Veterinary Parasitology, 43 (1992) 105-113 Elsevier Science Publishers B.V., Amsterdam
105
vaccination of mice with liposome encapsulated antigen
Ascaris suum
S. Lukes Institute o f Parasitology, CSA V, Brani~ovskd 31, 37005 Ceskk Bud~jovice, Czechoslovakia
(Accepted 28 November 1991 )
ABSTRACT Lukes, S., 1992. Ascaris suum - - vaccination of mice with liposome encapsulated antigen. Vet. Parasitol., 43:105-113. Vaccination with liposome encapsulated adult crude antigen with and without coencapsulated immunomodulator (levamisole) in a mice/larval Ascaris suum model provided protection against a challenge infection (2000 eggs) in mice immunised by immobilised antigen. The best results (88.9% protection) were obtained with a combination of two doses of liposome entrapped antigen with levamisole. Vaccination with liposome vaccine without modulator was slightly less effective (78.7% protection ). A single dose of vaccine was ineffective ( 14.3% protection). Application of the soluble antigen without any adjuvants led to the enhancement of worm yield in lungs and liver.
INTRODUCTION
The common trend in vaccine development is to identify the component of infective agents that are responsible for stimulating protective immune responses. Usually these soluble antigens are poor immunogens and require strong adjuvants or appropriate use of immunostimulators. A safe and completely harmless adjuvant has not yet been developed (Bomford, 1989). One possibility for a safe and effective delivery system is the use of liposomes (Bangham, 1980). Since it was first realised that liposomes have immunoadjuvant action (Allison and Gregoriadis, 1974), they have been used with many antigens (Van Rooijen and Van Nieuwmegen, 1983; Gregoriadis et al., 1989 ), including parasites (Richards et al., 1988; Alwing and Richards, 1990). Previous experiments demonstrated encouraging results with cestode infections in model (mouse, Taenia crassiceps) or natural (sheep, Moniezia ) (Lukes, 1991 ) infections. In this paper we report the supportive effect of liposomes on the model Correspondence to: S. Lukes, Institute of Parasitology, CSAV, Branigovsk~i 31, 37005 Cesk6 Bud6jovice, Czechoslovakia.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
106
s. LUKES
nematode infection in mice with and without immunostimulative agents, i.e. levamisole. MATERIAL AND METHODS
Animals
Outbred female ICR mice (VELAZ, Prague), weighing 20-25 g at the beginning of the experiment, were used. Mice were maintained in a vivarium at the Institute in standard plastic cages with eight to ten individuals per group. They were fed a Larsen diet (LD, VELAZ, Prague) and provided with water ad libitum. Immunomodulation
Levamisole (7.5% solution) (NILVERM, inj. ad usum veterinarium, BIOVETA, Ivanovice na Hane) was used as modulator of the i m m u n e response during vaccination. The dose used corresponds to 4 mg kg-~ body weight (for immunostimulation in domestic animals, doses of 3-10 mg kghave been used) (Euzeby, 1986). Parasite antigen
Adult worms ofAscaris s u u m obtained in local abattoirs from infected pigs were repeatedly washed carefully with tap water until surface contamination was removed. The last washing was carried out in phosphate buffered saline (PBS, pH 7.2, 0.15 M, at room temperature). The same solution was used for extraction of soluble antigens from the whole body homogenate. After dissection into small pieces and homogenisation on a vortex mixer for 5 min, the crude fraction was sedimented by low speed (3000 rev m i n - 1, 15 min) centrifugation and the homogenate purified by high speed centrifugation in a cooled centrifuge ( 10 000 rev m i n - ~, 1 h). The protein content was determined by Folin reagent (Lowry et al., 1951 ) with use of bovine serum albumin as standard. Infective eggs
Infective eggs of A. s u u m were prepared as previously described (Lukes, 1982). Eggs liberated from dissected uteri of female worms were incubated at room temperature for 3 h under shaking in 0.5% HC1, washed with water and gently centrifuged (up to 2000 rev min-1, 15 min), transferred onto filter paper placed on a watch glass and connected with paper bridges to the bottom part of a Petri dish filled with 0.05% potassium dichromate. In these
VACCINATION AGAINST ASCARIS SUUM IN MICE
| 07
dishes the eggs were incubated for at least 28 days at 27 °C and then observed microscopically for developing larvae. Before inoculation the eggs were washed with water and the number of full developed larvae determined.
Vaccine preparation The liposome preparation described by Claassen et al. (1987) with minor modification was used. Soybean phosphatidyl choline (Sigma Chemical Co., St. Louis, M O ) and cholesterol (Lachema, CSFR) (molar ratio 3.5:1 ) were dissolved in chloroform, evaporated in thin film on the wall of the tubes (5 ml of phospholipid solution per tube) and then rehydrated with PBS solution of the antigen ( 10X 100 mg in 1 ml of PBS per tube, groups AS3-AS6) and i m m u n o m o d u l a t o r (groups AS4-AS6). The process of preparing the vesicle suspension was supported by mixing on the shaker. After full dispersion the lipid film was left to stand in the tubes for 2 h at room temperature and then the suspension was sonicated in an ultrasonic bath with intensive cooling (ice cubes) for 3 X 1 min. After sonication the suspension was stored in a freezer before inj ection. All solutions (antigen and modulator) were sterilised before preparation of vaccine by filtration. Before use, the suspension was diluted with sterile PBS to a concentration of 100 mg of protein in one dose of vaccine.
