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Avian cytokines — the natural approach to therapeutics
Developmental and Comparative Immunology 24 (2000) 355±365 www.elsevier.com/locate/devcompimm
Avian cytokines Ð the natural approach to therapeutics John W. Lowenthal a,*, Benedicte Lambrecht b, Thierry P. van den Berg b, Marion E. Andrew a, A. David G. Strom a, Andrew G.D. Bean a b
a CSIRO Animal Health, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia Section of Avian Virology & Biotechnology, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, B1180, Brussels, Belgium
Accepted 1 November 1999
Abstract While the eective use of antibiotics for the control of human disease has saved countless lives and has increased life expectancy over the past few decades, there are concerns arising from their usage in livestock. The use of antibiotic feed additives in food production animals has been linked to the emergence in the food chain of multiple drug-resistant bacteria that appear impervious to even the most powerful antimicrobial agents. Furthermore, the use of chemical antimicrobials has led to concerns involving environmental contamination and unwanted residues in food products. The imminent banning of antibiotic usage in livestock feed has intensi®ed the search for environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators and regulators of the immune response, oer exciting new alternatives to conventional chemical-based therapeutics. The utilisation of cytokines is becoming more feasible, particularly in poultry, with the recent cloning of a number of avian cytokine genes. Chickens oer an attractive small animal model system with which to study the eectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review the status of avian cytokines and focus on our recent studies involving the therapeutic potential of chicken interferon gamma (ChIFN-g ) as a vaccine adjuvant and a growth promoter. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Interferon-g; Cytokines; Therapeutics; Chicken
Abbreviations: ACG, Avian Cytokine Group; ChiFN-g, chicken interferon g; cMGF, chicken myelomonocytic growth factor; ELISA, enzyme linked immunosorbent assay; FAV, fowl adenovirus; IFN, interferon; IFN-g, interferon g; IL-1, interleukin 1; LPS, lipopolysaccharide; NDV, Newcastle disease virus; TGF-b, transforming growth factor b; TMV, tobacco mosaic virus; WHO, World Heath Organization. * Corresponding author. Tel.: +61-3-5227-5759; fax: +61-3-5227-5331. E-mail address: [email protected] (John W. Lowenthal). 0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 9 9 ) 0 0 0 8 3 - X
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1. Introduction: the need for alternative natural therapeutics The sheer size of the poultry broiler industry is staggering: almost 40 billion chickens are hatched worldwide every year. Chicken meat represents approximately 40% of all meat consumed and is a US$150 billion per annum retail market, as well as supporting a multi-billion dollar poultry health market. Chickens are reared under intensive conditions which are conducive to infection by opportunistic pathogens. This is especially critical during the ®rst weeks of life, a time at which the immune system has not yet fully matured [1] and when levels of maternal antibody are declining. A major problem faced by intensive livestock industries, such as the poultry industry, is loss of productivity due to disease, therefore considerable resources are required in order to maintain the health status of these animals. The mechanism by which infectious disease is controlled involves the combined use of vaccines, antibiotics and chemicals. Vaccines are designed to provide long term immunity and provide speci®c protection against a particular pathogen following immunisation. Usually the most eective form of vaccination is immunisation with a live (attenuated) organism, however, the risks of virulent mutant strains arising from vaccine strains is a constant concern. Vaccination with killed organisms or recombinant antigens, which are generally less immunogenic, requires the use of powerful adjuvants in order to develop a longterm protective immune response. Unfortunately, most commercial adjuvants are oil-based and their use can result in adverse local reactions resulting in downgrading of meat quality. The livestock industries now face the planned phaseout of oil-based vaccines which is a driving force for the search for eective alternative, natural vaccine adjuvants. In contrast to vaccines, antibiotics provide short-term, broad-spectrum protection and are provided continuously to livestock as feed additives. Antibiotics have become very popular in the livestock industries because, in addition to being an antimicrobial, they also have growth
promoting activity [2]. In fact, up to half of the world's production of antibiotics is used in agriculture. As recently reported from a World Health Organisation (WHO) meeting in Berlin on the medical impact of the use of antimicrobials in food animals (see http://www.who.int/ emc-documents/antimicrobial_resistance/whoemczoo974c.html), the extensive use of antibiotics, particularly those that are used in human medicine, has resulted in human health concerns. The WHO has recently urged meat producers to stop using the same antibiotics that are used in humans or those which select for cross-resistance, however, these are usually the most eective in controlling disease. Furthermore, the WHO has strongly recommended the development and use of alternative, environmentally-friendly methods to control disease. Similarly, the long term use of chemicals, such as coccidiostats, results in emergence of resistant strains of pathogens such as Eimeria, increases environmental contamination and results in the deposition of residues in meat products. Some European countries have already banned the use of antibiotic and chemical feed additives in food production animals and more plan to follow suit. Livestock industries now acknowledge that withdrawal of antimicrobials from feed is inevitable and we must now develop strategies to cope with this. 2. Cytokines and their in¯uence on immunity and growth Cytokines are a diverse family of proteins that play a crucial role in controlling the immune system. They determine both the type and extent of an immune response that is generated following infection with a pathogen or after vaccination. Depending on the combination of cytokines produced, a protective immune response can be generated as either an antibody-mediated (Th2) response or a cell-mediated (Th1) response [3]. Cytokines therefore represent excellent candidates as naturally occurring therapeutics as well as vaccine adjuvants [4,5]. Of particular relevance to poultry is the susceptibility of newly hatched chickens to infection by opportunistic pathogens,
