The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities. RESULTS: Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45-71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: effect of IL-12, dose, and route.

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.

Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses.

Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.

Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.