Brucella abortus genes identified following constitutive growth and macrophage infection.

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.

Bovine herpesvirus-1 infects activated CD4+ lymphocytes.

Acute virus infections can induce immune deficiencies, as shown by immunosuppression to a variety of antigens and mitogens. Previously we observed that live bovine herpesvirus-1 (BHV-1) induced considerable lymphocyte death in culture, suggesting that the virus infected one or more cell populations. Our goal was to identify the cells infected by BHV-1 and the mechanism resulting in cell death. ConA activated cells were cultured with BHV-1 and stained with monoclonal antibodies specific for virus envelope glycoproteins (gB, gC and gD) and lymphocyte surface proteins (CD2, CD4 and CD8) and a molecule associated with gamma/delta cells. Two-colour immunofluorescence revealed that virus glycoproteins were preferentially expressed on T lymphocytes of the CD4+ phenotype. Live virus was required for virus glycoprotein expression, and by 48 h considerable loss of CD4 expression was observed. To confirm virus replication, RNA was isolated from cells, reverse transcribed and amplified using primers to a 342 bp region of immediate-early and early genes (IER2.9/ER2.6) or a 392 bp region of an early gene (gD). Immediate-early/early gene products were detected in CD4+T lymphocytes but not in infectious virions. Lymphocyte apoptosis was observed by 7 h post-infection with increasing levels of cell death at 24-48 h after infection. These findings suggest that the loss of proliferating CD4+ T cells during infection or vaccination with modified live vaccines provides the opportunity for secondary infections that commonly occur following BHV-1 infection.

Truncated bovine herpesvirus-1 glycoprotein I (gpI) initiates a protective local immune response in its natural host.

Current modified live and killed BHV-1 vaccines have not reduced the incidence of bovine herpesvirus-1 (BHV-1), the principal viral agent in bovine respiratory disease complex. The requirement for production of viral proteins for immune study has resulted in the establishment of a cell line which constitutively expresses BHV-1 gpI. A truncated BHV-1 envelope gpI protein was secreted into the culture supernatant of D17 cells transfected with the gpI gene lacking the coding sequence for the transmembrane region (TMR). The transmembrane domain is essential for gpI stability in the envelope, virus infectivity and, most probably, natural killer cell recognition; however, we have tested the possibility that this domain is not required for inducing an adaptive, protective immune response. Immunization of calves with this truncated gpI protein induced gpI-specific nasal IgA, IgG1, serum neutralizing antibodies and gpI-specific peripheral lymphocyte proliferation. All immunized calves were protected from clinical disease after BHV-1 challenge. Further, nine of ten immunized calves had no intranasal viral shedding. One animal shed a minimal amount of virus following challenge, but produced no antibodies to other viral proteins as evidenced by immunoprecipitation of 35S-labelled viral proteins by sera from virus-challenged animals. This study represents the first evidence that a recombinant truncated gpI subunit vaccine can confer local mucosal immunity and establish a strong protective barrier against disease caused by BHV-1 in the natural host. Also, these data demonstrate the feasibility of preventing initial viral replication in the host and distinguishing vaccinated from wild-type virus-infected animals.

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.

Effect of monoclonal antibodies on in vitro function of T-cell subsets.

We have examined the functional heterogeneity of 43 monoclonal antibodies which recognised CD1, CD2, CD4, CD5, CD6 or CD8. Blocking of the proliferative response to alloantigen, virus, and phytoheamagglutinin (PHA) was studied. The majority of anti-CD2 and CD4 antibodies could inhibit an alloantigen response. Some of the anti-CD5 and CD8 antibodies could also block this response. Only a few antibodies to CD2 and CD4 could prevent proliferation of lymphocytes to PHA or virus.

Biochemical analysis of bovine T cell antigens with workshop monoclonal antibodies.

The relative molecular weight (Mr) of the CD1, CD2, CD4, CD5, CD6, CD8 and additional molecules identified by monoclonal antibodies (mAb) submitted to the workshop was determined using 125I surface-labelled leukocytes. Of 37 mAb precipitating lymphocyte surface molecules, 33 had Mr correlating with their clusters of differentiation. Further, we were able to determine Mr for three "undefined T lymphocyte" antibodies. Of the 20 mAb that did not precipitate a molecule from the surface of leukocytes, one was non-reactive as determined by flow microfluorimetry. The inability to precipitate a molecule from the remaining cells was probably due to lower affinities of those antibodies and conformational changes in the antigen(s) following lysis of the leukocytes.

Cloned bovine cytolytic T cells recognize bovine herpes virus-1 in a genetically restricted, antigen-specific manner.

The inability to demonstrate bovine herpes virus-1 (BHV-1)-specific lymphocyte responses from BHV-1-infected cattle has been a major difficulty in confirming the importance of cellular effector mechanisms during BHV-1 infection. We have examined the capacity of bovine cytolytic T-cell clones to lyse BHV-1-infected, concanavalin A-stimulated blast cells. Cytolysis was as high as 58% at an effector to target (E:T) ratio of 1:1. All cytolytic T-cell clones produced were genetically restricted in killing cells of the autologous genotype. Cytotoxic T-lymphocyte (CTL) clones were specific for BHV-1 but not related herpes viruses, i.e. BHV-2 and pseudorabies virus. These results provide evidence that cytolytic T lymphocytes have an antigen-specific role in the immune response of cattle against BHV-1, and that CTL may serve as effector cells in the identification of glycoproteins useful in recombinant vaccine preparation since only determinants of BHV-1 were recognized.

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production.

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.

Enhancement of T cell cytotoxic responses by purified human fibroblast interferon.

Purified polyribonucleotide-induced human fibroblast interferon (HFIF) was tested for its effects on proliferative and cytotoxic human T cell responses to alloantigens. The addition of HFIF (100 to 400 IFU/ml) to mixed leukocyte cultures decreased alloantigen-induced lymphocyte proliferative responses as determined by both recovery of responding cells and by 3H-thymidine incorporation into responding cells. However, HFIF, but not the mock interferon preparation, increased the cytotoxic response of T cells to allogeneic cells by 4- to 5-fold when expressed in terms of lytic units. Although fibroblast and leukocyte interferons have different physicochemical and biologic properties, the results reported here are in concert with previous findings concerning the effects of virus-induced leukocyte interferon on human T cell functions.