Resumo
Background: Foot and mouth disease (FMD) is the causative agent and one of the most transmissible livestock diseaseswhich cause important economic losses. The genome of FMD virus is a positive-sense, single-stranded RNA and it wrappedwith 60 copies of 4 structural proteins, VP1, VP2, VP3 and VP4. The VP1 is a major immunogenic antigen with criticalepitopes for inducing immune responses. The current vaccine, which successfully prevents disease, includes inactivatedwhole virus antigen. However, it is not without problems. The aim of this study is enhancement of immune responsesagainst Iranian isolate of FMD-type O/IRN/1/2010 based on VP1 and human HSP70 fusion protein in BALB/c mice.Materials, Methods & Results: In this study FMD virus type O/IRN/2010 was isolated from vesicles of the infected cattlein Qoum, and serotyped as a new linage of type O (PanAsia-linage 2) in Iran. The isolated FMD virus was propagated onIBRS2 cell line and whole RNA of the infected cells was extracted by commercial kit instruction. The extracted RNA wasamplifi ed using VP1 gene-specifi c primer pairs by means of one-step RT-PCR. The specifi c primer pair was designed byAllelID6 software. There are sequences of Kpn I and BamH I restriction enzymes and three overhanging nucleotides atthe start of forward and reverse primers, respectively. The VP1 nucleotide sequence was deposit in Genbank-NCBI database under accession number JN 676146. The purifi ed VP1 gene was sub-cloned into PTZ57R/T vector. Then digestedVP1 gene by KpnI and BamHI enzymes was ligated in pcDNA3.1+ vector as a DNA vaccine. Also, the improved DNAimmunization system was constructed using pcDNA3.1+ plasmid contains VP1 gene of Iranian isolate FMD virus typeO/IRN/1/2010 and human HSP70 gene and expression of VP1-HSP70 fusion protein confi rmed in BHKT7 cell line byGuinea pig specifi c polyclonal antibody against FMD virus type O and conjugated rabbit...(AU)
Assuntos
Animais , Camundongos , Genoma Viral , Proteínas Virais/isolamento & purificação , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Vacinas de DNA/análise , Camundongos Endogâmicos BALB C , Proteínas de Choque Térmico HSP70Resumo
Background: Foot and mouth disease (FMD) is the causative agent and one of the most transmissible livestock diseaseswhich cause important economic losses. The genome of FMD virus is a positive-sense, single-stranded RNA and it wrappedwith 60 copies of 4 structural proteins, VP1, VP2, VP3 and VP4. The VP1 is a major immunogenic antigen with criticalepitopes for inducing immune responses. The current vaccine, which successfully prevents disease, includes inactivatedwhole virus antigen. However, it is not without problems. The aim of this study is enhancement of immune responsesagainst Iranian isolate of FMD-type O/IRN/1/2010 based on VP1 and human HSP70 fusion protein in BALB/c mice.Materials, Methods & Results: In this study FMD virus type O/IRN/2010 was isolated from vesicles of the infected cattlein Qoum, and serotyped as a new linage of type O (PanAsia-linage 2) in Iran. The isolated FMD virus was propagated onIBRS2 cell line and whole RNA of the infected cells was extracted by commercial kit instruction. The extracted RNA wasamplifi ed using VP1 gene-specifi c primer pairs by means of one-step RT-PCR. The specifi c primer pair was designed byAllelID6 software. There are sequences of Kpn I and BamH I restriction enzymes and three overhanging nucleotides atthe start of forward and reverse primers, respectively. The VP1 nucleotide sequence was deposit in Genbank-NCBI database under accession number JN 676146. The purifi ed VP1 gene was sub-cloned into PTZ57R/T vector. Then digestedVP1 gene by KpnI and BamHI enzymes was ligated in pcDNA3.1+ vector as a DNA vaccine. Also, the improved DNAimmunization system was constructed using pcDNA3.1+ plasmid contains VP1 gene of Iranian isolate FMD virus typeO/IRN/1/2010 and human HSP70 gene and expression of VP1-HSP70 fusion protein confi rmed in BHKT7 cell line byGuinea pig specifi c polyclonal antibody against FMD virus type O and conjugated rabbit...