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due primarily to declining maternal antibody levels at a time when their immune system is still relatively immature [1]. The production of IgA antibodies at the mucosal surface is a ®rst line of defence against respiratory and gastrointestinal pathogens [6]. It is therefore feasible to elevate mucosal IgA levels in newly hatched chicks by appropriately administering IgA-inducing cytokines, thereby enhancing their resistance to disease (see Muir et al., this issue). The ecacy of cytokine therapy has been demonstrated in several human and animal studies. Interferons (IFN) are a family of cytokines that share potent antiviral as well as immunostimulatory properties. IFN-g has been shown to be an eective adjuvant in immunocompromised mice when delivered with a malarial vaccine [7] and also enhanced antibody responses and protection from disease when administered with a subunit vesicular stomatitis virus vaccine [8] or in¯uenza vaccine [9]. These eects are likely to be mediated through the ability of IFN-g to induce T cell help for antibody production. Other studies have demonstrated that T cell responses such as delayed type hypersensitivity and T cell help for antibody production were enhanced following the co-administration of IFN-g with a vaccine [10]. Yet other studies have shown that IFN-g is capable of activating cells such as macrophages to directly kill intracellular Eimerian oocysts [11]. In a human clinical trial, aerosol delivery of IFN-g to tuberculosis patients was shown to be an eective therapeutic in the treatment of multi-drug resistant bacteria [12]. Another member of the interferon family, IFN-a, is an eective antiviral agent when administered as a recombinant protein [13±15]. In a recent study, Marcus et al. [15] showed that oral administration of ChIFN-a protein to one day old chicks via drinking water oered protection from challenge with La Sota strain Newcastle disease virus (NDV). They found that ChIFN-a treatment delayed the onset and severity of clinical symptoms and prevented the histopathological changes (in¯ammation, hyperplasia, deciliation and lymphocyte in®ltration) normally seen in the trachea following NDV infection. Cytokines are also thought to play a direct
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role in the growth of animals. It is known that both illness and stress reduce an animal's ability to grow at an optimal rate. It is now becoming evident that growth is in¯uenced by interactions between the immune and endocrine systems and the brain [16]. Whereas the local action of cytokines involves activation of the immune system, their systemic eects include, amongst other things, alterations in body metabolism and neuroendocrine functions. For example, cytokines such as IL-1 and TNF can accelerate muscle degradation, and reduce food and water intake [17,18]. Treatment of animals with combinations of certain cytokines, or alternatively with antagonists of cytokines, may in¯uence their growth rate, resulting in an increase in productivity which would obviously be a commercial bene®t. The utilisation of cytokines in poultry is becoming more feasible with the recent cloning of a number of avian cytokine genes (see Table 1). The major limiting factor for cytokine therapy has been the mode of delivery since cytokines typically persist for a short time in vivo due to rapid utilisation and/or degradation. Delivery of cytokines to chickens is now commercially feasible using live viral vectors (see Johnson et al., this issue), which allows long term in vivo production. 3. Cloning of avian cytokine genes A variety of cytokine genes have now been cloned in several avian species (Table 1). The majority of avian cytokine genes so far cloned share limited sequence identity with their mammalian counterparts [19,20], and the expressed proteins rarely cross react on mammalian cells. In the case of chicken IFN-g (ChIFN-g ), 3D protein modeling has revealed that the homodimer structure is surprisingly similar to human and bovine IFN-g [21], whereas the level of amino acid identity is very low (about 35%). As a consequence of this, the full repertoire of genes must be identi®ed, cloned and expressed in order to fully test the therapeutic potential of avian cytokines. A number of laboratories involved in avian
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cytokine research have recently formed the Avian Cytokine Group (ACG) in order to facilitate the exchange of basic information and reagents for research purposes. A web site has now been developed; for further information on the ACG see: www.ah.csiro.au/AvianCytokines/. It was also agreed that certain laboratories would be reference laboratories for particular cytokines. The aim is for international standard preparations to be adopted and distributed to interested laboratories to be used as the positive control and standard to which all other preparations of that cytokine will be compared. This should allow standardisation of results obtained from dierent laboratories. 4. Production of recombinant cytokines Several procaryotic and eucaryotic expression systems are available for the production of recombinant cytokines; each have their particular advantages as well as disadvantages. If recombinant cytokines are to be used as commercial therapeutics, particularly in the poultry industry, there are several critical constraints that have to be considered in order for a product to be considered cost-eective. The protein must be able to be produced on a commercial scale (multi-billion doses per annum), easily puri®ed, inexpensive to produce, stable upon storage, and retain bio-activity in vivo. Various avian cytokines have been successfully expressed in E. coli. This system
allows large amounts of biologically active recombinant protein to be produced, which can be puri®ed by relatively simple procedures. Disadvantages of this system are that these type of proteins are non-glycosylated and may not be folded in authentic native forms, thereby having less than optimal speci®c activities or reduced half-lives in vivo. Cytokines have also been expressed in a variety of eucaryotic systems, including COS, CHO, and yeast, as well as virusbased systems such as baculovirus, tobacco mosaic virus (TMV), fowlpox virus and fowl adenovirus (see Johnson et al., this issue). Recombinant proteins expressed from these systems more closely resemble naturally produced proteins in terms of glycosylation patterns and folding. Perhaps the most crucial consideration is one of biological activity. One important immunomodulatory eect of IFN-g is its ability to induce the expression of MHC Class II molecules on the surface of antigen presenting cells. When dierent preparations of ChIFN-g (expressed by either E. coli, COS, FAV or baculovirus) were compared for their ability to induce Class II expression on HD11 chicken macrophages, similar levels in up-regulation of expression were observed. HD11 cells express relatively low levels of class II molecules on their cell surface when stained with the CIa antibody (Southern Biotechnology) and this level of expression was not in¯uenced by the addition of low levels of LPS (20 ng/ml). Exposure of HD11 cells to ChIFN-g (10 U/ml for 24 h) resulted in an increase in the pro-
Table 1 Cloned avian cytokine genes Species cloneda
Cytokine Interferon-a Interferon-b Interferon-g Interleukin-1b Interleukin-2 Chemokines (CEF4, K60) Chicken myelomonocytic growth factor Stem cell factor Transforming growth factor-b a
IFN-a IFN-b IFN-g IL-1b IL-2 cMGF SCF TGFb
Ch Ch Ch Ch Ch Ch Ch Ch Ch
[22,23], Tu [24], Du [25] [23] [26], Tu [20], Jq [20], Ph [20], Gf [20], Du [27,28] [29] [30] [31,32] [33] [34] [35]
Ch, chicken; Tu, turkey; Jq, Japanese quail; Ph, pheasant; Gf, guinea fowl; Du, duck.
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Fig. 1. (a) Induction of MHC Class II expression by ChIFNg. HD11 cells were incubated for 24 h in the presence of 10 U/ml of ChIFN-g expressed from either COS, E. coli, FAV or baculovirus, with or without the presence of 20 ng/ml of LPS. Cells were harvested, stained with anti-Class II Mab (CIa), and analysed by ¯ow cytometry. Numbers in each panel refer to the percentage of cells expressing Class II. (b) Eect of ChIFN-g dose on the expression of MHC Class II by HD11 cells. Cells were incubated in the presence of indicated doses of ChIFN-g for 24 h and measured for Class II expression. Approximately 15% of cells were positive in the absence of ChIFN-g.
