The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants

Veterinary Immunology and Immunopathology 85 (2002) 119±128

Mini-review

The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants Louise S. Hilton, Andrew G.D. Bean, John W. Lowenthal* CSIRO Livestock Industries, Australian Animal Health Laboratories, Private Bag 24, Geelong, Vic. 3220, Australia Accepted 7 November 2001

Abstract The use of antibiotic feed additives and chemical antimicrobials in food production animals is a double-edged sword. On one hand, it helps to prevent the outbreak of disease and promotes the growth of animals, but on the other hand, concerns are mounting over the emergence of antibiotic-resistant bacteria. As a consequence, some countries have already banned the use of in-feed antibiotics which has resulted in meat producers urgently seeking environmentally friendly alternative methods to control disease. Cytokines are proteins that control the type and extent of an immune response following infection or vaccination. They therefore represent excellent naturally occurring therapeutics. The use of cytokines in poultry has become more feasible with the discovery of a number of avian cytokine genes. Since the immune system of chickens is similar to that of mammals, they offer an attractive model system to study the effectiveness of cytokine therapy in the control of disease in livestock. This review will focus on the recent advances made in avian cytokines, with a particular focus on their assessment as therapeutic agents and vaccine adjuvants. Crown Copyright # 2002 Published by Elsevier Science B.V. All rights reserved. Keywords: Avian cytokines; Immunotherapeutics; Vaccine adjuvants

1. Introduction Antibiotics have been used to treat and control diseases in livestock and poultry for more than 50 years. In-feed antibiotics are generally used at low levels and result in improvements in growth rate and feed-conversion ef®ciency. Thus in-feed antibiotics are usually referred to as growth promotants. For many years, problems with antibiotic-resistant bacteria in human medicine have been linked to the use of antibiotics in livestock feed (Swann Committee, 1969). The long term use of chemicals to control bacterial and parasitic diseases such as coccidiosis *

Corresponding author. Tel.: ‡61-3-5227-5759; fax: ‡61-3-5227-5555. E-mail address: [email protected] (J.W. Lowenthal).

in poultry has also created problems. For example, chemical resistance has emerged in certain strains of Eimeria, thereby rending them dif®cult to treat. Furthermore, chemical residues in meat and the risk of chemical contamination of the environment through water runoff are also important issues. Public concern over these issues has resulted in reviews of the current practices involving the use of medication in livestock feed. In 1986, Sweden became the ®rst country to ban growth promoters for food production animals and allowed the use of antimicrobials in livestock on veterinary prescription only (reviewed in Williams, 2001). This led the way for further bans on prophylactic antibiotic use with the European Union prohibiting the use of four antibiotic substances as in-feed additives for the promotion of growth in 1998.

0165-2427/02/$ ± see front matter. Crown Copyright # 2002 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 4 1 4 - 7

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The World Health Organisation (WHO) extensively reviewed the use of in-feed antibiotics and released a series of recommendations urging stringent regulatory controls to be put on certain antibiotics and to implement infection prevention strategies and hygienic measures for livestock (World Health Organisation, 1998). The WHO strongly advised that alternative environmentally friendly methods should be used to control disease. Other countries soon followed suit by issuing guidelines controlling the prudent use of antibiotics in food production animals. With the imminent and widespread ban of in-feed antibiotics and chemicals, alternative measures need to be sought to ensure that the livestock industry will not be adversely affected. It is expected that without substitutes for prophylactic antibiotics, microorganisms currently inhibited by in-feed medication will be able to survive and cause disease. Based on recent experiences in poultry, it is anticipated that gastrointestinal problems will increase through the increased prevalence of Clostridium perfringens, which are currently controlled by antibiotics, and the result may be a decrease in production along with downgrading of meat quality. Discovering replacements for current growth promoters in livestock is therefore vital.