Assuntos
Animais , Camundongos , Genoma Viral , Proteínas Virais/isolamento & purificação , Vacinas de DNA/análise , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Camundongos Endogâmicos BALB CResumo
This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.(AU)
Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.(AU)
Assuntos
Animais , Bovinos , Herpesvirus Bovino 1/isolamento & purificação , Herpesvirus Bovino 5/isolamento & purificação , Glicoproteínas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Testes Imunológicos/métodos , Vacinas de DNA , Ensaio de Imunoadsorção Enzimática , Células ProcarióticasResumo
Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.(AU)
O circovírus suíno 2 (PCV2) é geralmente associado à síndrome da circovirose suína, que é considerada uma importante doença de suínos e possui um sério impacto econômico na suinocultura mundial. Este trabalho descreve a construção de um plasmídeo recombinante que expressa a proteína estrutural do PCV2 e a avaliação das respostas imune humoral e celular por meio de vacinação em camundongos BALB/c. O candidato vacinal foi submetido a análises in vivo, determinando a capacidade de induzir resposta imune específica em camundongos. O DNA de um isolado brasileiro de PCV2 foi extraído e o gene que codifica para a proteína do capsídeo foi amplificado por PCR e inserido num plasmídeo de expressão. Grupos de camundongos BALB/c foram inoculados por via intramuscular e intradérmica a cada 15 dias, com 100µg e 50µg da construção vacinal, respectivamente. Outro grupo foi inoculado com 100µg do plasmídeo original, correspondente ao grupo controle. A soroconversão e a resposta celular dos grupos de camundongos BALB/c vacinados foram comparados como parâmetros de avaliação vacinal. A soroconversão foi avaliada por um teste de ELISA. Após 3 imunizações, as células esplênicas dos animais imunizados foram utilizadas nos ensaios de linfoproliferação. A soroconversão para o PCV2 foi detectada por ELISA nos animais inoculados com a construção vacinal quando comparados com o grupo controle. Nos ensaios de linfoproliferação foi observada uma grande proliferação celular nos animais inoculados comparados ao grupo controle. Portanto, o candidato vacinal demonstrou ser capaz de induzir tanto uma resposta humoral e celular nos camundongos inoculados.(AU)
Assuntos
Animais , Circovirus/isolamento & purificação , Vacinas de DNA/efeitos adversos , Ensaio de Imunoadsorção Enzimática/métodos , CamundongosResumo
The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08x106, 4x106, and 200x106) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100µg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200x106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100µg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1x104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.(AU)
Assuntos
Tuberculose , Vacinas de DNA , Mycobacterium avium , Imunidade , AnticorposResumo
A genetic vaccine consisting of the bovine herpesvirus-1.2a (BHV-1.2a) glycoprotein D (gD) gene under the control of the cytomegalovirus immediate-early promoter/enhancer was generated and administered to sheep intramuscularly in the neck. All animals developed serum antibodies which recognized the homologous antigen (BHV-1.2a strain BH-83) and also exhibited cross-reactivity against the heterologous antigen (BHV-5 strain EVI-190). Three intramuscularly injections were given but serological responses were not improved after the second inoculation. Specific antibodies were detected against BHV-1.2a until at least 12 months after the first inoculation. However, the capacity to induce antibodies against BHV-5 was lower and of shorter duration than to BHV-1.2a.(AU)
Uma vacina geneticamente preparada com o gene da glicoproteína D do herpesvirus bovino-1.2a (BHV-1.2a) controlado com o promotor/potenciador imediatamente precoce de citomegalovírus foi administrada no pescoço de ovinos. Todos os animais produziram anticorpos séricos que reconheceram, em ELISA, o antígeno homólogo (BHV-1.2a, amostra BH-83) e também mostraram reatividade cruzada contra um antígeno heterólogo (BHV-5 amostra EVI-190). Foram aplicadas três inoculações, mas não houve melhora de resposta com a terceira inoculação. Resposta específica contra BHV-1.2a foi detectada pelo menos até 12 meses após a primeira inoculação, entretanto, a resposta sorológica contra o BHV-5 foi de menor intensidade e duração do que aquela contra o BHV-1.2a.(AU)