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portion of cells expressing Class II molecules and also the intensity of staining, irrespective of whether ChIFN-g was glycosylated (expressed by COS, FAV or baculovirus) or expressed by E. coli (Fig. 1a). Fig. 1b shows that the eect of ChIFN-g on Class II expression titrates with a dose response curve that is similar to that seen in the nitrite release assay. Plant viruses have been successfully used to produce recombinant foreign proteins in plants. One of the best studied systems is tobacco mosaic virus (TMV), a single stranded RNA virus that is capable of inducing high level transient expression of non-viral foreign proteins within a few days of introduction into the plant [36]. The TMV expression vectors have been shown to be easily and quickly generated and the level and timing of gene expression can be controlled. Commercial manufacture of recombinant proteins in plants is becoming an attractive alternative to expression in yeast and E. coli. Large scale production in tobacco plants via TMV vectors has been shown to be both commercially feasible and inexpensive. Recent examples include the production of malarial vaccine antigens [37], a-trichosanthin [38] as well as antibiotics and milk proteins. The TMV expression system oers the advantages of minimal environmental impact due to the generation of relatively little waste, ability to produce large amounts of protein, relatively simple processing requirements and the ability to produce soluble or membrane bound proteins. Commercial products derived from TMV expression are already on the market. A TMV vector producing ChIFN-g has been developed [39]. The plant-expressed recombinant ChIFN-g protein is identical to ChIFN-g derived from procaryotic (E. coli ) and eucaryotic (COS cells) expression systems. It is also similar to naturally occurring ChIFN-g as measured by biological activity, sensitivity to heat treatment and reactivity to anti-ChIFN-g antibodies [39]. 5. The search for alternative, natural adjuvants Current vaccine research involves the development of recombinant subunit vaccines which are
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intended to replace the traditional forms of vaccination which use live or killed whole organisms. Unfortunately many of these new generation vaccines are, by themselves, poorly immunogenic and require several rounds of immunisation. They usually require the use of powerful adjuvants in order to elicit an eective immune response. However, the move away from the use of oil-based adjuvants, due to adverse site reactions, has led to a focus on the application of cytokines, which might make these new generation vaccines safer and more eective [40]. Some of the practical considerations of vaccine development, especially for poultry, are cost, eectiveness, ease of production, and delivery. When used as a vaccine adjuvant, cytokine proteins can simply be mixed together with the vaccine antigen prior to immunisation. For this type of function relatively small amounts of cytokine are required as they would act locally, at or near the site of injection. If used as an immunomodulator, cytokines would act mucosally or systemically and hence require considerably larger amounts of protein to be administered. However, for therapeutic or growth promoting activities, frequent injections over a period of time would be required, which is not feasible or cost-eective in a commercial setting. For these uses viral vector delivery may come to the forefront. Nevertheless, for smaller experimental trials assessing feasibility, injection of cytokines is practical. 6. ChIFN-gg as a vaccine adjuvant Birds, like mammals, express a family of interferon proteins whose genes have been cloned [41]. The gene for ChIFN-g has been cloned and recombinant protein of high speci®c activity produced [26,42±44]. The potential of ChIFN-g as a vaccine adjuvant has been assessed in experimental trials [45]. Co-administration of recombinant ChIFN-g with antigen resulted in enhanced secondary antibody responses that persisted at higher levels and for longer periods compared to antigen injected in the absence of ChIFN-g. In addition, ChIFN-g treatment allowed a lower dose of antigen to be used more eectively while
signi®cantly increasing the proportion of birds within a group that sero-converted [45]. The ability of an adjuvant to enhance the ecacy of lower doses of antigen or to reduce the number of immunisations required to induce long term protection, may overcome the often prohibitive cost of livestock vaccines. Furthermore, the ability of cytokines to prolong immune responsiveness in individuals as well as increase the proportion of animals that respond to vaccination can be employed in strategies to reduce multi-dose regimes required for many existing vaccines, thereby reducing the overall cost of vaccination. It is feasible that cytokines may eventually replace the need for conventional oil-based adjuvants that often induce adverse site reactions, which lead to downgrading of meat quality. 7. ChIFN-gg as a growth promoter Conditions of stress and illness have a negative impact on the ability of animals to grow at their optimal rate. It is also becoming evident that growth is in¯uenced by interactions between the immune system and other systems within the body such as the neurological and endocrine systems and that cytokines may play a central role regulating this [16]. It has been reasoned that reduction of stress and illness would allow a redirection of the body's resources toward growth. There is now an emerging area of research examining the eects of utilising a combination of anti-in¯ammatory and immunostimulatory agents to achieve this, with cytokines being key players. We have performed experiments to assess the growth promoting activity of ChIFN-g in chickens [42]. Seven-day old broiler chickens were injected with ChIFN-g protein on two consecutive days while control birds were injected with diluent alone and body weight was recorded daily. Birds treated with ChIFN-g displayed enhanced weight gain over a 12 day period, relative to those injected with diluent alone or control birds that were not treated. The increase in body weight ranged from 3±8%. Similar increases in growth rates were observed over a 3± 8 week period in subsequent experiments which
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were performed on larger group sizes with ChIFN-g delivered via a fowl adenovirus (FAV) vector (see Johnson et al., this issue). These results clearly indicate the potential use of ChIFN-g as an eective, naturally occurring growth promoter. The underlying mechanisms are unknown, but may be due to the IFN-g mediated enhancement of the immune system resulting in healthier and more productive birds.
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9. Development of an ELISA for ChIFN-gg Up to now the only way to detect and quantitate the biological activity of ChIFN-g was to use a bioassay. ChIFN-g shares with other members of the interferon family the capacity to inhibit
8. Eect of ChIFN-gg on weight gain during infection with Eimeria acervulina ChIFN-g has been shown to prevent the development of Eimeria parasites in vitro, presumably through its ability to activate macrophages [12,46]. The therapeutic potential of ChIFN-g has also been demonstrated in vivo using a coccidial challenge model [42]. Infection of young broilers with Eimeria acervulina normally results in weight loss beginning on day 4 after infection with weight gain resuming 2±3 days later. In these studies, broiler chickens were inoculated on two consecutive days with either E. coli-derived ChIFN-g or diluent and then infected with E. acervulina oocytes one day later. Body weight was recorded daily for 12 days following challenge. ChIFN-g treated birds lost less weight early in infection (day 4±5 post challenge) and recovered more quickly as indicated by a larger weight gain between days 6 and 8 post challenge when compared to controls. This result indicates that ChIFN-g reduced the eect of coccidiosis on growth performance. In two separate trials, treatment of infected birds with ChIFN-g resulted in a consistent increase in weight gain ranging from 2.7 to 12.5% relative to non-treated infected birds. In a later independent study, ChIFN-g therapy was shown to also reduce oocyst production following Eimeria challenge [47]. These results have now been reproduced in larger trials performed under commercial farm conditions, where ChIFN-g was delivered by live FAV vectors (see Johnson et al., this issue).
Fig. 2. (a) ELISA detection of ChIFN-g preparations expressed by either E. coli, COS or FAV. Plates were coated with anti-ChIFN-g Mab 1E12 [49] and serial dilutions of ChIFN-g were incubated in the plate at room temperature. Plates were washed and incubated with biotinylated antiChIFN-g Mab 80 (J.W. Lowenthal, 1999, unpublished), followed by the addition of HRP-labelled goat-anti-mouse serum. Plates were read in an ELISA reader at 450 nm. (b) In vivo half-life of ChIFN-g. Chickens were injected intravenously with E. coli-derived ChIFN-g. Serum samples were taken over a 24 h period and the level of ChIFN-g was determined by the HD11 (q) bioassay [47] and by ELISA (Q).
