RESUMEN

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/genética

RESUMEN

Brucella spp. are gram-negative bacteria that cause the most frequent zoonotic disease worldwide, with more than 500,000 human infections yearly; however, no human vaccine is currently available. As with other intracellular organisms, cytotoxic mechanisms against infected cells are thought to have an important role in controlling infection and mediating long-term immunity. Live attenuated strains developed for use in animals elicit protection but retain unacceptable levels of virulence. Thus, the optimal design for a brucellosis vaccine requires a nonliving vaccine that confers effective immunity. Historically, inactivation methods such as chemical or heat treatment successfully impair Brucella reproductive capacity; nevertheless, metabolically inactive vaccines (subunit or killed) present very limited efficacy. Hence, we hypothesized that bacterial metabolism plays a major role in creating the proper antigenic and adjuvant properties required for efficient triggering of protective responses. Here, we demonstrate that inactivation of Brucella melitensis by gamma-irradiation inhibited its replication capability and yet retained live-Brucella protective features. Irradiated Brucella possessed metabolic and transcriptional activity, persisted in macrophages, generated antigen-specific cytotoxic T cells, and protected mice against virulent bacterial challenge, without signs of residual virulence. In conclusion, pathogen metabolic activity has a positive role in shaping protective responses, and the generation of inactivated and yet metabolically active microbes is a promising strategy for safely vaccinating against intracellular organisms such as B. melitensis.

Asunto(s)

Vacuna contra la Brucelosis/uso terapéutico , Brucella melitensis/efectos de la radiación , Brucelosis/prevención & control , Rayos gamma , Animales , Vacuna contra la Brucelosis/inmunología , Brucella melitensis/fisiología , Citotoxicidad Inmunológica , Ratones , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/uso terapéutico , Factores de Virulencia/biosíntesis , Factores de Virulencia/efectos de la radiación

RESUMEN

Nitric oxide (NO), produced by the iNOS protein, is known as a defense mechanism against various pathogens and an apoptotic inducer of cells. Apoptosis can also be a host protective mechanism against intracellular bacteria. The intracellular survival of Brucella abortus in RAW264.7 macrophages was examined under conditions of the apoptotic inducer, NO. Since B. abortus does not induce high output of NO, Escherichia coli LPS and IFN-gamma, as potential therapeutic modalities, were added to increase the expression of iNOS, and thus NO. Using 10 ng/ml E. coli LPS and 25 U/ml IFN-gamma, nitrite production was as high as 140 microM by 72 h. However, when macrophages were infected with B. abortus, the nitrite concentration was 60 microM after 72 h post infection, greater than a two-fold decrease. The number of surviving bacteria decreased, from 6 to 24 h, in the presence of nitrite accumulation. In the absence of B. abortus there was an increase in apoptotic cells at 72 h with high nitrite accumulation. In contrast, the number of macrophage apoptotic bodies decreased in the presence of B. abortus. The data suggest that: (i) NO accelerates the killing of intracellular B. abortus, but not to completion during the first 24 h of infection; (ii) B. abortus can prevent apoptosis as an advantage for bacterial survival inside macrophages and (iii) surviving intracellular bacteria then replicate steadily after 24 h. B. abortus probably expresses genes that counteract the effect of a high NO environment or activates genes to utilize NO as a nitrogen source, as the Brucella genome codes for nitric and nitrous oxide reductase genes.

Asunto(s)

Brucella abortus/crecimiento & desarrollo , Brucelosis/microbiología , Isotiuronio/análogos & derivados , Macrófagos/microbiología , Óxido Nítrico/biosíntesis , Animales , Apoptosis , Brucella abortus/patogenicidad , Bovinos , Línea Celular , Recuento de Colonia Microbiana , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo

RESUMEN

The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for Brucella. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B. abortus genes. Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively produced gfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K(+) transport gene), and site-specific recombination (xerD gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5' phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of B. abortus genes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.

