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Cytokines as adjuvants for ruminant vaccines
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International Journal for Parasitology, Vol. 26, No. 8/9, pp. 835 842, 1996 Copyright © 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd Printed in Great Britain
Pergamon
PII: S0020-7519(96)00070-7
0020-7519/96$15.00+0.00
Cytokines as Adjuvants for Ruminant Vaccines S. A . L O F T H O U S E , A . E. A N D R E W S , M . J. E L H A Y , V. M . B O W L E S , E. N . T . M E E U S E N and A. D. NASH*
Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3052, Australia
Abstract--S. A. Lofthouse, M. J. Elhay, V. M. Bowles, E. N. T. Meeusen, A. E. Andrews & A. D. Nash. Cytokines as adjuvants for ruminant vaccines. InternationalJournalfor Parasitology26: 835~42. Successful vaccination against any potential pathogen is critically dependant on inducing an appropriate immune response. The pivotal role of cytokines in the immune response to vaccination suggests that non-protective responses or responses that exacerbate disease subsequent to infectious challenge may be the result of limiting or preferential production of one or a number of these mediators. This suggests that the use of recombinant cytokines as vaccine adjuvants may offer a mechanism whereby the magnitude and phenotype of the immune response to vaccination can be specifically modified. In mice, recombinant cytokines have been used extensively as therapeutics, while studies describing vaccine adjuvant activity are more limited. Recombinant (r) interleukin (IL)-I, IL-2 and interferon (IFN)T have been used primarily to enhance humoral responses with enhanced protection assessed where appropriate. Cytokine adjuvant studies in ruminants have been restricted to recombinant ovine (roy) and bovine (rbov) IL-1 and IL-2. In sheep, their application has been optimised with respect to dose, route of delivery and formulation, for induction of humoral and cell mediated immunity (DTH and cytotoxicity) to the model protein antigen (Ag) avidin. The level of adjuvant activity of IL-I in particular compares favourably to that of a variety of other traditional and new chemical adjuvants and detailed analysis has indicated no adverse local or systemic side-effects. Recent studies in our laboratory demonstrating the effectiveness of rovIL-1 as an adjuvant in single and multi-component bacterial toxoid vaccines, and studies from other laboratories demonstrating the application of rbovIL-1 as an adjuvant for the response in cattle to live attenuated viral vaccines, suggest that rIL-I may become the adjuvant of choice for diseases where protection is mediated by high levels of circulating antibody (Ab). With respect to helminth parasites, IL-1 may prove useful as a component of vaccines based on "hidden Ags" which rely on induction of Ab. Based on analysis in mouse models of helminth infection, other cytokines such as IL-4 and IL-10 may be appropriate for vaccines based on induction of mechanisms involved in natural immunity. Copyright © 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words." Cytokine; adjuvant; ruminant; interleukin-1; interleukin-2.
INTRODUCTION T h e complex a n d multi-faceted n a t u r e o f the a d a p tive i m m u n e response allows for d e v e l o p m e n t o f protective i m m u n i t y against a variety o f pathogens, each o f which m a y use a different strategy to infect a n d multiply within the host a n d evade i m m u n o logical rejection. Effective v a c c i n a t i o n against specific o r g a n i s m s is d e p e n d e n t u p o n inducing n o t only i m m u n e responses o f sufficient m a g n i t u d e , b u t also of a p h e n o t y p e t h a t correlates with rejection. W i t h the exception o f live organisms, a n d some whole cell *To whom correspondence should be addressed. Fax: 03 9347 4083; E-mail: [email protected].
formulations, effective v a c c i n a t i o n requires the use o f adjuvants. These are substances which act non-specifically to e n h a n c e or modify the i m m u n e response to a given vaccine, a n d were first d e m o n strated in the 1920s w h e n R a m o n used materials such as tapioca, agar, lecithin a n d s a p o n i n to e n h a n c e the response to d i p h t h e r i a a n d tetanus toxoid vaccination ( R a m o n , 1926). Subsequent studies o n a d j u v a n t s have s h o w n t h a t the use o f these i m m u n e m o d u l a t o r s is often h i n d e r e d by i n d u c t i o n o f toxic side-effects a n d site reactions in vaccinated individuals a n d their effective application is d e p e n d e n t o n finding a balance between efficacy a n d safety. The classic F r e u n d ' s a d j u v a n t s provide a n excellent example. 835
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Freund's complete adjuvant (FCA), a mineral oil emulsion containing killed mycobacterial fragments, induces excellent cellular and humoral immune responses to many Ags but is associated with chronic inflammation and ulceration at the injection site (Smith, 1992), sensitivity reactions, and severe pain and fever (Gupta et al., 1993). In contrast, Freund's incomplete adjuvant (FIA), which lacks the mycobacterial fraction, has fewer deleterious side-effects but is a relatively poor stimulator of cellular responses and induces only moderate Ab responses (Gupta et al., 1993). This form of the oil emulsion adjuvant has been acceptable for commercial use in animals, although they have been avoided in recent years due to prohibition of substances which persist within the tissues for long periods after injection (Stewart-Tull, 1991). Currently the only adjuvant licensed for use in man and the most commonly used adjuvant for animal vaccines is aluminium hydroxide gel, or alum. While this compound has been shown to be relatively safe, local granuloma formation at the injection site have been reported (Gupta & Relyveld, 1991). In addition, induction of cell-mediated immunity is limited and induction of humoral immunity is poor compared with induction by more effective adjuvants such as FCA. More recent studies have demonstrated that the response induced by alum is typical of the Th2 type, characterised by secretion of the cytokines IL-3, IL-4, IL-5, IL-6, IL-10 and G M - C S F (Grun & Maurer, 1989) and enhanced production of IgG1 and IgE antibodies (Lise & Audibert, 1989). While this may be the response of choice for vaccination against some pathogens this will certainly not always be the case. These limitations of alum, which are particularly relevant in the context of poorly immunogenic subunit vaccines, have led to investigation of a range of alternatives including mycobacterial fractions such as muramyl dipeptide, saponins and their derivatives, block-co-polymer gels and hydrocarbon derivatives such as dimethyl-dioctadecyl-ammonium bromide (reviewed in Cox & Coulter, 1992). Recent advances in our understanding of the mechanisms of immunological rejection of a diverse range of pathogens has provided a framework of specific physical and functional immune compartments each of which represents a potential target for adjuvant activity. These compartments include: the site of vaccination; traffic between the site of vaccination and local secondary lymphoid tissue (specifically, the transport of Ag); Ag presentation; and the expansion and/or differentiation (e.g. isotype switching and Th polarisation) and maturation (selection for increased Ab affinity) events that provide the basis for subsequent anamnestic B- and T-cell re-
