The influence of gamma inulin and Algammulin on the immune response in sheep to a recombinant antigen of Taenia ovis

Vaccine, Vol. 13, No. 5. pp. 429-433, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 02%410)(/95 $lo.oo+o.w

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,Papers The influence of gamma inulin and Algammulin on the immune response in sheep to a recombinant antigen of Taenia ovis Harjit S. Deol, Dieter G. Palmer*, Theda Dunsmore and Patrick R. Carnegie Gamma inulin and Algammulin, two new adjuvants, were examined and compared with alum and Freund’s CompletelIncomplete Adjuvant (FCAIFIA), for potentiation of cellmediated immunity (CMZ) and humoral immunity in sheep to a recombinant Taenia ovis antigen. The Qbility to protect sheep when challenged with live T. ovis eggs was also assessed. The results showed that gamma inulin and Algammulin induced a CA4I response which was comparable to the FCAIFIA and alum groups and significantly higher than the control saline group. While gamma inulin, Algammulin and alum performed similarly and induced a significantly higher humoral immune response than the saline group, FcAlFlA elicited a much higher humoral immune response. Algammulin did not show the synergistic effect seen in mice and performed similarly to gamma inulin and alum alone. All the adjuvant groups induced significantly higher IgGl and IgG2 levels than the saline group and they all favoured IgGl production. When the sheep were challenged with live T. ovis eggs, at 25 weeks after primary immunization, the only group to show significant protection was the one which received FCAIFIA. Keywords: Adjuvant;

sheep;

Tuenia ovis; inulin; vaccination

The advent of DNA recombinant technology has enhanced the prospects for the development of subunit vaccines based on the recombinant proteins. However, recombinant proteins are usually weakly as immunogenic, a safe and (effective adjuvant is necessary to amplify the immune response’. Adjuvants affect the immune system through a variety of actions and can selectively enhance specific cell-mediated immunity (CMI) or humoral immune responses to an antigen’, 2. Several classes of adjuvant formulations are available at present, but only a few are registered for either veterinary or human use and some are unsuitable for certain vaccinesi4. Oil-based adjuvants such as Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA) are good inducers of CM1 and humoral immune responses, but are too toxic for use in commercial vaccine preparations and are suitable for research use only’-5. Gamma inulin and! Algammulin are new polysaccharide adjuvant#. ‘. Gamma inulin is the highly insoluble form of the polyfructose inulin and activates

complement through the alternative pathway, which is believed to be the basis of its adjuvant activity’. Algammulin is a hybrid adjuvant and comprises l-2 pm particles of the gamma inulin in which aluminium hydroxide gel is embedded as a protein carrier. In mice’, Algammulin (in which gamma inulin is present on alum) is a more potent adjuvant than either gamma inulin or alum alone or a simple mixture of the two. Gamma inulin and Algammulin are non-toxic, non-antigenic and cheap to produce which makes them suitable candidates for use in farm animals. We therefore postulated that gamma inulin and Algammulin might show strong adjuvant activity in sheep and be able to confer protection, when given in combination with the well-defined recombinant Tuenia ovis antigen (GST45W)‘. In this study, we measured cellular and humoral immune responses and the protective effect in sheep vaccinated with GST-45W without and with the adjuvants alum, gamma inulin, Algammulin and FCA/ FIA. MATERIALS

Biotechnology Programme, School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia. *Animal Industries Division, Department of Agriculture Western Australia, Baron-Hay Court, South Perth, Western Australia 6151, Australia. (Received 13 May 1994; revised 14 Octolber 1994; accepted 19 October 1994)

AND

METHODS

Animals

Thirty Merino wethers, aged approximately 16 months, were purchased from a farm known to have a low incidence of T. ovis infection. They were kept under worm-free conditions in pens and fed a standard cubed diet.

