RESUMEN
VNP20009 is a very effective anti-cancer agent and can specifically target tumors and inhibit tumor growth. It was assumed that the tumor targeting ability of VNP20009 correlated to its anticancer capacity. However, our observation contradicted to this assumption. Three VNP20009 mutant strains (ΔslyA, ΔSTM3120 and ΔhtrA) with reduced fitness in normal tissues and unchanged fitness in tumors partially or completely lost their anti-cancer capacities. The genes slyA, STM3120 and htrA were required for survival within macrophages and were indispensable for tumor microenvironment remodeling by VNP20009. The infiltration of immune cells occurred less in the tumors of mice infected with the mutant strains. In addition, the mRNA levels of TNF-α and IL-1ß were significantly decreased in the tumors of mice treated with the mutant strains. Our results indicate that the immune responses elicited by bacteria rather than the bacterial titer in tumors play a "decisive" role in VNP20009-mediated bacterial cancer therapy, which provides a novel perspective for the underlying mechanism of bacterial cancer therapy.
Asunto(s)
Proteínas Bacterianas/genética , Vacunas Bacterianas/farmacología , Proteínas de Choque Térmico/genética , Melanoma Experimental/terapia , Proteínas Periplasmáticas/genética , Salmonella enterica/genética , Serina Endopeptidasas/genética , Neoplasias Cutáneas/terapia , Factores de Transcripción/genética , Animales , Proteínas Bacterianas/inmunología , Proteínas de Choque Térmico/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Melanoma Experimental/inmunología , Melanoma Experimental/microbiología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Periplasmáticas/inmunología , Células RAW 264.7 , Salmonella enterica/inmunología , Serina Endopeptidasas/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/microbiología , Neoplasias Cutáneas/patología , Factores de Tiempo , Factores de Transcripción/inmunología , Carga Tumoral , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity.
Asunto(s)
Aglutinación/inmunología , Antígenos Bacterianos/inmunología , Brucella melitensis/inmunología , Brucelosis/diagnóstico , Látex/inmunología , Proteínas Periplasmáticas/inmunología , Enfermedades de las Ovejas/diagnóstico , Pruebas de Aglutinación/métodos , Animales , Brucelosis/inmunología , Femenino , Masculino , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Ovinos , Enfermedades de las Ovejas/inmunologíaRESUMEN
Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.
Asunto(s)
Antígenos Bacterianos/análisis , Antígenos de Superficie/análisis , Proteómica , Shigella flexneri/inmunología , Shigella sonnei/inmunología , Antígenos Bacterianos/genética , Antígenos de Superficie/genética , Vacunas Bacterianas , Membrana Celular/inmunología , Membrana Celular/ultraestructura , Disentería Bacilar/prevención & control , Proteínas de la Membrana/inmunología , Proteínas Periplasmáticas/inmunología , Shigella flexneri/ultraestructura , Shigella sonnei/ultraestructuraRESUMEN
Because of the serious economic and medical consequences of brucellosis, efforts are to prevent infection of domestic animals through vaccines. Many disadvantages are associated with the current Brucella melitensis Rev.1 vaccine prompting development of alternative vaccines and delivery. Escherichia coli (DH5α) was engineered to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes. These recombinant invasive E. coli expressing B. melitensis outer membrane proteins (Omp31 or 16) or the periplasmic protein BP26 were evaluated for protection of mice against virulent B. melitensis. Importantly, these invasive E. coli vaccines induced significant protection against B. melitensis challenged mice. Invasive E. coli may be an ideal vaccine platform with natural adjuvant properties for application against B. melitensis since the E. coli delivery system is non-pathogenic and can deliver antigens to antigen-presenting cells promoting cellular immune responses.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Vacuna contra la Brucelosis/inmunología , Brucella melitensis/inmunología , Brucelosis/prevención & control , Portadores de Fármacos/administración & dosificación , Escherichia coli/genética , Proteínas Periplasmáticas/inmunología , Animales , Carga Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Vacuna contra la Brucelosis/administración & dosificación , Vacuna contra la Brucelosis/genética , Brucella melitensis/genética , Brucelosis/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Leucocitos Mononucleares/inmunología , Listeria monocytogenes/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Periplasmáticas/genética , Bazo/inmunología , Bazo/microbiología , Yersinia pseudotuberculosis/genéticaRESUMEN
Edwardsiella tarda is a severe aquaculture pathogen with a broad host range that includes humans, animal, and fish. A gene (degP(Et)) encoding a DegP homologue was cloned from TX01, a pathogenic E. tarda strain isolated from diseased fish. DegP(Et) shares high sequence identities with the DegP proteins of several bacterial species. Functional analyses showed that degP(Et) could complement the temperature-sensitive phenotype of an Escherichia coli degP null mutant. Expression of degP(Et) in TX01 was modulated by growth phase and temperature, the latter possibly through the action of the sigma(E)-like factor. Overexpression of degP(Et) (i) enhanced the ability of TX01 to disseminate in fish blood at the advanced stage of infection, (ii) heightened the activity of type 2 autoinducer, and (iii) increased the expression of luxS and the genes encoding components of the virulence-associated type III secretion system. Recombinant DegP(Et) purified from E. coli was a serine protease that exhibited maximum activity at 40 degrees C and pH8.0. The proteolytic activity of recombinant DegP(Et) depended on the catalytic triad and the PDZ domains. Immunoprotective analyses showed that purified recombinant DegP(Et) was a protective immunogen that could induce the production of specific serum antibodies and elicit strong protective immunity in fish vaccinated with DegP(Et).