Experimental design Mice were vaccinated with the same doses of antigen (100 mg) either in liposomes with or without i m m u n o m o d u l a t o r or without the liposome adjuvant combination. Vaccination was done subcutaneously in all cases (0.5 ml of suspension per mouse). Group AS 1 served as non-immunised controls and were injected with PBS only on Days 1 and 14. Group AS2 served as a soluble antigen control and was injected with two doses of soluble antigen in PBS on Days 1 and 14. Group AS3 was immunised by two doses of liposome entrapped antigen without modulator, and group AS4 with two doses of soluble antigen mixed with levamisole on Days 1 and 14. Groups AS5 and AS6 were i m m u n i s e d with two (AS5) or one (AS6) (Days 1 and 14, and Day 1, respectively) dose of antigen coencapsulated with i m m u n o m o d u l a t o r into liposomes. On Day 14 after the last injection, mice were challenged by 2000 infective eggs ofA. suum per os (by feeding needle). Mice were bled under light anaesthesia from the orbital sinus using fine Pasteur pipettes before injection, reimmunisation and challenge on Day 28. The last serum samples were taken immediately before killing. Serum samples were stored at - 20 oC before evaluation of antibody reactivity.
S. LUKES
108
Assessment of antibody production For the measurement of the specific antibodies in the serum of experimental animals the (ELISA) technique (Engvall and Perlmann, 1979) was used. We used polystyrene plates (GAMA, Dalecin), coated with crude antigen preparation in coating buffer (0.625 mg ml-~ of buffer, determined by titration). Samples were diluted in PBS with 0.05% Tween-20 (PBS-T-20) 1 : 100, incubated for 1 h at 37 ° C. All washing steps (repeated three times) were done using PBS-T-20 solution. Swine anti-mouse Ig peroxidase conjugate (SWAM/ Px, 1 : 2000, SEVAC, Prague) was diluted in PBS-T-20 and incubated for 1 h at 37 ° C. The substrate was 40 mg ortho-phenylenediaminein 100 ml citrate buffer (pH 5.5) with 40 ml of hydrogen peroxide. The reaction was stopped with 2.5 M sulphuric acid and evaluated by MR 580 (Dynatech) at 492 nm. The reactivity o f the serum samples was recorded as optical density of sample at 492 nm at 1 : I00 serum dilution. TABLE 1 Effectiveness of various vaccine preparations against A. munisation of mice Group
Vaccine
Dose ~
No. of infected mice 2
ASI
0 (PBS)
2M 4
10of 10
AS2
ag
2X
10 of 10
AS3
Liposome ag Levamisole ag Liposome Levamisolc ag Liposome Levamisole ag
2×
7 of 8
2×
7of10
AS4 AS5
AS6
2×
1×
4 of 9
7 of 9
suum
infection and worm burdens after im-
No. of larvae recorded (mean and range)
Protection 3 (%)
Lungs
Liver
Total
14.0 (0-40) 43.2 (0-192) 30.5 (0-92) 2.6
10.4 (4-24) 30.0 (0-108) 3.5 (0-12) 1.6
24.4 (8-64) 73.2 (16-196) 34.0 (0-92) 5.2
(0-16)
(0-8)
(0-16)
2.4 (0-8)
2.8 (0-8)
88.9
(0-4) 14.2 (0-20)
6.7 (0-44)
20.9 (0-48
14.3
0.4
78.7
PBS, phosphate buffered saline, pH 7.2. ag, Total worm homogenate. Levamisole: one dose corresponds to 4 mg kg l body weight. ~One dose of antigen corresponds to 100 mg of protein. 2Mice challenged with 2000 infective eggs per os 14 days after last injection of vaccine. 3protection = (mean number of larvae in control g r o u p - m e a n number of larvae in treated group )/ mean number of larvae in control group × 100 (Tendler et al., 1986 ). 41njections on Days I and 14.