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viral replication, albeit with a lower speci®c activity than other interferons [48]. This function forms the basis of the virus protection assay. A more reliable and less demanding assay is the HD11 nitrite assay [49], which measures the ability of ChIFN-g to activate macrophages through their release of nitric oxide [48]. This assay is very sensitive, allowing the quantitation of subnanogram level of ChIFN-g. In other animal species, ELISAs have been developed and have proven a very reliable and simple way to measure cytokine levels, particularly in blood and tissue samples which are dicult to measure by bioassay. We have recently developed a panel of monoclonal antibodies (Mabs) and polyclonal rabbit sera against ChIFN-g and used these to develop a sandwich ELISA that has a similar level of sensitivity to the HD11 bioassay (Figs. 2a and b). A similar ELISA has been developed by Lambrecht et al. [50], using independently-derived antibodies. Collaborative work is now in progress to compare both ELISAs and to develop a standardized assay for quantitative
detection of ChIFN-g. Amongst a collection of samples of ChIFN-g that were produced by dierent expression systems, there is a very tight correlation between the level of biological activity (biological titre) and detectibility by ELISA (ELISA titre). Furthermore, heating samples of ChIFN-g or exposure to low pH conditions results in a concomitant decrease in both biological and ELISA titres (Fig. 3). Taken together, the data indicate that this ELISA detects only biologically active molecules (i.e. homodimers) of ChIFN-g and not inactive molecules, and therefore can be used to reliably quantitate the biological activity of ChIFN-g with a sensitivity comparable to that of the conventional HD11 bioassay (see Fig. 2b). We have used this ELISA to measure the levels of E. coli-derived ChIFN-g following injection into chickens. ChIFN-g was injected either intravenously (i.v.) or subcutaneously (s.c.) and serum samples were taken over time and ChIFN-g was measured by ELISA. Following i.v. injection, the level of ChIFN-g reached peak serum levels within 3±5 min and thereafter declined rapidly, with a t1/2 of approximately 10±15 min (Fig. 2b), a value similar to that reported for mammalian IFN-g [51,52]. Following s.c. injection ChIFN-g appeared slowly in the serum, reaching peak levels at about 1 h and remained relatively constant for about 8 h and then declined to low levels within 24 h (J.W. Lowenthal, 1999, unpublished). Following s.c. injection, the peak serum level was lower than that obtained by i.v. injection, but detectable levels were sustained for a considerably longer period. It is not known for how long and at what level IFN-g needs to be present in order to manifest biological activity in vivo. 10. Where to now? Ð bring out the crystal ball
Fig. 3. Correlation between biological activity and ELISA titre. ChIFN-g preparations expressed by either E. coli, COS, baculovirus, FAV or ConA stimulated chicken spleen cells were assayed by the HD11 bioassay [47] and by ELISA. W, E coli; R, COS; Q, FAV; w, baculo; q, native.
The ability of ChIFN-g to combat infection and enhance vaccine ecacy in chickens makes it an excellent candidate as a therapeutic agent and adjuvant. Other cytokines, once optimally expressed and characterised, are also likely to have therapeutic value. For example, cytokines
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such as TGF-b and cMGF oer the possibility of enhancing mucosal immunity and IL-1 receptor antagonists may be able to reduce in¯ammation. Furthermore, potentially type 1 interferons are potent antiviral therapeutics and their recently described ability to synergise with ChIFN-g [53] warrants further exploration. The concept of synergy is an extremely attractive one for therapeutic strategies since it is fundamental to the way cytokines normally work in controlling the immune response. As we build on our understanding of how cytokines control the immune system, we will gain further insight on how to optimise immune responses to vaccination. Administration of recombinant cytokine proteins by injection is not feasible in commercial poultry. The rapid degradation and clearance of cytokines in vivo may necessitate multiple injections of protein which is also not commercially viable. Recent developments in live viral vectors and DNA vaccination technologies now provide realistic alternatives. Viral vector technology has allowed a variety of cytokines to be administered and expressed in animals [54±59]. This provides a simple, eective and inexpensive commercial delivery method via feed, water or aerosol. These new generation delivery mechanisms also allow the administration of single or multiple cytokines, in combination with vaccine antigens. The choice of particular viruses will allow antigen and cytokine targeting to speci®c sites such as gut, thereby allowing the most appropriate type of immune responses to be generated. Therapeutics can also be directly administered to the embryo prior to hatching via in ovo injection of protein or targeted vectors. This allows cytokines and vaccines to be inexpensively delivered by an automated egg injector system and allows other potentially eective technologies such as in ovo DNA vaccination to be explored. Since the chicken's immune system and its response to disease and vaccination is similar to that of mammals, it oers an attractive model system with which to study the eectiveness of cytokine therapy in the control of disease in
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intensive livestock. Chickens are relatively easy and inexpensive to raise, have fast generation times and are easily handled in large numbers. Furthermore, there are a large number of research reagents available such as antibodies to cell surface markers, cytokine genes and proteins as well as several well established disease models. Field trials for candidate therapeutics can be inexpensively performed on large numbers of animals, thereby allowing generic type technologies such as live vector or DNA based delivery of vaccine antigens and/or cytokines to be assessed.
Acknowledgements We thank Terri O'Neil, Mary Broadway and Matthew Bruce for their technical expertise. This work is supported by the Australian Rural Industries Research and Development Corporation through its Chicken Meat and Egg Programs.
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[26] Digby MR, Lowenthal JW. Cloning and expression of the chicken interferon-g gene. J Interferon Cytokine Res 1995;15:939±45. [27] Huang A, Scongall CA, Lowenthal JW, Jilbert AR, Kotlarski I. Cloning and expression of duck interferon gamma. 1999; submitted for publication. [28] Schultz U, Chisari FV. Recombinant duck interferon gamma inhibits duck hepatitis B virus replication in primary hepatocytes. J Virol 1999;73:3162±8. [29] Weining KC, Sick C, Kaspers B, Staeheli P. A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and puri®cation of active recombinant protein. Eur J Biochem 1998;258:994±1000. [30] Sundick RS, Gill-Dixon C. A cloned chicken lymphokine homologous to both mammalian IL-2 and IL-15. J Immun 1997;159:720±5. [31] Bedard PA, Alcorta D, Simmons DL, Luk KC, Erikson RL. Constitutive expression of a gene encoding a polypeptide homologous to a biologically active human platelet protein in Rous sarcoma virus-transformed ®broblasts. Proc Natl Acad Sci USA 1987;84:6715±9. [32] Staeheli P. 1998: personal communication. [33] Leutz A, Damm K, Sterneck E, Kowenz E, Ness S, Frank R, Gausepohl H, Pan YCE, Smart J, Hayman M, Graf T. Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationships to interleukin 6 and granulocyte colony stimulating factor. EMBO 1989;8:175±81. [34] Zhou JH, Ohtaki M, Sakuri M. Sequence of a cDNA encoding chicken stem cell factor. Gene 1993;127:269±70. [35] Jakowlew SB, Dillard PJ, Kondaiah P, Sporn MB, Roberts AB. Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-beta messenger ribonucleic acid from chick embryo chondrocytes. Molec Endocr 1988;2:747±55. [36] Dawson WO. Tobamovirus-plant interactions. Virology 1992;186:359±67. [37] Turpen TH, Reinl SJ, Charoenvit Y, Homan SL, Fallaeme V, Grill LK. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology 1995;13:53±7. [38] Kumagi MH, Turpen TH, Weinzettl N, Della-Cioppa G, Turpen AM, Donson J, Hilf ME, Grantham GL, Dawson WO, Chow TP, Piatak JR, Grill LK. Rapid high-level expression of biologically active a-trichosanthin in transfected plants by an RMA viral vector. Proc Natl Acad Sci USA 1993;90:427±30. [39] Lowenthal JW, Hanley K, Andrew ME, O'Neil TE, Pogue GP. Expression and characterization of chicken interferon-gamma in tobacco plants. 1999; submitted for publication. [40] Lowenthal JW, O'Neil TE, Strom ADG, Andrew ME. Cytokine therapy: a natural alternative for disease control. Vet Immun Immunopath 1999;72:183±8. [41] Lowenthal JW, Staeheli PJ, Schultz U, Sekellick MJ, Marcus PI. Nomenclature of avian interferons, J. Interferon Cytokine Res. 2000; (in press).
