Injectable silicone implants as vaccine delivery vehicles.

Injectable silicone implants were assessed as vaccine delivery vehicles in sheep, using either the model antigen avidin or Clostridium tetani and Clostridium novyi toxoids. Two types of implant were compared, the matrix type, that has been shown to deliver antigen in vitro in a first-order profile over approximately 1 month, and the covered rod type, that delivers antigen for several months in a zero-order profile. The implants were prepared using lyophilized antigen and adjuvant (in this case, recombinant ovine interleukin-1beta; rovIL-1beta) and manufactured in the absence of extremes of temperature or pH or the use of organic solvents. Use of the matrix type implant was capable of inducing antibody responses equivalent to those induced by conventional vaccination with aluminium hydroxide adjuvant ("alum"). The use of the covered rod implants, that release very low levels of antigen over a long period, induced responses that were markedly enhanced over the alum control groups. The covered rod implant also favoured production of both IgG1 and IgG2 isotypes in contrast to responses of matrix-vaccinated sheep and conventionally vaccinated control sheep in which IgG1 predominated. Prolonged duration of the antibody response was also observed following vaccination with covered rod implants. Dose-response analysis using the matrix implant demonstrated a trend towards improved responses for lower antigen doses. Clostridial vaccination of sheep showed that protective antibody titres up to 4-fold higher than for alum-adjuvanted groups could be induced by administering the antigen in the covered rod implant. Responses elicited by all implant groups were dependent on the inclusion of adjuvant into the implant formulation.

Isolation and characterization of a novel inducible mammalian galectin.

A novel mammalian galectin cDNA (ovgal11) was isolated by representational difference analysis from sheep stomach (abomasal) tissue infected with the nematode parasite, Haemonchus contortus. The mRNA is greatly up-regulated in helminth larval infected gastrointestinal tissue subject to inflammation and eosinophil infiltration. Immunohistological analysis indicates that the protein is localized in the cytoplasm and nucleus of upper epithelial cells of the gastrointestinal tract. The protein is also detected in mucus samples collected from infected abomasum but not from uninfected tissue. The restricted and inducible expression of ovgal11 mRNA and limited secretion of the protein support the hypothesis that OVGAL11 may be involved in gastrointestinal immune/inflammatory responses and possibly protection against infection.

Parameters related to the application of recombinant ovine interleukin-1 beta as an adjuvant.

This paper describes aspects of the safety and efficacy of recombinant ovine interleukin-1 beta (rovIL-1 beta) as an immunological adjuvant. A dose-response relationship was established using the intramuscular route, and significant adjuvant activity was observed following delivery of 10 or 100 micrograms of the cytokine delivered either in PBS or in combination with alum. Similar doses of rovIL-1 beta also showed adjuvant activity when delivered via the subcutaneous route. In experiments in both mice and sheep, rovIL-1 beta-mediated adjuvant activity was neutralised by a monoclonal antibody (mAb), 3.41, confirming that the adjuvant effect was due to the biological activity of the cytokine. Serum clearance rates and physiological responses to intravenous, intramuscular or subcutaneous administration of rovIL-1 beta in sheep were also determined. RovIL-1 beta was shown to have a serum half-life of 2 min. Transient body temperature increases of 2 degrees C following intravenous or subcutaneous delivery, or 1 degrees C following intramuscular delivery, were observed. White blood cell counts also fluctuated post-injection, which was shown to be due to changes in the number of circulating neutrophils. The action of the neutralising mAb on serum clearance, body temperatures and white cell counts was also determined. Co-injection of rovIL-1 beta with the mAb 3.41 prevented rapid clearance of the cytokine from the serum, and was associated with an extension in elevated body temperature. The mAb appeared to have no significant influence on the white blood cell profile induced following injection with rovIL-1 beta.

Cloning and expression of a cDNA encoding ovine interleukin 7.

Using the polymerase chain reaction (PCR) and primers based on regions of homology between the human and murine interleukin 7 (IL-7)-encoding cDNAs, we have amplified an ovine (ov) IL-7 cDNA from reverse-transcribed RNA extracted from concanavalin A (Con A)-activated ovine lymph-node cells. The nucleotide sequence of the cDNA and the predicted amino acid (aa) sequence showed significant homology to those of the human and murine molecules. The ovIL-7 cDNA encodes a 176-aa polypeptide that, based on analysis of murine IL-7, is processed to a protein of 151 aa. The cDNA was demonstrated to encode a protein with IL-7 biological activity. Supernatants from COS or CHO-K1 cells transfected with an expression vector containing the ovIL-7 cDNA were able to synergise with a suboptimal level of Con A to induce proliferation of ovine thymocytes. In addition, both supernatants were able to induce thymocyte proliferation, albeit at a reduced level, in the absence of Con A. Further experiments demonstrated that for induction of ovine thymocyte proliferation, recombinant (re)-ovIL-7 was able to synergise with re-human (h) IL-2 but not re-hIL-6 or tumor necrosis factor-alpha (re-hTNF alpha).