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Antigen The fusion protein, GST-45W9 (kindly supplied by Dr G. Harrison, Pitman-Moore New Zealand Ltd) was used as an antigen for vaccination and for the measurement of the humoral immune response. The cleaved recombinant protein 45W9 was used for the in vitro measurement of cell proliferation responses. Adjuvants The adjuvants used were alum (Alhydrogel, CSL, Australia), gamma inulin, Algammulin (both gamma inulin and Algammulin were kindly supplied by Dr P.D. Cooper, Australia National University, Australia) and FCA/FlA (Sigma, USA). Saline was used as an adjuvant-free control. Controls using adjuvants only were not included in this experiment because it has previously been shown that injections of adjuvant without specific antigen did not confer protectiong. Immunization and challenge The sheep were divided into five groups of six sheep each, namely saline, alum, gamma inulin, Algammulin and FCAIFIA. Each vaccination consisted of 200 pg of GST-45W mixed with saline or the respective adjuvants and injected into the left semitendinosus muscle. Each animal in the respective groups received 1.6 ml of saline, 3.34 mg of alum, 67.5 mg of gamma inulin and Algammulin, and 0.90 ml of FCA/FlA per vaccination. Animals were given the primary vaccination on week 0 and were boosted with the same preparation at week 4, except that FCA was replaced by FIA. All sheep were infected with 500 viable T. ovis eggs (kindly supplied by Dr M. Lightowlers, Melbourne University, Melbourne, Australia) at week 25, killed 4 weeks after the infection and examined for the presence of cysts in skeletal muscles, the diaphragm and the heart. Sheep were bled at weekly intervals for the first 8 weeks, at the time of challenge infection (week 25) and two weeks after challenge infection (weeks 27 and 28) for the collection of peripheral blood mononuclear cells (PBMC) and plasma.

a further 2 h, after which 100 ~1 of extraction buffer (20% sodium dodecyl sulphate in 50% aqueous N, Ndimethyl formamide, pH 4.6) was added to dissolve formazan crystals. The plates were incubated overnight at 37°C and the optical density (OD) was read in a BioRad Microplate reader at 570 nm wavelengthlO. The cell proliferation responses were expressed as the OD difference between antigen-stimulated cultures and unstimulated control cultures (AOD 570). Enzyme linked immunosorbent (ELISA) assay Anti GST-45W antibodies were measured by ELISA. ELISA plates (Titertek/Flow) were coated with GST45W (10 pg ml-‘) in coating buffer (100 mM bicarbonate buffer, pH 9.6), blocked with bovine serum albumin (1% BSA, in coating buffer) and washed with washing buffer (0.1% BSA, 0.05% Tween 20 in 10 mM phosphate buffered saline, pH 7.4). Sheep sera diluted in washing buffer were applied and plates incubated at 4°C for 2 h. Antigen-specific antibodies were visualized with peroxidase-conjugated rabbit anti-sheep immunoglobulins (Dakopatts, Denmark) and the substrate ABTS [2, 2’-azino-bis (3-ethylben-thiazoline-6-sulfonic acid) diammonium salt, Sigma, USA]. The antibody activity was expressed as a multiple of normal activity (MONA) units”. Total antibody activity was determined in samples collected at weeks 0, 4, 8, 25 and 28. IgGl and lgG2 subclasses were also measured with ELISA using monoclonal antibodies against sheep IgGl and lgG2 (kindly supplied by Dr Beh, CSIRO, Australia), followed by peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dakopatts, Denmark). The antibody activity of IgGl and lgG2 subclasses were expressed as ELISA units which are defined as the ratio of the optical density (OD) reading of a test serum to a standard serum”. The ratio was then multiplied by the dilution of the sera, which was adjusted according to the slope of the standard curve of the respective isotype (ratio x dilution x slope of the standard curve). Antibody activity for IgGl and lgG2 was determined on weeks 8 and 25.

