Microdiversity of Echinococcus granulosus sensu stricto in Australia.

Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep.

Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing.

Using a large animal model, we evaluated whether delivery of influenza vaccine via its mucosal site of infection could improve vaccine effectiveness. Unexpectedly, pulmonary immunization with extremely low antigen doses (0.04 microg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 microg unadjuvanted influenza, subcutaneously) and vastly superior lung mucosal antibodies. Induction of this potent response following lung vaccination was dependent on addition of ISCOMATRIX adjuvant and deep lung delivery. Functional antibody activity, marked by hemagglutination inhibition, was only present in the lungs of animals that received adjuvanted vaccine via the lungs, suggesting this approach could potentially translate to improved protection. The 375-fold reduction in antigen dose and improved mucosal antibody responses, compared to the current vaccine, suggests that mucosal delivery via the pulmonary route may be particularly relevant in the event of an influenza pandemic, when vaccine supplies are unlikely to meet demand.

Activation of NF-kappaB transcription factor in the preterm ovine brain and placenta after acute LPS exposure.

Intrauterine infection may be causally related to inflammation and injury of the fetal brain, however the mechanisms by which this occurs are unclear. We have investigated whether nuclear factor (NF)-kappaB, a transcription factor for proinflammatory cytokines, is activated in the fetal brain after acute LPS-exposure. At 95 days of gestation (term = approximately 147 days), 5 fetuses received a single intravenous bolus dose of LPS (1 microg/kg); 6 fetuses served as controls. Fetal blood samples were taken hourly for 6 hr post LPS-exposure to assess physiological status. Ewes and fetuses were then euthanased, placental and brain tissue examined histologically, and NF-kappaB activation assessed in several regions of the fetal brain using an electromobility shift assay (EMSA). Oxidative stress was measured using lipid peroxidation and 8-isoprostane biochemical assays and brain cytokine concentrations analysed by enzyme linked immunosorbent assay (ELISA). LPS-exposed fetuses (relative to controls) were hypoxemic and the haematocrit and lactate levels had increased. In the brains of LPS-exposed fetuses compared to controls, NF-kappaB binding activity was elevated in the hippocampus and the thalamus/basal ganglia; 8-isoprostane levels were elevated overall (P < 0.05) in the parietal/occipital/temporal lobes and thalamus/basal ganglia. TNF-alpha and IL-6 concentrations were not elevated, however, there was a tendency for an elevation of IFN-gamma concentrations in the thalamus/basal ganglia. IFN-gamma concentration was elevated (P < 0.05) in the plasma 4 hr after LPS-exposure. In the placenta, NF-kappaB binding activity was increased (P < 0.05). We conclude that acute systemic administration of LPS leads to increased binding activity of NF-kappaB subunits in specific regions of the fetal brain and in the placenta, but that there is no clear-cut relationship between this elevation and vulnerability to endotoxic damage.

Characterisation of the rapid release of activin A following acute lipopolysaccharide challenge in the ewe.

A series of experiments were conducted in adult ewes to delineate the release profile of activin A and its relationship to other cytokines following an i.v. injection of the bacterial cell wall component, lipopolysaccharide (LPS). Following this challenge, plasma activin A increased rapidly and appeared to be released in a biphasic manner, slightly preceding the release of tumour necrosis factor-alpha (TNFalpha) and before elevation of interleukin (IL)-6 and follistatin levels. The concentration of activin A was correlated with body temperature during the response to LPS. A second experiment compared cytokine concentrations in matched blood and cerebrospinal fluid (CSF) samples. This revealed that activin A was not released centrally in the CSF following a peripheral LPS injection, nor was TNFalpha or the activin binding protein, follistatin, but IL-6 showed a robust elevation. In a third experiment, the stimulus for activin A release was examined by blocking prostaglandin synthesis. Flurbiprofen, a prostaglandin synthesis inhibitor, effectively attenuated the fever response to LPS and partly inhibited cortisol release, but the cytokine profiles were unaffected. Finally, the bioactivity of TNFalpha and/or IL-1 was blocked using soluble receptor antagonists. These treatments did not affect the initial release of activin A, but blockade of TNFalpha depressed the second activin peak. These studies define more rigorously the release of activin A into the circulation following acute inflammatory challenge. The response is rapid and probably biphasic, independent of prostaglandin- mediated pathways and does not depend upon stimulation by TNFalpha or IL-1. The data suggest that activin A release is an early event in the inflammatory cascade following the interaction of LPS with its cellular receptor.

In vivo electroporation improves immune responses to DNA vaccination in sheep.

In vivo electroporation was utilised to enhance plasmid DNA expression in sheep muscle to improve the immune response to DNA vaccination. DNA encoding enhanced green fluorescence protein expressed at higher levels in sheep muscle following in vivo electroporation which caused minimal muscle damage. Groups of seven sheep were then given three intramuscular injections of plasmids encoding two Haemonchus contortus Ag, with and without electroporation at 0, 3 and 7 weeks. Humoral responses were enhanced in electroporated sheep. Four weeks after vaccination, all groups were injected subcutaneously with recombinant Ag formulated in Quil A. Induction of vaccine-specific immune memory was demonstrated in DNA-vaccinated sheep.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

The immune response to a DNA vaccine can be modulated by co-delivery of cytokine genes using a DNA prime-protein boost strategy.

