Arna E. Andrews

Characterization of monoclonal antibodies to ovine tumor necrosis factor-α and development of a sensitive immunoassay

Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TNF9 and 10, completely blocked the activity of rovTNF alpha and macrophage derived native ovTNF alpha, as measured by their ability to inhibit TNF alpha-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNF alpha (rhuTNF alpha). However, when tested for the ability to inhibit TNF alpha induced thymocyte proliferation, only mAbs TNF1, -3, -5, -7, -9 and -10 could completely block activity. Competitive binding analysis using unlabelled and horseradish peroxidase (HRPO) labelled mAbs indicated that the mAbs could be divided into five groups based on their reactivity with rovTNF alpha. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNF alpha. All combinations of mAbs and the polyclonal antiserum were tested to determine which pair of antibodies gave the most sensitive assay. The combination of TNF5 as the capture antibody and the polyclonal antiserum gave the most sensitive result, detecting less than 0.24 ng rovTNF alpha ml-1. A similar sensitivity was obtained when TNF4 was used as the capture antibody and TNF10 HRPO labelled mAb as the second antibody. The immunoassay was more sensitive than the WEHI-164 bioassay which had a detection limit of 1 ng ml-1 for rovTNF alpha. This immunoassay also detected glycosylated ovTNF alpha in the supernatant of COS-7 cells which had been transfected with an ovTNF alpha cDNA.

Humoral and cellular responses induced by intradermally administered cytokine and conventional adjuvants

Serum antibody responses to the model protein antigen avidin were monitored in sheep following intradermal injection of avidin formulated with a range of commercially available and experimental adjuvants, including muramyl dipeptide (MDP), aluminium hydroxide gel (alum), recombinant ovine interleukin1 beta (rovIL-1 beta), rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus. The highest antibody responses were recorded for animals immunised with avidin in rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus, with moderate responses resulting from use of rovIL-1 beta or alum alone as adjuvants. Lower antibody responses to avidin were recorded when avidin was administered alone or with MDP. Delayed-type hypersensitivity (DTH) responses to avidin indicated that the most pronounced cellular response occurred in animals immunised with rovIL-1 beta + alum. Local cellular changes induced after primary and secondary intradermal injections indicated that distinct patterns of cellular recruitment were induced by the different adjuvants. Avidin with MDP resulted in an elevation of CD4+ T cells in the upper dermis while Emulsigen Plus induced an infiltration of large numbers of neutrophils throughout the dermis and reticular layers. CD4+, CD8+ and gamma delta + T cells increased in number and were found evenly distributed throughout these regions. Alum-based adjuvants resulted in the development of distinct cellular accumulations comprising primarily CD4+ T cells and CD45R + B cells arranged in distinct foci in the reticular layer. These cells were strongly class II positive as were the majority of macrophage like cells surrounding the foci. Staining for factor VIII related antigen indicated the presence of endothelial venules in the T and B cell foci and surrounding tissues.

Vaccination of sheep against larvae of the sheep blowfly (Lucilia cuprina)

Four first stage larval antigens from the sheep blowfly were identified using supernatants from cultures of antibody secreting cells. These partially purified larval antigens, when added to Montanide ISA-25 containing recombinant ovine IL-1 beta (rovIL-1 beta) were used to successfully vaccinate sheep against larvae of the sheep blowfly. Significantly less strikes were recorded on vaccinated sheep compared to controls (P < 0.033) with surviving larvae from vaccinated sheep up to 85% smaller than larvae from control sheep. RovIL-1 beta was found to be an important component of the vaccine. Vaccinated sheep showed both humoral and cellular immune responses to the larval antigens. Antibody levels generally correlated directly with delayed-type hypersensitivity (DTH) responses, but neither antibody nor DTH correlated positively with protection in vaccinated sheep. Skin sections removed from individual sheep immediately after challenge revealed aggregations of CD4+, gamma delta-TCR+ and CD1+ cells located directly under the epidermis in vaccinated sheep.