Production and application of monoclonal antibodies to ovine interleukin-1 alpha and interleukin-1 beta.

Monoclonal antibodies (mAbs) were raised against recombinant ovine interleukin-1 alpha and beta (ovIL-1 alpha and ovIL-1 beta). Five ovIL-1 alpha specific mAbs and three ovIL-1 beta specific mAbs, all of the IgG1 isotype, were characterized. Four of the five ovIL-1 alpha specific mAbs, designated 10.36, 10.49, 10.82 and 5.16, fell into two distinct groups based on several criteria. MAbs 10.36, 10.49 and 10.82 reacted with recombinant ovIL-1 alpha in Western blot analysis, were potent in neutralizing ovIL-1 alpha biological activity in vitro and bound to the same or a closely related epitope. MAb 5.16 also bound ovIL-1 alpha in Western blot analysis, but was less potent in neutralizing ovIL-1 alpha biological activity and bound to a different epitope. A fifth ovIL-1 alpha specific mAb, 5.01, had some characteristics of antibodies from both groups. While the combination of mAb 5.16 with any of 10.36, 10.49 and 10.82 was suitable for detection of ovIL-1 alpha in a sandwich immunoassay, the most sensitive detection of ovIL-1 alpha utilized mAb 10.82 for capture and a rabbit polyclonal anti-ovIL-1 alpha antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent. This combination of reagents had a detection limit for ovIL-1 alpha of 5 pg ml-1 and could detect both recombinant and native ovIL-1 alpha. Of the three ovIL-1 beta specific mAbs, (designated 2.93, 3.41 and 5.60) 3.41 and 5.60 recognized the same or a closely related epitope while 2.93 recognized an epitope more accessible on denatured ovIL-1 beta and proved most useful in Western blot analysis. Only mAb 3.41 was potent in neutralizing ovIL-1 beta biological activity in vitro. A sandwich immunoassay using mAb 3.41 to capture ovIL-1 beta and a rabbit polyclonal anti-ovIL-1 beta antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent had a sensitivity of 5 ng ml-1. The immunoassays were used to assess the relative proportions of IL-1 alpha and IL-1 beta in the supernatant of lipopolysaccharide stimulated ovine alveolar macrophages with IL-1 beta found to be the predominant secreted species of ovIL-1.

Recombinant cytokines as immunological adjuvants.

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin-2 (rovIL-2), interleukin-1 alpha (rovIL-1 alpha) and tumour necrosis factor alpha. These purified proteins had specific activities in appropriate bioassays of 1 x 10(7) 1 x 10(7) and 1 x 10(5) U/mg, respectively. Recombinant ovIL-1 alpha was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL-1 alpha significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 micrograms per sheep enhanced the humoral response to a similar extent. Recombinant ovIL-1 beta had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL-1 beta into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Molecular cloning and characterization of a ruminant interleukin-6 cDNA.

By hybridization with a human interleukin-6 (IL-6) cDNA fragment a corresponding ruminant (ovine) cDNA was isolated from a lipopolysaccharide (LPS)-stimulated alveolar macrophage library. The nucleotide sequence of the cDNA and the predicted amino acid sequence of the protein showed significant homology to the human and murine molecules. Ovine IL-6 cDNA encodes a polypeptide of 208 amino acids that, based on analysis of human IL-6, is processed to a protein of 180 amino acids. Northern blot analysis and the 7TD1 bioassay were used to analyse regulatory aspects of IL-6 production by primary ovine fibroblasts. Both LPS and recombinant ovine IL-1 alpha were shown to induce IL-6 mRNA with peak levels occurring at 1 h post-stimulation and declining thereafter. When fibroblasts were pretreated with cyclohexamide prior to stimulation the level of induction by LPS and IL-1 alpha increased dramatically and peak levels were observed at 5 h post-stimulation. The level of secreted IL-6 increased rapidly over the first 24 h and continued to increase over the next 48 h.