damage caused by species of gut parasites such as Eimeria (Bedford, 2000). This in turn inhibits secondary infections by opportunistic C. perfringens and reduces the incidence of necrotic enteritis. Further to this approach is the use of bacteriocins, small proteins produced by certain bacteria for the purpose of eliminating other competing bacteria (Jack et al., 1995). Particular types of bacteriocins have been identi®ed that speci®cally kill species of bacteria linked to necrotic enteritis in chickens. Treatment with recombinant bacteriocins may provide alternatives to in-feed antibiotics by limiting the growth of C. perfringens and other disease-causing bacteria in chickens. Chicken cytokines are also being examined as replacements for in-feed antibiotics in poultry. Cytokines are proteins that are produced by the immune system immediately following infection or vaccination. Cytokines regulate immune responses by mediating a multitude of effects ranging from activation and differentiation of immune cells to enhancing the immune function and production of other cytokines. This review will focus on the emerging role of avian cytokines as potential commercial therapeutics in poultry.

2. Alternatives to antibiotic growth promoters

The identi®cation, cloning and characterisation of cytokine genes in chickens has lagged somewhat behind similar work in mammals. Progress in isolating chicken homologues of mammalian cytokines has been slowed by the generally low level of sequence similarity, typically in the range 30±50% (Table 1). As a consequence, their identi®cation by homologous probes and simple PCR ampli®cation of counterpart cytokine genes using primers based on mammalian sequences has generally been very dif®cult and unproductive. Genes with a relatively high level of nucleotide identity to mammalian genes were able to be cloned this way, with a rare example being stem cell factor (SCF) (Zhou et al., 1993). Interferon alpha (ChIFN-a) was cloned by PCR using primers based on small regions that are highly conserved amongst mammalian homologues, despite the overall level of similarity being low (Sekellick et al., 1994). Other methods such as identifying clones from cDNA libraries based on functional expression have also

Alternatives to in-feed antibiotics and chemicals in livestock will need to be safe, easy to administer and cost-effective. They need to guarantee that food production animals such as poultry are able to reach their growth potential without a concomitant increase in the food conversion ratios and higher costs to the farmer. A number of alternatives have been suggested, however, most of these will not on their own compensate fully for the removal of antibiotics. One such alternative is the use of in-feed enzymes such as Avizyme which acts by increasing the rate of diet digestibility and sugar provision, ultimately changing the substrate quality and quantity available to intestinal ¯ora (Bedford, 2000). Digested nutrients can then be absorbed by the chicken for energy and growth, instead of feeding the intestinal organisms. Another suggested alternative is betaine which acts as an osmolyte (Allen et al., 1998) to minimise intestinal

3. Cloning of avian cytokine genes

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Table 1 Avian cytokine genes: a comparison to mammalian homologues Cytokine gene

Species cloneda

% Identityb

Amino acidsc

IFN-a

C (Sekellick et al., 1994), T (Suresh et al., 1995), D (Schultz et al., 1995) C (Sick et al., 1996) C (Digby and Lowenthal, 1995), T (Kaiser et al., 1988), J (Kaiser et al., 1988), P (Kaiser et al., 1988), G (Kaiser et al., 1988), D (Schultz and Chisari, 1999) C (Weining et al., 1998) C (Sundick and Gill-Dixon, 1997), T (Lawson et al., 2000) C (Schneider et al., 2001; P. Kaiser, unpublished results) C (Kaiser et al., 1999) C (J. Burnside, L. Sofer, GenBank accession number AF152927) C (P. Kaiser, Personal communication) C (Schneider et al., 2000), D (J. Burnside, L. Sofer, GenBank accession number AF336122) C (Zhou et al., 1993), J (Petitte and Kulik, 1996) C (Leutz et al., 1989) C (Jakowlew et al., 1988) C (Rossi et al., 1999) C (Petrenko et al., 1995) C (Sick et al., 2000)

18±22

193

18±20 22±35

203 145

25±29 16±24 35±39 28±48 34±36 ± 30

267 143 241 103 187 ± 198

45±52 20±30 72±79 25±28 75±80 38±48

287 201 412 97 90 104, 89

IFN-b IFN-g

Interleukin-1b Interleukin-2 Interleukin-6 Interleukin-8 Interleukin-15 Interleukin-16 Interleukin-18 SCF MGF TGFb Lymphotactin MIP-1b CXC and CC chemokines a

