently, serotype 4 fowl adenovirus (FAdV-4) has spread widely and caused huge economic loss to poultry industry. However, little is known about the molecular pathogenesis of FAdV-4. Fiber protein is thought to be vital for its infection and pathogenesis. RESULTS: Two novel monoclonal antibodies (mAbs) targeting the fiber-1 protein of FAdV-4 were generated, designated as mAb 3B5 and 6H9 respectively. Indirect immunofluorescence assay (IFA) showed that both mAbs only reacted with the FAdV-4 and FAdV-10, not with other serotypes including FAdV-1, FAdV-5, FAdV-6, FAdV-7, FAdV-8 and FAdV-9 tested. Although both mAbs did not recognize the linear epitopes, they could efficiently immunoprecipitate the fiber-1 protein in LMH cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber-1. Moreover, mAb 3B5 as a capture antibody and HRP-conjugated mAb 6H9 as a detection antibody, a novel sandwich ELISA for efficient detection of FAdV-4 was generated. The limit of detection of the ELISA could reach to 1000 TCID50/ml of FAdV-4 and the ELISA could be efficiently applied to detect FAdV-4 in the clinical samples. CONCLUSION: The two mAbs specific targeting fiber-1 generated here would pave the way for further studying on the role of fiber-1 in the infection and pathogenesis of FAdV-4, and the established mAb based sandwich ELISA would provide an efficient diagnostics tool for detection of FAdV-4/10.


Assuntos
Infecções por Adenoviridae/diagnóstico , Anticorpos Monoclonais/metabolismo , Aviadenovirus/fisiologia , Proteínas do Capsídeo/imunologia , Doenças das Aves Domésticas/diagnóstico , Infecções por Adenoviridae/virologia , Animais , Anticorpos Antivirais/metabolismo , Aviadenovirus/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Galinhas , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Limite de Detecção , Camundongos Endogâmicos BALB C , Doenças das Aves Domésticas/virologia
7.
Virol J ; 16(1): 75, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159841

RESUMO

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Parvovirus Suíno/imunologia , Animais , Antígenos Virais/química , Mapeamento de Epitopos , Epitopos/química , Masculino , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologia , Suínos
8.
Microb Pathog ; 134: 103568, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31195113

RESUMO

Hand, foot, and mouth disease (HFMD) is a major public health concern, especially among infants and young children. The primary pathogen of HFMD is enterovirus 71 (EV71), whose capsid assembly mechanism including capsid protein processing has been widely studied. However, some of its mechanisms remain unclear, such as the VP0 cleavage. This study aimed to identify the cleavage site of the EV71 VP0 capsid protein and to elucidate the effects of EV71 VP0 cleavage on viral infectivity and assembly. A mass spectrometry analysis indicated that the cleavage site of EV71 VP0 is located between residues Lys69 and Ser70. To analyze the importance of either residue to cleavage, we designed single mutations of Lys69, Ser70 and double mutations respectively and implemented these genomes to encapsulation. The results indicated that Ser70 is more important for VP0 cleavage and EV71 infectivity. In addition, exogenous expression of EV71 protease 2A and 3C was used to verify whether they play roles in VP0 cleavage. Analyses also showed that none of them participate in this process. This study provides novel insights into the mechanisms of EV71 capsid maturation, which may be a potential target to improve the productivity and immunogenicity of EV71 vaccines.


Assuntos
Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/metabolismo , Infecções por Enterovirus/virologia , Clivagem do RNA/fisiologia , Montagem de Vírus , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Células HEK293 , Humanos , Proteínas Virais/metabolismo , Vacinas Virais
9.
Appl Microbiol Biotechnol ; 103(18): 7467-7480, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31253999

RESUMO

Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes 152FQEAFWLEDG161, 168LIYPHQ173, and 46DATSVD51 reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope 152FQEAFWLEDG161 is exposed on surface of the virion and epitopes 46DATSVD51 and 168LIYPHQ173 are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope 168LIYPHQ173 is completely conserved in all BEV strains. Epitope 46DATSVD51 is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope 152FQEAFWLEDG161 is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Bovino/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Bovinos , Epitopos de Linfócito B/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Homologia de Sequência
10.
Eur J Pharm Biopharm ; 141: 221-231, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154067