sponses. Many of the molecular and cellular events within each of these compartments are regulated by the action of soluble polypeptide cytokines and this has led to the recent interest in the application of recombinant cytokines as vaccine adjuvants. The accumulation of data on the role of specific cytokines within each of these compartments is in stark contrast to our poor understanding of the mode of action of most chemical adjuvants, and this may allow for a rational approach to vaccine design in which the most appropriate immune response to vaccination may be generated by selection of the most relevant cytokines for co-injection with specific Ags. RECOMBINANT CYTOKINES AS IMMUNOLOGICAL ADJUVANTS IN LABORATORY ANIMALS A large number of studies have examined the application of recombinant cytokines as therapeutic agents, often as agents able to modify the outcome of infectious challenge in normal or genetically susceptible/resistant inbred mice. While such studies are often significant in the context of adjuvant activity and vaccination, the more limited number of studies directly addressing adjuvant activity are more relevant. Initial studies of the potential for cytokines as adjuvants utilised the mouse model and focused on the action of IL-2, presumably due to its role in the growth and proliferation of Ag-activated T-cells (Nunberg et al., 1989). This cytokine was shown to enhance responses to viral vaccines, including an inactivated rabies vaccine (Nunberg et al., 1989) and a herpes simplex virus Ag (HSV; Rouse et al., 1985) and to overcome genetic non-responsiveness to a malaria sporozooite vaccine when used in conjunction with an oil-emulsion adjuvant (Good et al., 1988). IL-2 has also been used to protect guinea-pigs against HSV when administered as an adjuvant for immunisation with either a crude extract of HSV or a recombinant glycoprotein D subunit Ag (Weinberg & Merigan, 1988). A second cytokine often used in adjuvant studies is IFNT, which upregulates MHC class I and MHC class II expression on Ag-presenting cells (Rosa & Fellous, 1984) and is a primary regulator of various compartments of cell-mediated immunity including Th-cell differentiation and NK-cell and macrophage activation (Morris, 1988; Mosmann & Coffman, 1989). Study of this cytokine in the mouse model has demonstrated its utility for inducing protection and enhancing Ab responses, T-cell help and DTH responses against malarial challenge following coadministration with a Triton-X lysate of whole parasitised erythrocytes (Playfair & deSouza, 1987; Heath
Cytokine adjuvants Table l--Cloned and expressed ovine cytokines for adjuvant analysis Recombinant product Cytokine for adjuvant studies* References IL-la
yes
IL-lfl
yes
IL-2 IL-3 IL-7 IL-8 IL-10 TNFa
yes no no yes no yes
Nash et al., 1993 Fiskerstrand et al., 1992 Andrews et al., 1994 Fiskerstrand et al., 1992 Scow et al., 1994a Nash et al., 1993 McInnes et al., 1993 Barcham et aL, 1995 Seow et al., 1994b Martin et al., 1995 Nash et al., 1993 Green et al., 1993
* Purified recombinant material of demonstrated biological activity and in sufficient quantity. Table excludes cloned cytokine cDNA from which no biologically active material has been expressed.
et al., 1989a, 1989b; Heath & Playfair, 1991). IFN 7
has shown particular advantages as an adjuvant in low Ab responder mouse strains, mice depleted of CD4+ T-cells (Heath et al., 1989b) and mice with genetic defect in Ab affinity maturation (Holland, Holland & Steward, 1990). Examination of the adjuvant action of IL-1 in mice demonstrated that Ab responses to BSA could be enhanced up to 50-fold by the co-injection of 200 unit doses of this cytokine with the Ag, although maximal responses were observed when the cytokine was delivered 2 h after the injection of Ag (Staruch & Wood, 1983). Further studies have shown that doses of 50,000 units of recombinant human IL-la significantly enhanced the generation of splenic plaque forming cells to sheep red blood cells in vitro a n d / n vivo in mice (Reed et al., 1989) and that recombinant human IL-lfl effectively increased Ab responses to ovalbumin in rabbits when delivered in saline at doses of 30,000 units (Sagara et al., 1990). RECOMBINANT CYTOKINES AS IMMUNOLOGICAL ADJUVANTS IN RUMINANTS R e c o m b i n a n t interleukin-1
Analysis of the adjuvant activity of cytokines in large animal models has been hindered by the lack of available species-specific recombinant cytokines. In recent years, however, a large number of ruminant cytokines have been cloned and a limited number expressed in quantities sufficient for application in adjuvant trials (summarised in Table 1). Recombinant ovine (Rov) IL-la and IL-lfl have been
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studied extensively as adjuvants for use in sheep (Table 2), with many of the initial experiments utilising the model protein Ag avidin and demonstrating an effective dose range of 10 to 100 ~tg using either intramuscular, subcutaneous or intradermal delivery (Nash et al., 1993; Andrews et al., 1994). Secondary anti-avidin responses were at least equivalent to those induced using alum and the combination of alum and rovIL-1 resulted in Ab titres up to 8-fold higher than those induced by administration of either adjuvant alone. Indeed, responses induced by the combination of alum and IL-1 were subsequently shown to be equivalent or superior to those induced by a variety of other experimental adjuvants including Quil A, MDP and Emulsigen plus (oil in water emulsion) and in addition represented the only formulation that was able to evoke significant avidin-specific DTH responses (Lofthouse et al., 1995a). Experiments using a rovIL-lfl neutralising monoclonal Ab demonstrated that rovIL-lb-mediated adjuvant activity was directly attributable to the biological activity of the cytokine and not to contaminating material, such as LPS, in the recombinant protein preparation (Lofthouse et al., 1995b). The demonstration that IL-1 and Ag could be delivered separately provided that the injection sites were drained by the same regional lymphoid tissue suggested that the adjuvant activity of IL-1 was mediated within this tissue rather than at the site of administration. While this suggested that direct conjugation of the Ag to rovIL-lfl may be beneficial, as has been previously reported for other cytokines used as adjuvants for the response to avidin (Heath et al., 1989b), this proved not to be the case (Lofthouse, Andrews & Nash, unpublished). The efficacy of rovIL-lfl in stimulating the humoral immune response suggested that it may be ideal for inclusion in vaccines against diseases where Ab is the primary mediator of protective immunity. From the perspective of ruminant vaccination, the commercial multi-component toxoid vaccine against the various pathogenic clostridial species (CI. novyi, Cl. tetani, Cl. p e r f r i n g e n s etc) represents perhaps the best example of such a vaccine. The pathogenicity of these bacteria is dependent on toxin production, and host-protection on the level of neutralising anti-toxin antibodies. We have included rovIL-lfl in vaccine formulations containing one or a number of different clostridial toxoids and compared the efficacy of these formulations to those formulated with no adjuvant or with alum (the adjuvant used in commercial preparations; Elhay et al., unpublished). For a single component Cl. n o v y i vaccine the humoral response induced 14 days after secondary vaccination with rovIL-1B as the adjuvant was superior (2- to 6-fold
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S. A. Lofthouse et aL Table 2--Recombinant cytokines as immunological adjuvants in livestock species Species
Cytokine
Vaccine/antigen
Parameters assessed References
Bovine
bovlL-lfl
BHV-I BVD PI-3 BHV-I BVD PI-3 avidin
Serum Ab Tc, virus shedding Serum Ab Tc, virus shedding Serum Ab DTH
bovlL-2 Ovine
ovlL-la/fl
H. contortus
clostridials L. cuprina
Porcine
ovlL-2
avidin clostridials
bovlL- lfl bovlL-2
Strep. suis
Serum Ab DTH survival growth, NK cells, serunq Ab