C: chicken; T: turkey; J: Japanese quail; P: pheasant; G: guinea fowl; D: duck. Percent amino acid identity to mammalian cytokine homologues. c No. of amino acids predicted to be coding for the full-length protein. b

been successfully employed. This approach identi®ed three of the better characterised chicken cytokine genes, ChIFN-g (Digby and Lowenthal, 1995), interleukin-1b (ChIL-1b) (Weining et al., 1998) and ChIL-2 (Sundick and Gill-Dixon, 1997). A recent advance in the cloning of avian cytokine genes is the establishment of expressed sequence tag (EST) libraries. ESTs are short single-pass DNA sequences obtained from either end of cDNA clones. Although these ESTs are relatively short, typically less than 500 bases, this amount of sequence is usually suf®cient to obtain most of the open reading frame for cytokine genes. A number of groups have sequenced chicken cDNA derived from ESTs generated from different cell types. Avian immunologists now have access to databases containing these EST sequences (for more information, refer to: http:// www.chickest.udel.edu, http://genetics.hpi.uni-hamburg.de/estonline.html, http://www.ri.bbsrc.ac.uk/ chickmap/Submissions.html). In evolutionary distinct organisms, cytokine homologues are often dif®cult to identify by simple sequence comparison. The use of genomics has been

successful in identifying avian cytokine genes when sequence comparisons were not conclusive. By examining the ratio of exons to introns and the number of nucleotides in each region, as well as conserved regions upstream of the promoter, it is possible to identify avian genes based on their mammalian homologues. This approach has been used to distinguish ChIL-2 from a ChIL-15 homologue (Kaiser and Mariani, 1999), and helped in con®rming the existence of a ChIL-8 gene (Kaiser et al., 1999). Kaiser et al. (1988) also used genetic techniques to compare IFN-g exon structures from different avian species and deduced the evolutionary relationships between these genes. Examining the gene structure and mapping of cytokine genes to speci®c chromosomes will play a critical role in the characterisation of new avian cytokine genes. 4. Avian cytokines: a growing list Cytokines can be generally characterised according to the type of response they generate. Th1 cytokines

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include IL-2, IFN-g, tumor necrosis factor (TNF) and lymphotoxin (LT) (Mosmann and Sad, 1996) and are mainly involved in the generation of cell-mediated immunity. IFN-g and TNF are macrophage activating cytokines, whilst LT is directly cytotoxic for some cells. Th2 cytokines are generally involved in the activation of B cells and therefore regulate antibody production. IL-4, IL-5, IL-6, IL-10 and IL-13 are Th2 cytokines and function mainly to regulate humoral immune responses (Mosmann and Sad, 1996). Virtually all of the cytokines cloned in the chicken to date are categorised as being Th1-like. The exception is the recent identi®cation of IL-6 (Schneider et al., 2001; P. Kaiser, unpublished results). It remains to be determined whether other classical Th2 cytokines can also be found in the chicken. Table 1 lists chicken cytokine genes that have been cloned to date. Recent advances have meant that some of these cytokines have also been cloned in other avian species, including turkey, Japanese quail, duck, pheasant and guinea fowl. With high amino acid identity between chicken and other avian species (70±98%), the cloning of many of these cytokine genes is relatively straightforward by using PCR with primers derived from the corresponding chicken sequence. Once cloned, these gene products can be characterised and assessed for their immuno-enhancing activities. A number of laboratories involved in avian cytokine research have recently formed the avian cytokine group (ACG) in order to facilitate the exchange of information and reagents for research purposes. Several laboratories have been appointed as reference laboratories for particular cytokines and are willing to provide international standards to be used as experimental controls. A web site has been developed (for further information on the ACG, see: http:// www.geel.li.csiro.au/aviancytokines). 4.1. ChIFN-g One of the more extensively characterised chicken cytokines is ChIFN-g. Identi®cation of this gene (Digby and Lowenthal, 1995) was made possible due to the properties shared by both ChIFN-g and mammalian IFN-g, including inactivation by exposure to pH 2 and a capacity to induce nitrite production by macrophages. IFN-g is a member of the interferon family of cytokines that share the capacity to modulate