RESUMO

Thimerosal has been widely used as a preservative in human vaccines for decades. Thimerosal, a thiol capping agent with ethyl mercury being the active degradant, could have impacts on the vaccine potency due to potential thiol modification. The effects on the antigenicity and immunogenicity of human papillomavirus (HPV) virus-like particles (VLPs) in the presence of thimerosal was studied. In general, reduced binding activity was observed between HPV antigens and monoclonal antibodies (mAbs) upon thimerosal treatment, accompanied by reduced protein conformational stability. The immunogenicity of a pentavalent vaccine formulation (HPV6, HPV11, HPV16, HPV18 and hepatitis E virus) with or without thimerosal was studied in mice. The functional antibody titres, as well as the binding titres, were determined, showing a substantial decrease for vaccine formulations containing thimerosal for HPV16/18. Similarly, epitope-specific competition assays using specific and functional mAbs as tracers also showed a significant reduction in immunogenicity for HPV16/18 in the presence of thimerosal. Structural alterations in the capsid protein for HPV18 were observed with cryo-electron microscopy and 3-dimensional reconstruction in the comparative structural analysis. The results should alert scientists in formulation development field on the choice for vaccine preservatives, in particular for thiol-containing antigens.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Antígenos Virais/imunologia , Papillomaviridae/imunologia , Timerosal/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinação/métodos
11.
Virology ; 533: 145-154, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31170612

RESUMO

Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic diversity among its serotypes, requiring several anti-FMDV antibodies for its laboratory diagnosis, which complicated the used techniques. To conquer this cumbersome, we developed a new panel of different single-chain fragment variable (scFv) for serotype-independent detection of FMDV. The recombinant VP2 capsid protein, as a relatively conserved protein among FMDV serotypes, was expressed in E. Coli, and injected in mice. Spleen's RNA was extracted for isolating the coding sequences of IgG variable domains that were assembled into repertoires of scFv. Phage library displaying scFv was constructed with ∼1.9 × 108 plaque forming units. Characterization of the library showed eight of unique scFvs, which were expressed as bacterial periplasmic proteins with apparent molecular weight of ∼27 kDa. Our data revealed the broad-spectrum binding affinity of the eight scFvs as both coating and tracing antibodies to FMDV serotypes A, O, and SAT 2.


Assuntos
Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , Anticorpos de Cadeia Única/análise , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Feminino , Febre Aftosa/diagnóstico , Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Sorogrupo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(5): 459-464, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31223114

RESUMO

Objective To prepare a polyclonal antibody against S3 of Nelson Bay virus (NBV). Methods The E.coli BL21 (DE3) competent cells were transfected with the constructed Pris His MB-S3 recombinant plasmid. Protein expression was induced by IPTG. The target protein was purified by Ni-NTA column affinity chromatography to obtain a large amount of fusion recombinant protein, which was tested and used as the antigen for producing the S3 polyclonal antibody in rats. Results The relative molecular mass (Mr) of Pris His MB-S3 protein was around 39 000, in the form of inclusion bodies. NBV S3 polyclonal antibody was successfully produced from the immunized rats. The titer of the antibody was up to 1:64 000 determined by indirect ELISA. The indirect immunofluorescence assay verified that the S3 protein was successfully expressed in cells and distributed in a granular form in the cytoplasm. Conclusion The highly reactive and specific S3 protein polyclonal antibody is successfully prepared.


Assuntos
Anticorpos , Proteínas do Capsídeo/isolamento & purificação , Orthoreovirus , Proteínas de Ligação a RNA/isolamento & purificação , Animais , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Plasmídeos , Proteínas de Ligação a RNA/imunologia , Ratos , Proteínas Recombinantes de Fusão
13.
Virol J ; 16(1): 57, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046793

RESUMO

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important viral pathogen for swine industry worldwide. However, current PCV2 vaccines provide incomplete protection against the PCV2d, which has recently emerged as the predominant pathogenic form of PCV2. METHODS: To develop a novel DNA vaccine with high efficacy against PCV2d virus, we fused the ORF2 of PCV2d to three copies of the minimum-binding domain of the complement C3 cascade terminal component, C3d-P28. Expression of ORF2 alone (pVO) or fused C3d-P28 (pVOC3) were verified by immunofluorescent assay. Vaccine efficacy was tested by measured the DNA copy and T and B cell immune response. RESULTS: Vaccination with pVOC3 reduced the levels of PCV2 genomic DNA after pigs were infected with either PCV2b or PCV2d genotypes, produced potent antibodies against PCV2, and stimulated PCV2-specific interferon-γ secreting cells. CONCLUSION: Results suggested pVOC3 would be a safe and effective DNA vaccine to confer cross-protection against both PCV2b and PCV2d genotypes in pigs.


