RESUMEN
Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Quitosano , Leptospira/inmunología , Leptospirosis/prevención & control , Lipoproteínas/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/química , Línea Celular , Quitosano/química , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Lipoproteínas/administración & dosificación , Lipoproteínas/química , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/química , Proteínas Recombinantes , Linfocitos T/inmunología , Linfocitos T/metabolismo , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas Sintéticas/administración & dosificaciónRESUMEN
BACKGROUND: The development of recombinant house dust mite (HDM) allergens opened the way for the in-depth characterization of these molecules but also provided new opportunities to refine the diagnostic procedures of HDM allergy as well as the allergen-specific immunotherapy through tailor-made treatments. OBJECTIVE: In the present study, the HDM allergen Der p 21 was expressed in Pichia pastoris under a secreted form. The physico-chemical as well as the allergenic characterizations of recombinant Der p 21 (rDer p 21) were performed. METHODS: Purified rDer p 21, secreted from recombinant P. pastoris was characterized by CD and MS analysis and the frequency of IgE reactivity was determined by ELISA using 96 sera of HDM-allergic patients from Bangkok. The direct airway epithelial cell activation by rDer p 21 was also evaluated. RESULTS: rDer p 21 was highly expressed under a secreted form in P. pastoris. The physico-chemical characterization of purified rDer p 21 showed that the allergen displayed appropriate α-helix secondary structure content although a two amino acids truncation at the N-terminus of the protein was evidenced by MS. The prevalence of IgE reactivity to rDer p 21 reached 25% in the cohort of the HDM-allergic patients. rDer p 21 could trigger IL-8 production in airway epithelial cells through TLR2-dependent signaling. CONCLUSION: Properly folded rDer p 21 produced in P. pastoris is appropriate for HDM allergy diagnosis as well for future recombinant allergen-based specific immunotherapy.
Asunto(s)
Alérgenos/inmunología , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Pichia/genética , Alérgenos/biosíntesis , Alérgenos/genética , Animales , Antígenos Dermatofagoides/biosíntesis , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Clonación Molecular , Humanos , Inmunoglobulina E/inmunología , Interleucina-8/biosíntesis , Pichia/metabolismo , Estructura Secundaria de Proteína , Pyroglyphidae/genética , Proteínas Recombinantes/genética , Mucosa Respiratoria/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 2/inmunologíaRESUMEN
Bacterial outer membrane lipoproteins represent potent immunogens for the design of recombinant subunit vaccines. However, recombinant lipoprotein production and purification could be a challenge notably in terms of expression yield, protein solubility, and post-translational acylation. Together with the cost effectiveness, facilitated production, and purification as well as good stability, DNA-based vaccines encoding lipoproteins could become an alternative strategy for antibacterial vaccinations. Although the immunogenicity and the efficacy of DNA-based vaccines can be demonstrated in small rodents, such vaccine candidates could request concrete optimization as they are weak immunogens in primates and humans and particularly when administered by conventional injection. Therefore, the goal of the present study was to optimize the immunogenicity of a DNA vaccine encoding an outer membrane lipoprotein. LipL32, the major outer membrane protein from pathogenic Leptospira, was selected as a model antigen. We evaluated the influence of antigen secretion, the in vivo DNA delivery by electroporation, the adjuvant co-administration, as well as the heterologous prime-boost regimen on the induction of anti-LipL32 specific immune responses. Our results clearly showed that, following transfections, a DNA construct based on the authentic full-length LipL32 gene (containing leader sequence and the N-terminus cysteine residue involved in the protein anchoring) drives antigen secretion with the same efficiency as a plasmid-encoding anchor-less LipL32 and for which the bacterial leader sequence was replaced with a viral signal peptide. The in vivo DNA delivery by electroporation drastically enhanced the production of strong Th1 responses characterized by specific IgG2a antibodies and the IFNγ secretion in a restimulation assay, regardless of the DNA constructs used. In comparison with the heterologous prime-boost regimen, the homologous prime-boost vaccinations with DNA co-administrated with polyinosinic-polycytidylic acid (poly I:C) generated the highest specific IgG and IgG2a titers as well as the greatest IFNγ production. Taken together, these data suggest that optimization of outer membrane lipoprotein secretion is not critical for the induction of antigen-specific responses through DNA vaccination. Moreover, the potent antibody response induced by DNA plasmid encoding lipoprotein formulated with poly I:C and delivered through electroporation provides the rationale for the design of new prophylactic vaccines against pathogenic bacteria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Lipoproteínas/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/química , Humanos , Inmunización Secundaria , Lipoproteínas/química , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Poli I-C/farmacología , TransfecciónRESUMEN
Broad-complex (Br-c) is the early ecdysone responsive gene encoding a family of zinc-finger transcription factors. In this study, the full-length cDNA of the Br-c gene of the giant tiger shrimp (Penaeus monodon) was identified. PmBr-c was 1897 bp in length containing an ORF of 1329 bp deducing to a polypeptide of 442 amino acids. PmBr-c was more abundantly expressed in ovaries than testes of P. monodon broodstock. The expression levels of PmBr-c mRNA in ovaries of juveniles was significantly greater than that in stages II (vitellogenic), IV (mature) and V (post-spawning) ovaries of intact broodstock. The expression level of PmBr-c was significantly increased in stage III (nearly mature) ovaries of intact wild broodstock and in stage IV ovaries of eyestalk-ablated broodstock (P<0.05). In domesticated broodstock, ovarian PmBr-c was expressed lower in 18-month-old shrimp compared to 6-month-old shrimp (P<0.05). In situ hybridization revealed that PmBr-c mRNA was localized in ooplasm of previtellogenic oocytes in various ovarian stages of P. monodon broodstock. Serotonin (5-HT, 50 µg/g body mass; 18-month-old shrimp) and progesterone (0.1 µg/g body mass; 14-month-old shrimp) injection significantly promoted the expression level of PmBr-c in ovaries of domesticated broodstock at 24 and 48-72 h post injection (hpi, P<0.05). The expression levels of PmBr-c in ovaries of juvenile P. monodon was significantly increased following 20-hydroxyecdysone treatment (1 µg/g body mass; 4-month-old shrimp) for 168 hpi (P<0.05). Taken together, PmBr-c seems to play an important role during ovarian development of P. monodon.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Penaeidae/metabolismo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ecdisterona/farmacología , Femenino , Datos de Secuencia Molecular , Oocitos/metabolismo , Especificidad de Órganos , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Penaeidae/crecimiento & desarrollo , Progesterona/metabolismo , Serotonina/metabolismoRESUMEN
This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.