J.W. Lowenthal et al. / Developmental and Comparative Immunology 24 (2000) 355±365 [42] Lowenthal JW, York JJ, O'Neil TE, Rhodes S, Prowse SJ, Strom ADG, Digby MR. In vivo eects of chicken interferon gamma during infection with Eimeria. J Interferon Cytokine Res 1997;17:551±8. [43] Weining KC, Schultz U, Munster U, Kaspers B, Staehli P. Biological properties of recombinant chicken interferon-g. Eur J Immun 1996;26:2440±7. [44] Lowenthal JW, O'Neil TE, Broadway M, Strom ADG, Digby MR, Andrew M, York JJ. Co-administration of interferon gamma enhances antibody responses in chickens. J Interferon Cytokine Res 1998;18:617±22. [45] Lambrecht B, Gonze M, Morales D, Meulemans G, van den Berg TP. Comparison of biological activities of natural and recombinant chicken interferon-gamma. Vet Immun Immunopath 1999;70:257±67. [46] Kogut MH, Lange C. Interferon-gamma-mediated inhibition of the development of Eimeria tenella in cultured cells. J Parasitol 1989;75:313±7. [47] Lillehoj HS, Choi KD. Recombinant chicken interferongamma-mediated inhibition of Eimeria tenella development in vitro and reduction of oocyst production and body weight loss following Eimeria acervulina challenge infection. Avian Dis 1998;42:307±14. [48] Lowenthal JW, Digby MR, York JJ. Production of interferon-g by chicken cells. J Interferon Cytokine Res 1995;15:933±8. [49] Sung-Jy, Hotchkiss JH, Austic RE, Dietert RR. L-arginine dependent production of reactive nitrogen intermediate by macrophages of a uricotelic species. J Leukocyte Biol 1991;50:49±56. [50] Lambrecht B, Gonze M, Meulemans G, van den Berg TP. Development of an ELISA for the quantitative detection of chicken interferon-gamma. Vet Immunol Immunopath 1999; (in press). [51] Foon KA, Sherwin SA, Abrams PG, Stevenson HC, Holmes P, Maluish AE, Oldham RK, Herberman RB. A phase I trial of recombinant gamma interferon in
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Avian cytokines Ð the natural approach to therapeutics John W. Lowenthal a,*, Benedicte Lambrecht b, Thierry P. van den Berg b, Marion E. Andrew a, A. David G. Strom a, Andrew G.D. Bean a b
a CSIRO Animal Health, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia Section of Avian Virology & Biotechnology, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, B1180, Brussels, Belgium
Accepted 1 November 1999
Abstract While the eective use of antibiotics for the control of human disease has saved countless lives and has increased life expectancy over the past few decades, there are concerns arising from their usage in livestock. The use of antibiotic feed additives in food production animals has been linked to the emergence in the food chain of multiple drug-resistant bacteria that appear impervious to even the most powerful antimicrobial agents. Furthermore, the use of chemical antimicrobials has led to concerns involving environmental contamination and unwanted residues in food products. The imminent banning of antibiotic usage in livestock feed has intensi®ed the search for environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators and regulators of the immune response, oer exciting new alternatives to conventional chemical-based therapeutics. The utilisation of cytokines is becoming more feasible, particularly in poultry, with the recent cloning of a number of avian cytokine genes. Chickens oer an attractive small animal model system with which to study the eectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review the status of avian cytokines and focus on our recent studies involving the therapeutic potential of chicken interferon gamma (ChIFN-g ) as a vaccine adjuvant and a growth promoter. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Interferon-g; Cytokines; Therapeutics; Chicken
Abbreviations: ACG, Avian Cytokine Group; ChiFN-g, chicken interferon g; cMGF, chicken myelomonocytic growth factor; ELISA, enzyme linked immunosorbent assay; FAV, fowl adenovirus; IFN, interferon; IFN-g, interferon g; IL-1, interleukin 1; LPS, lipopolysaccharide; NDV, Newcastle disease virus; TGF-b, transforming growth factor b; TMV, tobacco mosaic virus; WHO, World Heath Organization. * Corresponding author. Tel.: +61-3-5227-5759; fax: +61-3-5227-5331. E-mail address: [email protected] (John W. Lowenthal). 0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 9 9 ) 0 0 0 8 3 - X
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1. Introduction: the need for alternative natural therapeutics The sheer size of the poultry broiler industry is staggering: almost 40 billion chickens are hatched worldwide every year. Chicken meat represents approximately 40% of all meat consumed and is a US$150 billion per annum retail market, as well as supporting a multi-billion dollar poultry health market. Chickens are reared under intensive conditions which are conducive to infection by opportunistic pathogens. This is especially critical during the ®rst weeks of life, a time at which the immune system has not yet fully matured [1] and when levels of maternal antibody are declining. A major problem faced by intensive livestock industries, such as the poultry industry, is loss of productivity due to disease, therefore considerable resources are required in order to maintain the health status of these animals. The mechanism by which infectious disease is controlled involves the combined use of vaccines, antibiotics and chemicals. Vaccines are designed to provide long term immunity and provide speci®c protection against a particular pathogen following immunisation. Usually the most eective form of vaccination is immunisation with a live (attenuated) organism, however, the risks of virulent mutant strains arising from vaccine strains is a constant concern. Vaccination with killed organisms or recombinant antigens, which are generally less immunogenic, requires the use of powerful adjuvants in order to develop a longterm protective immune response. Unfortunately, most commercial adjuvants are oil-based and their use can result in adverse local reactions resulting in downgrading of meat quality. The livestock industries now face the planned phaseout of oil-based vaccines which is a driving force for the search for eective alternative, natural vaccine adjuvants. In contrast to vaccines, antibiotics provide short-term, broad-spectrum protection and are provided continuously to livestock as feed additives. Antibiotics have become very popular in the livestock industries because, in addition to being an antimicrobial, they also have growth
promoting activity [2]. In fact, up to half of the world's production of antibiotics is used in agriculture. As recently reported from a World Health Organisation (WHO) meeting in Berlin on the medical impact of the use of antimicrobials in food animals (see http://www.who.int/ emc-documents/antimicrobial_resistance/whoemczoo974c.html), the extensive use of antibiotics, particularly those that are used in human medicine, has resulted in human health concerns. The WHO has recently urged meat producers to stop using the same antibiotics that are used in humans or those which select for cross-resistance, however, these are usually the most eective in controlling disease. Furthermore, the WHO has strongly recommended the development and use of alternative, environmentally-friendly methods to control disease. Similarly, the long term use of chemicals, such as coccidiostats, results in emergence of resistant strains of pathogens such as Eimeria, increases environmental contamination and results in the deposition of residues in meat products. Some European countries have already banned the use of antibiotic and chemical feed additives in food production animals and more plan to follow suit. Livestock industries now acknowledge that withdrawal of antimicrobials from feed is inevitable and we must now develop strategies to cope with this. 2. Cytokines and their in¯uence on immunity and growth Cytokines are a diverse family of proteins that play a crucial role in controlling the immune system. They determine both the type and extent of an immune response that is generated following infection with a pathogen or after vaccination. Depending on the combination of cytokines produced, a protective immune response can be generated as either an antibody-mediated (Th2) response or a cell-mediated (Th1) response [3]. Cytokines therefore represent excellent candidates as naturally occurring therapeutics as well as vaccine adjuvants [4,5]. Of particular relevance to poultry is the susceptibility of newly hatched chickens to infection by opportunistic pathogens,