the immune response and inhibit viral replication. With these properties in mind, the ability of ChIFNg to act as an immunoenhancer and vaccine adjuvant has been assessed. There are concerns over the use of live vaccines in the poultry industry in terms of emergence of hypervirulent strains. Currently there is a need for alternative vaccines, however, killed and recombinant subunit vaccines do not offer an adequate level of protection and often require the use of adjuvants. Oilbased adjuvants, however, induce adverse site reactions resulting in decreased meat quality and animal discomfort. At this time there are no suitable, costeffective adjuvants for use in poultry, particularly broilers. ChIFN-g is one cytokine that has been assessed for its ability to enhance antibody responses. When co-administered to birds with antigen, recombinant ChIFN-g produced a prolonged secondary antibody response that persisted at higher levels and for longer periods compared to antigen injected alone (Lowenthal et al., 1998). Similarly, the immunoenhancing and therapeutic effects of ChIFN-g have been widely demonstrated using coccidiosis challenge models. Treatment with ChIFN-g resulted in protection from infection with Eimeria and reduced weight loss associated with this disease. Detailed results are described in Lowenthal et al. (1997), Lillehoj and Choi (1998), Lowenthal et al. (2000). ChIFN-g was also found to act as a natural growth promoter, with treatment resulting in an increase in body weight of 3±8%. The results for the growth promoting potential of ChIFN-g have been described fully elsewhere (Lowenthal et al., 1997, 2000). 4.2. ChIL-1b IL-1b in mammals has a diverse range of effects including induction of fever, elevation of corticosterone levels and general activation of the cytokine network (Durum et al., 1990). Mammalian IL-1b stimulates T cell proliferation via IL-2 induction and induces B cell maturation and antibody production (Durum et al., 1990). A cDNA expression library from a stimulated chicken macrophage cell line was used to identify and clone the gene for ChIL-1b (Weining et al., 1998). Recombinant ChIL-1b showed biological activities similar to that of its mammalian

L.S. Hilton et al. / Veterinary Immunology and Immunopathology 85 (2002) 119±128

homologue in that it induces ®broblasts to secrete chemokines and upregulates corticosterone production (Weining et al., 1998). ChIL-1b has been examined for its immunoadjuvant activities using tetanus toxoid as an antigen. When administered as recombinant protein, ChIL-1b was found to increase antibody responses compared to administering the antigen alone (Schijns et al., 2000). Co-administration of ChIL-1b, ChIFN-a and ChIFN-g showed an additive effect on the antibody response to the tetanus toxoid. This suggests that combinations of cytokines may be more effective adjuvants. 4.3. ChIFN-a ChIFN-a was originally cloned from chicken ®broblasts (Sekellick et al., 1994). Type 1 IFN (IFN-a/b) shares potent anti-viral and anti-tumor effects with type 2 IFN (IFN-g), and plays a pivotal role as an immunomodulator (reviewed in Mannering and Deloria, 1986). The therapeutic potential and antiviral activities of ChIFN-a has been assessed by administering recombinant ChIFN-a in drinking water to 1-day-old chickens and then challenging with Newcastle disease virus (NDV) 1 day later (Marcus et al., 1999). Chickens infected with NDV normally show signs of respiratory stress such as railing (a rattling in the throat) as well as weight loss and lethargy. Compared to untreated controls, birds receiving a high dose of recombinant ChIFN-a and then challenged with NDV had a higher mean body weight and were less lethargic than untreated birds. More importantly, birds receiving ChIFN-a were fully protected from viral replication in the trachea. The therapeutic potential of ChIFN-a was further demonstrated when Karaca et al. (1998) showed that co-expression of NDV and ChIFN-a in a fowl pox virus (FPV) vector protected birds from challenge with NDV. NDV genes delivered by FPV alone induced protection from challenge with NDV, however, the weight of chickens was compromised due to the fowl pox virus. When ChIFN-a was co-administered with NDV gene in FPV, there was no subsequent loss of body weight compared to unvaccinated birds, indicating that ChIFN-a can be used to prevent certain avian diseases whilst maintaining vaccination potential. It will be important to further determine the antiviral effects of co-delivering ChIFN-a and ChIFN-g as