109
VACCINATION AGAINSTASC4RIS SUUM IN MICE
Effectiveness of vaccination against challenge infection Mice were killed by cervical dislocation on Day 7 post-infection with 2000 infective eggs of A. suum. The lungs and liver were prepared and digested separately in pepsin solution after being minced to small pieces of tissue. After 3 h of agitation at 37°C in a water bath, the rest of the tissue block was discarded by gauze filtration and larvae in five 50-ml aliquots were counted under the dissecting microscope. Larvae numbers were enumerated on the full volume of digestive fluid obtained from the sample. All animals of each group were observed separately. Effectivity of vaccination was assessed by larval counts in lungs and liver on Day 7 post-infection (Table 1 ). RESULTS
Immunisation of mice with soluble A. suum antigen and immobilised and potentiated antigen preparations elicited different degrees of resistance among experimental groups. The results of the trial are summarised in Tables 1 and 2. We found significant degrees of induced protection after vaccination with soluble antigens incorporated into liposomal particles and combined with levamisole (group AS5, Table 1 ), vaccinated twice 14 days apart. In these cases both parameters (effectiveness of infection and decrease of worm burden) were better than in other groups. The effectiveness of infection decreased to 44.4% and protection was 88.9%. Other combinations of either antigen alone in liposomes (group AS3) or soluble antigen with levamisole (group AS4) gave lower values for both parameters (effectiveness of infection 87.5% and 70.0%, respectively, and protection in AS4, 78.7%). The solTABLE 2 Measurement of antibody production against A. s u u m in vaccinated and infected mice (mean optical density value of group and SD) Day
AS 1
AS2
AS3
AS4
AS5
AS6
1
0.244 0.030 0.210 0.026 0.133 0.048 0.198 0.053
0.250 Ns 0.041 0.381"* 0.085 0.718*** 0.152 0.735*** 0.110
0.226 Ns 0.077 0.317 ys 0.121 0.575*** 0.202 0.765*** 0.298
0.316" 0.107 0.320 Ns 0.109 0.505*** 0.166 0.464*** 0.208
0.294 Ns 0.110 0.257 Ns 0.070 0.512*** 0.227 0.467*** 0.124
0.287 Ns 0.077 0.280** 0.070 0.212** 0.063 0.208*** 0.066
Before immunisation Before reimmunisation Before infection
28
After challenge
35
14
Significance was determined by non-paired Student's test. *P< 0.05; **P< 0.01; ***P< 0,001; NS, non-significant.
110
S. LUKES
uble antigen alone and antigen with liposomes without modulator (groups AS2 and AS3, respectively) induced some degree of tolerance to parasite infection, as demonstrated by enhanced number of liver and lung larvae in groups AS2 and AS3 (Table 1 ). Some changes exist in reactivity in group AS3 (antigen in liposomes) demonstrable by comparison of effectivity of infection (see Table 1 ). Little protection was induced in group AS6, immunised once with the liposome/levamisole/antigen combination.
Antibody production in experimental mice after vaccination Results of measurement of the antibody production in vaccinated and infected mice are summarised in Table 2. With the exception of group AS4, groups were without difference in antibody reactivity before the initiation of the trial (ELISA mean optical density (+_SD) ranged from 0.226, ( + 0 . 0 7 0 ) to 0.316 ( + 0 . 1 0 7 ) ). The difference shown by group AS4 at a minor degree of significance is probably a result of non-specific reaction with crude antigen. After first injection of antigens and vaccine combination we detected various degrees of reactivity with crude antigen, depending on the type of vaccine used. We found a surprisingly high production of antibodies in mice injected with crude, non-treated extract of the adult worms (group AS2) after the second injection on Day 28 (0.718 +_0.150), but these antibodies were not protective. We found some degree of tolerance and an enhanced worm burden in this group. Differences between groups before challenge were not significant. After the second injection in groups AS2-AS5 a significant increase of antibody reactivity was detectable. These differences were prolonged before the time of challenge. After challenge in groups AS2 and AS3 we detected further increase of antibody reactivity. Most prominent was the increase in group AS2 (0.919 + 0.110) (Table 2). In liposome vaccinated groups (AS4-AS6) no further increase of antibody levels was observed. DISCUSSION
Many authors have examined the possibility of immunisation against nematode infection, including the A. suum model. Most of the trials were based on the use of soluble extracts or secreted products of worms. In these experiments only minor resistance was induced without the use of strong adjuvants (Bindseil, 1969; Guerrero and Silverman, 1972; Stromberg and Soulsby, 1977). Irradiated infective eggs were partially effective (Gaur and Dutt, 1976). Further experiments (Urban and Tromba, 1984) showed the possibility of induced protective responses after multiple inoculation of UV-irradiated infective eggs (protection up to 94% in pigs), but for field use the system is too complicated and impractical.