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due primarily to declining maternal antibody levels at a time when their immune system is still relatively immature [1]. The production of IgA antibodies at the mucosal surface is a ®rst line of defence against respiratory and gastrointestinal pathogens [6]. It is therefore feasible to elevate mucosal IgA levels in newly hatched chicks by appropriately administering IgA-inducing cytokines, thereby enhancing their resistance to disease (see Muir et al., this issue). The ecacy of cytokine therapy has been demonstrated in several human and animal studies. Interferons (IFN) are a family of cytokines that share potent antiviral as well as immunostimulatory properties. IFN-g has been shown to be an eective adjuvant in immunocompromised mice when delivered with a malarial vaccine [7] and also enhanced antibody responses and protection from disease when administered with a subunit vesicular stomatitis virus vaccine [8] or in¯uenza vaccine [9]. These eects are likely to be mediated through the ability of IFN-g to induce T cell help for antibody production. Other studies have demonstrated that T cell responses such as delayed type hypersensitivity and T cell help for antibody production were enhanced following the co-administration of IFN-g with a vaccine [10]. Yet other studies have shown that IFN-g is capable of activating cells such as macrophages to directly kill intracellular Eimerian oocysts [11]. In a human clinical trial, aerosol delivery of IFN-g to tuberculosis patients was shown to be an eective therapeutic in the treatment of multi-drug resistant bacteria [12]. Another member of the interferon family, IFN-a, is an eective antiviral agent when administered as a recombinant protein [13±15]. In a recent study, Marcus et al. [15] showed that oral administration of ChIFN-a protein to one day old chicks via drinking water oered protection from challenge with La Sota strain Newcastle disease virus (NDV). They found that ChIFN-a treatment delayed the onset and severity of clinical symptoms and prevented the histopathological changes (in¯ammation, hyperplasia, deciliation and lymphocyte in®ltration) normally seen in the trachea following NDV infection. Cytokines are also thought to play a direct
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role in the growth of animals. It is known that both illness and stress reduce an animal's ability to grow at an optimal rate. It is now becoming evident that growth is in¯uenced by interactions between the immune and endocrine systems and the brain [16]. Whereas the local action of cytokines involves activation of the immune system, their systemic eects include, amongst other things, alterations in body metabolism and neuroendocrine functions. For example, cytokines such as IL-1 and TNF can accelerate muscle degradation, and reduce food and water intake [17,18]. Treatment of animals with combinations of certain cytokines, or alternatively with antagonists of cytokines, may in¯uence their growth rate, resulting in an increase in productivity which would obviously be a commercial bene®t. The utilisation of cytokines in poultry is becoming more feasible with the recent cloning of a number of avian cytokine genes (see Table 1). The major limiting factor for cytokine therapy has been the mode of delivery since cytokines typically persist for a short time in vivo due to rapid utilisation and/or degradation. Delivery of cytokines to chickens is now commercially feasible using live viral vectors (see Johnson et al., this issue), which allows long term in vivo production. 3. Cloning of avian cytokine genes A variety of cytokine genes have now been cloned in several avian species (Table 1). The majority of avian cytokine genes so far cloned share limited sequence identity with their mammalian counterparts [19,20], and the expressed proteins rarely cross react on mammalian cells. In the case of chicken IFN-g (ChIFN-g ), 3D protein modeling has revealed that the homodimer structure is surprisingly similar to human and bovine IFN-g [21], whereas the level of amino acid identity is very low (about 35%). As a consequence of this, the full repertoire of genes must be identi®ed, cloned and expressed in order to fully test the therapeutic potential of avian cytokines. A number of laboratories involved in avian
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cytokine research have recently formed the Avian Cytokine Group (ACG) in order to facilitate the exchange of basic information and reagents for research purposes. A web site has now been developed; for further information on the ACG see: www.ah.csiro.au/AvianCytokines/. It was also agreed that certain laboratories would be reference laboratories for particular cytokines. The aim is for international standard preparations to be adopted and distributed to interested laboratories to be used as the positive control and standard to which all other preparations of that cytokine will be compared. This should allow standardisation of results obtained from dierent laboratories. 4. Production of recombinant cytokines Several procaryotic and eucaryotic expression systems are available for the production of recombinant cytokines; each have their particular advantages as well as disadvantages. If recombinant cytokines are to be used as commercial therapeutics, particularly in the poultry industry, there are several critical constraints that have to be considered in order for a product to be considered cost-eective. The protein must be able to be produced on a commercial scale (multi-billion doses per annum), easily puri®ed, inexpensive to produce, stable upon storage, and retain bio-activity in vivo. Various avian cytokines have been successfully expressed in E. coli. This system
allows large amounts of biologically active recombinant protein to be produced, which can be puri®ed by relatively simple procedures. Disadvantages of this system are that these type of proteins are non-glycosylated and may not be folded in authentic native forms, thereby having less than optimal speci®c activities or reduced half-lives in vivo. Cytokines have also been expressed in a variety of eucaryotic systems, including COS, CHO, and yeast, as well as virusbased systems such as baculovirus, tobacco mosaic virus (TMV), fowlpox virus and fowl adenovirus (see Johnson et al., this issue). Recombinant proteins expressed from these systems more closely resemble naturally produced proteins in terms of glycosylation patterns and folding. Perhaps the most crucial consideration is one of biological activity. One important immunomodulatory eect of IFN-g is its ability to induce the expression of MHC Class II molecules on the surface of antigen presenting cells. When dierent preparations of ChIFN-g (expressed by either E. coli, COS, FAV or baculovirus) were compared for their ability to induce Class II expression on HD11 chicken macrophages, similar levels in up-regulation of expression were observed. HD11 cells express relatively low levels of class II molecules on their cell surface when stained with the CIa antibody (Southern Biotechnology) and this level of expression was not in¯uenced by the addition of low levels of LPS (20 ng/ml). Exposure of HD11 cells to ChIFN-g (10 U/ml for 24 h) resulted in an increase in the pro-
Table 1 Cloned avian cytokine genes Species cloneda
Cytokine Interferon-a Interferon-b Interferon-g Interleukin-1b Interleukin-2 Chemokines (CEF4, K60) Chicken myelomonocytic growth factor Stem cell factor Transforming growth factor-b a
IFN-a IFN-b IFN-g IL-1b IL-2 cMGF SCF TGFb
Ch Ch Ch Ch Ch Ch Ch Ch Ch
[22,23], Tu [24], Du [25] [23] [26], Tu [20], Jq [20], Ph [20], Gf [20], Du [27,28] [29] [30] [31,32] [33] [34] [35]
Ch, chicken; Tu, turkey; Jq, Japanese quail; Ph, pheasant; Gf, guinea fowl; Du, duck.