increase) to that induced by alum, using either subcutaneous or intramuscular vaccination. Furthermore, the anti-C/, n o v y i response induced by rovIL-1 persisted so that, 37 days after vaccination, Ab titres remained at least 2-fold greater than the maximum response induced with alum. Further experiments have since demonstrated that increasing the number of vaccine components (3 or 5 components) does not diminish the adjuvant activity of IL-1 for the Cl. n o v y i response and that the response induced to the additional components was also superior to that induced by alum. Serum transfer experiments have demonstrated that the rovlL-1 induced toxin specific Ab is neutralising. In addition to toxin mediated disease, protection against viral disease is also dependent on neutralising Ab (as well as cytotoxic T-cell activity in some cases). Recombinant bovine IL-lfl has been used in cattle in conjunction with a commercial modified live bovine herpes virus (BHV)-I vaccine (Table 2; Reddy et al., 1990), a BHV-l/parainfluenza(PI)-3 vaccine and a killed bovine diarrhoea virus (BVD) vaccine (Reddy et al., 1993). In each case, cattle treated with vaccine adjuvanted with rbolL-lfl exhibited improved serum neutralising Ab titres as well as decreased virus shedding and increased protection following challenge. Taken together with the ovine toxoid vaccine data described above, these results suggest that recombinant ruminant IL-1 may find application in the reformulation of existing vaccine preparations in addition to application with new vaccines. Recombinant
interleukin-2
Interleukin-2 has also been investigated for its adjuvant properties in ruminants (Table 2). In cattle,
Reddy et al., 1990 Reddy et al., 1993 Reddy et al., 1989 Reddy et al., 1993 Nash et al., 1993 Andrews et al., 1994 Lofthouse et al., 1995a Elhay et al., unpublished Bowles et al., 1995 Andrews et al., unpublished Elhay et al., unpublished Blecha et al., 1995
intramuscular administration of 0.25 pg.kg 1 to 25 pg.kg i of recombinant bovine IL-2 daily for 5 days has been used to augment immune responses to a commercial BHV vaccine, resulting in a 6-fold enhancement in neutralising Ab titre, a 4-fold reduction in virus shedding and increased protection to challenge. Cytotoxic responses also increased in an IL-2 dose-dependent manner (Reddy et al., 1989). Significant increase in cytotoxicity, lymphocyte proliferative activity and Ab titres were also recorded following co-injection of multiple doses (0.5 /~g.kg 1) of IL-2 with a subunit glycoprotein Ag of BHV-1 (Hughes et al., 1991, 1992). We have demonstrated the activity of recombinant ovine IL-2 as an adjuvant using the model protein avidin (Table 2). When 40 pg of rovIL-2 was administered intramuscularly to sheep with Ag at primary and secondary immunisation, a 4-fold enhancement in secondary Ab titre was observed. Multiple administration of IL-2 on days 0, 2 and 4 or daily for 5 days resulted in a further significant increase in Ab titres with the former inducing the highest Ab levels (4-fold over single-dose administration). This administration regime utilising soluble |L-2 resulted in Ab levels exceeding those induced with alum as adjuvant. Similar to the case of IL-1, the combination of alum and IL-2 was more effective for induction of Ab than use of these adjuvants individually. In contrast to IL-1 however, IL-2 alone or in combination with alum did not appear to enhance DTH responses. Recently we have extended this analysis to assess the adjuvant activity of combinations of rov|L-2 and rovlL-lfl. The response to avidin induced by intramuscular administration of 1L-lfl or IL-2 at the time
Cytokine adjuvants of vaccination and then IL-2 again on days 2 and 4 was compared to that induced through coadministration of both IL-1 and IL-2 according to these protocols. Somewhat surprisingly the influence of the cytokines appeared to be entirely additive if not synergistic with Ab titre induced by the combination of cytokines some 4- to 8-fold greater than that induced using IL-1 alone. In addition, sheep receiving both cytokines exhibited strong avidin specific DTH on subsequent challenge (Nash, Barcham & Andrews, unpublished). While examples presented here have highlighted the requirement for multiple dosing of IL-2 when used as an adjuvant, techniques developed in drug delivery science, such as chemical modification (Katre, Knauf & Laird, 1987) or the incorporation of cytokine into liposomes (Joffret et al., 1990; Ho, Burke & Merigan, 1992), microspheres (Singh-Hora et al., 1990), minipellets (Fujiwara et al., 1991) or pluronic gels (Morikawa et al., 1987) have the potential to increase the short serum half-life of IL-2 and enhance its adjuvant potential.
Recombinant cytokines as adjuvants for helminth vaccines Currently, approaches to vaccination against helminth parasites are based on the induction of 2 distinct forms of protective immunity. The "hidden Ag" approach relies on the induction of what is essentially neutralising humoral immunity against critical components of the parasite not normally seen by the host. Alternative vaccination strategies are based on induction of effector mechanisms that are involved in the development of natural protective immunity against these parasites. If mouse models of helminth immunity are relevant to ruminant helminth immunity then these mechanisms are almost certain to be dependant on the generation of Th2-mediated immune responses. There is clear evidence that both vaccination strategies may benefit from the application of recombinant cytokines as adjuvants. Vaccination with H 1 l, an integral membrane protein of the intestinal microvilli of H. contortus, represents an excellent example of the "hidden Ag" approach (Munn et al., 1993; Tavernor et al., 1992). Cloning and sequencing of the gene encoding HI 1 revealed homology with mammalian aminopeptidases and subsequent enzyme assays using purified H11 confirmed its role in the cleavage of dipeptide products of digestion, to amino acids for transport across the plasma membrane (Graham et al., 1993). Sheep vaccinated with purified H11 emulsified in F C A show, on average, a 90% reduction in FEC and
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a 75% reduction in worm burdens (Newton, 1995). Protection is presumed to be a result of Ab-mediated inhibition of aminopeptidase activity leading to starvation of worms. On this basis rovlL-1 which, when used alone or in combination with other adjuvants (including rovlL-2) has been shown to induce high titre Ab responses in the absence of any adverse reactions, may be an ideal replacement for the commercially unacceptable FCA. When candidate vaccine Ags aimed at inducing natural immunity are formulated with rovlL-1, high A b titres accompany exacerbation of disease as indicated by increased FEC in vaccinates compared with controls (Nash et al., 1993). Given the observations that rovlL-1 adjuvanted responses are associated with induction of DTH, a Thl-mediated response, this is perhaps not surprising. Extensive analysis of murine models of helminth infection (Trichuris muris, Heligmosomoides polygyrus and Nippostrongylus brasiliensis for example) have indicated that, in mice at least, natural protective immunity is based on induction of a Th2 response that mediates mucosal mastocytosis and eosinophilia in conjunction with preferential switching to IgG1 and IgE isotypes. For example, inbred mice normally susceptible to T. muris can be converted to a resistant phenotype after treatment with cytokines that promote a Th2 response (IL-4) or with antibodies that neutralise Thl-inducing cytokines (anti-IFNT; Else et al., 1994). Conversely, mice treated with IL-12, a cytokine which stimulates IFN7 production and promotes a Thl response are susceptible to more severe N. brasiliensis infection (Finkelman et al., 1994). With respect to intentionally directing Th polarisation along these lines there are 2 related points to consider. Firstly, differentiation of the Th response appears to be dependent on the milieu of cytokines present when naive T-cells first encounter Ag. Secondly, and presumably as a consequence of this, an ongoing response to one Ag can profoundly influence the phenotype of developing immunity to another (Kullberg et al., 1992: Curry et al., 1995). Thus, if the response to any immunodominant Ag can be biased towards Th2 at the time of vaccination, subsequent responses to other parasite Ags during infectious challenge should be preferentially directed towards a Th2 phenotype, independent of the response they might induce as isolated molecules. Given that appropriate effector mechanisms are regulated by similarly differentiated T-helper cells in ruminants, it is possible that cytokines which support development of Th2 responses (IL-4) or antagonise development of Thl responses (IL-4 and possibly |L-10) may be of use in helminth vaccine formulations.