Asunto(s)

Brucella abortus/crecimiento & desarrollo , Brucella abortus/genética , Genes Bacterianos , Proteínas de Choque Térmico/genética , Macrófagos/microbiología , Medios de Cultivo , Ambiente , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Biblioteca Genómica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transcripción Genética

RESUMEN

Tyrosine phosphorylation has been shown to play a role in the replication of several herpesviruses. In this report, we demonstrate that bovine herpesvirus 1 infection triggered tyrosine phosphorylation of proteins with molecular masses similar to those of phosphorylated viral structural proteins. One of the tyrosine-phosphorylated viral structural proteins was the tegument protein VP22. A tyrosine 38-to-phenylalanine mutation totally abolished the phosphorylation of VP22 in transfected cells. However, construction of a VP22 tyrosine 38-to-phenylalanine mutant virus demonstrated that VP22 was still phosphorylated but that the phosphorylation site may change to the C terminus rather than be in the N terminus as in wild-type VP22. In addition, the loss of VP22 tyrosine phosphorylation correlated with reduced incorporation of VP22 compared to that of envelope glycoprotein D in the mutant viruses but not with the amount of VP22 produced during virus infection. Our data suggest that tyrosine phosphorylation of VP22 plays a role in virion assembly.

Asunto(s)

Herpesvirus Bovino 1/fisiología , Proteínas Estructurales Virales/fisiología , Animales , Bovinos , Fosforilación , Tirosina , Virión/fisiología , Ensamble de Virus

RESUMEN

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.

Asunto(s)

Núcleo Celular/metabolismo , Herpesvirus Bovino 1/metabolismo , Histonas/metabolismo , Proteínas Estructurales Virales/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Perros , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/genética , Datos de Secuencia Molecular , Transfección , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética

RESUMEN

DNA vaccines must induce a greater immune response to be effective in the biomedical industry. Therefore, we tested the trafficking trait of the bovine herpesvirus 1 (BHV-1) protein VP22 (BVP22) fused to an antigen and applied this unique trait to genetic immunization. DNA immunization with BVP22-antigen stimulates immune responses superior to that of standard DNA immunization. Mice were injected intramuscularly with gene constructs expressing the antigen yellow fluorescent protein (YFP), YFP fused to BVP22, or YFP fused to BHV-1 tegument protein VP16 (BVP16). The results revealed a significantly enhanced YFP antibody response with BVP22-YFP DNA immunization compared with either YFP or BVP16-YFP gene immunization. Notably, the BVP22-YFP DNA construct induced a stronger T helper 1 (Th1) response, based on IFN-gamma and IL-4 cytokine levels, and IgG2a/IgG1 ratios. Furthermore, BVP22-YFP genetic immunization induced a greater cytotoxic T lymphocyte response. The genetic adjuvant properties of BVP22 can make DNA vaccines much more effective clinically.

Asunto(s)

Antígenos/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vacunas de ADN , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Animales , Western Blotting , Línea Celular , Citocinas/metabolismo , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas Fluorescentes Verdes , Proteína Vmw65 de Virus del Herpes Simple/genética , Humanos , Inmunoglobulina G/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección

RESUMEN

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-gamma mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Biolística/métodos , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/inmunología , Vacunas de ADN/administración & dosificación , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Animales , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Citocinas/genética , Cartilla de ADN/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/veterinaria , Inmunoglobulina G/biosíntesis , Pruebas de Neutralización , Plásmidos/genética , Vacunas de ADN/genética , Vacunas Virales/genética

RESUMEN

Brucella abortus actively secretes materials and uptakes nutrients to maintain the survival and multiplication of the bacteria in host cells. ATP-binding cassette (ABC) transporters can uptake or secrete diverse materials across the bacterial membrane, and thus, ABC transporters may be important for survival of the pathogen in the host. In the present study, the B. abortus genes encoding tandem repeated Brucella ATP-binding proteins, BapA and BapB, were identified. The deduced amino acid sequences of these two genes place BapA and BapB into group 6 containing RTX toxin transporters and cyclic beta-1,2-glucan transporters, one of 25 ABC transporter ortholog groups. One of the ortholog group 6 proteins, Haemophilus influenzae LktB, shows the highest similarity and identity with these two Brucella proteins. To test the role of these putative tandem repeated ABC transporters in Brucella pathogenesis, a bap deletion mutant was constructed and used to infect murine RAW 264.7 macrophages and mice. The number of cfu from RAW 264.7 cells and spleens of BALB/c mice infected with wild type or the bap deletion mutant was similar during the course of infection, suggesting the bap genes are not necessary to maintain the pathogenesis of B. abortus, or alternative compensatory mechanisms may exist to permit the intracellular survival of B. abortus in vitro and in vivo. This is the first molecular approach to investigate the role of putative ABC transporters classified into ortholog group 6 in Brucella pathogenesis.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Brucella abortus/genética , Proteínas de Transporte de Membrana , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Brucella abortus/química , Brucella abortus/patogenicidad , Línea Celular , Femenino , Haemophilus influenzae/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación Puntual , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Virulencia