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Fig. 1. (a) Induction of MHC Class II expression by ChIFNg. HD11 cells were incubated for 24 h in the presence of 10 U/ml of ChIFN-g expressed from either COS, E. coli, FAV or baculovirus, with or without the presence of 20 ng/ml of LPS. Cells were harvested, stained with anti-Class II Mab (CIa), and analysed by ¯ow cytometry. Numbers in each panel refer to the percentage of cells expressing Class II. (b) Eect of ChIFN-g dose on the expression of MHC Class II by HD11 cells. Cells were incubated in the presence of indicated doses of ChIFN-g for 24 h and measured for Class II expression. Approximately 15% of cells were positive in the absence of ChIFN-g.
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portion of cells expressing Class II molecules and also the intensity of staining, irrespective of whether ChIFN-g was glycosylated (expressed by COS, FAV or baculovirus) or expressed by E. coli (Fig. 1a). Fig. 1b shows that the eect of ChIFN-g on Class II expression titrates with a dose response curve that is similar to that seen in the nitrite release assay. Plant viruses have been successfully used to produce recombinant foreign proteins in plants. One of the best studied systems is tobacco mosaic virus (TMV), a single stranded RNA virus that is capable of inducing high level transient expression of non-viral foreign proteins within a few days of introduction into the plant [36]. The TMV expression vectors have been shown to be easily and quickly generated and the level and timing of gene expression can be controlled. Commercial manufacture of recombinant proteins in plants is becoming an attractive alternative to expression in yeast and E. coli. Large scale production in tobacco plants via TMV vectors has been shown to be both commercially feasible and inexpensive. Recent examples include the production of malarial vaccine antigens [37], a-trichosanthin [38] as well as antibiotics and milk proteins. The TMV expression system oers the advantages of minimal environmental impact due to the generation of relatively little waste, ability to produce large amounts of protein, relatively simple processing requirements and the ability to produce soluble or membrane bound proteins. Commercial products derived from TMV expression are already on the market. A TMV vector producing ChIFN-g has been developed [39]. The plant-expressed recombinant ChIFN-g protein is identical to ChIFN-g derived from procaryotic (E. coli ) and eucaryotic (COS cells) expression systems. It is also similar to naturally occurring ChIFN-g as measured by biological activity, sensitivity to heat treatment and reactivity to anti-ChIFN-g antibodies [39]. 5. The search for alternative, natural adjuvants Current vaccine research involves the development of recombinant subunit vaccines which are
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intended to replace the traditional forms of vaccination which use live or killed whole organisms. Unfortunately many of these new generation vaccines are, by themselves, poorly immunogenic and require several rounds of immunisation. They usually require the use of powerful adjuvants in order to elicit an eective immune response. However, the move away from the use of oil-based adjuvants, due to adverse site reactions, has led to a focus on the application of cytokines, which might make these new generation vaccines safer and more eective [40]. Some of the practical considerations of vaccine development, especially for poultry, are cost, eectiveness, ease of production, and delivery. When used as a vaccine adjuvant, cytokine proteins can simply be mixed together with the vaccine antigen prior to immunisation. For this type of function relatively small amounts of cytokine are required as they would act locally, at or near the site of injection. If used as an immunomodulator, cytokines would act mucosally or systemically and hence require considerably larger amounts of protein to be administered. However, for therapeutic or growth promoting activities, frequent injections over a period of time would be required, which is not feasible or cost-eective in a commercial setting. For these uses viral vector delivery may come to the forefront. Nevertheless, for smaller experimental trials assessing feasibility, injection of cytokines is practical. 6. ChIFN-gg as a vaccine adjuvant Birds, like mammals, express a family of interferon proteins whose genes have been cloned [41]. The gene for ChIFN-g has been cloned and recombinant protein of high speci®c activity produced [26,42±44]. The potential of ChIFN-g as a vaccine adjuvant has been assessed in experimental trials [45]. Co-administration of recombinant ChIFN-g with antigen resulted in enhanced secondary antibody responses that persisted at higher levels and for longer periods compared to antigen injected in the absence of ChIFN-g. In addition, ChIFN-g treatment allowed a lower dose of antigen to be used more eectively while
signi®cantly increasing the proportion of birds within a group that sero-converted [45]. The ability of an adjuvant to enhance the ecacy of lower doses of antigen or to reduce the number of immunisations required to induce long term protection, may overcome the often prohibitive cost of livestock vaccines. Furthermore, the ability of cytokines to prolong immune responsiveness in individuals as well as increase the proportion of animals that respond to vaccination can be employed in strategies to reduce multi-dose regimes required for many existing vaccines, thereby reducing the overall cost of vaccination. It is feasible that cytokines may eventually replace the need for conventional oil-based adjuvants that often induce adverse site reactions, which lead to downgrading of meat quality. 7. ChIFN-gg as a growth promoter Conditions of stress and illness have a negative impact on the ability of animals to grow at their optimal rate. It is also becoming evident that growth is in¯uenced by interactions between the immune system and other systems within the body such as the neurological and endocrine systems and that cytokines may play a central role regulating this [16]. It has been reasoned that reduction of stress and illness would allow a redirection of the body's resources toward growth. There is now an emerging area of research examining the eects of utilising a combination of anti-in¯ammatory and immunostimulatory agents to achieve this, with cytokines being key players. We have performed experiments to assess the growth promoting activity of ChIFN-g in chickens [42]. Seven-day old broiler chickens were injected with ChIFN-g protein on two consecutive days while control birds were injected with diluent alone and body weight was recorded daily. Birds treated with ChIFN-g displayed enhanced weight gain over a 12 day period, relative to those injected with diluent alone or control birds that were not treated. The increase in body weight ranged from 3±8%. Similar increases in growth rates were observed over a 3± 8 week period in subsequent experiments which
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were performed on larger group sizes with ChIFN-g delivered via a fowl adenovirus (FAV) vector (see Johnson et al., this issue). These results clearly indicate the potential use of ChIFN-g as an eective, naturally occurring growth promoter. The underlying mechanisms are unknown, but may be due to the IFN-g mediated enhancement of the immune system resulting in healthier and more productive birds.