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SafeO, considerations
An important consideration in the use of any adjuvants in food animal species is the safety of the material for injection, both from an animal ethics perspective and for protection of humans consuming animal products. N u m e r o u s adjuvants are restricted in usage due to their ability to induce localised pain and inflammation at the injection site, systemic reactions such as fever, or destruction of tissue due to retention of toxic materials such as oils. In addition, many adjuvants are suspected carcinogens or have the ability to induce disease such as adjuvant arthritis (Gupta et al., 1993). While, as biologicals, cytokines are likely to be inherently less harmful their application must be subject to all routine safety considerations. This is especially important in view of the fact that not all known biological properties of any given cytokine may be compatible with administration of exogenous recombinant material. Excessive endogenous IL-1 synthesis, for example, is associated with chronic inflammatory diseases and (along with T N F and IL-6) fatal septic shock. The safety of adjuvant-active doses of r o v I L - l a , rovIL-lfl and IL-2 in sheep has been examined by analysis of a variety of parameters including body temperature, total and differential white blood cell counts and analysis of the site of injection, both by gross assessment and immunohistological examination. In addition, the serum half-lives of these cytokines have been determined (Lofthouse et al., 1995b). Intravenous administration of 100 ¢tg of r o v I L - l a or r o v I L - l f l resulted in body temperature increases of up to 2°C which returned to pre-injection levels by 24 h post-injection. This represented the most severe route of delivery however, and when the cytokine was delivered by the subcutaneous or intramuscular route the maximum recorded rise was only I°C, with temperatures returning to normal by 24 h post-injection. Goff et al. (1992) reported similar transient changes in body temperatures of cattle following intramuscular injection with recombinant bovine IL-lfl at doses of approximately 30/~g per animal. Immediately following injection of sheep with either r o v l L - l a or r o v l L - l f l total white cell counts dropped to one-quarter normal levels within 2 h, which was followed by an immediate increase in cell count to reach 3-times normal levels by 20 h post-injection. White cell counts declined thereafter to reach pre-injection levels again by 3 days postinjection. These fluctuations in white cell count were shown to be entirely due to the change in the number of circulating neutrophils. Similar, although less severe, changes in body temperature and white cell counts have been observed after administration of rovlL-2. Such changes are often observed following
immunisation with many commercial live vaccines (Tuckwell, 1993) and are considered acceptable providing that the fluctuations are transient. Problems of long-term retention of adjuvants within tissue are probably not applicable with respect to cytokines used as components of soluble formulations. Intradermal and intramuscular administration of soluble rovlL-lfl, for example, has little or no impact on local immunohistology with no palpable site reactions and only a minor influx of C D 4 + cells observed (Lofthouse et aL, 1995a). This was in contrast to a parallel study using alum, where alum residue was evident and a massive infiltration of cells, including B-cells, T-cells and macrophages into skin or muscle was observed. In contrast to alum, injected cytokine probably drains rapidly to local lymphoid tissue, where its adjuvant activity is manifested. The short serum half-life of r o v l L - l f l (2 ~ , min) would ensure that any excess material entering the bloodstream via the thoracic duct would be rapidly cleared. Clearly, however, additional problems may be encountered if cytokines are incorporated as components of more complex formulations (additional adjuvants, delivery vehicles) and their safety will need to be considered in each individual circumstance. This work was supported by grant UME93 from the International Wool Secretariat and by the Australian Research Council. Acknowledgements
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Cytokine adjuvants Else K. J., Finkelman F. D., Maliszewski C. R. & Grencis R. K. 1994. Cytokine-mediated regulation of chronic intestinal helminth infection. Journal of Experimental Medicine 179:347 351. Finkelman F. D., M a d d e n K. B., Cheever A. W., K a t o n a 1. M., Morris S. C., Gately M. K., H u b b a r d B. R., Gause W. C. & U r b a n J. F. 1994. Effects of interleukin 12 on imune responses and host protection in mice infected with intestinal nematode parasites. Journal ~/'Experhnental Medicine 179:1563 1572. Fiskerstrand C. E., Roy D. J., Green I. & Sargan D. R. 1992. Cloning, expression and characterisation of ovine interleukins la and [L C)~tokine 4: 4 1 8 4 2 8 . Fujiwara T., Sakantigenami K., M a t s u o k a J., Shiozaki S., Fujioka K., T a k a d a Y., Uchida S., O n o d a T. & Orita K. 1991. A u g m e n t a t i o n of anti-tumour effect on syngeneic murine solid t u m o u r s by an interleukin-2 slow delivery system, the IL-2 minipellet. Biotherapy 3:203 209. Goff J. P., Naito Y., Kehru M. E., Hayes P. & Daley M. 1992. Physiologic effects of administration of interleukin-lfl in cows. American Journal of Veterinao~ Research 53:1983 1987. Good M. F., P o m b o D., Lunde M. N., Maloy W. L., Halenbeck R., Koths K, Miller L. H. & Berzofsky J. A. 1988. Recombinant h u m a n IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides. Correlation of effect with biologic activity of IL-2. Journal of hnmunoh)gy 141:972 977. G r a h a m M., Smith T. S., M u n n E. A., Knox D. P., Oliver J. J. & Newton S. E. 1993. Recombinant D N A molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections. Patent no. W O 93/23542. Green I. R., Fiskerstrand C., Bertoni G., Roy D. J., Peterhans E. & Sargan D. R. 1993. Expression and characterisation of bioactive recombinant ovine TNFcz: some species specificity in cytotoxic response to T N F . Cvtokine 5:213 223. G r u n J. E. & Maurer P. H. 1989. Different T helper cell subsets elicited in mice utilising two different adjuvant vehicles: the role of endogenous interleukin-I in proliferative responses. Celhdar b~lmunology 121:135 145. G u p t a R. K. & Relyveld E. H. 1991. Adverse reactions after injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are not due only to pertussis organisms or pertussis components of the vaccine. Vaccine 9: 699 702. G u p t a R. J., Relyveld E. H., Lindblad E. B., Bizzini B., Ben-Effraim B. & G u p t a C. K. 1993. Adjuvants- a balance between toxicity and adjuvanticity. VacehTe 11: 293 306. Heath A. W., Haque N. A., deSouza J. B. & Playfair J. H. L. 1989a. Interferon g a m m a as an effective immunological adjuvant. In Vaccines 89: 43~,6. Cold Spring H a r b o u r Laboratories, New York. Heath A. W., Devy M. E., Brown I. N., Richards C. E. & Playfair J. H. L. 1989. Interferon g a m m a as an adjuvant in i m m u n o c o m p r o m i s e d mice. hnmunology 67: 520 524. Heath A. W. & Playfair J. H. L. 1991. Effects of added cytokines on i m m u n e response and memory. In Vaccines (Edited by G. Gregoriadis), pp. 77 84. Plenum Press, New York. Ho R. J. Y., Burke R. L. & Merigan T. C., 1992. Liposomeformulated interleukin-2 as an adjuvant of recombinant HSV glycoprotein gD for the treatment of recurrent genital HSV-2 in guinea pigs. Vaccine 10:209 213.