RESUMEN

To maintain survival in macrophages, Brucella must overcome a hostile phagosomal environment defined as low pH, limited nutrition and low oxygen tension. The specific mechanisms utilized by Brucella to surmount such unfavorable environmental factors in phagosomes are not well understood. In general, to adapt to a change in environmental oxygen tension, bacteria use different terminal oxidases that have different oxygen affinity. To survive in phagosomes where low oxygen tension exists, Brucella, like other bacteria, may require high oxygen affinity terminal oxidases that can accept electrons through a cytochrome bc(1)complex dependent or independent pathway. Using a Brucella abortus cytochrome bc(1)complex deficient mutant, delta fbcF, the requirement for a high oxygen affinity terminal oxidase governed by the cytochrome bc(1)complex dependent pathway was tested. The number of cfu from RAW 264.7 macrophage cells and spleens of BALB/c mice infected with wild-type or the cytochrome bc(1)complex deficient mutant was similar during the course of infection. These results suggest that B. abortus contains no essential terminal oxidase utilized at low oxygen tension in phagosomes requiring the cytochrome bc(1)complex. Alternatively, other branched cytochrome bc(1)complex independent respiratory mechanisms that contain the high oxygen affinity terminal oxidases likely exist to facilitate Brucella survival in phagosomes. This is the first investigation regarding the Brucella respiratory system at the molecular level and the involvement of a respiratory system in Brucella pathogenesis.

Asunto(s)

Brucella abortus/patogenicidad , Brucelosis/microbiología , Complejo III de Transporte de Electrones/fisiología , Eliminación de Gen , Macrófagos/microbiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Brucella abortus/enzimología , Brucella abortus/genética , Línea Celular , Modelos Animales de Enfermedad , Complejo III de Transporte de Electrones/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Operón , Fagosomas/microbiología , Análisis de Secuencia de ADN , Bazo/microbiología , Virulencia

RESUMEN

Bovine leukemia virus (BLV) is a complex B-lymphotrophic retrovirus of cattle and the causative agent of enzootic bovine leukosis. Serum antibody in infected animals does not correlate with protection from disease, yet only some animals develop severe disease. While a cytotoxic T-lymphocyte response may be responsible for directing BLV pathogenesis, this possibility has been left largely unexplored, in part since the lack of readily established cytotoxic target cells in cattle has hampered such studies. Using long-term naturally infected alymphocytic (AL) cattle, we have established the existence of cytotoxic T-lymphocyte response against BLV envelope proteins (Env; gp51/gp30). In vitro-expanded peripheral blood mononuclear (PBM) cell effector populations consisted mainly of gammadelta(+) (>40%), CD4(+) (>35%), and CD8(+) (>10%) T lymphocytes. Specific lysis of autologous fibroblasts infected with recombinant vaccinia virus (rVV) delivering the BLV env gene ranged from 30 to 65%. Depletion studies indicated that gammadelta(+) and not CD8(+) T cells were responsible for the cytotoxicity against autologous rVVenv-expressing fibroblasts. Additionally, cultured effector cells lysed rVVenv-expressing autologous fibroblasts and rVVenv-expressing xenogeneic targets similarly, suggesting a lack of genetic restricted killing. Restimulation of effector populations increased the proportion of gammadelta(+) T cells and concomitantly Env-specific cytolysis. Interestingly, culture of cells from BLV-negative or persistently lymphocytic cattle failed to elicit such cytotoxic responses or increase in gammadelta(+) T-cell numbers. These results imply that cytotoxic gammadelta(+) T lymphocytes from only AL cattle recognize BLV Env without a requirement for classical major histocompatibility complex interactions. It is known that gammadelta(+) T lymphocytes are diverse and numerous in cattle, and here we show that they may serve a surveillance role during natural BLV infection.