Assuntos
Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/genética , Complemento C3d/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Infecções por Circoviridae/prevenção & controle , Circovirus/imunologia , Complemento C3d/genética , Proteção Cruzada , DNA Viral/genética , Genoma Viral , Genótipo , Masculino , Suínos , Doenças dos Suínos/virologia , Vacinação , Vacinas de DNA/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/genética
14.
PLoS One ; 14(5): e0216799, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086415

RESUMO

Evidence has indicated that viral infection increases the risk of developing asthma. Although the association of human parvovirus B19 (B19V) or human bocavirus (HBoV) with respiratory diseases has been reported, little is known about the influence of the B19V-VP1u and HBoV-VP1u proteins on the symptoms of asthma. Herein, we investigated the systemic influence of subcutaneously injected B19V-VP1u and HBoV-VP1u recombinant proteins in an OVA-sensitized asthmatic mouse model. A significantly higher Penh ratio and IgE level were detected in the serum, bronchoalveolar lavage fluid (BALF) and the supernatant of a lymphocyte culture from mice treated with HBoV-VP1u or B19V-VP1u than in a lymphocyte culture from OVA-sensitized mice. Significantly higher levels of serum and BALF IgE, total IgG, IgG1, OVA-specific IgE and OVA-specific IgG1 were detected in mice treated with HBoV-VP1u or B19V-VP1u than in OVA-sensitized mice. Conversely, a significantly lower IgG2a level was detected in mice from the HBoV-VP1u or B19V-VP1u groups than in mice from the OVA group. The mice treated with HBoV-VP1u or B19V-VP1u exhibited more significant lung inflammatory indices, including elevated serum and BALF IL-4, IL-5, IL-10 and IL-13 levels; BALF lymphocyte, neutrophil and eosinophil counts, MMP-9 and MMP-2 activity; and the amount of lymphocyte infiltration, relative to those in the control mice or in those sensitized with OVA. These findings demonstrate that the subcutaneous injection of HBoV-VP1u or B19V-VP1u proteins in OVA-sensitized mice result in elevated asthmatic indices and suggest that human parvoviruses may increase the risk of developing airway inflammation in a mouse model of asthma.


Assuntos
Asma/virologia , Proteínas do Capsídeo/imunologia , Bocavirus Humano/imunologia , Infecções por Parvoviridae/complicações , Parvovirus B19 Humano/imunologia , Animais , Asma/etiologia , Asma/imunologia , Proteínas do Capsídeo/química , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Parvoviridae/imunologia
15.
Gene Ther ; 26(6): 264-276, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110296

RESUMO

The prevalence of adeno-associated virus (AAV) has been investigated in bat populations, but little is known about the biological properties of this virus. In this study, four full-length bat AAV capsid genes were isolated in China, with their amino acid sequences sharing 61% identity with those of AAV2 on average. These capsid genes could package AAV particles in combination with AAV2 rep and ITRs, albeit at a lower efficiency. Bat AAVs could only slightly infect mouse liver but could transduce mouse muscle to some extent after systemic administration with a higher muscle/liver ratio than that of primate AAVs. Bat AAV 10HB showed moderate muscle transduction, similar to that of AAV2, during direct intramuscular injection and, compared with other AAV serotypes, was also relatively efficient in resisting human antibody neutralization after intramuscular injection. Evolutionary analysis revealed a number of codons in bat AAV capsid genes subject to positive selection, with sites corresponding to V259 and N691 in 10HB capsids being localized on the surface of the AAV2 capsid. Mutagenesis studies indicated that the positive selection in bat AAV capsids is driven by their tropism evolution in host species. Taken together, the results of this study indicate that bat AAV 10HB vector has the possible applications for muscular gene therapy, especially in the presence of human AAV neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Dependovirus/imunologia , Terapia Genética/métodos , Vetores Genéticos/imunologia , Evasão da Resposta Imune , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Quirópteros , Dependovirus/genética , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Células HEK293 , Células HT29 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Músculo Esquelético/metabolismo
16.
Poult Sci ; 98(10): 4426-4432, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111922