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Holland G. P., Holland N. & Steward M. W. 1990. Interferon g a m m a potentiates antibody affinity in mice with a genetically controlled defect in affinity maturation. Clinical and Experimental bnmunology 82: 221-226. Hughes H. P. A., C a m p o s M., Godson P. L., Van Drunen Littel -Van Denhurk S., McDougall L., Rapin N., Z a m b T. & Babuik L. A. 1991. lmmunopotentiation of bovine herpes virus subunit vaccination by interleukin-2. hnmunology 74: 4 6 1 4 6 6 . Hughes H. P. A., C a m p o s M., Van Drunen Littel-Van Denhurk S., Z a m b T., Sordillo L. M., G o d s o n D. & Babuik L. A. 1992. Multiple administration of interleukin-2 potentiates antigen-specific responses to subunit vaccination with bovine herpes virus-I glycoprotein IV. Vaccine 10: 226-230. Joffret M. L., Morgeaux S., LeClerc C., Oth D., Zanetti C., Sureau P. & Perrin P. 1990. Enhancement of interleukin-2 activity by liposomes. Vaccine 8: 385-389. Katre N. V., K n a u f M. J. & Laird W. J. 1987. Chemical modification of recombinant interleukin 2 by polethylene glycol increases its potency in the murine Meth A sarcoma model. Proceedings of the National Acadamy ~71' Science USA 84: 1487-1491. Kullberg M. C., Pearce E. J., Hieny S. A., Sher A. & Berzofsky J. A. 1992. Infection with Schistosoma mansoni alters T h l / T h 2 cytokine responses to a non-parasite antigen. Journal oflmmunology 148: 3264-3270. Lise L. & Audibert F. 1989. I m m u n o a d j u v a n t s and analogues of i m m u n o m o d u l a t o r y bacterial structures. Current Opinions in hnmunology 2:269 274. Lofthouse S. A., Andrews A. E., Nash A. D. & Bowles V. M. 1995. Humoral and cellular responses to intradermally administered cytokine and conventional adjuvants. Vaccine 13:1131 1137. Lofthouse S. A., Andrews A. E., Barcham G. J. & Nash A. D. 1995. Parameters related to the application of recombinant ovine interleukin-lfl as an adjuvant. Vaccine (Adjuvant Research) 13:1277 1287. Martin H. M., Nash A. D. & Andrews A. E. 1995. Cloning and characterisation of an ovine interleukin-10-encoding c D N A . Gene 159:187 191. Mclnnes C., Haig D. & Logan M. 1993. The cloning and expression of the gene for ovine interleukin 3 (multiCSF) and a comparison of the in vitro hematopoietic activity of ovine IL-3 with ovine G M - C S F and h u m a n M-CSF. Experimental Hematology 21:1528 1534. Morikawa K., Okada F., Hosokawa M. O. & Koboyashi H. 1987. Enhancement of the therapeutic effects of recombinant interleukin-2 on a transplantable rat fibrosarcoma by the use of a sustained release vehicle, pluronic gel. Cancer Research 47: 3 7 ~ 1 . Morris A. G. 1988. Interferons. Immunology Suppl. 1, pp. 4 3 ~ 5 . M o s m a n n T. R. & Coffman R. L. 1989. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Advances in h,munology 46:111 142. M u n n E. A., Smith T. S., G r a h a m S., Tavernor A. S. & Greenwood C. A. 1993. The potential value of integral m e m b r a n e proteins in vaccination of lambs against
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International Journal for Parasitology, Vol. 26, No. 8/9, pp. 835 842, 1996 Copyright © 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd Printed in Great Britain
Pergamon
PII: S0020-7519(96)00070-7
0020-7519/96$15.00+0.00
Cytokines as Adjuvants for Ruminant Vaccines S. A . L O F T H O U S E , A . E. A N D R E W S , M . J. E L H A Y , V. M . B O W L E S , E. N . T . M E E U S E N and A. D. NASH*
Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3052, Australia
Abstract--S. A. Lofthouse, M. J. Elhay, V. M. Bowles, E. N. T. Meeusen, A. E. Andrews & A. D. Nash. Cytokines as adjuvants for ruminant vaccines. InternationalJournalfor Parasitology26: 835~42. Successful vaccination against any potential pathogen is critically dependant on inducing an appropriate immune response. The pivotal role of cytokines in the immune response to vaccination suggests that non-protective responses or responses that exacerbate disease subsequent to infectious challenge may be the result of limiting or preferential production of one or a number of these mediators. This suggests that the use of recombinant cytokines as vaccine adjuvants may offer a mechanism whereby the magnitude and phenotype of the immune response to vaccination can be specifically modified. In mice, recombinant cytokines have been used extensively as therapeutics, while studies describing vaccine adjuvant activity are more limited. Recombinant (r) interleukin (IL)-I, IL-2 and interferon (IFN)T have been used primarily to enhance humoral responses with enhanced protection assessed where appropriate. Cytokine adjuvant studies in ruminants have been restricted to recombinant ovine (roy) and bovine (rbov) IL-1 and IL-2. In sheep, their application has been optimised with respect to dose, route of delivery and formulation, for induction of humoral and cell mediated immunity (DTH and cytotoxicity) to the model protein antigen (Ag) avidin. The level of adjuvant activity of IL-I in particular compares favourably to that of a variety of other traditional and new chemical adjuvants and detailed analysis has indicated no adverse local or systemic side-effects. Recent studies in our laboratory demonstrating the effectiveness of rovIL-1 as an adjuvant in single and multi-component bacterial toxoid vaccines, and studies from other laboratories demonstrating the application of rbovIL-1 as an adjuvant for the response in cattle to live attenuated viral vaccines, suggest that rIL-I may become the adjuvant of choice for diseases where protection is mediated by high levels of circulating antibody (Ab). With respect to helminth parasites, IL-1 may prove useful as a component of vaccines based on "hidden Ags" which rely on induction of Ab. Based on analysis in mouse models of helminth infection, other cytokines such as IL-4 and IL-10 may be appropriate for vaccines based on induction of mechanisms involved in natural immunity. Copyright © 1996 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words." Cytokine; adjuvant; ruminant; interleukin-1; interleukin-2.
INTRODUCTION T h e complex a n d multi-faceted n a t u r e o f the a d a p tive i m m u n e response allows for d e v e l o p m e n t o f protective i m m u n i t y against a variety o f pathogens, each o f which m a y use a different strategy to infect a n d multiply within the host a n d evade i m m u n o logical rejection. Effective v a c c i n a t i o n against specific o r g a n i s m s is d e p e n d e n t u p o n inducing n o t only i m m u n e responses o f sufficient m a g n i t u d e , b u t also of a p h e n o t y p e t h a t correlates with rejection. W i t h the exception o f live organisms, a n d some whole cell *To whom correspondence should be addressed. Fax: 03 9347 4083; E-mail: [email protected].
formulations, effective v a c c i n a t i o n requires the use o f adjuvants. These are substances which act non-specifically to e n h a n c e or modify the i m m u n e response to a given vaccine, a n d were first d e m o n strated in the 1920s w h e n R a m o n used materials such as tapioca, agar, lecithin a n d s a p o n i n to e n h a n c e the response to d i p h t h e r i a a n d tetanus toxoid vaccination ( R a m o n , 1926). Subsequent studies o n a d j u v a n t s have s h o w n t h a t the use o f these i m m u n e m o d u l a t o r s is often h i n d e r e d by i n d u c t i o n o f toxic side-effects a n d site reactions in vaccinated individuals a n d their effective application is d e p e n d e n t o n finding a balance between efficacy a n d safety. The classic F r e u n d ' s a d j u v a n t s provide a n excellent example. 835
836
S.A. Lofthouse et al.