VACCINATION AGAINST ASCARIS SUUM IN MICE
| ]1
We have demonstrated some degree of protection after injection of liposome immobilised antigen (groups AS3-AS6), especially after repeated injection of levamisole potentiated vaccine (group AS5). In these groups not only the number of larvae (mean of 2.7 per mouse), but even the overall success of the infection (44.4%) was depressed. Liposomes have one advantage over other effective adjuvants in that no granulomata are formed at the site of infection (Allison and Gregoriadis, 1974 ). A further advantage of the use of liposomes is the avoidance of hypersensitivity reactions to unentrapped antigen (Gregoriadis and Allison, 1974). It has been shown that the immune response against liposome associated protein antigens is T-lymphocyte (Beatty et al., 1984) and macrophage (Shek and Lukovich, 1982) dependent. As demonstrated by Bindseil ( 1971 ), the protective effect of acquired immunity to A. s u u m larvae is based on T-cell immunity. Further confirmation of the hypothesis was provided by Hermanek and Koudela (1990) who demonstrated the supportive role of thymic preparations on larval ascaridosis in mice. Our hypothesis of the construction of the liposome-levamisole potentiated vaccine was based on the supportive effect oflevamisole in vaccination against cestodes in a model system (Lukes, 1988). Because levamisole can also increase T-lymphocyte proliferation (Renoux and Renoux, 1977a,b; Symoens et al., 1979 ) the combination of adjuvant effect ofliposome alone and proliferative effect of modulator may be advantageous. The danger of direct action of levamisole on migrating larvae is avoided by its short half-life (about 4 h) and quick metabolism in mammalian organisms - - total elimination within 2 days (Symoens et al., 1979). Because we challenged the mice after 14 days, sufficient time elapsed for the clearance of the drug before challenge. The prolonged influence of levamisole necessary for stimulation of lymphocytes, may be a result of production of thymus-like substances (Renoux and Renoux, 1977c). There has been only one previous attempt to use liposomes in vaccination against A. s u u m infection in pigs (Rhodes et al., 1988 ). A low degree of resistance was induced only after a priming dose of eggs and consequent vaccination with the liposome preparation. No reaction was observed without priming. It is possible that the peroral or intraintestinal route of application of vaccine was not optimal for inducing reaction (Yasui and Ohwaki, 1983). From the point of view of monitoring of degree of protection, it is interesting that in the A. s u u m model infection of mice antibody reactivity does not correlate with the degree of protection, as no correlation between antibody reactivity and resistance was noted. The comparatively large variation in individual antibody levels in the groups of mice depend on the individual genetically based reactivity to the antigens (Kennedy, 1989).
112
s. LUKES
CONCLUSION
Liposomes with incorporated soluble A. s u u m antigen are able to elicit a protective immune response in mice against migrating larvae ofA. s u u m . The effect of liposomes was further potentiated by coencapsulation of the levamisole as immunomodulator. Soluble antigens without modulator or liposome induced tolerance of enhanced worm burdens in the hosts. ACKNOWLEDGEMENTS
Thanks are due to R. Vtelenska and R. Bruzkova for excellent technical assistance.
REFERENCES Allison, A.C. and Gregoriadis, G., 1974. Liposomes as immunological adjuvants. Nature, 252: 252. Alwing, C.R. and Richards, R.L., 1990. Liposomes containing lipid-A: A potent nontoxic adjuvant for a human malaria sporozoite vaccine. Immunol. Lett., 25: 1-3. Bangham, A.D., 1980. Development of the liposome concept. In: G. Gregoriadis and A.C. Allison (Editors), Liposomes in Biological Systems. John Wiley, New York, pp. 1-24. Beatty, J.D., Beatty, B.G., Paraskevas, F. and Froese, E., 1984. Liposomes as immune adjuvants: T cell dependence. Surgery, 96: 345-351. Bindseil, E., 1969. Immunity to A s c a r i s s u u m . 1. Immunity induced in mice by means of material from adult worms. Acta Pathol. Microbiol. Scand., 77:218-222. Bindseil, E., 1971. Immunity to A s c a r i s s u u m . 5. The effect of X-radiation and neonatal thymectomy on a primary infection in mice. Acta Pathol. Microbiol. Scand., 79:511-518. Bomford, R., 1989, Adjuvants for anti-parasite vaccines. Parasitol. Today, 5: 41-46. Claassen, E., Kors, N. and van Rooijen, N., 1987. Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphone and surface associated antigen as hapten. Immunology, 60:509-515. Engvall, E. and Perlmann, P., 1979. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry, 8: 871-874. Euzeby, J.P., 1986. Propri6t6s immunostimulantes du Levamisole. 