RESUMO

Currently, both goose astrovirus (GoAstV) and goose-origin Newcastle disease virus (NDV) are widely infectious agents for goslings. There is no vaccine for GoAstV. Capsid protein can elicit a neutralizing antibody in human astroviruses (HAstV). Molecular analysis of the genomic region encoding the capsid protein(ORF2) of goose astrovirus has revealed that it contains neutralizing epitopes. Goose-origin NDV is also an infectious agent. A wide range of NDV strains exist that can be commonly used as vaccine vectors. In the present study, the fusion protein cleavage site RRQKR↓F in a backbone of the virulent goose-origin NDV SH-12 was changed into an avirulent motif GRQGR↓L. The modified goose-origin NDV recombinant vaccine virus expressing the Capsid protein (Cap) of GoAstV was generated as a bivalent vaccine using a reverse-genetics approach. The recombinant virus, rNDV/GoAstV-Cap, was attenuated and similar growth dynamics, cytopathic effects, and virus titers in vitro were maintained when compared to the LaSota strain. Expression of the GoAstV-Cap protein in rNDV/GoAstV-Cap infected cells was detected by an immunofluorescence assay and Western blotting. Goslings inoculated with rNDV/GoAstV-Cap showed no apparent signs of disease and induced GoAstV-Cap-specific immune responses and NDV-specific serum antibody responses to a LaSota vaccination control. Complete protection against a pathogenic GoAstV challenge and avelogenic NDV challenge was conferred. The results of the study suggested that rNDV/GoAstV-Cap viruses have the potential to be the safe, stable, and effective bivalent vaccines.


Assuntos
Avastrovirus/imunologia , Gansos , Doença de Newcastle/terapia , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/terapia , Vacinas Virais/imunologia , Animais , Proteínas do Capsídeo/imunologia , Doença de Newcastle/virologia , Doenças das Aves Domésticas/virologia
17.
Medicine (Baltimore) ; 98(20): e15726, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31096528

RESUMO

RATIONALE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most frequent autoimmune encephalitis in children, and its presentation is various. The disease can be triggered by various infections. PATIENT CONCERNS: Case 1 was a 7-year-old female with the presentation of seizure, repeated fever, language disorder, and decreased muscle strength of the right limbs; Case 2 was a 7-year-old male with the manifestation of repeated emesis, headache, involuntary movement, altered personality, seizures, and cognitive impairment; Case 3 was a 2-year-old female with repeated fever, emesis, seizures, coma, and decreased muscle strength of limbs. Anti-NMDAR antibody was identified in cerebrospinal fluid (CSF) in the 3 cases, confirming the diagnosis of anti-NMDAR encephalitis. Pathogenic examinations revealed positive serum Epstein-Barr virus (EBV)-nuclear antigen and EBV-capsid antigen (CA)-IgG antibodies in the 3 cases, as well as positive EBV-early antigen (EA)-IgG antibody in CSF. Case 1 also had positive EBV-CA-IgA antibody; Case 3 also had positive EBV-CA-IgA and EBV-CA-IgG antibodies. DIAGNOSES: Anti-NMDAR antibody and EBV-EA-IgG antibody in CSF were tested positive in the 3 cases. Thus, they were diagnosed as anti-NMDAR encephalitis associated with reactivated EBV infection. INTERVENTIONS: All of the 3 cases received immunoglobulin, corticosteroid, and ganciclovir treatment. Cases 2 and 3 also received antiepileptic drugs due to repeated seizures. In addition, Case 3 also received assistant respiration, plasma exchange, and rituximab. OUTCOMES: The 3 cases were substantially recovered after treatment. Repeat CSF analysis showed decreased titer of the anti-NMDAR antibody. LESSONS: Reactivated EBV infection may trigger anti-NMDAR encephalitis in children, which has not been reported previously. Related possible virology tests should be completed while diagnosing the disease.


Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato/tratamento farmacológico , Encefalite Antirreceptor de N-Metil-D-Aspartato/virologia , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Convulsões/tratamento farmacológico , Corticosteroides/uso terapêutico , Encefalite Antirreceptor de N-Metil-D-Aspartato/líquido cefalorraquidiano , Anticonvulsivantes/uso terapêutico , Autoanticorpos/líquido cefalorraquidiano , Proteínas do Capsídeo/imunologia , Criança , Pré-Escolar , Infecções por Vírus Epstein-Barr/líquido cefalorraquidiano , Infecções por Vírus Epstein-Barr/complicações , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Ganciclovir/uso terapêutico , Humanos , Imunoglobulinas/uso terapêutico , Masculino , Convulsões/líquido cefalorraquidiano , Convulsões/etiologia , Resultado do Tratamento
18.
Biologicals ; 60: 55-59, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103374

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock animals and control of the disease based on vaccination against serotypes O, A and Asia 1 is important. VP1 (structural) protein and 3A (non-structural) protein is the important antigen in FMDV and they can be used to design recombinant vaccines. In this study the bioinformatics characteristics of VP1 [141-160 and 23-42] and 3A [21-35] of Iranian serotypes O, A and Asia 1 was obtained using on-line predicting software. Then the sequence VP1 [141-160]-GS-VP1 [23-42]-GS-3A [21-35]-GS were codon-optimized and cloned onpHT43shuttle vector and finally expressed in Bacillus subtilis WB600 strain. We could predict VP1 [141-160] as a B cell epitope, VP1 [23-42] as a CTL epitope and 3A [21-35] as a Th cell epitope. The 20KD recombinant protein expressed by Bacillus subtilis were detected by SDS-PAGE. The results showed that this recombinant protein had epitope characteristics and it could be useful as a vaccine candidate to control all serotypes of FMD in Iran.


Assuntos
Bacillus subtilis , Proteínas do Capsídeo , Epitopos de Linfócito B , Vírus da Febre Aftosa , Febre Aftosa/prevenção & controle , Vacinas Virais , Animais , Bacillus subtilis/genética , Bacillus subtilis/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Febre Aftosa/genética , Febre Aftosa/imunologia , Febre Aftosa/patologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Vacinação , Vacinas Sintéticas , Vacinas Virais/genética , Vacinas Virais/imunologia
19.
Microb Cell Fact ; 18(1): 66, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947747

RESUMO

BACKGROUND: Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer's patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis. RESULTS: We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer's patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis. CONCLUSIONS: Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.


Assuntos
Proteínas do Capsídeo/imunologia , Enterovirus Humano B/imunologia , Miocardite/prevenção & controle , Vacinas Virais/imunologia , Administração Oral , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Enterovirus Humano B/química , Enterovirus Humano B/genética , Humanos , Imunidade nas Mucosas , Inteínas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/imunologia , Miocardite/virologia , Nanopartículas/química , Vacinas Virais/administração & dosagem , Vacinas Virais/química , Vacinas Virais/genética
20.
Microb Pathog ; 132: 30-37, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004723

RESUMO

Previous studies on vaccine development against foot-and-mouth disease (FMD) virus reported that application of the inactivated vaccines for FMD virus is not completely effective. Novel vaccinations based on immune-dominant epitopes showed they induced immune responses. In addition, for better and safer immunization, access to of efficient adjuvants against FMD virus seems to be critical. In this study, we produced epitope recombinant vaccines from the VP1 protein of the FMD virus for serotype O of Iran that conjugated with Fc Immunoglobulin (FcIgG) and Interleukin-2 (IL-2). Multiple-epitope constructs included Polytope, Polytope-IL2-FcIgG, Polytope-IL2, Polytope-FcIgG that cloned and expressed in E. coli BL21 (DE3). To evaluate whether these epitope recombinant vaccines induce immune responses, BALB/c mice were injected with the epitope recombinant vaccines and their immune responses were compared with a negative control group. The humoral and cellular immune responses were measured by ELISA. The results showed there were significant differences between the negative control group and other immunized mice with recombinant epitope proteins (p < 0.05). The results of total IgG, IgG1, IgG2a levels and secretion of IFN-γ, IL-4 and IL-10 revealed that immune responses were enhanced when the epitope recombinant vaccine of FMD virus coupled with IL-2 and FcIgG. Observations indicated that the epitope recombinant plasmid of the VP1 protein co-expressed with IL-2 and FcIgG was effective in inducing an enhanced immune response. Therefore, IL-2 and FcIgG could be recommended as a potential adjuvant for epitope recombinant vaccine of the VP1 protein from FMD virus.


Assuntos
Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Imunização , Epitopos Imunodominantes/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Interleucina-2/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Feminino , Vírus da Febre Aftosa/genética , Imunidade Celular , Imunidade Humoral , Imunoglobulina G , Interferon gama , Interleucina-10 , Interleucina-2/genética , Interleucina-4 , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Virais/genética
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