Cell proliferation assay Peripheral blood mononuclear cells were isolated from blood collected in EDTA. Briefly, after centrifugation (4OOg, 25 min), the buffy coat was collected, diluted with Ca2’/Mg2’-free balanced salt solution (BSS), layered over Ficoll-paque (Pharmacia, Sweden) and centrifuged (4OOg, 25 min). The interface was collected, washed in BSS and resuspended in 0.16 M Tris-NH,Cl buffer pH 7.2 to lyse the red blood cell. The cells were washed twice in BSS and finally resuspended in complete medium (RPM1 1640 supplemented with 5% foetal calf serum, 100 IU ml-’ penicillin, 100 pg ml-’ streptomycin, 2 mM L-glutamine, 25 mM HEPES buffer, 0.05 mM 2-mercaptoethanol). The cells were incubated in 96-well flat bottomed tissue culture plates at 25~10~ cells well“ in 200 ~1 total volume and a final antigen (45W) concentration of 10 pg ml-‘. The cultures were incubated in humidified atmosphere containing 7% CO, at 37°C for 5 days. On day 5, 100 ~1 of the supernatant was removed from each well and 20 ~1 of tetrazolium salt (MTT, 5 mg ml-’ in PBS) was added to each well. The cells were cultured for

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Statistical analysis The statistical analysis of the cell proliferation responses, humoral responses, and of the number of cysts at necropsy, was performed by analysis of variance (ANOVA) after log transformation of the data. The four adjuvant groups were compared at each sampling time, with the saline group and among themselves by the Fisher PLSD test. Statistical significance was defined at the p < 0.05 level. RESULTS Cell proliferation responses The means of cell proliferation responses to 45W for the different groups are shown in Figure 1. All adjuvant groups had significantly higher responses than the saline group at week 2 only @ c 0.05). There was no significant difference in the response to the booster injection. The cell proliferation response between all groups showed no significant difference at the time of challenge infection.

Influence of gamma inulin and Algammulin:

Challenge infection di’d not induce significant differences in cell proliferation responses between the different groups. Humoral immune responses Total antibody responsles of sheep to GST-45W are shown in Table I. Total antibody response of all experimental animals to GST-45W prior to the start of the experiment (week 0) was 0.365*0.040 MONA units (x 10, mean k S.E.). All the adjuvant groups showed 0.200

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H.S. Deol et al.

primary and secondary antibody responses at weeks 4 and 8 which were significantly higher than those in the saline group (~~0.05). At week 25, the time of the challenge infection, the antibody titres had dropped in all adjuvant groups compared to week 8. However, the alum, Algammulin and FCA/FIA group still had significantly higher antibody titres than the saline group (p < 0.05). At week 28, all the groups except FCA/FIA showed a significant rise in the antibody titre following challenge infection, but only the alum, Algammulin and FCA/FIA groups showed significantly higher titres than the saline group @ < 0.05). All the adjuvant groups produced higher IgGl and IgG2 levels at week 8 compared to the saline control group (p < 0.05, Table 2). At week 25, all the adjuvant groups still had significantly higher levels of IgGl than the saline control group and only alum and FCA/FIA groups had significantly higher IgG2 levels (p < 0.05). Number of cysts at necropsy and their relationship with the immune responses at the time of challenge

0

2

4

6

8

25

27 28

Weeks Figure 1

Mean cell proliferation responses to 45W expressed as AOD 570 nm In the saline (!I), alum (0) gamma inulin (0) Algammulin (A), and FCA/FlA (Cl) group. Animals were vaccinated at week 0, boosted at week 4 .and challenged at week 25. Alum, gamma inulin, Algammulin ancl FCAIFIA group had significantly (p< 0.05) higher responses than the control group at week 2 only

The number of T. ovis cysts at necropsy and the total antibody response at the time of challenge infection is shown in Table 3. The average number of cysts was 27 in the saline group, 15 cysts in the alum group, 14 cysts in the gamma inulin group, 20 cysts in the Algammulin group, and 0.5 cysts in the FCA/FIA group. However, only FCA/FIA group differed significantly from the saline group @ < 0.05). The number of cysts at necropsy correlated with the total antibody titre (r= -0.82, p < O.OOl), IgGl titre (r = -0.82, p < O.OOl), IgG2 titre (Y= -0.81, p c 0.001) but not with the cell proliferation responses (r = -0.32, p > 0.05) at the time of challenge infection (week 25).