2. Applications en mddicinc vdtdrinaire et effects secondaires. Rev. Med. Vet., 137: 499-520. Gaur, S.N.S. and Dutt, S.C., 1976. Studies on irradiated form of A s c a r i s s u u m : immunizing effect of irradiated A. s u u m eggs. Indian Vet. J., 53: 305-306. Gregoriadis, G. and Allison, A.C., 1974. Entrapment of proteins in liposomes prevents allergy reactions in preimmunized mice. FEBS Lett., 45:71-75. Gregoriadis, G., Davis, D., Garcon, N., Tan, L., Weissig, V. and Xiao, Q., 1989. The immunoadjuvant action of liposomes. In: G. Lopez-Berenstein (Editor), Liposomes in the Therapy of Infectious Diseases and Cancer. UCLA Symposia, Vol. 89, pp. 35-56. Guerrero, J. and Silverman, P.H., 1972. Active immunization against A s c a r i s s u u m minimum lethal dose in mice. Z. Parasitenkd., 39: 339-344. Hermanek, J. and Koudela, B., 1990. Influence of nonspecific immunomodulation with thymic
VACCINATION AGAINST ASCARIS SUUM IN MICE
113
preparations on the result of infection in some experimental parasitoses. In: K.N. Masihi and W. Lange (Editors), Immunotherapeutic Prospects of Infectious Diseases. Springer, Berlin, pp. 71-76. Kennedy, M.W., 1989. Genetic control of the immune repertoire in Nematode Infections. Parasitol. Today, 56: 316-324. Lowry, O.H., Rosebrough, N.R., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Lukes, S., 1982. Immunological methods in experimental migration of larvae of some ascarids in rabbits. Ph.D. Thesis, Institute of Parasitology, CSAV, Czechoslovakia, 266 pp. Lukes, S., 1988. Levamisole - - biological response modifier in Taenia c r a s s i c e p s - mouse experimental system, Folia Parasitol., 35: 37-40. Lukes, S., 1991. Preliminary results of the experiments with vaccines against helminths on basis of immobilized antigens. In: Proceedings of a Conference on Parasitoses of Domestic Animals, 1989, C. Budejovice, (in Czech), pp. 123-126. Renoux, G. and Renoux, M., 1977a. Influence du levamisole sur la production ~ anticorps. Ann. Immunol. Inst. Pasteur, 128c: 273-274. Renoux, G. and Renoux, M., 1977b. MOchanismes d'action du levamisole, stimulant des responses d'immunit6 cellulaire. Ann. Immunol. Inst. Pasteur, 128c: 275-277. Renoux, G. and Renoux, M., 1977c. Thymus-like activities of sulphur derivatives on T-cell differentiation. J. Exp. Med., 145: 466-471. Rhodes, M.B., Baker, P.K., Christensen, D.L. and Anderson, G.A., 1988. Ascaris suum antigens incorporated into liposomes used to stimulate protection to migrating larvae. Vet. Parasitol., 26: 343-349. Richards, R.L., Hayre, M.D., Hockmeyer, W.T. and Alving, C.R., 1988. Liposomes, lipid A, and aluminium hydroxide enhance the immune response to a synthetic malaria sporozoite antigen. Infect. Immun., 56: 682-686. Shek, P.N. and Lukovich, S., 1982. The role of macrophages in promoting the antibody response mediated by liposome-associated protein antigens. Immunol. Lett., 5: 305-309. Stromberg, B.E. and Soulsby, E.J.L., 1977. Ascaris suum: immunization with soluble antigens in the guinea pigs. Int. J. Parasitol., 7: 287-291. Symoens, J., Rosenthal, M., de Brabander, M. and Goldstein, G., 1979. Immunoregulation with levamisol. Springer Semin. Immunopathol., 2: 49-59. Tendler, M., Pinto, R.N., Lima, A.O., Gebara, G. and Katz, N., 1986. Schistosoma mansoni: vaccination with adult worm antigens. Int. J. Parasitol., 16: 347-352. Urban, J.F. and Tromba, F.G., 1984. An ultraviolet-attenuated egg vaccine for swine ascariasis: Parameters affecting the development of protective immunity. Am. J. Vet. Res., 45: 21042108. Van Rooijen, N. and van Nieuwmegen, R., 1983. Use ofliposomes as biodegradable and harmless adjuvants. Methods Enzymol., 93: 83-95. Yasui, H. and Ohwaki, M., 1983. Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen. Microbiol. Immunol., 27:1107-1116.
105
vaccination of mice with liposome encapsulated antigen
Ascaris suum
S. Lukes Institute o f Parasitology, CSA V, Brani~ovskd 31, 37005 Ceskk Bud~jovice, Czechoslovakia
(Accepted 28 November 1991 )
ABSTRACT Lukes, S., 1992. Ascaris suum - - vaccination of mice with liposome encapsulated antigen. Vet. Parasitol., 43:105-113. Vaccination with liposome encapsulated adult crude antigen with and without coencapsulated immunomodulator (levamisole) in a mice/larval Ascaris suum model provided protection against a challenge infection (2000 eggs) in mice immunised by immobilised antigen. The best results (88.9% protection) were obtained with a combination of two doses of liposome entrapped antigen with levamisole. Vaccination with liposome vaccine without modulator was slightly less effective (78.7% protection ). A single dose of vaccine was ineffective ( 14.3% protection). Application of the soluble antigen without any adjuvants led to the enhancement of worm yield in lungs and liver.