Freund's complete adjuvant (FCA), a mineral oil emulsion containing killed mycobacterial fragments, induces excellent cellular and humoral immune responses to many Ags but is associated with chronic inflammation and ulceration at the injection site (Smith, 1992), sensitivity reactions, and severe pain and fever (Gupta et al., 1993). In contrast, Freund's incomplete adjuvant (FIA), which lacks the mycobacterial fraction, has fewer deleterious side-effects but is a relatively poor stimulator of cellular responses and induces only moderate Ab responses (Gupta et al., 1993). This form of the oil emulsion adjuvant has been acceptable for commercial use in animals, although they have been avoided in recent years due to prohibition of substances which persist within the tissues for long periods after injection (Stewart-Tull, 1991). Currently the only adjuvant licensed for use in man and the most commonly used adjuvant for animal vaccines is aluminium hydroxide gel, or alum. While this compound has been shown to be relatively safe, local granuloma formation at the injection site have been reported (Gupta & Relyveld, 1991). In addition, induction of cell-mediated immunity is limited and induction of humoral immunity is poor compared with induction by more effective adjuvants such as FCA. More recent studies have demonstrated that the response induced by alum is typical of the Th2 type, characterised by secretion of the cytokines IL-3, IL-4, IL-5, IL-6, IL-10 and G M - C S F (Grun & Maurer, 1989) and enhanced production of IgG1 and IgE antibodies (Lise & Audibert, 1989). While this may be the response of choice for vaccination against some pathogens this will certainly not always be the case. These limitations of alum, which are particularly relevant in the context of poorly immunogenic subunit vaccines, have led to investigation of a range of alternatives including mycobacterial fractions such as muramyl dipeptide, saponins and their derivatives, block-co-polymer gels and hydrocarbon derivatives such as dimethyl-dioctadecyl-ammonium bromide (reviewed in Cox & Coulter, 1992). Recent advances in our understanding of the mechanisms of immunological rejection of a diverse range of pathogens has provided a framework of specific physical and functional immune compartments each of which represents a potential target for adjuvant activity. These compartments include: the site of vaccination; traffic between the site of vaccination and local secondary lymphoid tissue (specifically, the transport of Ag); Ag presentation; and the expansion and/or differentiation (e.g. isotype switching and Th polarisation) and maturation (selection for increased Ab affinity) events that provide the basis for subsequent anamnestic B- and T-cell re-
sponses. Many of the molecular and cellular events within each of these compartments are regulated by the action of soluble polypeptide cytokines and this has led to the recent interest in the application of recombinant cytokines as vaccine adjuvants. The accumulation of data on the role of specific cytokines within each of these compartments is in stark contrast to our poor understanding of the mode of action of most chemical adjuvants, and this may allow for a rational approach to vaccine design in which the most appropriate immune response to vaccination may be generated by selection of the most relevant cytokines for co-injection with specific Ags. RECOMBINANT CYTOKINES AS IMMUNOLOGICAL ADJUVANTS IN LABORATORY ANIMALS A large number of studies have examined the application of recombinant cytokines as therapeutic agents, often as agents able to modify the outcome of infectious challenge in normal or genetically susceptible/resistant inbred mice. While such studies are often significant in the context of adjuvant activity and vaccination, the more limited number of studies directly addressing adjuvant activity are more relevant. Initial studies of the potential for cytokines as adjuvants utilised the mouse model and focused on the action of IL-2, presumably due to its role in the growth and proliferation of Ag-activated T-cells (Nunberg et al., 1989). This cytokine was shown to enhance responses to viral vaccines, including an inactivated rabies vaccine (Nunberg et al., 1989) and a herpes simplex virus Ag (HSV; Rouse et al., 1985) and to overcome genetic non-responsiveness to a malaria sporozooite vaccine when used in conjunction with an oil-emulsion adjuvant (Good et al., 1988). IL-2 has also been used to protect guinea-pigs against HSV when administered as an adjuvant for immunisation with either a crude extract of HSV or a recombinant glycoprotein D subunit Ag (Weinberg & Merigan, 1988). A second cytokine often used in adjuvant studies is IFNT, which upregulates MHC class I and MHC class II expression on Ag-presenting cells (Rosa & Fellous, 1984) and is a primary regulator of various compartments of cell-mediated immunity including Th-cell differentiation and NK-cell and macrophage activation (Morris, 1988; Mosmann & Coffman, 1989). Study of this cytokine in the mouse model has demonstrated its utility for inducing protection and enhancing Ab responses, T-cell help and DTH responses against malarial challenge following coadministration with a Triton-X lysate of whole parasitised erythrocytes (Playfair & deSouza, 1987; Heath
Cytokine adjuvants Table l--Cloned and expressed ovine cytokines for adjuvant analysis Recombinant product Cytokine for adjuvant studies* References IL-la
yes
IL-lfl
yes
IL-2 IL-3 IL-7 IL-8 IL-10 TNFa
yes no no yes no yes
Nash et al., 1993 Fiskerstrand et al., 1992 Andrews et al., 1994 Fiskerstrand et al., 1992 Scow et al., 1994a Nash et al., 1993 McInnes et al., 1993 Barcham et aL, 1995 Seow et al., 1994b Martin et al., 1995 Nash et al., 1993 Green et al., 1993
* Purified recombinant material of demonstrated biological activity and in sufficient quantity. Table excludes cloned cytokine cDNA from which no biologically active material has been expressed.