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production.

Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFN gamma) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFN gamma increased surface expression of MHC class I and class II (DR alpha, DP alpha, and DQ alpha) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN gamma specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN gamma required for increased class II expression was 0.1 U ml-1 and for increased class I expression, 1 U ml-1. Northern blot analysis indicated that rIFN gamma mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human cDNA probes we failed to detect mRNA encoding the cytokines IL-1 alpha, IL-1 beta, and TNF alpha in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNF alpha mRNA was induced to maximal levels within 1 h, declining thereafter. IL-1 alpha mRNA was detected at 1 h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-1 beta mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-1 induced detectable levels of TNF alpha mRNA while levels as low as 10 pg ml-1 induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.

Molecular cloning and characterization of ovine IL-1 alpha and IL-1 beta.

Interleukin-1 (IL-1) is a cytokine with a wide range of effects on a variety of cell types. By hybridization with human IL-1 alpha and IL-1 beta cDNA probes, the corresponding ovine cDNAs were isolated from a stimulated alveolar macrophage cDNA library. The sequences of these cDNAs showed that ovine IL-1 alpha and IL-1 beta encode proteins of 268 and 266 amino acids, respectively, with both the nucleotide and amino acid sequences showing a high degree of homology with their human, mouse and bovine equivalents. In a mammalian COS cell-expression system these cDNAs produced biologically active IL-1. Further experiments demonstrated the importance of sequences within the 3' untranslated portion of the cDNAs in determining the level of expression of these molecules. The analysis of expression of IL-1 alpha- and IL-1 beta-specific mRNA in response to endotoxin, phorbol myristic acid (PMA) or PMA plus ionomycin revealed a distinct pattern of differential regulation of the two genes. From genomic analysis both IL-1 alpha and IL-1 beta appear to exist as single copies in the ovine genome.

Molecular cloning, expression and characterization of ovine TNF alpha.

Tumour necrosis factor alpha (TNF alpha) is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. By hybridization with a human TNF alpha cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNF alpha encodes a polypeptide of 234 amino acids that, based on analysis of human TNF alpha, is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin-D treated WEHI-164 cells and induce COS cells to produce and secrete interleukin 6 (IL-6). Further experiments demonstrated the importance of sequences within the 3' untranslated region of the cDNA in determining the level of expression of ovine TNF alpha. Northern blot analysis was used to analyse the kinetics of induction of ovine TNF alpha mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNF alpha at 1 h and 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNF alpha mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNF alpha appears to exist as a single copy.

Cysticercosis: cellular immune responses during primary and secondary infection.

Immune reactions to cysticercosis have been extensively studied in mice. The lack of significant lymphocyte infiltration into the livers of infected mice and the obvious role of antibodies in rejection has led to the general conclusion that cellular reactions do not play a role in protection against this disease. In contrast, the present study examining the immune response to cestode infections in a large animal model (sheep) revealed the presence of a massive and highly organized cellular infiltration in the livers after a secondary Taenia hydatigena infection. The majority of the infiltrating lymphocytes were of the CD4+ phenotype with much fewer CD8+ cells present. While most gamma delta-TCR+ cells in peripheral blood are SBU-T19+, the majority of gamma delta-TCR+ lymphocytes in the liver lesions are SBU-T19- suggesting selective migration of these cells into the lesions. In contrast to the diffuse distribution of T cells in the lesions, B cells were present as distinct aggregates. In primary T. hydatigena infections, host class I and class II MHC antigens were shown, for the first time in cestode infections, to be absorbed onto the surface of the metacestode bladderwall indicating their possible involvement in parasite survival. No immune reactions were observed close to the parasite although lymphocytes and eosinophils were infiltrating the adjacent portal tract areas. Most lymphocytes in both primary and secondary infections were positive for MHC class II antigens suggesting selective recruitment of activated cells to the site of infection. Significant changes in relative and absolute numbers of lymphocyte subpopulations were also observed in the draining hepatic lymph nodes dominated by a massive increase of B cells. In contrast, at the peak of local cellular infiltration, no changes in lymphocyte subpopulations were observed in peripheral blood showing the limited usefulness of this compartment in studying cellular changes in localized infections. The vigorous cellular response observed in the livers of sheep contrasts sharply with the lack of lymphocyte infiltration reported in mice indicating that small animal models may not be appropriate to study cellular responses to cysticercosis in large animals and man.