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9. Development of an ELISA for ChIFN-gg Up to now the only way to detect and quantitate the biological activity of ChIFN-g was to use a bioassay. ChIFN-g shares with other members of the interferon family the capacity to inhibit
8. Eect of ChIFN-gg on weight gain during infection with Eimeria acervulina ChIFN-g has been shown to prevent the development of Eimeria parasites in vitro, presumably through its ability to activate macrophages [12,46]. The therapeutic potential of ChIFN-g has also been demonstrated in vivo using a coccidial challenge model [42]. Infection of young broilers with Eimeria acervulina normally results in weight loss beginning on day 4 after infection with weight gain resuming 2±3 days later. In these studies, broiler chickens were inoculated on two consecutive days with either E. coli-derived ChIFN-g or diluent and then infected with E. acervulina oocytes one day later. Body weight was recorded daily for 12 days following challenge. ChIFN-g treated birds lost less weight early in infection (day 4±5 post challenge) and recovered more quickly as indicated by a larger weight gain between days 6 and 8 post challenge when compared to controls. This result indicates that ChIFN-g reduced the eect of coccidiosis on growth performance. In two separate trials, treatment of infected birds with ChIFN-g resulted in a consistent increase in weight gain ranging from 2.7 to 12.5% relative to non-treated infected birds. In a later independent study, ChIFN-g therapy was shown to also reduce oocyst production following Eimeria challenge [47]. These results have now been reproduced in larger trials performed under commercial farm conditions, where ChIFN-g was delivered by live FAV vectors (see Johnson et al., this issue).
Fig. 2. (a) ELISA detection of ChIFN-g preparations expressed by either E. coli, COS or FAV. Plates were coated with anti-ChIFN-g Mab 1E12 [49] and serial dilutions of ChIFN-g were incubated in the plate at room temperature. Plates were washed and incubated with biotinylated antiChIFN-g Mab 80 (J.W. Lowenthal, 1999, unpublished), followed by the addition of HRP-labelled goat-anti-mouse serum. Plates were read in an ELISA reader at 450 nm. (b) In vivo half-life of ChIFN-g. Chickens were injected intravenously with E. coli-derived ChIFN-g. Serum samples were taken over a 24 h period and the level of ChIFN-g was determined by the HD11 (q) bioassay [47] and by ELISA (Q).
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viral replication, albeit with a lower speci®c activity than other interferons [48]. This function forms the basis of the virus protection assay. A more reliable and less demanding assay is the HD11 nitrite assay [49], which measures the ability of ChIFN-g to activate macrophages through their release of nitric oxide [48]. This assay is very sensitive, allowing the quantitation of subnanogram level of ChIFN-g. In other animal species, ELISAs have been developed and have proven a very reliable and simple way to measure cytokine levels, particularly in blood and tissue samples which are dicult to measure by bioassay. We have recently developed a panel of monoclonal antibodies (Mabs) and polyclonal rabbit sera against ChIFN-g and used these to develop a sandwich ELISA that has a similar level of sensitivity to the HD11 bioassay (Figs. 2a and b). A similar ELISA has been developed by Lambrecht et al. [50], using independently-derived antibodies. Collaborative work is now in progress to compare both ELISAs and to develop a standardized assay for quantitative
detection of ChIFN-g. Amongst a collection of samples of ChIFN-g that were produced by dierent expression systems, there is a very tight correlation between the level of biological activity (biological titre) and detectibility by ELISA (ELISA titre). Furthermore, heating samples of ChIFN-g or exposure to low pH conditions results in a concomitant decrease in both biological and ELISA titres (Fig. 3). Taken together, the data indicate that this ELISA detects only biologically active molecules (i.e. homodimers) of ChIFN-g and not inactive molecules, and therefore can be used to reliably quantitate the biological activity of ChIFN-g with a sensitivity comparable to that of the conventional HD11 bioassay (see Fig. 2b). We have used this ELISA to measure the levels of E. coli-derived ChIFN-g following injection into chickens. ChIFN-g was injected either intravenously (i.v.) or subcutaneously (s.c.) and serum samples were taken over time and ChIFN-g was measured by ELISA. Following i.v. injection, the level of ChIFN-g reached peak serum levels within 3±5 min and thereafter declined rapidly, with a t1/2 of approximately 10±15 min (Fig. 2b), a value similar to that reported for mammalian IFN-g [51,52]. Following s.c. injection ChIFN-g appeared slowly in the serum, reaching peak levels at about 1 h and remained relatively constant for about 8 h and then declined to low levels within 24 h (J.W. Lowenthal, 1999, unpublished). Following s.c. injection, the peak serum level was lower than that obtained by i.v. injection, but detectable levels were sustained for a considerably longer period. It is not known for how long and at what level IFN-g needs to be present in order to manifest biological activity in vivo. 10. Where to now? Ð bring out the crystal ball
Fig. 3. Correlation between biological activity and ELISA titre. ChIFN-g preparations expressed by either E. coli, COS, baculovirus, FAV or ConA stimulated chicken spleen cells were assayed by the HD11 bioassay [47] and by ELISA. W, E coli; R, COS; Q, FAV; w, baculo; q, native.
The ability of ChIFN-g to combat infection and enhance vaccine ecacy in chickens makes it an excellent candidate as a therapeutic agent and adjuvant. Other cytokines, once optimally expressed and characterised, are also likely to have therapeutic value. For example, cytokines
J.W. Lowenthal et al. / Developmental and Comparative Immunology 24 (2000) 355±365
such as TGF-b and cMGF oer the possibility of enhancing mucosal immunity and IL-1 receptor antagonists may be able to reduce in¯ammation. Furthermore, potentially type 1 interferons are potent antiviral therapeutics and their recently described ability to synergise with ChIFN-g [53] warrants further exploration. The concept of synergy is an extremely attractive one for therapeutic strategies since it is fundamental to the way cytokines normally work in controlling the immune response. As we build on our understanding of how cytokines control the immune system, we will gain further insight on how to optimise immune responses to vaccination. Administration of recombinant cytokine proteins by injection is not feasible in commercial poultry. The rapid degradation and clearance of cytokines in vivo may necessitate multiple injections of protein which is also not commercially viable. Recent developments in live viral vectors and DNA vaccination technologies now provide realistic alternatives. Viral vector technology has allowed a variety of cytokines to be administered and expressed in animals [54±59]. This provides a simple, eective and inexpensive commercial delivery method via feed, water or aerosol. These new generation delivery mechanisms also allow the administration of single or multiple cytokines, in combination with vaccine antigens. The choice of particular viruses will allow antigen and cytokine targeting to speci®c sites such as gut, thereby allowing the most appropriate type of immune responses to be generated. Therapeutics can also be directly administered to the embryo prior to hatching via in ovo injection of protein or targeted vectors. This allows cytokines and vaccines to be inexpensively delivered by an automated egg injector system and allows other potentially eective technologies such as in ovo DNA vaccination to be explored. Since the chicken's immune system and its response to disease and vaccination is similar to that of mammals, it oers an attractive model system with which to study the eectiveness of cytokine therapy in the control of disease in
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intensive livestock. Chickens are relatively easy and inexpensive to raise, have fast generation times and are easily handled in large numbers. Furthermore, there are a large number of research reagents available such as antibodies to cell surface markers, cytokine genes and proteins as well as several well established disease models. Field trials for candidate therapeutics can be inexpensively performed on large numbers of animals, thereby allowing generic type technologies such as live vector or DNA based delivery of vaccine antigens and/or cytokines to be assessed.
Acknowledgements We thank Terri O'Neil, Mary Broadway and Matthew Bruce for their technical expertise. This work is supported by the Australian Rural Industries Research and Development Corporation through its Chicken Meat and Egg Programs.
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