INTRODUCTION
The common trend in vaccine development is to identify the component of infective agents that are responsible for stimulating protective immune responses. Usually these soluble antigens are poor immunogens and require strong adjuvants or appropriate use of immunostimulators. A safe and completely harmless adjuvant has not yet been developed (Bomford, 1989). One possibility for a safe and effective delivery system is the use of liposomes (Bangham, 1980). Since it was first realised that liposomes have immunoadjuvant action (Allison and Gregoriadis, 1974), they have been used with many antigens (Van Rooijen and Van Nieuwmegen, 1983; Gregoriadis et al., 1989 ), including parasites (Richards et al., 1988; Alwing and Richards, 1990). Previous experiments demonstrated encouraging results with cestode infections in model (mouse, Taenia crassiceps) or natural (sheep, Moniezia ) (Lukes, 1991 ) infections. In this paper we report the supportive effect of liposomes on the model Correspondence to: S. Lukes, Institute of Parasitology, CSAV, Branigovsk~i 31, 37005 Cesk6 Bud6jovice, Czechoslovakia.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
106
s. LUKES
nematode infection in mice with and without immunostimulative agents, i.e. levamisole. MATERIAL AND METHODS
Animals
Outbred female ICR mice (VELAZ, Prague), weighing 20-25 g at the beginning of the experiment, were used. Mice were maintained in a vivarium at the Institute in standard plastic cages with eight to ten individuals per group. They were fed a Larsen diet (LD, VELAZ, Prague) and provided with water ad libitum. Immunomodulation
Levamisole (7.5% solution) (NILVERM, inj. ad usum veterinarium, BIOVETA, Ivanovice na Hane) was used as modulator of the i m m u n e response during vaccination. The dose used corresponds to 4 mg kg-~ body weight (for immunostimulation in domestic animals, doses of 3-10 mg kghave been used) (Euzeby, 1986). Parasite antigen
Adult worms ofAscaris s u u m obtained in local abattoirs from infected pigs were repeatedly washed carefully with tap water until surface contamination was removed. The last washing was carried out in phosphate buffered saline (PBS, pH 7.2, 0.15 M, at room temperature). The same solution was used for extraction of soluble antigens from the whole body homogenate. After dissection into small pieces and homogenisation on a vortex mixer for 5 min, the crude fraction was sedimented by low speed (3000 rev m i n - 1, 15 min) centrifugation and the homogenate purified by high speed centrifugation in a cooled centrifuge ( 10 000 rev m i n - ~, 1 h). The protein content was determined by Folin reagent (Lowry et al., 1951 ) with use of bovine serum albumin as standard. Infective eggs
Infective eggs of A. s u u m were prepared as previously described (Lukes, 1982). Eggs liberated from dissected uteri of female worms were incubated at room temperature for 3 h under shaking in 0.5% HC1, washed with water and gently centrifuged (up to 2000 rev min-1, 15 min), transferred onto filter paper placed on a watch glass and connected with paper bridges to the bottom part of a Petri dish filled with 0.05% potassium dichromate. In these
VACCINATION AGAINST ASCARIS SUUM IN MICE
| 07
dishes the eggs were incubated for at least 28 days at 27 °C and then observed microscopically for developing larvae. Before inoculation the eggs were washed with water and the number of full developed larvae determined.
Vaccine preparation The liposome preparation described by Claassen et al. (1987) with minor modification was used. Soybean phosphatidyl choline (Sigma Chemical Co., St. Louis, M O ) and cholesterol (Lachema, CSFR) (molar ratio 3.5:1 ) were dissolved in chloroform, evaporated in thin film on the wall of the tubes (5 ml of phospholipid solution per tube) and then rehydrated with PBS solution of the antigen ( 10X 100 mg in 1 ml of PBS per tube, groups AS3-AS6) and i m m u n o m o d u l a t o r (groups AS4-AS6). The process of preparing the vesicle suspension was supported by mixing on the shaker. After full dispersion the lipid film was left to stand in the tubes for 2 h at room temperature and then the suspension was sonicated in an ultrasonic bath with intensive cooling (ice cubes) for 3 X 1 min. After sonication the suspension was stored in a freezer before inj ection. All solutions (antigen and modulator) were sterilised before preparation of vaccine by filtration. Before use, the suspension was diluted with sterile PBS to a concentration of 100 mg of protein in one dose of vaccine.
Experimental design Mice were vaccinated with the same doses of antigen (100 mg) either in liposomes with or without i m m u n o m o d u l a t o r or without the liposome adjuvant combination. Vaccination was done subcutaneously in all cases (0.5 ml of suspension per mouse). Group AS 1 served as non-immunised controls and were injected with PBS only on Days 1 and 14. Group AS2 served as a soluble antigen control and was injected with two doses of soluble antigen in PBS on Days 1 and 14. Group AS3 was immunised by two doses of liposome entrapped antigen without modulator, and group AS4 with two doses of soluble antigen mixed with levamisole on Days 1 and 14. Groups AS5 and AS6 were i m m u n i s e d with two (AS5) or one (AS6) (Days 1 and 14, and Day 1, respectively) dose of antigen coencapsulated with i m m u n o m o d u l a t o r into liposomes. On Day 14 after the last injection, mice were challenged by 2000 infective eggs ofA. suum per os (by feeding needle). Mice were bled under light anaesthesia from the orbital sinus using fine Pasteur pipettes before injection, reimmunisation and challenge on Day 28. The last serum samples were taken immediately before killing. Serum samples were stored at - 20 oC before evaluation of antibody reactivity.
