Analysis of Ehrlichia ruminantium-specific T1/T2 responses during vaccination with a protective killed vaccine and challenge of goats.

Ehrlichia ruminantium is an obligate intracellular bacterium that causes heartwater in ruminants and for which T-cell-mediated immunity is believed to play an important role in protection. To better characterize protective cellular immunity, E. ruminantium-specific IFN-gamma and IL-4 recall responses in major T-cell subsets were analysed by flow cytometry during immunization of goats with a killed vaccine and following a virulent challenge. The killed vaccine elicited both CD8+ and CD4+ subsets to produce cytoplasmic IFN-gamma in the absence of IL-4, thus indicating a biased T1 response. The relative capacity of CD8+ T-cells to produce IFN-gamma was significantly higher than CD4+ T-cells but the final contribution of both subsets was comparable. Circulating ER-specific CD4 and CD8 effectors substantially decreased in numbers after the booster injection and could not be detected in most animals during challenge, which warrants further investigation in immune compartments other than blood. Since IFN-gamma inhibits the growth of the pathogen in target cells, the information provided in this study on E. ruminantium-specific T1 responses will be valuable to develop cellular tools for the identification of potential protective antigens.

IFN-gamma as an indicator of successful immunization of goats vaccinated with a killed Cowdria ruminantium vaccine.

Cowdria ruminantium-induced production of IFN-gamma was measured by ELISA on a weekly basis during the course of vaccination with killed organisms emulsified in ISA50. Upon challenge, all (3/3) vaccinated animals that gave the lowest IFN-gamma response died of peracute cowdriosis. On the other hand, only one of three animals showing high IFN-gamma responses to vaccination died, but with a delay of 4 days in comparison with naïve controls. Thus, there seems to be a threshold level of IFN-gamma below which the probability for vaccinated animals to survive a lethal challenge is very low. During challenge, a much lower, but still physiologically meaningful production of IFN-gamma was detected using the 24-hour whole blood assay on day 5 after infection in animals controlling the infection. In contrast, IFN-gamma production was absent or negligible in naïve and vaccinated animals that died within 8-10 days after infection. Although these results need to be validated on a larger number of animals, they strongly suggest that IFN-gamma is a useful indicator of protective immunity in animals immunized with killed COWDRIA:

Cowdria ruminantium antigens of around 15 kDa are potent inducers of IFN-gamma.

Cellular responses induced in two Creole goats by vaccination with killed Cowdria ruminantium (Cowdria) were confirmed by IFN-gamma production and interleukin-2 receptor (IL-2R) expression. Both CD4+ and CD8+ but not WC1+ T cells showed a substantial increase in cell surface expression of IL-2R molecules in response to whole Cowdria lysate. Cowdria (Welgevonden strain) proteins were fractionated using continuous-flow electrophoresis and tested for their ability to induce IFN-gamma production by PBMC collected three weeks after the first inoculation and one week after the booster injection. Pooled fractions of around 15, 22, and 24 kDa were found to induce significant IFN-gamma production in both vaccinated animals on one of the two occasions. Antigens of around 15 kDa induced substantially higher IFN-gamma production than any other fractions in both animals. These pilot experiments pave the way towards the identification of proteins/genes that have potential for the development of a recombinant vaccine against heartwater.

A highly sensitive, non-radioactive assay for T cell activation in cattle: applications in screening for antigens recognised by CD4(+) and CD8(+) T cells.

We describe a highly sensitive, non-radioactive assay for T cell activation, based on the rapid induction of class II MHC expression by constitutively negative bovine endothelial cells, when cultured in the presence of supernatants derived from activated bovine T cells. We demonstrate the effectiveness of this assay in detecting rBoIFNgamma and activation of immune CD4(+) and CD8(+) T cell lines and clones in response to specific antigen and transfected COS-7 cells, respectively. We also demonstrate its utility in identifying purified pathogen fractions that activate immune CD4(+) T cell clones.

Effect of isolation techniques, in vitro culture and IFNgamma treatment on the constitutive expression of MHC Class I and Class II molecules on goat neutrophils.

Previous studies on the ability of human neutrophils to synthesize cytokines and express MHC Class I and inducible Class II molecules have suggested a possible role of these cells as accessory or antigen presenting cells (APC). There is no information available to date concerning this aspect in ruminants. Therefore, as the first step of these investigations, we have studied the cell surface expression of MHC molecules on goat neutrophils. We show that goat neutrophils can be distinguished from eosinophils with monoclonal antibody (MoAb) ILA-24 which recognizes cattle monocytes and neutrophils. Goat neutrophils constitutively express MHC Class II molecules. However, cell surface expression of MHC Class I and Class II molecules is dramatically reduced on neutrophils purified by density gradient centrifugation in comparison to neutrophils obtained from whole blood after lysis of erythrocytes. Also, the level of expression of MHC Class I antigens is seasonal and donor-dependent and rapidly decreases after in vitro culture despite negligible necrosis and apoptosis of neutrophils. Although treatment with IFNgamma partially prevents the loss of MHC Class I molecules on neutrophils, it fails to induce MHC Class II antigens. Implications of these results for further studies on the potential role of neutrophils as APC are discussed.

