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ABSTRACTWe have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginaleinduces a functionally exhausted CD4 T-cell response to the A. marginaleimmunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were sporadic and transient during persistent infection. The induction of an exhausted T-cell response following infection presumably facilitates pathogen persistence. In the current study, we hypothesized that the loss of epitope-specific T-cell responses requires the presence of the immunizing epitope on the pathogen, and T-cell dysfunction correlates with the appearance of regulatory T cells. In limited studies in cattle, regulatory T cells have been shown to belong to ?d T-cell subsets rather than be CD4 T cells expressing forkhead box protein P3 (FoxP3). Cattle expressing the DRB3*1101 haplotype were immunized with a truncated A. marginalemajor surface protein (MSP) 1a that contains a DRB3*1101-restricted CD4 T-cell epitope, F2-5B. Cattle either remained unchallenged or were challenged with A. marginalebacteria that express the epitope or with A. marginalesubsp. centralethat do not. Peripheral blood and spleen mononuclear cells were monitored for MSP1a epitope F2-5B-specfic T-cell proliferative responses and were stained for ?d T-cell subsets or CD4+CD25+FoxP3+T cells before and during infection. As hypothesized, the induction of T-cell exhaustion occurred only following infection with A. marginale, which did not correlate with an increase in either CD4+CD25+FoxP3+T cells or any ?d T-cell subset examined.

ABSTRACTInfection of cattle with Anaplasma marginalefails to prime sustained effector/memory T-cell responses, and high bacterial load may induce antigen-specific CD4 T exhaustion and deletion. We tested the hypothesis that clearance of persistent infection restores the exhausted T-cell response. We show that infection-induced T-cell exhaustion, characterized as loss of antigen-specific proliferation, and gamma interferon (IFN-?) and tumor necrosis factor alpha (TNF-a) production are partially restored in cattle following clearance of persistent infection with tetracycline.

ABSTRACTThe prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective, they are difficult and expensive to isolate and standardize and thus are often impractical for development and implementation in vaccination programs. In contrast, individual proteins, which are easily adapted for use in subunit vaccines, tend to be poorly protective. Consequently, identification of the specific characteristics of outer membrane-based immunogens, in terms of the antigen contents and contexts that are required for protective immunity, represents a major gap in the knowledge needed for bacterial vaccine development. Using as a model Anaplasma marginale, a persistent tick-borne bacterial pathogen of cattle, we tested two sets of immunogens to determine whether membrane context affected immunogenicity and the capacity to induce protection. The first immunogen was composed of a complex of outer membrane proteins linked by covalent bonds and known to be protective. The second immunogen was derived directly from the first one, but the proteins were individualized rather than linked. The antibody response induced by the linked immunogen was much greater than that induced by the unlinked immunogen. However, both immunogens induced protective immunity and an anamnestic response. These findings suggest that individual proteins or combinations of proteins can be successfully tested for the ability to induce protective immunity with less regard for overall membrane context. Once protective antigens are identified, immunogenicity could be enhanced by cross-linking to allow a reduced immunogen dose or fewer booster vaccinations.

ABSTRACTPrevious studies suggest that smooth Brucellaorganisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucellaorganisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortusS2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortusS2308 or pretreated with B. abortusO polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucellarough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.

Anaplasma marginaleis a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2pseudogenes.

ABSTRACTWithin the protective outer membrane (OM) fraction of Anaplasma marginale, several vaccine candidates have emerged, including a family of OM proteins (OMPs) 7 to 9, which share sequence identity with each other and with the single protein OMP7 in the vaccine strain A. marginalesubsp. centrale.A. marginaleOMPs 7 to 9 are logical vaccine candidates because they are surface exposed, present in the OM immunogen and protective cross-linked OM proteins, recognized by immune serum IgG2 and T cells in cattle immunized with OM, and recognized by immune serum IgG2 from cattle immunized with the A. centralevaccine strain. We report the identification of a globally conserved 9-amino-acid T-cell epitope FLLVDDAI/VV shared between A. centralevaccine strain OMP7 and the related A. marginaleOMPs 7 to 9, where position 8 of the peptide can be isoleucine or valine. The epitope is conserved in American A. marginalestrains, in the Australia Gypsy Plains strain, and in multiple field isolates from Ghana. This epitope, together with additional T-cell epitopes that are present within these proteins, should be considered for inclusion in a multivalent vaccine for A. marginalethat can provide protection against disease caused by globally distributed bacterial strains.