Table 1 Total antibody responses of sheep to GST-45W (mean f SE.) expressed in MONA units (x 10). Animals were given the primary immunization at week 0, the booster at week 4 and the challenge infection at week 25 Total antibody titre (MONA units x 10) at week Group

4

8

25

28

Saline Alum Gamma inulin Algammulin FCAfFIA

0.33*0.08” .I3.86i4.97” 3.34*1 .68b 9.21*2.01 G 66.09i19.25d

0.53*0.13” 20.3Oill. 18” 17.00*10.86” 15.64*2.37” 121.23e31.75’

0.24*0.04” 2.03il.18” 1.5Oil .03”,b 1.22*0.17” 38.1Oe9.65’

2.28iO.59” 15.OO~tlO.56” 5.19*1.51”,* 13.51~6.34” 54.58*12.62*

a.b.c.dMeans in a column with no common superscripts

differ significantly

(p< 0.05)

Table2 IgGl and lgG2 antibody responses of sheep to GST-45W expressed as ELISA units (mean * S.E.). Animals were given the primary immunization at week 0, the booster at week 4 and the challenge infection at week 25

-

IgGl

lgG2

Group

Week 8

Week 25

Week 8

Week 25

Saline Alum Gamma inulin Algammulin FCA/FIA

32.35i4.73” 1;!70.55i277.16’ 1;!39 94*338.68b 1158:91*127.72b 9;!02.88*706.63”

17.01*2.53” 158.61 i43.40b 115.48*43.99’ 131.48i18.32’ 2674.48k336.31 c

4.83kO.53” 16.03*2.726 13.68k3.08’ 14.28i1.15’ 31.85k1.62’

5.59il.45” 1 2.05*2.33b 8.411~2.98”,~ 7.62*1 35”,b 28.96*2:39”

e,b~cMeans in a column with no common superscripts

differ significantly

(~~0.05)

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Number of

and Algammulin:

H.S. Deal et al.

Taenia014scysts at necropsy and total antibody titre (MONA units x IO) at the time of challenge infection

Group

Mean

Saline Alum Gamma inulin Algammulin FCA/FIA

27” 15” 14” 20” 0.50b

Number of cysts at necropsy 8(0.39) 3(1.59) 7(0.54) 7(1.21) O(63.48)

a,b Means in a column with no common superscripts

13(0.09) g(7.86) 8(0.14) 14(0.72) O(52.83) differ significantly

DISCUSSION The present study examined the relative efficiency of two new adjuvants, gamma inulin and Algammulin, in augmenting CM1 and antibody responses in sheep to a recombinant T. ovis antigen. The ability to protect sheep from challenge with live T. ovis eggs was also assessed and the two new adjuvants were compared with conventional adjuvants such as alum and FCA/FIA. The measurement of CM1 is considered important for the assessment of new adjuvants’ and was measured in the present study by in vitro stimulation of PBMC with the specific antigen. Gamma inulin has been shown to increase CMI responses in mice”. In the present study in induced sheep, gamma inulin and Algammulin significantly higher CM1 responses than saline two weeks after primary immunization. The CM1 response was similar for both preparations and was comparable in magnitude to FCA/FIA group. FCA is known to be an excellent inducer of CMI’, 4. Alum also induced a significantly higher CM1 response than saline, which is contrary to findings by others” “, but it has been reported in sheep previously’3. Gamma inulin and Algammulin boost antigenspecific antibody responses in mice6,‘. Algammulin has a synergistic effect in mice because it is 6- to 9-fold more potent for inducing antibody responses than its individual components’. In our study with sheep, gamma inulin, Algammulin and alum performed similarly and induced significantly higher primary and secondary antibody responses than saline at weeks 4 and 8. Immunological memory at week 25 was similar alum inulin, Algammulin and for gamma (approximately 10% of peak values at week 8) however, the recall memory (at week 28) after experimental challenge infection at week 25, was 2- to 3-fold higher for alum and Algammulin than for gamma inulin. FCAI FIA’s adjuvant group outperformed all other adjuvants by 6- to &fold in respect to peak responses at week 8, by 19- to 3 l-fold in respect to immunological memory at week 25, and by 4- to lo-fold in respect to recall memory at week 28. Investigation of the effect of adjuvants on the antibody activity of IgG subclasses is important for vaccines intended for use in ruminants. Colostral antibodies which are predominantly of the subclass IgG114 can protect offspring of vaccinated dams. T. ovis vaccine has been shown to protect lambs of vaccinated ewes”. The regulatory mechanisms for IgGl and IgG2 production in sheep are not fully understood, therefore direct comparison of adjuvant activity on IgG subclassses between different animal species is not possible. Furthermore, the effect of adjuvants on the immune responses can also depend on the antigen used,