Bovine CD4+ T-cells lines reactive with soluble and membrane antigens of Cowdria ruminantium.

Cowdria-specific CD4+ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-gamma in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-gamma by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4+ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.

Immune responses to Cowdria ruminantium infections.

Understanding the basis of protective immunity to Cowdria ruminantium will facilitate the development of an effective subunit vaccine against heartwater in ruminants and contribute to a better definition of protective immune mechanisms to obligate intracellular pathogens in general. Until recently, immunological studies of heartwater in ruminants concentrated solely on antibody responses. Since 1995, the mechanisms underlying cell-mediated immunity of heartwater have been analysed. Progress achieved in these areas is discussed here by Philippe Totté and colleagues, with special emphasis on ruminants, the natural hosts of C. ruminantium.

Analysis of cellular responses to native and recombinant proteins of Cowdria ruminantium.

The response of PBMC collected from cattle immunized with killed Cowdria to MAP1, MAP1-B, MAP2 and to a lysate of Cowdria was characterized in vitro. CD4+ T lymphocytes responded strongly to Cowdria lysates while B cells and gamma delta T cells proliferated in response to recombinant proteins. Short-term cell lines derived from PBMC using Cowdria lysates were more than 90% CD4+ and did not respond to any of the recombinant proteins. Restimulation of PBMC with MAP1, MAP1-B, and MAP2 proved difficult, and cultures usually stopped proliferating after 2 to 3 weeks. Only one cell line could be generated using MAP1 and it was composed of 85% gamma delta T cells. This study indicates that in animals immunized with killed Cowdria, proteins other than the serologically immunodominant MAP1 and MAP2 are strongly immunogenic for T lymphocytes.

Inhibitory effect of Cowdria ruminantium on the expression of MHC class I and class II molecules on bovine endothelial cells.

Bovine endothelial cells constitutively express MHC class I molecules, whereas MHC class II molecules can be induced by interferon gamma (IFN gamma) treatment. Endothelial cells may play a role as antigen presenting cells (APC), but their role in the immune response against Cowdria ruminantium is unknown. We have studied the effect of C. ruminantium infection on the expression of MHC class I and class II molecules on the surface of bovine endothelial cells. Bovine brain endothelial cells (BBEC) from the microvasculature and bovine umbilical endothelial cells (BUEC) from the macrovasculature were cultured in vitro, infected with different concentration of Cowdria, and treated or not with IFN gamma. We observed that Cowdria induced a reduction of MHC molecules expression on the surface of BUEC and BBEC. This inhibitory effect of Cowdria on MHC molecules could affect the capacity of endothelial cells to act as efficient antigen-presenting cells.

Inhibition of MHC class I and class II cell surface expression on bovine endothelial cells upon infection with Cowdria ruminantium.

Endothelial cells constitute a main target for Cowdria ruminantium (CR) and can potentially play a role as antigen presenting cells (APC). Therefore, we measured, in vitro, the effect of CR infections on the expression of MHC class I and class II molecules on bovine umbilical endothelial cells (BUEC) and on bovine brain endothelial cells (BBEC). A dramatic inhibition of the expression of IFNgamma induced MHC class II molecules was observed on BUEC and to a lesser extent on BBEC upon CR infection. This inhibitory effect was also observed on constitutively expressed MHC class I molecules. Part of the reduction of cell surface MHC molecules could be ascribed to their accumulation in intracellular compartments pinpointing a disruption in the transit of these molecules to the surface of the cells. The exact mechanisms of inhibition are not yet known but, as opposed to what is described in other models, the involvement of prostaglandin E2 can be excluded. The results obtained in this study show that endothelial cells have a decreased capacity to express both MHC class I and class II molecules on their surface upon CR infection, thus favouring the escape of this pathogen from the host immune system.

Analysis of T-cell responses in cattle immunized against heartwater by vaccination with killed elementary bodies of Cowdria ruminantium.

Cattle were successfully immunized against heartwater with a lysate of Cowdria ruminantium formulated in Freund's adjuvant. Vaccinated animals proved fully resistant to virulent challenge 3 and 10 months after vaccination. For the first time a helper T lymphocyte response to Cowdria antigens was observed and characterized. Cowdria-specific T-cell lines generated from vaccinated animals by in vitro restimulation with Cowdria lysates are 95 to 100% CD4+, are MHC class II restricted, and produce gamma interferon. They proliferate in response to autologous monocytes infected with live Cowdria but not in response to uninfected monocytes. These T-cell lines will facilitate the search for Cowdria antigens that are immunogenic for T cells and will therefore be of relevance in the development of a subunit vaccine against the disease.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells.

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Uptake of recombinant myeloperoxidase, free or fused to Fc gamma, by macrophages enhances killing activity toward micro-organisms.