Asunto(s)
Vacunas Bacterianas/inmunología , Edwardsiella tarda/genética , Edwardsiella tarda/inmunología , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/prevención & control , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Análisis Mutacional de ADN , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/prevención & control , Escherichia coli/metabolismo , Lenguado , Proteínas Recombinantes/inmunología , Homología de Secuencia de Ácido Nucleico , Temperatura , Factores de TiempoRESUMEN
Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish.
Asunto(s)
Vacunas Bacterianas/inmunología , Edwardsiella tarda/inmunología , Infecciones por Enterobacteriaceae/prevención & control , Enfermedades de los Peces/prevención & control , Lenguado/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Infecciones por Enterobacteriaceae/inmunología , Enfermedades de los Peces/inmunología , Flagelina/inmunología , Lenguado/microbiología , Proteínas Periplasmáticas/inmunología , Plásmidos , Vacunas de ADN/inmunologíaRESUMEN
Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Femenino , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Regiones Constantes de Inmunoglobulina/aislamiento & purificación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Masculino , Periplasma/genética , Periplasma/inmunología , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Proteínas Periplasmáticas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Development of a single diagnostic test for brucellosis in animals is the top priority of present-day research in the field. There is currently a battery of serological tests relying mainly on the use of LPS (lipopolysaccharide) as an antigen, culminating in false positives due to serological cross-reactivity. Other problems include difficulties in antigen production and the associated biohazard risk. This has prompted the need to develop an alternative antigen to replace LPS. In the present study, we cloned and expressed a BP26 (26 kDa periplasmic protein) antigen gene (bp26) of Brucella abortus. The recombinant periplasmic protein [rBP26 (recombinant BP26)] was expressed to high levels in Escherichia coli and purified in a single step. The purified rBP26 was examined for its binding activity with antibodies in a serum derived from a rabbit immunized intramuscularly with whole-cell lysate of B. abortus, as well as with commercial Brucella antibody (Difco). The purified rBP26 was used to develop an in-house plate ELISA and was further tested with a panel of 75 bovine brucellosis sera samples characterized previously by conventional serological tests. The results of both were in excellent agreement. The results show that rBP26 has potential use in the diagnosis of brucellosis, both in the laboratory and in field-based conditions with high levels of sensitivity and specificity.
Asunto(s)
Brucelosis Bovina/diagnóstico , Proteínas de la Membrana/aislamiento & purificación , Proteínas Periplasmáticas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/sangre , Brucella abortus , Brucelosis Bovina/sangre , Bovinos , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Escherichia coli/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
In the present study, hybridomas were developed for the production of monoclonal antibodies (MAb) against recombinant 26-kDa periplasmic protein (rBP26) of Brucella abortus. A set of six stabilized hybridoma cell lines were generated. Monoclonal antibodies secreted by all of these clones exhibited reaction for rBP26, as well as with the protein of 26-kDa, derived from whole cell lysate of B. abortus 544. Three out of six MAbs were IgG1, two were IgM, and one was IgG2b in nature. These MAbs did not show any cross-reaction with whole cell lysate of Yersinia enterocolitica O: 9, Vibrio cholerae, Salmonella typhimurium, and Escherichia coli O157 in Western blot analysis. Reactivity of these MAbs was further assessed with other organisms of Brucella species, namely, B. abortus 544, B. canis, B. melitensis 16M, and B. suis. Collectively, these data suggest that these MAbs may have the potential for their use in the detection of Brucella species with high specificity.
Asunto(s)
Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Antígenos Bacterianos/inmunología , Brucella abortus/inmunología , Proteínas de la Membrana/inmunología , Proteínas Periplasmáticas/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Bovinos , Femenino , Hibridomas , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunologíaRESUMEN
The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.
Asunto(s)
Holosporaceae/fisiología , Paramecium/microbiología , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Secuencia de Bases , Proliferación Celular , Electroforesis en Gel Bidimensional , Holosporaceae/citología , Holosporaceae/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Peso Molecular , Paramecium/citología , Paramecium/metabolismo , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Unión Proteica , Transporte de Proteínas , Análisis de Secuencia de ADN , SimbiosisRESUMEN
Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a Delta aroC Delta aroD Delta htrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4(+) and CD8(+) S. Typhi immune responses. Potent CTL responses and IFN-gamma secretion by CD8(+) T cells were detected following immunization with CVD 908-htrA in high (4.5 x 10(8) CFU) and low (5 x 10(7) CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-gamma spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 +/- 41 SFC/10(6) PBMC and 233 +/- 87 SFC/10(6) PBMC, in the high and low dose groups, respectively. Strong CD4(+) T cell responses were also observed. Increases in the IFN-gamma production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-gamma SFC to stimulation with S. Typhi-infected cells and IFN-gamma released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-gamma production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Choque Térmico/inmunología , Activación de Linfocitos/inmunología , Proteínas Periplasmáticas/inmunología , Salmonella typhi/inmunología , Serina Endopeptidasas/inmunología , Vacunas Tifoides-Paratifoides/inmunología , Administración Oral , Adolescente , Adulto , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Estudios Cruzados , Pruebas Inmunológicas de Citotoxicidad , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas de Choque Térmico/administración & dosificación , Proteínas de Choque Térmico/genética , Humanos , Interferón gamma/metabolismo , Lipopolisacáridos/inmunología , Masculino , Proteínas Periplasmáticas/administración & dosificación , Proteínas Periplasmáticas/genética , Salmonella typhi/genética , Serina Endopeptidasas/administración & dosificación , Serina Endopeptidasas/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/microbiología , Vacunas Tifoides-Paratifoides/administración & dosificación , Vacunas Tifoides-Paratifoides/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.