S. LUKES
108
Assessment of antibody production For the measurement of the specific antibodies in the serum of experimental animals the (ELISA) technique (Engvall and Perlmann, 1979) was used. We used polystyrene plates (GAMA, Dalecin), coated with crude antigen preparation in coating buffer (0.625 mg ml-~ of buffer, determined by titration). Samples were diluted in PBS with 0.05% Tween-20 (PBS-T-20) 1 : 100, incubated for 1 h at 37 ° C. All washing steps (repeated three times) were done using PBS-T-20 solution. Swine anti-mouse Ig peroxidase conjugate (SWAM/ Px, 1 : 2000, SEVAC, Prague) was diluted in PBS-T-20 and incubated for 1 h at 37 ° C. The substrate was 40 mg ortho-phenylenediaminein 100 ml citrate buffer (pH 5.5) with 40 ml of hydrogen peroxide. The reaction was stopped with 2.5 M sulphuric acid and evaluated by MR 580 (Dynatech) at 492 nm. The reactivity o f the serum samples was recorded as optical density of sample at 492 nm at 1 : I00 serum dilution. TABLE 1 Effectiveness of various vaccine preparations against A. munisation of mice Group
Vaccine
Dose ~
No. of infected mice 2
ASI
0 (PBS)
2M 4
10of 10
AS2
ag
2X
10 of 10
AS3
Liposome ag Levamisole ag Liposome Levamisolc ag Liposome Levamisole ag
2×
7 of 8
2×
7of10
AS4 AS5
AS6
2×
1×
4 of 9
7 of 9
suum
infection and worm burdens after im-
No. of larvae recorded (mean and range)
Protection 3 (%)
Lungs
Liver
Total
14.0 (0-40) 43.2 (0-192) 30.5 (0-92) 2.6
10.4 (4-24) 30.0 (0-108) 3.5 (0-12) 1.6
24.4 (8-64) 73.2 (16-196) 34.0 (0-92) 5.2
(0-16)
(0-8)
(0-16)
2.4 (0-8)
2.8 (0-8)
88.9
(0-4) 14.2 (0-20)
6.7 (0-44)
20.9 (0-48
14.3
0.4
78.7
PBS, phosphate buffered saline, pH 7.2. ag, Total worm homogenate. Levamisole: one dose corresponds to 4 mg kg l body weight. ~One dose of antigen corresponds to 100 mg of protein. 2Mice challenged with 2000 infective eggs per os 14 days after last injection of vaccine. 3protection = (mean number of larvae in control g r o u p - m e a n number of larvae in treated group )/ mean number of larvae in control group × 100 (Tendler et al., 1986 ). 41njections on Days I and 14.
109
VACCINATION AGAINSTASC4RIS SUUM IN MICE
Effectiveness of vaccination against challenge infection Mice were killed by cervical dislocation on Day 7 post-infection with 2000 infective eggs of A. suum. The lungs and liver were prepared and digested separately in pepsin solution after being minced to small pieces of tissue. After 3 h of agitation at 37°C in a water bath, the rest of the tissue block was discarded by gauze filtration and larvae in five 50-ml aliquots were counted under the dissecting microscope. Larvae numbers were enumerated on the full volume of digestive fluid obtained from the sample. All animals of each group were observed separately. Effectivity of vaccination was assessed by larval counts in lungs and liver on Day 7 post-infection (Table 1 ). RESULTS
Immunisation of mice with soluble A. suum antigen and immobilised and potentiated antigen preparations elicited different degrees of resistance among experimental groups. The results of the trial are summarised in Tables 1 and 2. We found significant degrees of induced protection after vaccination with soluble antigens incorporated into liposomal particles and combined with levamisole (group AS5, Table 1 ), vaccinated twice 14 days apart. In these cases both parameters (effectiveness of infection and decrease of worm burden) were better than in other groups. The effectiveness of infection decreased to 44.4% and protection was 88.9%. Other combinations of either antigen alone in liposomes (group AS3) or soluble antigen with levamisole (group AS4) gave lower values for both parameters (effectiveness of infection 87.5% and 70.0%, respectively, and protection in AS4, 78.7%). The solTABLE 2 Measurement of antibody production against A. s u u m in vaccinated and infected mice (mean optical density value of group and SD) Day
AS 1
AS2
AS3
AS4
AS5
AS6
1
0.244 0.030 0.210 0.026 0.133 0.048 0.198 0.053
0.250 Ns 0.041 0.381"* 0.085 0.718*** 0.152 0.735*** 0.110
0.226 Ns 0.077 0.317 ys 0.121 0.575*** 0.202 0.765*** 0.298
0.316" 0.107 0.320 Ns 0.109 0.505*** 0.166 0.464*** 0.208
0.294 Ns 0.110 0.257 Ns 0.070 0.512*** 0.227 0.467*** 0.124
0.287 Ns 0.077 0.280** 0.070 0.212** 0.063 0.208*** 0.066
Before immunisation Before reimmunisation Before infection
28
After challenge
35
14
Significance was determined by non-paired Student's test. *P< 0.05; **P< 0.01; ***P< 0,001; NS, non-significant.
110
S. LUKES
uble antigen alone and antigen with liposomes without modulator (groups AS2 and AS3, respectively) induced some degree of tolerance to parasite infection, as demonstrated by enhanced number of liver and lung larvae in groups AS2 and AS3 (Table 1 ). Some changes exist in reactivity in group AS3 (antigen in liposomes) demonstrable by comparison of effectivity of infection (see Table 1 ). Little protection was induced in group AS6, immunised once with the liposome/levamisole/antigen combination.
