RESUMO
Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLDâ¯+â¯rCP09720 (G3), and rPLDâ¯+â¯rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21â¯days after the last immunization. The animals were evaluated daily for 40â¯days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (pâ¯<â¯.05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (pâ¯<â¯.05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLDâ¯+â¯rCP09720), and 50% (rPLDâ¯+â¯rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Fosfolipase D/imunologia , Proteínas Recombinantes/imunologia , Fosfatase Ácida/administração & dosagem , Fosfatase Ácida/genética , Fosfatase Ácida/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/genética , Esterases/administração & dosagem , Esterases/genética , Esterases/imunologia , Cabras/microbiologia , Imunidade Celular , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interferon gama/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Camundongos , Fosfolipase D/administração & dosagem , Fosfolipase D/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Ovinos/microbiologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controle , Células Th1/imunologia , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
PURPOSE: We tested the efficacy of the esterase encoded by cp1002_RS09720 from Corynebacteriumpseudotuberculosis in recombinant subunit and DNA caseous lymphadenitis (CLA) vaccines. This target was predicted as one of the best CLA vaccine candidates by mature epitope density analysis. METHODOLOGY: Gene cp1002_RS09720 was cloned into two different vectors (pAE for subunit vaccine and pTARGET for DNA vaccine). Four groups of 15 mice each were immunized with the recombinant esterase rCP09720 associated with aluminium hydroxide adjuvant (G1), pTARGET/cp09720 DNA vaccine (G2), a naked pTARGET (G3) or PBS as a negative control (G4). Immunization occurred in two doses intercalated by a 21 day interval. Twenty-one days after the last dose administration, animals were challenged with a virulent C. pseudotuberculosis MIC-6 strain. RESULTS: G1 showed high levels of IgG1 and IgG2a on days 21 and 42 post-immunization and a significant level of IFN-γ (P<0.05), suggesting a Th1 response. The protection levels obtained were 58.3 and 16.6â% for G1 and G2, respectively. CONCLUSION: The subunit vaccine composed of the recombinant esterase rCP09720 and Al(OH)3 is a promising antigenic formulation for use against CLA.
Assuntos
Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/imunologia , Esterases/genética , Linfadenite/prevenção & controle , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Citocinas/metabolismo , Esterases/administração & dosagem , Esterases/imunologia , Imunoglobulina G/sangue , Interferon gama/imunologia , Linfadenite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.
Assuntos
Anticorpos Neutralizantes/imunologia , Antídotos , Antivenenos/imunologia , Bothrops/imunologia , Venenos de Crotalídeos/imunologia , Proteínas de Répteis/imunologia , Mordeduras de Serpentes/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Antídotos/farmacologia , Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Western Blotting , Bothrops/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/toxicidade , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/prevenção & controle , Eletroforese em Gel Bidimensional , Esterases/imunologia , Esterases/metabolismo , Fosfolipases A2 do Grupo II/imunologia , Fosfolipases A2 do Grupo II/metabolismo , Hemorragia/sangue , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Masculino , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/metabolismo , Proteólise , Ratos Wistar , Proteínas de Répteis/metabolismo , Proteínas de Répteis/toxicidade , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/enzimologia , Fatores de TempoRESUMO
Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.
Assuntos
Antígenos de Protozoários/sangue , Esterases/sangue , Leishmania donovani/enzimologia , Leishmaniose Visceral/parasitologia , África Oriental , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Sequência de Bases , Southern Blotting , Brasil , Linhagem Celular , Cromatografia de Afinidade , Cães , Esterases/imunologia , Esterases/metabolismo , Humanos , Imunoprecipitação , Índia , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmania donovani/isolamento & purificação , Leishmania infantum/enzimologia , Leishmania infantum/genética , Leishmaniose Visceral/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The three-dimensional model built for the major latex allergen Hev b 13 consists of the typical organization of plant esterases made of a central bundle of five parallel beta-strands surrounded by five alpha-helices associated to two shorter alpha-helical segments. Up to 12 sets of sequential IgE-binding peptides were identified in SPOT experiments along the amino acid sequence of Hev b 13. They correspond in fact to eight IgE-binding epitopic stretches exposed on the surface of the allergen. With the exception of epitope #5, all other epitopes contain charged residues. Epitope #8 contains the 3rd putative N-glycosylation site of Hev b 13 and should consist of a glycotope, whereas all other identified IgE-binding areas occur outside the two remaining putative N-glycosylation sites. Accordingly, the allergenicity of Hev b 13 does not primarily depends on its carbohydrate moiety.