et al., 1989a, 1989b; Heath & Playfair, 1991). IFN 7
has shown particular advantages as an adjuvant in low Ab responder mouse strains, mice depleted of CD4+ T-cells (Heath et al., 1989b) and mice with genetic defect in Ab affinity maturation (Holland, Holland & Steward, 1990). Examination of the adjuvant action of IL-1 in mice demonstrated that Ab responses to BSA could be enhanced up to 50-fold by the co-injection of 200 unit doses of this cytokine with the Ag, although maximal responses were observed when the cytokine was delivered 2 h after the injection of Ag (Staruch & Wood, 1983). Further studies have shown that doses of 50,000 units of recombinant human IL-la significantly enhanced the generation of splenic plaque forming cells to sheep red blood cells in vitro a n d / n vivo in mice (Reed et al., 1989) and that recombinant human IL-lfl effectively increased Ab responses to ovalbumin in rabbits when delivered in saline at doses of 30,000 units (Sagara et al., 1990). RECOMBINANT CYTOKINES AS IMMUNOLOGICAL ADJUVANTS IN RUMINANTS R e c o m b i n a n t interleukin-1
Analysis of the adjuvant activity of cytokines in large animal models has been hindered by the lack of available species-specific recombinant cytokines. In recent years, however, a large number of ruminant cytokines have been cloned and a limited number expressed in quantities sufficient for application in adjuvant trials (summarised in Table 1). Recombinant ovine (Rov) IL-la and IL-lfl have been
837
studied extensively as adjuvants for use in sheep (Table 2), with many of the initial experiments utilising the model protein Ag avidin and demonstrating an effective dose range of 10 to 100 ~tg using either intramuscular, subcutaneous or intradermal delivery (Nash et al., 1993; Andrews et al., 1994). Secondary anti-avidin responses were at least equivalent to those induced using alum and the combination of alum and rovIL-1 resulted in Ab titres up to 8-fold higher than those induced by administration of either adjuvant alone. Indeed, responses induced by the combination of alum and IL-1 were subsequently shown to be equivalent or superior to those induced by a variety of other experimental adjuvants including Quil A, MDP and Emulsigen plus (oil in water emulsion) and in addition represented the only formulation that was able to evoke significant avidin-specific DTH responses (Lofthouse et al., 1995a). Experiments using a rovIL-lfl neutralising monoclonal Ab demonstrated that rovIL-lb-mediated adjuvant activity was directly attributable to the biological activity of the cytokine and not to contaminating material, such as LPS, in the recombinant protein preparation (Lofthouse et al., 1995b). The demonstration that IL-1 and Ag could be delivered separately provided that the injection sites were drained by the same regional lymphoid tissue suggested that the adjuvant activity of IL-1 was mediated within this tissue rather than at the site of administration. While this suggested that direct conjugation of the Ag to rovIL-lfl may be beneficial, as has been previously reported for other cytokines used as adjuvants for the response to avidin (Heath et al., 1989b), this proved not to be the case (Lofthouse, Andrews & Nash, unpublished). The efficacy of rovIL-lfl in stimulating the humoral immune response suggested that it may be ideal for inclusion in vaccines against diseases where Ab is the primary mediator of protective immunity. From the perspective of ruminant vaccination, the commercial multi-component toxoid vaccine against the various pathogenic clostridial species (CI. novyi, Cl. tetani, Cl. p e r f r i n g e n s etc) represents perhaps the best example of such a vaccine. The pathogenicity of these bacteria is dependent on toxin production, and host-protection on the level of neutralising anti-toxin antibodies. We have included rovIL-lfl in vaccine formulations containing one or a number of different clostridial toxoids and compared the efficacy of these formulations to those formulated with no adjuvant or with alum (the adjuvant used in commercial preparations; Elhay et al., unpublished). For a single component Cl. n o v y i vaccine the humoral response induced 14 days after secondary vaccination with rovIL-1B as the adjuvant was superior (2- to 6-fold
838
S. A. Lofthouse et aL Table 2--Recombinant cytokines as immunological adjuvants in livestock species Species
Cytokine
Vaccine/antigen
Parameters assessed References
Bovine
bovlL-lfl
BHV-I BVD PI-3 BHV-I BVD PI-3 avidin
Serum Ab Tc, virus shedding Serum Ab Tc, virus shedding Serum Ab DTH
bovlL-2 Ovine
ovlL-la/fl
H. contortus
clostridials L. cuprina
Porcine
ovlL-2
avidin clostridials
bovlL- lfl bovlL-2
Strep. suis
Serum Ab DTH survival growth, NK cells, serunq Ab
increase) to that induced by alum, using either subcutaneous or intramuscular vaccination. Furthermore, the anti-C/, n o v y i response induced by rovIL-1 persisted so that, 37 days after vaccination, Ab titres remained at least 2-fold greater than the maximum response induced with alum. Further experiments have since demonstrated that increasing the number of vaccine components (3 or 5 components) does not diminish the adjuvant activity of IL-1 for the Cl. n o v y i response and that the response induced to the additional components was also superior to that induced by alum. Serum transfer experiments have demonstrated that the rovlL-1 induced toxin specific Ab is neutralising. In addition to toxin mediated disease, protection against viral disease is also dependent on neutralising Ab (as well as cytotoxic T-cell activity in some cases). Recombinant bovine IL-lfl has been used in cattle in conjunction with a commercial modified live bovine herpes virus (BHV)-I vaccine (Table 2; Reddy et al., 1990), a BHV-l/parainfluenza(PI)-3 vaccine and a killed bovine diarrhoea virus (BVD) vaccine (Reddy et al., 1993). In each case, cattle treated with vaccine adjuvanted with rbolL-lfl exhibited improved serum neutralising Ab titres as well as decreased virus shedding and increased protection following challenge. Taken together with the ovine toxoid vaccine data described above, these results suggest that recombinant ruminant IL-1 may find application in the reformulation of existing vaccine preparations in addition to application with new vaccines. Recombinant
interleukin-2
Interleukin-2 has also been investigated for its adjuvant properties in ruminants (Table 2). In cattle,
Reddy et al., 1990 Reddy et al., 1993 Reddy et al., 1989 Reddy et al., 1993 Nash et al., 1993 Andrews et al., 1994 Lofthouse et al., 1995a Elhay et al., unpublished Bowles et al., 1995 Andrews et al., unpublished Elhay et al., unpublished Blecha et al., 1995
intramuscular administration of 0.25 pg.kg 1 to 25 pg.kg i of recombinant bovine IL-2 daily for 5 days has been used to augment immune responses to a commercial BHV vaccine, resulting in a 6-fold enhancement in neutralising Ab titre, a 4-fold reduction in virus shedding and increased protection to challenge. Cytotoxic responses also increased in an IL-2 dose-dependent manner (Reddy et al., 1989). Significant increase in cytotoxicity, lymphocyte proliferative activity and Ab titres were also recorded following co-injection of multiple doses (0.5 /~g.kg 1) of IL-2 with a subunit glycoprotein Ag of BHV-1 (Hughes et al., 1991, 1992). We have demonstrated the activity of recombinant ovine IL-2 as an adjuvant using the model protein avidin (Table 2). When 40 pg of rovIL-2 was administered intramuscularly to sheep with Ag at primary and secondary immunisation, a 4-fold enhancement in secondary Ab titre was observed. Multiple administration of IL-2 on days 0, 2 and 4 or daily for 5 days resulted in a further significant increase in Ab titres with the former inducing the highest Ab levels (4-fold over single-dose administration). This administration regime utilising soluble |L-2 resulted in Ab levels exceeding those induced with alum as adjuvant. Similar to the case of IL-1, the combination of alum and IL-2 was more effective for induction of Ab than use of these adjuvants individually. In contrast to IL-1 however, IL-2 alone or in combination with alum did not appear to enhance DTH responses. Recently we have extended this analysis to assess the adjuvant activity of combinations of rov|L-2 and rovlL-lfl. The response to avidin induced by intramuscular administration of 1L-lfl or IL-2 at the time
Cytokine adjuvants of vaccination and then IL-2 again on days 2 and 4 was compared to that induced through coadministration of both IL-1 and IL-2 according to these protocols. Somewhat surprisingly the influence of the cytokines appeared to be entirely additive if not synergistic with Ab titre induced by the combination of cytokines some 4- to 8-fold greater than that induced using IL-1 alone. In addition, sheep receiving both cytokines exhibited strong avidin specific DTH on subsequent challenge (Nash, Barcham & Andrews, unpublished). While examples presented here have highlighted the requirement for multiple dosing of IL-2 when used as an adjuvant, techniques developed in drug delivery science, such as chemical modification (Katre, Knauf & Laird, 1987) or the incorporation of cytokine into liposomes (Joffret et al., 1990; Ho, Burke & Merigan, 1992), microspheres (Singh-Hora et al., 1990), minipellets (Fujiwara et al., 1991) or pluronic gels (Morikawa et al., 1987) have the potential to increase the short serum half-life of IL-2 and enhance its adjuvant potential.