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these cytokines have been reported to act synergistically in vitro (Sekellick et al., 1998). 4.4. ChIL-15 and ChIL-18 The genes for ChIL-15 and ChIL-18 have only recently been described (Schneider et al., 2000; J. Burnside, L. Sofer, GenBank accession number AF152927). In mammals, IL-15 shares many in vitro functions with IL-2, including T cell proliferation, which is thought to be due to their shared receptor components (IL-2/IL-15R/bg) (Grabstein et al., 1994; Bamford et al., 1994). However, both cytokines bind to receptors with unique a chains enabling IL-15 and IL-2 to mediate very different functions in vivo. A major role of IL-15 in mammals is in NK cell development and proliferation (Carson et al., 1994). IL-15 co-stimulates cytokine production by NK cells and regulates interactions between macrophages and NK cells. In our laboratory, ChIL-15 is being examined for its ability to stimulate innate immune responses and activation of NK cells when administered to birds. ChIL-15 also plays a main role in activating NK cells and CD8‡ memory T cells, and may therefore have potential as a vaccine adjuvant. IL-18 in mammals is produced by activated macrophages (reviewed in Sugawara, 2000). The primary function of IL-18 is upregulation of IFN-g production by Th1, NK cells and NK T cells (Okamura et al., 1995; Kohno et al., 1997). IL-18 also acts on T, B and NK cells which results in the production of a variety of other cytokines. 4.5. ChIL-2 The existence in chickens of a T cell growth factor with IL-2-like properties was known for many years (Schnetzler et al., 1983; Myers et al., 1992; Kaplan et al., 1993), however, it was some time before the gene was cloned (Sundick and Gill-Dixon, 1997). Due to the evolutionary distance between chickens and mammals, screening of chicken cDNA libraries by hybridisation with mammalian IL-2 probes was unsuccessful (Kaplan et al., 1993). Efforts to purify ChIL-2 from ConA-activated spleen cell supernatants also failed to yield protein of suf®cient purity for sequencing (Schnetzler et al., 1983; Myers et al., 1992). Finally, a chicken lymphokine gene was cloned

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from an activated chicken spleen cDNA library, and the expressed product showed T cell proliferative activity (Sundick and Gill-Dixon, 1997). This gene was found to share identity with both mammalian IL-2 and IL-15 showing 24 and 25% amino acid identity to bovine IL-2 and IL-15, respectively (Sundick and GillDixon, 1997). This ®nding was unusual as it had been reported that there was no signi®cant identity at the nucleotide or amino acid level between mammalian IL-15 and IL-2 sequences (Grabstein et al., 1994). This chicken cytokine shared similar properties with mammalian IL-2 by being expressed exclusively by activated T cells, having a 20-nucleotide 50 -untranslated region and containing a short signal peptide. However, the presence of four conserved cysteines is a characteristic of IL-15, but not IL-2. Gene structure analysis subsequently showed the gene to be the chicken homologue of mammalian IL-2 (Kaiser and Mariani, 1999). The gene structure more closely resembled that of IL-2 rather than IL-15 whereby it is comprised of four exons and three introns, similar to the genes for mouse and human IL-2. The promoter was also highly homologous to the promoter of mammalian IL-2, with conservation of most of the transcriptional factor binding sites both in nucleotide sequence and order. This gene mapped to chicken chromosome 4 linked to a gene encoding annexin V, with synteny to mouse chromosome 3 and human chromosome 4 (Kaiser and Mariani, 1999). Finally amino acid sequence comparisons showed that ChIL-2 had a similar protein structure to its mammalian counterparts (Kaiser and Mariani, 1999). Mammalian IL-2 is an essential cytokine for many types of immune responses including T cell differentiation and activation, B cell development, and NK cell stimulation (Smith, 1988; Farner et al., 1997). In light of this, we have studied the effects of ChIL-2 on immune cell populations in vitro and in vivo. ChIL-2 displays a similar biological activity in vitro to that reported for mammalian IL-2 by inducing the proliferation of T cells following ConA activation (Stepaniak et al., 1999). This is further supported by the ®nding that the addition of a monoclonal antibody (mAb) speci®c for the ChIL-2 receptor (ChIL2R) a chain (Hala et al., 1986; Schauenstein et al., 1988) blocks this activity (Hilton et al., unpublished results). Furthermore, we have identi®ed the target cells for ChIL-2 activity by examining the expression