Production and in vivo use of recombinant ovine IL-1β as an immunological adjuvant

To determine the potential of ovine interleukin 1 (IL-1) as a vaccine adjuvant in sheep, we have expressed and purified recombinant ovine IL-1 beta (rovIL-1 beta) from bacterial cultures using a modified form of the ovine IL-1 beta cDNA. Adjuvant trials using the model protein avidin demonstrated that rovIL-1 beta when administered in association with a compound providing a slow-release mechanism, resulted in significant enhancement of specific serum antibody levels in both mice and sheep. In a dose-response experiment in sheep, intradermal immunization with avidin plus either 10 or 100 micrograms of rovIL-1 beta in aluminium hydroxide resulted in antibody levels four- to eightfold higher than immunizations without rovIL-1 beta. The addition of rovIL-1 beta also resulted in a more severe DTH response to avidin indicating that rovIL-1 beta is able to enhance both humoral and cell-mediated responses to avidin. The highest antibody titres were observed when sheep received rovIL-1 beta in both the primary and secondary immunizations although the addition of rovIL-1 beta in only one of the immunizations still resulted in a significant increase in antibody levels. Additional experiments showed that rovIL-1 beta and avidin must be administered in a site drained by the same lymph node for the adjuvant effect of rovIL-1 beta to be observed.

Cloning and characterisation of an ovine interleukin-10-encoding cDNA

Expression of the interleukin 10-encoding (IL-10) mRNA by ovine (ov-) cells, in response to mitogenic stimulation, was assessed by Northern blot and polymerase chain reaction (PCR) analyses using a human (hu) IL-10 cDNA probe and oligodeoxyribonucleotide primers based on homologous regions of the human and murine IL-10 cDNA sequences. A 315-bp cDNA generated by the PCR analysis was cloned and used to screen a lipopolysaccharide-stimulated alveolar ov-macrophage cDNA library. The full-length ov-cDNA sequence isolated translates to a protein of 177 amino acids (aa) with a predicted 18-aa leader sequence and molecular mass of 20,165 Da. Expression in a mammalian system demonstrated that the ov-cDNA encoded a protein with the expected IL-10 biological activity. Both recombinant huIL-10 and supernatants from COS cells transfected with an expression vector containing the ovIL-10 cDNA inhibited production of IL-1 and tumour necrosis factor-alpha by ov-alveolar macrophages. Genomic DNA analysis indicated ovIL-10 exists as a single gene within the ov-genome.

Parameters related to the application of recombinant ovine interleukin-1β 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.

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.

Production and application of monoclonal antibodies to ovine interleukin-1α and interleukin-1β

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.

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).

Cytokine mRNA expression in skin in response to ectoparasite infection

Cellular infiltration and local cytokine mRNA levels were examined during the first 48 h of infection of skin by larvae of the sheep blowfly Lucilia cuprina. At the cellular level the response involved a dramatic influx of leucocytes (CD45+ cells). Among these infiltrating cells were large numbers of granulocytes, including neutrophils and eosinophils, as well as macrophage‐like cells and lymphocytes. Many of the lymphocytes expressed cell surface markers characteristic of T cells including CD4, CD8 and the γδ TCR. The numbers of each of these cell types increased progressively as infection continued so that by 48 h the lesions were densely populated. Expression of mRNA for IL‐6 could be detected by Northern blot analysis while mRNA for other inflammatory cytokines including IL‐1α, IL‐1β, IL‐8 and TNFα was detected using the polymerase chain reaction. Coincident with the influx of granulocytes and other cells there was an increase in the level of mRNA for the cytokines IL‐lα, IL‐1β, IL‐6 and IL‐8. In the skin of the sheep there appeared to be constitutive expression of message for the cytokines IL‐1β, IL‐6 and TNFα, with the level of the latter not found to increase during the 48 h of infection examined. In situ hybridization was used to determine the location of IL‐6 and TNFα mRNA within resting and infected skin. During infection, fibroblasts, macrophage‐like cells and endothelium appeared to produce high levels of IL‐6 mRNA. Expression of the T cell dependent cytokines IL‐2 and IFN‐γ but not IL‐4, increased in expression as time progressed and the population of infiltrating cells, including T cells, expanded.