A large-scale DNA vaccination trial was performed in sheep to investigate whether co-delivery of the cytokine genes IL-4, IL-5, IL-15, GM-CSF or IFN-gamma could modulate the immune response generated to an antigen, in a DNA prime-recombinant protein boost regime. Vaccination with the recombinant EG95 protein has been shown to induce protection in sheep from Echinococcus granulosus infection, the causative agent of hydatid disease. Here we demonstrate that vaccination with DNA encoding EG95 effectively primed the humoral response, as judged by high IgG anti-EG95 titres detected one-week after a boost with the recombinant protein. However, by two weeks after protein-boost the titres in the control group had reached levels similar to the groups primed with EG95 DNA. Priming with two doses of DNA vaccine followed by boosting with recombinant protein induced a predominantly IgG1 response. In contrast, priming and boosting with the protein vaccine generated a strong IgG2 response. Co-delivery of the EG95 DNA vaccine with DNA encoding GM-CSF enhanced the antibody titre to EG95 while co-delivery of IFN-gamma or IL-4 encoding DNA appeared to reduce the ability of the DNA vaccine to prime an IgG antibody response. This study has demonstrated the efficacy of the co-delivery of cytokines to modulate immune responses generated in a DNA prime-protein boost strategy.

Immune responses to ISCOM formulations in animal and primate models.

ISCOMs are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs will also be discussed.

Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.

Ovine interleukin-12: analysis of biologic function and species comparison.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.

The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation.

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA.

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.

Functional and structural comparison of cytokines in different species.

As the number of recombinant cytokines increases, so does our knowledge of their structure and function in different species. The biological cross-reactivity of cytokines from one species on cells from a different species has been reported on in the literature but this information is scattered over many publications and some of it has not yet been published. Comparing sequence information combined with three-dimensional and receptor-binding information (i.e. biological cross-reactivity) in different species provides insight into the underlying rules governing cross-reactivity and conservation. It was observed that there is quite a strict threshold of 60% amino acid identity above which cytokines tend to cross-react. Below this threshold few cytokines cross-react on cells from a different species. When comparing frequencies of reported species cross-reactivities between cytokines belonging to different cytokine-folding families it is obvious that not all cytokines within these folding families are equally cross-reactive. The underlying reason for these differences may lay in the ability of certain folding families to accumulate more mutations and still produce a protein, which is able to fold in the desired tridimensional structure. For example, cytokines belonging to the short 4-alpha-helix bundle can accumulate mutations in the 4-alpha-helices and large loops connecting the 4-alpha-helices and are the least cross-reactive. In contrast, cytokines belonging to the beta-sheet based folds (beta-trefoil and beta-sandwich) are the most cross-reactive and also the most conserved cytokines amongst the different species studied.

Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.

Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

Effective in vivo depletion of T cell subpopulations and loss of memory cells in cattle using mouse monoclonal antibodies.

Conditions were established to obtain depletion of T lymphocyte subsets in lymphoid tissues of calves by injection of mouse monoclonal antibodies to T cell antigens. Adverse reactions were avoided by injecting small quantities of antibody, until target cells had disappeared from blood. Two different mechanisms appeared to be responsible for elimination of the target cells. Rapid depletion of T cells was associated with complement-binding antibody isotypes (IgG2a, IgM), suggesting a complement-mediated mechanism. Clearance of T cells after several days was observed with a non complement-binding isotype (IgG1), suggesting phagocytosis or induction of apoptosis as possible mechanisms. Clearance of the cells in peripheral blood and spleen was obtained with 10-20 mg of anti-CD4 or anti-CD8, but almost ten times as much was needed to obtain depletion of the cells in lymph nodes and Peyer's patches. Depletion lasted for 12 days for CD4 T cells and 3 weeks for CD8 T cells. Successful and lasting depletion (at least 2 weeks) was also obtained with other T cell reagents, such as anti-CD2 and anti-WC1 (gamma/delta T cells). Although B lymphocytes could be removed by a complement-binding antibody, complete depletion of these cells only lasted for a few hours, probably because B cells regenerate faster than T cells. T cell function was severely inhibited when CD4+ T cells were depleted. Stimulation of T cells with foot and mouth disease viral antigen (FMDV) in vaccinated calves was non-existent after depletion. Even 2 months after restoration of normal CD4 T cell levels in blood, activity to FMDV was low. This suggested that the depleted T cells were replaced by naive cells.

Ovine cytokines and their role in the immune response.

Using a variety of expression systems the number of available recombinant ovine cytokines has increased steadily. This has led to the use of ovine cytokines as adjuvants to modulate the immune responses to vaccine antigens, both quantitatively and qualitatively. In addition DNA immunization, now common in mice, is being increasingly used in sheep. This may provide a unique avenue for the use of cytokines as immunomodulators, as it avoids preparing large quantities of biologically active recombinant protein and allows a slow, prolonged release of the cytokine at the same site as the antigen. As detection systems are developed their usefulness and shortcomings become apparent. The combination of cytokine detection, lymphatic cannulation and the in vivo neutralization of cytokines has allowed a greater understanding of the immune response during vaccination and of the interaction between pathogens and the immune system.

Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.