Fig. 1. Both CD4‡ and CD8‡ T cells rapidly express ChIL-2Ra after activation. Spleen cells were activated with ConA over 72h and were stained with an anti-ChIL-2a chain receptor antibody and either anti-CD4 or -CD8 mAbs. Cells were then analysed using ¯ow cytometry. Data are mean values for four chickens and are representative of two independent experiments.

of ChIL-2R by T cells following their activation by ConA. A low proportion of freshly isolated CD4‡ and CD8‡ spleen cells expressed ChIL-2R. Following activation, the vast majority of CD4‡ cells expressed ChIL-2R within 24h, whereas expression of receptors by CD8‡ T cells was delayed (Fig. 1). This identi®cation is particularly useful for the assessment of in vivo studies. Since mammalian IL-2 has been shown to be an effective vaccine adjuvant, we were interested to see if this also holds true in the chicken. As a ®rst step, it was crucial to develop tools to study and detect ChIL-2. One important issue was to determine how long ChIL2 persists in vivo following administration. We have raised mAbs to ChIL-2 and developed a sandwich ELISA to monitor recombinant ChIL-2 levels once injected into chickens. After intravenous injection, serum levels of ChIL-2 peaked within 1±2 min followed by a rapid rate of decline (half-life of <3 min, Fig. 2). This rapid clearance is characteristic of many cytokines, including mammalian IL-2 (Anderson and Sorenson, 1994) and ChIFN-g (Lowenthal et al., 1999), which suggests that the recombinant ChIL-2 protein is either rapidly degraded and/or binds to ChIL-2R bearing target cells. Despite being cleared rapidly, ChIL-2 treatment resulted in an increase in the proportion of both CD4‡ and CD8‡ peripheral blood

L.S. Hilton et al. / Veterinary Immunology and Immunopathology 85 (2002) 119±128

Fig. 2. Recombinant ChIL-2 protein injected into chickens has a very short half-life. Two hundred and thirty micrograms per kilogram of recombinant ChIL-2 protein was injected intravenously into chickens. Blood was taken at various time points and serum rChIL-2 levels were measured by ELISA. Data points represent mean value for seven chickens.

T cells within 48 h compared to control birds (Table 2). We are in the process of determining whether these changes in T cell populations are due to active proliferation of these cells (by measuring BrdU incorporation) or a result of changes in cell mobilisation (due to recruitment, homing or thymus export). Taken together, the biological activities displayed by ChIL-2 suggest that it may be able to augment cell-mediated immune responses when co-delivered with vaccines. 4.6. ChIL-6 IL-6 has been shown in mammals to have a wide range of activities affecting virtually all cells of the Table 2 The proportion of CD4‡ and CD8‡ peripheral blood T cells increases following ChIL-2 treatmenta Hours post-injection

CD4‡ cells (%)

CD8‡ cells (%)

0 24 48

5 11 31

10 13 29

a

Chickens were injected intravenously with 230 mg/kg of recombinant ChIL-2 protein, and then bled at 0, 24 and 48 h. T cells were stained with either anti-CD4 or anti-CD8 mAbs and analysed by ¯ow cytometry. Percent of peripheral blood lymphocytes staining positive for CD4 or CD8 at each time point are indicated. The staining of samples with isotype control antibodies was used as a reference to determine positive and negative populations.