Asunto(s)

Enfermedades de los Bovinos/inmunología , Leucosis Bovina Enzoótica/inmunología , Virus de la Leucemia Bovina/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Bovinos , Enfermedades de los Bovinos/virología , Línea Celular , Citotoxicidad Inmunológica , Leucosis Bovina Enzoótica/virología , Citometría de Flujo , Virus de la Leucemia Bovina/patogenicidad , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Depleción Linfocítica , Linfopenia/inmunología , Linfopenia/virología , Proteínas Oncogénicas de Retroviridae/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Prostaglandin E(2) (PGE(2)), produced by macrophages, has important immune regulatory functions, suppressing a type 1 immune response and stimulating a type 2 immune response. Type 1 cytokines (interleukin-2 [IL-2], IL-12, and gamma interferon) increase in freshly isolated peripheral blood mononuclear cells (PBMCs) of animals with an early disease stage of bovine leukemia virus (BLV) infection, while IL-10 increases in animals with a late disease stage. Although IL-10 has an immunosuppressive role in the host immune system, IL-10 also inhibits BLV tax and pol mRNA levels in vitro. In contrast, IL-2 stimulates BLV tax and pol mRNA and p24 protein expression in cultured PBMCs. The inhibitory effect of IL-10 on BLV expression depends on soluble factors secreted by macrophages. Thus, we hypothesized that PGE(2), a cyclooxygenase 2 (COX-2) product of macrophages, may regulate BLV expression. Here, we show that the level of COX-2 mRNA was decreased in PBMCs treated with IL-10, while IL-2 enhanced the level of COX-2 mRNA. Addition of PGE(2) stimulated BLV tax and pol mRNA levels and reversed the IL-10 inhibition of BLV mRNA. In addition, the specific COX-2 inhibitor, NS-398, inhibited the amount of BLV mRNA detected. Addition of PGE(2) increased BLV tax mRNA regardless of NS-398 addition. PGE(2) inhibited antigen-specific PBMC stimulation, suggesting that stimulation of BLV tax and pol mRNA levels by PGE(2) is independent of cell proliferation. These findings suggest that macrophage-derived COX-2 products, such as PGE(2), regulate virus expression and disease progression in BLV infection.

Asunto(s)

Dinoprostona/farmacología , Productos del Gen pol/genética , Productos del Gen tax/genética , Interleucina-10/farmacología , Interleucina-2/farmacología , Isoenzimas/genética , Virus de la Leucemia Bovina/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Comunicación Autocrina , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interleucina-10/antagonistas & inhibidores , Isoenzimas/metabolismo , Isoenzimas/farmacología , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/inmunología , Virus de la Leucemia Bovina/fisiología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Activación de Linfocitos/efectos de los fármacos , Modelos Biológicos , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Sulfonamidas/farmacología

RESUMEN

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.

Asunto(s)

Herpesvirus Bovino 1/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Núcleo Celular/metabolismo , ADN Viral , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética

RESUMEN

The relationship between the putative bovine leukemia virus receptor gene (BVLRcp) and the susceptibility of human cells to BLV infection was studied. Three cDNA clones encoding different portions of the human equivalent of bovine BLVRcp1 were isolated by DNA-DNA hybridization by comparison of the human cDNA clones to bovine BLVRcp1. Amino acid sequence indicated that the human sequence encodes the delta subunit of the AP-3 adaptor-related protein. When the recombinant human homologue BLVRcp2 was expressed in E. coli, it failed to bind the BLVgp51. However, the BVLVgp51 binding ability was restored when the chimerical BLVRcp molecule was prepared by exchanging 5' ends between bovine and human BLVRcp cDNAs. This finding implies that this BLVgp51 binding site is present only on the bovine BLVRcp and therefore its human homologue cannot be recognized by BLVgp51. This might also explain the resistance of human cells to BLV infection.

