Nondividing but metabolically active gamma-irradiated Brucella melitensis is protective against virulent B. melitensis challenge in mice.

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.

Tyrosine phosphorylation of bovine herpesvirus 1 tegument protein VP22 correlates with the incorporation of VP22 into virions.

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.

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization.

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.

A genetic immunization adjuvant system based on BVP22-antigen fusion.

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.

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host.

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.

Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations.

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.

Regulation of transgene expression in genetic immunization.

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.

Regulation of transgene expression in genetic immunization

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

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection.

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.

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

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.

Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs.

Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium. As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B. Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR. Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside. The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody. Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins. In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion. Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb. The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants. The recombinant B. abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs. Moreover, the results of this study suggest that B. abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.

Loss of MHC I transcription trans-activator in the bovine B-LCL, BL3.1.

BL3.1, a variant derived from the BLV infected bovine B cell line, BL3, is distinguished by a loss of expression of MHC class I. All surface MHC I products were down-regulated in BL3.1 compared with BL3 correlating with a diminution in MHC I heavy chain transcription. Class I genes, including regulatory elements, showed no aberrations. The variant, BL3.1, did not differ from the parent cell line in expression of Bovine Leukemia Virus (BLV) or oncogene, c-myc. Transient transfection experiments determined the defect was trans rather than cis, and was due to loss of a trans-activator rather than gain of a trans-suppressor as evidenced by transient heterokaryon fusions. Southwestern blot analysis indicated that two DNA binding proteins associated with the MHC class I promoter were missing in BL3.1 cells. The specific response elements for these proteins in BL3 did not appear to be within the enhancerA region, the major enhancer region of the MHC I promoter.

Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter.

The use of mammalian gene expression vectors has become increasingly important for transgenics and gene therapy as well as basic research. Essential for the success of these vectors in medical research applications is the proper choice of promoter linked to the gene of interest. Many mammalian expression vectors use promoter elements from pathogenic viruses, including simian virus 40 (SV40) and cytomegalovirus (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 interferon-gamma (IFN-gamma) on transgene expression driven by a 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 greater or lesser levels than the MHC I promoter, treatment with IFN-gamma caused inhibition of 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 immune response applications or in patients where IFN levels may be elevated. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

CD8+ lymphocytes that kill allogeneic and xenogeneic major histocompatibility complex class I targets.

CD8+ CTLs generated in a two-way MLR should lyse target cells only if these targets share a class I MHC allele with the original stimulators. Using cattle PBMCs in a two-way MLR, we generated CD8+ CTLs that kill allogeneic and xenogeneic cell lines. We have named these cells MLK cells. PBMCs isolated from two unrelated animals were cultured together. After 14 days microfluorimetry analysis was performed on the MLK cells with results showing > 90% CD8+ cells. RFLP analysis revealed these cells to be predominately of one animal. MLK cells were then used as effector cells in cytotoxicity assays with syngeneic, allogeneic, and xenogeneic target cells. MLK cells were able to kill all targets. Incubating MLK cells with mAbs to CD8 markedly reduced killing, suggesting a TCR-mediated cytolytic pathway. Effective cytolysis of these targets by MLK cells was dependent on class I expression. MHC class I expression-impaired mutants of allogeneic and xenogeneic targets were not susceptible to cytolysis. Comparisons to LAK cells revealed similarities in phenotype and function to the NK1.1-, CD8+ subset.

The cattle major histocompatibility complex (MHC) class-I possesses HLA-like promoters.

To study the genetic regulation of the cattle major histocompatibility complex (MHC) class-I, a cattle MHC class-I promoter DNA fragment was isolated and characterized for the first time. Semi-degenerate PCR was performed on cattle genomic DNA and the resulting product was isolated, subcloned and sequenced. Sequence comparison of the HLA-A, -B and -C promoters to the cloned product, designated BL3-6prmtr, revealed the cattle MHC class-I promoter to have close homology to human MHC class-I promoters. To address the ability of the cattle MHC class-I promoter to initiate transcription, BL3-6prmtr was subcloned into a luciferase reporter vector and transiently transfected into cattle and human B-lymphoblastoid cell lines. A strong transcription initiation ability of BL3-6prmtr was observed, including the ability of the enhancer A and interferon response sequence (IRS) to upregulate transcription initiation.

Impairment of MHC class I transcription in a mutant bovine B cell line.

To better define the regulation and expression of bovine major histocompatibility complex (MHC) antigens, the bovine B lymphoblastoid cell line, BL3, was exposed to gamma-irradiation and surviving cells were immunoselected for MHC class I antigen loss. The resulting class I expression loss variant, BL3.1, was characterized at both the protein and genetic levels to ascertain the nature of the defect. Microfluorimetry analysis revealed a 3--5-fold surface density reduction of all class I products on BL3.1 cells relative to the parental BL3 cells. This decreased surface expression was specific for MHC class I and not for MHC class II or the non-MHC-linked gene product, immunoglobulin (Ig). Northern and quantitative slot blot analyses demonstrated a corresponding diminution of class I RNA in BL3.1 suggesting a transcriptional level defect. Nuclear run-off and transcription inhibition experiments confirmed no post-transcriptional changes while Southern blot analysis provided no evidence for alterations within or near the class I genes. To help elucidate the mechanism of altered class I expression, the parent, BL3, and variant, BL3.1, were cultured with factors known to enhance MHC class I transcription. Interferon (IFN)-gamma, lipopolysaccharide (LPS), and activated peripheral blood lymphocyte (PBL) supernatant cultured with both cell lines induced MHC class I transcription and surface expression 2--3-fold greater than the untreated controls. It is likely, therefore, that a genetic alteration outside of the class I genes has occurred within BL3.1 impairing expression of MHC class I.