A chimeric antibody-like molecule consisting of the human myeloperoxidase (rMPO) fused to the second and third constant-sequence (CH2 and CH3) Fc domains of human immunoglobulin G-1 has been constructed and expressed in Chinese hamster ovary (CHO) cells. This fusion molecule was designed to combine the binding specificity of Fc with the antimicrobial properties of rMPO. The rMPO-Fc fusion dimerized through the Fc fragment, while retaining the enzymatic activity of rMPO. The chimeric molecule was glycosylated and most of the propeptide was eliminated, indicating a better processing of the polypeptide than for rMPO alone. Both rMPO and rMPO-Fc bound to and were internalized by macrophage-like U937 promonocytic cells. Unexpectedly, the chimera failed to bind to the Fc receptor but interacted with a higher affinity than rMPO with the same binding sites. The presence of the Fc fragment in the chimera, in addition, did not extend the plasma half-life of the fusion protein. In vitro, rMPO-Fc exhibited a stronger killing effect than rMPO toward Candida albicans in the presence of either H202 alone or human macrophages. In vivo, rMPO-Fc similarly conferred a better protection than rMPO in a lethal model of murine cowdriosis. These properties could be related to the Fc-induced dimerization of the fusion protein in CHO cells.

Production of alpha interferon in Cowdria ruminantium-infected cattle and its effect on infected endothelial cell cultures.

Cattle that resisted experimental heartwater infection caused by the rickettsia Cowdria ruminantium produced significant levels of circulating alpha interferon (IFN-alpha), whereas animals that died from heartwater did not. In vitro, recombinant bovine IFN-alpha was found to significantly reduce the yield of Cowdria organisms in bovine endothelial cells, but even at a high concentration (1,000 U/ml), IFN-alpha did not completely prevent the growth of Cowdria organisms in these cells. This limited inhibitory effect of IFN-alpha is in agreement with the in vivo situation where an infectious process has to take place to induce a protective immune response. Our results suggest that IFN-alpha produced in vivo in response to Cowdria infection may represent an efficient way to slow down the infection and allow the animal to mount a protective immune response. IFN-alpha is the first endogenously produced factor shown to have anti-Cowdria activity.

Bovine and human endothelial cell growth on collagen microspheres and their infection with the rickettsia Cowdria ruminantium: prospects for cells and vaccine production.

We successfully cultivated the rickettsia Cowdria ruminantium, in bovine endothelial cell lines (Bovine Umbilical Endothelial Cells/BUEC and Bovine microvasculature Cells/BMC) and also in primary endothelial cells of bovine origin (Bovine Aorta Endothelial cells/BAEC) and more surprisingly in cells of human origin--Human Umbilical Vein Endothelial Cells/HUVEC--and Human Endothelial Cells from the Microvasculature/HEMEC. This first evidence of the pathogenicity of this bovine rickettsia in the human cell system gene-rates new interest as regards its possible relevance for human health. It provides also further possibilities for the attenuation of Cowdria ruminantium isolates, and therefore brings new prospects for vaccine preparation. In vaccine production, mass cell culture is essential. Our results indicate that endothelial cells attach efficiently on collagen microspheres. As BAEC cells grow well on them in a batch mode, and if the process could be optimized for the different cell types (using appropriate adhesion and growth factors) our observations offer interesting prospects for the future development of a Cowdria ruminantium vaccine production in the fluidized-bed reactor VERAX System one, which provides easy control of growth conditions.

Inhibition of Cowdria ruminantium infectious yield by interferons alpha and gamma in endothelial cells.

We have shown before that there is a positive correlation between resistance of cattle against Cowdria infection and early IFN production. Our in vitro studies demonstrated an activity of rBoIFN alpha 2C and rBoIFN gamma against Cowdria in bovine endothelial cells of brain microvasculature (BMEC). rBoIFN gamma is much more active in this respect than rBoIFN alpha 2C. These results suggest a role of IFNs in the resistance against the disease. Strikingly, in the same conditions rBoIFN alpha 2C has no effect on the yield of Cowdria from infected bovine endothelial cells of umbilical artery origin (BUEC). Similarly we showed that HuIFNa had no effect on the multiplication of Cowdria in human vein umbilical endothelial cells (HUVEC). We found no differences in the capacity of BUE and BME cells to bind rBoIFN alpha 2C. This may reflect a true difference between capillary and large blood vessels.

Role of interferons in infectious diseases in the bovine species: effect on viruses and rickettsias.

Successful protection was obtained with interferon treatment in experimental viral infections in the bovine species in a number of cases. The efficacy of the treatment against vaccinia virus infection and against rotavirus infection have been demonstrated. On the contrary, bovine herpes virus 1 (BHV 1-causing rhinotracheitis and part of the shipping fever complex) infections were not inhibited by interferon (IFN). The authors have undertaken a study in cattle in Zimbabwe to assess the role of interferon in the resistance of the animals to Cowdria ruminantium. A good correlation between production of interferon by the animal following the infection, and the resistance of the animals against the rickettsia was demonstrated. This pointed out the possible "adjuvant" role of interferons and other cytokines.