Asunto(s)

Virus de la Leucemia Bovina/metabolismo , Proteínas de la Membrana , Receptores Virales/metabolismo , Factores de Transcripción/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Complejo 3 de Proteína Adaptadora , Subunidades delta de Complexo de Proteína Adaptadora , Animales , Southern Blotting , Western Blotting , Bovinos , ADN Complementario/genética , Humanos , Virus de la Leucemia Bovina/patogenicidad , Masculino , Receptores Virales/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genética

RESUMEN

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.

Asunto(s)

Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen pol/genética , Productos del Gen tax/genética , Interleucina-10/farmacología , Interleucina-2/farmacología , Virus de la Leucemia Bovina/genética , Animales , Bovinos , Femenino , Productos del Gen pol/biosíntesis , Productos del Gen tax/biosíntesis , Virus de la Leucemia Bovina/metabolismo , ARN Mensajero/biosíntesis

RESUMEN

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-gamma on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-gamma indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-gamma inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Asunto(s)

Regulación de la Expresión Génica , Vectores Genéticos , Transgenes , Vacunas de ADN , Citomegalovirus , Elementos de Facilitación Genéticos , Genes MHC Clase I , Luciferasas , Regiones Promotoras Genéticas , Virus 40 de los Simios

RESUMEN

In B lymphocytes, the down-regulatory phosphatase SHP-1 associates with CD22 and CD32b (also known as FcgammaRIIB) and acts as a critical negative regulator of B-cell receptor signaling. Bovine leukemia virus, a retrovirus of the HTLV/BLV group, causes persistently increased numbers of peripheral blood B lymphocytes, known as persistent lymphocytosis (PL) and, in some animals, progression to B-cell leukemia and/or lymphoma. Here, we show that SHP-1 associates with the bovine leukemia virus transmembrane protein, gp30. This interaction is either direct or indirect. The interaction is dependent on tyrosine phosphorylation, and the interaction increases after cell stimulation with sodium pervanadate. The gp30-SHP-1 interaction is seen in all of the BLV-infected, PL animals tested, but is not seen in uninfected animals or in most BLV-infected, non-PL animals, which do not express significant quantities of gp30. However, one BLV-infected, non-PL animal expressed large quantities of gp30, yet no gp30-SHP-1 interaction was detected, suggesting that there may be other factors in cells from the PL animals that facilitate the gp30-SHP-1 interaction. The association of gp30 and SHP-1 suggests the hypothesis that gp30 may act as a decoy to sequester SHP-1, resulting in up-regulation of B-cell receptor signaling. The implication of this could be a novel mechanism of viral activation of lymphocytes by removal of a down-regulatory phosphatase.

Asunto(s)

Linfocitos B/inmunología , Leucosis Bovina Enzoótica/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Regulación hacia Abajo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6

RESUMEN

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system

Asunto(s)

Regulación de la Expresión Génica , Vectores Genéticos , Transgenes , Vacunas de ADN , Citomegalovirus , Elementos de Facilitación Genéticos , Genes MHC Clase I , Luciferasas , Regiones Promotoras Genéticas , Virus 40 de los Simios

RESUMEN

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAbeta-/-) mice, which are unable to mediate CD4(+) T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAbeta-/- mice inoculated with 10(4) PFU of attenuated Mengo virus (vMC24) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8(+) T lymphocytes prior to lethal EMCV challenge ablated protection in vMC24-immunized RHAbeta-/- mice. The CD8(+) T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8(+) T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8(+) T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC24-immunized RHAbeta-/- mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC24-immunized RHAbeta-/- mice survived when rechallenged 90 days later. The present study demonstrating CD8(+) T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC24-specific CD4(+) T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.

Asunto(s)

Anticuerpos Antivirales/sangre , Infecciones por Cardiovirus/prevención & control , Mengovirus/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Cardiovirus/sangre , Infecciones por Cardiovirus/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Susceptibilidad a Enfermedades/inmunología , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización

RESUMEN

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer.

Asunto(s)

Infecciones Bacterianas/inmunología , Citocinas/fisiología , Subgrupos de Linfocitos T/fisiología , Animales , Ratones , Subgrupos de Linfocitos T/patología