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.

Modulation by pentobarbital of neutrophil responses to inhaled E. coli endotoxin in sheep: role of lung epithelium.

Neutrophils (PMNs) are implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). The role of the epithelium in the modulation of PMN migration within the lungs was examined. Epithelial integrity and PMN concentrations in the lung air spaces and lymph were measured in sheep anaesthetized with either halothane (1-2.5%) or intravenous pentobarbital (12+/-4 mg x kg(-1) x h(-1)). Ventilation with an aerosol containing 25 mg Escherichia Coli endotoxin (lipopolysaccharide; LPS) effected neutrophil recruitment to the air spaces. Lymphatic clearance of aerosolized 99mTc-DTPA provided an index of epithelial integrity. Three hours after the deposition of LPS, the lung lining fluid of sheep anaesthetized with halothane (n=7) had 4.9+/-3.2x10(6) PMN.mL(-1), but the lung lymph had almost no PMNs (3+/-8%). Sheep anaesthetized with pentobarbital (n=6) had fewer PMNs in the air spaces (2.4+/-1.2X10(6) mL(-1)) and more PMNs in the lung lymph (30+/-20%). Control sheep (n=5) that received no LPS had almost no PMNs in the airspaces or lung lymph, regardless of the anaesthesia. Three additional sheep that remained awake after receiving LPS also had no PMNs in the lung lymph. The PMN fraction in the lung lymph correlated well with the extra-alveolar epithelial permeability measured by lymphatic clearance of aerosolized diethylenetriamine penta-acetic acid (r=0.81, p<0.001). Aerosolized lipopolysaccharide recruits neutrophils into the lungs of sheep, but they appear to remain in the airspaces unless extra-alveolar permeability is increased by agents such as pentobarbital.

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.

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.

Cytokine release by ovine macrophages following infection with Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen that causes abortion in both sheep and humans. The disease in sheep (but not humans) is characterized by a long-term persistent phase that appears to be under the control of interferon-gamma. However, nothing is known about cytokine induction that precedes the persistent phase in sheep. Primary alveolar lavage cells recovered from normal adult sheep were used to study cytokine production in the first 72 h of infection with C. psittaci. These cells were phenotypically characteristic of macrophages, being adherent, phagocytic, CD14+ and staining positive for non-specific esterase. In vitro infection of the macrophages with C. psittaci resulted in the release of IL-1beta, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as measured by ovine-specific ELISAs. Heat-treated chlamydiae (1 h at 65 degrees C) did not induce the release of IL-1beta, but the release of IL-8 was similar to that induced by untreated organisms. The cells from different sheep varied most notably in their patterns of GM-CSF release in response to heat-treated and untreated organisms.

Characterization of a bioassay for detection of recombinant and native ovine interleukin-5.

In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.

Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants.

The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.

In vivo effects of chicken myelomonocytic growth factor: delivery via a viral vector.

We have constructed a recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (cMGF). Administration of this construct (fp/cMGF) to 1-day-old chicks resulted in a marked and sustained increase in the number of circulating blood monocytes compared with chicks infected with the parental FPV strain (fp/M3). Blood monocyte numbers were elevated within 4 days of fp/cMGF infection, reached maximal levels at day 9, and returned to normal levels by day 16. During the peak response, approximately 35% of blood leukocytes were monocytes, compared with 4 to 7% in uninfected control birds. Infection with fp/M3 also resulted in a detectable increase in monocyte numbers; however, the effect was less dramatic. Compared with fp/M3, fp/cMGF consistently induced two- to threefold higher monocyte numbers, and the period of monocytosis was longer (10 vs 5 days). No other specific changes in white blood cell populations were observed. Associated with the increase in the number of monocytes was an increase in their state of activation, as measured by the ability to produce nitric oxide (NO) and to phagocytose latex beads. Blood monocytes from birds infected with fp/cMGF produced about 6 times as much NO per cell compared with monocytes from fp/M3-infected birds. Monocytes from normal birds failed to produce detectable levels of NO. Furthermore, cMGF treatment specifically induced enhanced phagocytic activity in blood monocytes. Overall, these results indicate that viral vectors are suitable for the delivery of biologically active cytokines and that they allow an assessment of cytokine activities in vivo.

In vitro characterization of a novel avian haemopoietic growth factor derived from stromal cells.

To better define the role of the chicken haemopoietic microenvironment in supporting haemopoiesis, a continuous cell line was generated by RSV transformation of avian spleen stromal cells (SSL-1). Supernatants from this line were found to contain haemopoietic growth factor activity as measured by the ability to induce proliferation and differentiation of precursor cells present in embryonic and post-hatched haemopoietic tissues. Comparison of cultures grown in the presence of cMGF and SSL-1 conditioned media (CM) revealed that both cytokine sources induced similar types of cell populations. Both sources supported the proliferation of predominantly macrophage-like cells based on colony morphology, differential staining, non-specific esterase staining, and phagocytosis activity. Interestingly, SSL-1 does not express any message for cMGF, nor does it secrete any IL-2 or interferon activities suggesting that the growth factor activities seen in SSL-1 are novel.