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(total antibody titre at the time of challenge 23(0.3) 1O(0.72) 12(0.19) 1 a(O.90) O(26.40)

33(0.21) 17(0.33) 16(6.63) lg(1.85) O(60.45)

infection) in each animal

34(0.29) 24(0.64) 17(0.57) 20(1.05) l(10.95)

52(0.15) 24( 1.02) 23(0.92) 41 (I .58) 2(14.48)

(p< 0.05)

because adjuvants can modulate immune responses in an “antigen-dependent” manner16, 17. Gamma inuli#* 7 and Algammulin’ have been shown to favour IgG2 over IgGl production in mice. In sheep it is known that alum does not favour either IgGl or IgG2 production and FIA favours IgGl over IgG213. In our study with sheep, all adjuvant preparations boosted IgGl more than IgG2. We observed a relative increase of IgG2 in the second half of the immunization experiment (week 25) a finding shared by others”. This indicates either an increased degradation of IgGl over the 17 week period or more likely an increased production of IgG2 in the later period of the immune response. In a vaccine adjuvant study, challenge is the ultimate test for relevant immune responses. In this study, only the FCA/FIA group was protected from the challenge infection. This confirms the findings of a previous report’ that a significant protection can be achieved by incorporating FCA in the T. ovis vaccine. We achieved a higher level of protection (98O> than in the previous report (79%)‘. There was no significant correlation between the T-cell proliferation responses at the time of challenge infection and the number of cysts at necropsy. However, a significant correlation was found between the antibody titres (total antibody, IgGl, IgG2) at the time of challenge infection and the number of cysts at necropsy. Our results suggest that protection against T. ovis is antibody (IgG) mediated as reported by others’. “. I’. The present study shows that immunization with gamma inulin, Algammulin or alum failed to protect sheep against challenge infection with T. ovis eggs, probably because they failed to induce a high enough antibody response. Contrary to findings in mice’, no synergism was observed in sheep between the two elements of Algammulin, i.e. gamma inulin and alum. Algammulin behaved more like alum in respect to antibody responses and like gamma inulin in respect to CM1 responses. The differences between the immune responses in sheep and mice with Algammulin could be due to species differences in the activation of complement. It is possible that in sheep the activation of complement, which is the basis for the adjuvant activity of gamma inulin and Algammulin, is only important for CM1 responses but not for antibody responses (P. D. Cooper, personal communication). The antigen load of the Algammulin particles is also critical for the optimization of the immune response in mice’. We used the optimal antigen load for mice in our sheep vaccination trial because the most effective antigen load for sheep is not known. Our results highlight the varied responses adjuvants induce in different animal species. These findings

Influence of gamma inulin and Algammulin:

support the argument that the immunomodulatory effect of an adjuvant should be studied in the target species before being used in vaccination trials.