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immune system (Van Snick, 1990). Its major activities involve acute-phase protein responses but has also been implicated in the development of Th2 type responses. The recent cloning of ChIL-6 and the description of its biological activity (Schneider et al., 2001) is a signi®cant advancement in the ®eld of avian cytokines. Previously cloned avian cytokines are predominantly involved in Th1 type responses. The availability of ChIL-6 allows the opportunity to study its effect on the generation of IgA antibody responses at mucosal sites, as has been shown for IL-6 in mammals. Cytokines such as IL-4, IL-5, and IL-10 have shown to be important in the generation of mammalian Th2 type responses, but their homologues have not yet been described in avians. The existence of Th1/Th2 paradigm in avians therefore remains an open question. 5. Delivery methods for chicken cytokines For cytokines to be used as commercial therapeutics, we must consider certain aspects of the poultry and in particular the broiler (meat) industry. The poultry industry has continued to expand with 40 billion broiler chickens being hatched per annum throughout the world. The delivery methods for vaccines and other therapeutics are of prime importance and are required to be safe, easy to administer and cost-effective. Recombinant cytokines produced from baculovirus, yeast or E. coli expression systems have been successfully delivered via injection to larger high-value animals such as pigs and cattle. For use in chickens, the recombinant protein must be manufactured cheaply on a large scale (tens of billions of doses annually) and ideally be given as a single dose to be cost-effective. The delivery of cytokines and vaccines via live viral vectors expressing these proteins has been successful and often eliminates the need for multiple boosts. Live viral vectors such as fowl adenovirus (FAV) expressing cytokine genes (Johnson et al., 2000) can overcome the short half-life of recombinant cytokines in vivo because the cytokines are expressed over a period of many days until the virus is cleared. FAV can be delivered via drinking water or aerosol sprays, making it very easy to administer. Further advantages of FAV vectors include the production of native proteins rather than prokaryotically expressed proteins which may be less active.

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An additional safety aspect is that the virus is chicken-speci®c and will not replicate in other animal species. An FAV recombinant expressing ChIFN-g (FAV::ChIFN-g) has recently been described in detail (Johnson et al., 2000). Commercial chickens treated with FAV::ChIFN-g showed enhanced weight gain over control birds, even in the face of infection with Eimeria. The capacity of FAV to co-deliver a vaccine with a cytokine adjuvant such as ChIL-2, to obtain an enhanced immune response to vaccination is also being explored. 6. What does the future hold? Since the chicken's immune system is similar to that of mammals, chickens offer an attractive model system to study the effectiveness of cytokine therapy in controlling disease in 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 vaccine antigens and/or cytokine therapeutics can be inexpensively performed on large numbers of animals. 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. One of the remaining challenges involves a closer understanding of the nature of protective immune responses. As outlined above, acquired protection against pathogens in mammals generally falls into one of the two typesÐcell mediated or antibody mediated. However, a combination of cell mediated, antibody and innate responses are often generated during phases of an infection, making it dif®cult to determine which of these responses is responsible for protection. In chickens, it has not been established whether the same Th1/Th2 paradigm exists as it does in mammals. In order to rationally design therapeutics for a particular disease, it is critical to ®rst understand the nature of the protective immune response and then replicate that response during a vaccination strategy. This involves studying the cytokines produced during infection by the pathogen in question as well as the

immune cell populations affected. With the growing accessibility to a number of avian cytokine genes, and the recent development of Real Time PCR and TaqMan1 technology, cytokine pro®les can now be accurately measured during the course of an infection. Another remaining hurdle is the delivery of therapeutics and recombinant vaccines to poultry. Given that administration of recombinant cytokine proteins by injection is not feasible in commercial poultry, alternative methods are needed. FAV technology provides a simple, effective and inexpensive commercial delivery system. 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 effective technologies such as in ovo DNA vaccination to be explored. When considering therapeutic strategies, the concept of synergy is an extremely important one since it is fundamental to the way cytokines normally work in controlling the immune response. These new generation delivery mechanisms permit the administration of single or multiple cytokines in combination with vaccine antigens. The choice of particular vectors will enable antigen and cytokine targeting to speci®c sites such as the gut or respiratory tract, thereby allowing the most appropriate type of immune responses to be generated at the correct site. Cytokines offer a natural approach to therapeutics particularly in relation to the enhancement of protective immune responses produced by vaccines. With the escalating number of chicken cytokines being cloned, only time will tell just how important these regulatory immune proteins will be for the poultry industry. Acknowledgements We would like to thank the following people for their valuable contributions: Terri O'Neil, David Strom and Michael Johnson. We are grateful to Dr. Karel Hala for kindly providing the IL-2Ra chain mAb, and Drs. Peter Kaiser and Peter Staeheli for sharing unpublished information. This work is supported in part by the Australian Rural Industry Research and Development Corporation through its Chicken Meat and Egg Programs.

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