Cloning and sequencing of an ovine interleukin-5 cDNA.

Interleukin-5 (IL-5) is a T-cell derived cytokine which stimulates eosinophil production and activation in human, mice and sheep. IL-5 is active as a growth factor for mouse but not human B cells. The role of IL-5 on ruminant B cells has not been clearly defined. By hybridisation with human IL-5 cDNA, the ovine IL-5 gene was isolated from a liver genomic library. The IL-5 cDNA was obtained by reverse-transcriptase PCR using primers designed from the 5' and 3' coding sequence derived from the ovine IL-5 gene. The sequences of the cDNA shows that there is 79% and 73% nucleotide homology with the human and mouse sequences. The ovine IL-5 cDNA encoded a protein of 132 amino acids and the level of amino acid homology with human and mouse IL-5 is 64% and 56%, respectively. Two cysteine residues are conserved in ovine, human and mouse IL-5. There are two potential N-linked glycoyslation sites in ovine IL-5.

Development of T cell immune responsiveness in the chicken.

Chickens are highly susceptible to infection by opportunistic pathogens during the first few days after hatching. This observation has generally been attributed to an immaturity of the immune system; however, the mechanisms responsible are not known. This study investigated the ability of T cells from chickens of various ages to respond to immune stimulation. Splenic T cells were cultured in vitro and stimulated with various mitogens including Con A, PHA and monoclonal anti-CD3 antibody. T cells obtained from adult chickens proliferated extensively and produced high levels of IL-2, haemopoietic growth factors and IFN following stimulation. In contrast, it was found that T cells from 1 day old chickens failed to proliferate and secrete cytokines when similarly cultured. Reactivity to mitogens gradually developed between days 2 and 4, and by 1 week of age the level of responsiveness was equivalent to that observed with T cells obtained from adult chickens. Whereas T cells from 1 day old chicks were found to be phenotypically mature and capable of binding mitogens as effectively as T cells from adult birds, they were functionally immature as assessed by their inability to proliferate or produce cytokines following immune stimulation. In addition, cells present in the spleen of 1 day old chicks constitutively produced a soluble inhibitor that prevented the proliferation of stimulated adult T cells. The production of inhibitor decreased dramatically by the second day post-hatching which coincided with an enhanced ability of T cells to respond to immune stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies to three structural proteins of avian infectious bronchitis virus: characterization of epitopes and antigenic differentiation of Australian strains.

Ten monoclonal antibodies (MAbs) directed against three structural proteins of infectious bronchitis viruses (IBV), the peplomer (S), membrane (M) and nucleocapsid (N) proteins, were characterized and used to determine the antigenic relationship between Australian IBV strains. One MAb (MAb 5) was directed against an epitope on the S1 subunit of the peplomer, another (MAb 2) against an epitope on the M glycoprotein and eight MAbs (MAbs 1, 7, 9, 16, 24, 26, 27 and 51) were directed against seven non-overlapping epitopes on the N protein. None of the MAbs neutralized infectivity or inhibited haemagglutination of the virus. Conservation of the nine epitopes detected by these MAbs was determined in 13 serotypes of Australian IBV strains. Only epitope 5 on the S1 subunit of the peplomer was conserved in all strains. Epitope 2 on the M protein showed a high degree of conservation although this epitope was absent from four strains. None of the eight epitopes on the N proteins was conserved in all IBV strains but four epitopes (1, 16, 24 and 27) showed a high degree of conservation. Epitope 9 on the N protein was present only in IBV strains of one serotype whereas epitope 7 on the N protein distinguished vaccine viruses of serotype B from other IBV strains. The presence or absence of nine epitopes on three structural proteins differentiated IBV strains into five antigenic groups.

Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens.

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.

Production and characterization of monoclonal antibodies specific for bovine gamma-interferon.

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAbs reacted with the 16 kDa and 32 kDa polypeptides present in rBoIFN-gamma preparations. Competitive ELISA's showed that four MAbs bound to one epitope on rBoIFN-gamma, and the other five MAbs bound to a separate epitope. Two MAbs, each recognising different epitopes, were shown to neutralise the anti-viral activity of natural BoIFN-gamma.

Quantitation by ELISA of pili and sheep antibodies to the pili of Bacteroides nodosus.

ELISA systems have been developed to quantitate the isotypic antibody response of sheep naturally infected with B. nodosus isolate 198 or injected with pili from isolate 198 in oil emulsion vaccines. The predominant humoral antibody detected following vaccination was IgG1, with substantially lower amounts of IgG2 and IgM. The antibody response was relatively specific for the pilus antigen from isolate 198. Although weak cross reactivities were detected with antiserums to some other isolates, ELISA IgG antibody titres in excess of 200 offer a tentative identification of the isolates of B. nodosus involved in natural outbreaks of footrot. A related ELISA was also developed to quantitate the amount of pili in cell suspensions and crude preparations of pili used in vaccines.