10

ACKNOWLEDGEMENTS 11

This work was supported by a grant (UMUl 1) from the Wool Research and Development Corporation. The authors thank MS H. Kyme (Murdoch University, Murdoch) for her hel-p with the immunization of sheep. REFERENCES Warren, H.S. and Chedid, L.A. Future prospects for vaccine adjuvants. CRC Chit. Rev. Immunol. 1988, 8, 83-l 01 Vanselow, B.A. The application of adjuvants to veterinary medicine. Vet Bull 1987, 57, 881-896 Allison, A.C. and Byars, N.E. Adjuvants for a new generation of vaccines. In: New Generation Vaccines (Eds Woodrow, G.C. and Levine, M.M.). Marcel Dekker, New York, 1990, pp. 129-140 Smith, N.C. Concepts and strategies for anti-parasite immunoprophylaxis and therapy. lnt. J. Parasit. 1992, 22, 1047-l 082 Gupta, R.K., Relyveld, E.H., Lindblad, E.B., Bizzini, B., BenEfraim, S. and Gupta, C.K. Adjuvants-a balance between toxicity and adjuvanticity. Vaccine 1993, 11, 293-306 Cooper, P.D. and Steek?, E.J. The adjuvanticity of gamma inulin. Immunol. Cell Biol. 1988, 66, 345-352 Cooper, P.D., McComb, C. and Steele, E.J. The adjuvanticity of Algammulin, a new vaccine adjuvant. Vaccine 1991, 9, 408-415 Cooper, P.D. and Carter, M. Anticomplementary action of polymorphic “solubility forms” of particulate inulin. Mokc. Immunol. 1986, 23, 895-901 Johnson, KS., Harrison, G.B.L., Lightowlers, M.W., O’Hoy, K.L., Cougle, W.G., Dempster, R.P. et a/. Vaccination against

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ovine cysticercosis using a defined recombinant antigen. Nature 1989, 338, 585-587 Hansen, M.B., Nielsen, S.E. and Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Meth. 1989, 119, 203-210 Tijssen, P. Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology (Eds Burdon, R.M. and van Knppenberg, P.M.). Elsevier Science Publishers, Amsterdam, 1985, pp. 403-407 Reynolds, G.E., Suttie, J. and Griffin, J.F.T. Humoral immunity in the ewe 1. The influence of adjuvancy and immunogenic subsitution on immune reactivity following immunisation with protein antigens. Vet. lmmunol. Immunopathol. 1990, 25, 1-12 Emery, D.L., Rothel, J.S. and Wood, P.R. Influence of antigens and adjuvants on the production of gamma-interferon and antibody by ovine lymphocytes. Immunol. Cell Biol. 1990, 68, 127-l 36 Lascelles, A.K., Beh, K.J. and Husband, A.J. Origin of antibody in mammary secretion with particular reference to the IgA system. In: The Ruminant Immune System. Advances in Experimental Medicine and Biology (Eds Butler, J.E., Duncan, J.R. and Nielson, K.). Plenum Press, New York, 1980, 137, 493-511 Lightowlers, M. W. and Rickard, M.D. Vaccination against cestode parasites. Immunol. Cell Biol. 1993, 71, 443-451 Tiong, G.K.L., Gill, M.S., Lofthouse, S. and Puri, N.K. Comparsion of conventional adjuvants and “adjuvant-free” monoclonal antibody targeting for stimulating antibody responses against a conjugate of luteinizing hormone releasing hormone and avidin. Vaccine 1993, 11, 425-430 Fox, B.S. Antibody responses to a cytochrome c peptide do not correlate with lymphokine production patterns from helper T-cell subsets. Immunology 1992, 75, 164-169 Rickard, M.D. and Williams, J.F. Hydatidosis/cysticercosis: immune mechanisms and immunization against infection. In: Advances in Parasitology (Eds Baker, J.R. and Muller, R.). Academic Press, London, 1982, 21, pp. 230-296

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