Recombinant cytokines as adjuvants for helminth vaccines Currently, approaches to vaccination against helminth parasites are based on the induction of 2 distinct forms of protective immunity. The "hidden Ag" approach relies on the induction of what is essentially neutralising humoral immunity against critical components of the parasite not normally seen by the host. Alternative vaccination strategies are based on induction of effector mechanisms that are involved in the development of natural protective immunity against these parasites. If mouse models of helminth immunity are relevant to ruminant helminth immunity then these mechanisms are almost certain to be dependant on the generation of Th2-mediated immune responses. There is clear evidence that both vaccination strategies may benefit from the application of recombinant cytokines as adjuvants. Vaccination with H 1 l, an integral membrane protein of the intestinal microvilli of H. contortus, represents an excellent example of the "hidden Ag" approach (Munn et al., 1993; Tavernor et al., 1992). Cloning and sequencing of the gene encoding HI 1 revealed homology with mammalian aminopeptidases and subsequent enzyme assays using purified H11 confirmed its role in the cleavage of dipeptide products of digestion, to amino acids for transport across the plasma membrane (Graham et al., 1993). Sheep vaccinated with purified H11 emulsified in F C A show, on average, a 90% reduction in FEC and
839
a 75% reduction in worm burdens (Newton, 1995). Protection is presumed to be a result of Ab-mediated inhibition of aminopeptidase activity leading to starvation of worms. On this basis rovlL-1 which, when used alone or in combination with other adjuvants (including rovlL-2) has been shown to induce high titre Ab responses in the absence of any adverse reactions, may be an ideal replacement for the commercially unacceptable FCA. When candidate vaccine Ags aimed at inducing natural immunity are formulated with rovlL-1, high A b titres accompany exacerbation of disease as indicated by increased FEC in vaccinates compared with controls (Nash et al., 1993). Given the observations that rovlL-1 adjuvanted responses are associated with induction of DTH, a Thl-mediated response, this is perhaps not surprising. Extensive analysis of murine models of helminth infection (Trichuris muris, Heligmosomoides polygyrus and Nippostrongylus brasiliensis for example) have indicated that, in mice at least, natural protective immunity is based on induction of a Th2 response that mediates mucosal mastocytosis and eosinophilia in conjunction with preferential switching to IgG1 and IgE isotypes. For example, inbred mice normally susceptible to T. muris can be converted to a resistant phenotype after treatment with cytokines that promote a Th2 response (IL-4) or with antibodies that neutralise Thl-inducing cytokines (anti-IFNT; Else et al., 1994). Conversely, mice treated with IL-12, a cytokine which stimulates IFN7 production and promotes a Thl response are susceptible to more severe N. brasiliensis infection (Finkelman et al., 1994). With respect to intentionally directing Th polarisation along these lines there are 2 related points to consider. Firstly, differentiation of the Th response appears to be dependent on the milieu of cytokines present when naive T-cells first encounter Ag. Secondly, and presumably as a consequence of this, an ongoing response to one Ag can profoundly influence the phenotype of developing immunity to another (Kullberg et al., 1992: Curry et al., 1995). Thus, if the response to any immunodominant Ag can be biased towards Th2 at the time of vaccination, subsequent responses to other parasite Ags during infectious challenge should be preferentially directed towards a Th2 phenotype, independent of the response they might induce as isolated molecules. Given that appropriate effector mechanisms are regulated by similarly differentiated T-helper cells in ruminants, it is possible that cytokines which support development of Th2 responses (IL-4) or antagonise development of Thl responses (IL-4 and possibly |L-10) may be of use in helminth vaccine formulations.
840
S.A. Lofthouse et aL
SafeO, considerations
An important consideration in the use of any adjuvants in food animal species is the safety of the material for injection, both from an animal ethics perspective and for protection of humans consuming animal products. N u m e r o u s adjuvants are restricted in usage due to their ability to induce localised pain and inflammation at the injection site, systemic reactions such as fever, or destruction of tissue due to retention of toxic materials such as oils. In addition, many adjuvants are suspected carcinogens or have the ability to induce disease such as adjuvant arthritis (Gupta et al., 1993). While, as biologicals, cytokines are likely to be inherently less harmful their application must be subject to all routine safety considerations. This is especially important in view of the fact that not all known biological properties of any given cytokine may be compatible with administration of exogenous recombinant material. Excessive endogenous IL-1 synthesis, for example, is associated with chronic inflammatory diseases and (along with T N F and IL-6) fatal septic shock. The safety of adjuvant-active doses of r o v I L - l a , rovIL-lfl and IL-2 in sheep has been examined by analysis of a variety of parameters including body temperature, total and differential white blood cell counts and analysis of the site of injection, both by gross assessment and immunohistological examination. In addition, the serum half-lives of these cytokines have been determined (Lofthouse et al., 1995b). Intravenous administration of 100 ¢tg of r o v I L - l a or r o v I L - l f l resulted in body temperature increases of up to 2°C which returned to pre-injection levels by 24 h post-injection. This represented the most severe route of delivery however, and when the cytokine was delivered by the subcutaneous or intramuscular route the maximum recorded rise was only I°C, with temperatures returning to normal by 24 h post-injection. Goff et al. (1992) reported similar transient changes in body temperatures of cattle following intramuscular injection with recombinant bovine IL-lfl at doses of approximately 30/~g per animal. Immediately following injection of sheep with either r o v l L - l a or r o v l L - l f l total white cell counts dropped to one-quarter normal levels within 2 h, which was followed by an immediate increase in cell count to reach 3-times normal levels by 20 h post-injection. White cell counts declined thereafter to reach pre-injection levels again by 3 days postinjection. These fluctuations in white cell count were shown to be entirely due to the change in the number of circulating neutrophils. Similar, although less severe, changes in body temperature and white cell counts have been observed after administration of rovlL-2. Such changes are often observed following
immunisation with many commercial live vaccines (Tuckwell, 1993) and are considered acceptable providing that the fluctuations are transient. Problems of long-term retention of adjuvants within tissue are probably not applicable with respect to cytokines used as components of soluble formulations. Intradermal and intramuscular administration of soluble rovlL-lfl, for example, has little or no impact on local immunohistology with no palpable site reactions and only a minor influx of C D 4 + cells observed (Lofthouse et aL, 1995a). This was in contrast to a parallel study using alum, where alum residue was evident and a massive infiltration of cells, including B-cells, T-cells and macrophages into skin or muscle was observed. In contrast to alum, injected cytokine probably drains rapidly to local lymphoid tissue, where its adjuvant activity is manifested. The short serum half-life of r o v l L - l f l (2 ~ , min) would ensure that any excess material entering the bloodstream via the thoracic duct would be rapidly cleared. Clearly, however, additional problems may be encountered if cytokines are incorporated as components of more complex formulations (additional adjuvants, delivery vehicles) and their safety will need to be considered in each individual circumstance. This work was supported by grant UME93 from the International Wool Secretariat and by the Australian Research Council. Acknowledgements
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