Antibody production in experimental mice after vaccination Results of measurement of the antibody production in vaccinated and infected mice are summarised in Table 2. With the exception of group AS4, groups were without difference in antibody reactivity before the initiation of the trial (ELISA mean optical density (+_SD) ranged from 0.226, ( + 0 . 0 7 0 ) to 0.316 ( + 0 . 1 0 7 ) ). The difference shown by group AS4 at a minor degree of significance is probably a result of non-specific reaction with crude antigen. After first injection of antigens and vaccine combination we detected various degrees of reactivity with crude antigen, depending on the type of vaccine used. We found a surprisingly high production of antibodies in mice injected with crude, non-treated extract of the adult worms (group AS2) after the second injection on Day 28 (0.718 +_0.150), but these antibodies were not protective. We found some degree of tolerance and an enhanced worm burden in this group. Differences between groups before challenge were not significant. After the second injection in groups AS2-AS5 a significant increase of antibody reactivity was detectable. These differences were prolonged before the time of challenge. After challenge in groups AS2 and AS3 we detected further increase of antibody reactivity. Most prominent was the increase in group AS2 (0.919 + 0.110) (Table 2). In liposome vaccinated groups (AS4-AS6) no further increase of antibody levels was observed. DISCUSSION
Many authors have examined the possibility of immunisation against nematode infection, including the A. suum model. Most of the trials were based on the use of soluble extracts or secreted products of worms. In these experiments only minor resistance was induced without the use of strong adjuvants (Bindseil, 1969; Guerrero and Silverman, 1972; Stromberg and Soulsby, 1977). Irradiated infective eggs were partially effective (Gaur and Dutt, 1976). Further experiments (Urban and Tromba, 1984) showed the possibility of induced protective responses after multiple inoculation of UV-irradiated infective eggs (protection up to 94% in pigs), but for field use the system is too complicated and impractical.
VACCINATION AGAINST ASCARIS SUUM IN MICE
| ]1
We have demonstrated some degree of protection after injection of liposome immobilised antigen (groups AS3-AS6), especially after repeated injection of levamisole potentiated vaccine (group AS5). In these groups not only the number of larvae (mean of 2.7 per mouse), but even the overall success of the infection (44.4%) was depressed. Liposomes have one advantage over other effective adjuvants in that no granulomata are formed at the site of infection (Allison and Gregoriadis, 1974 ). A further advantage of the use of liposomes is the avoidance of hypersensitivity reactions to unentrapped antigen (Gregoriadis and Allison, 1974). It has been shown that the immune response against liposome associated protein antigens is T-lymphocyte (Beatty et al., 1984) and macrophage (Shek and Lukovich, 1982) dependent. As demonstrated by Bindseil ( 1971 ), the protective effect of acquired immunity to A. s u u m larvae is based on T-cell immunity. Further confirmation of the hypothesis was provided by Hermanek and Koudela (1990) who demonstrated the supportive role of thymic preparations on larval ascaridosis in mice. Our hypothesis of the construction of the liposome-levamisole potentiated vaccine was based on the supportive effect oflevamisole in vaccination against cestodes in a model system (Lukes, 1988). Because levamisole can also increase T-lymphocyte proliferation (Renoux and Renoux, 1977a,b; Symoens et al., 1979 ) the combination of adjuvant effect ofliposome alone and proliferative effect of modulator may be advantageous. The danger of direct action of levamisole on migrating larvae is avoided by its short half-life (about 4 h) and quick metabolism in mammalian organisms - - total elimination within 2 days (Symoens et al., 1979). Because we challenged the mice after 14 days, sufficient time elapsed for the clearance of the drug before challenge. The prolonged influence of levamisole necessary for stimulation of lymphocytes, may be a result of production of thymus-like substances (Renoux and Renoux, 1977c). There has been only one previous attempt to use liposomes in vaccination against A. s u u m infection in pigs (Rhodes et al., 1988 ). A low degree of resistance was induced only after a priming dose of eggs and consequent vaccination with the liposome preparation. No reaction was observed without priming. It is possible that the peroral or intraintestinal route of application of vaccine was not optimal for inducing reaction (Yasui and Ohwaki, 1983). From the point of view of monitoring of degree of protection, it is interesting that in the A. s u u m model infection of mice antibody reactivity does not correlate with the degree of protection, as no correlation between antibody reactivity and resistance was noted. The comparatively large variation in individual antibody levels in the groups of mice depend on the individual genetically based reactivity to the antigens (Kennedy, 1989).
112
s. LUKES
CONCLUSION
Liposomes with incorporated soluble A. s u u m antigen are able to elicit a protective immune response in mice against migrating larvae ofA. s u u m . The effect of liposomes was further potentiated by coencapsulation of the levamisole as immunomodulator. Soluble antigens without modulator or liposome induced tolerance of enhanced worm burdens in the hosts. ACKNOWLEDGEMENTS
Thanks are due to R. Vtelenska and R. Bruzkova for excellent technical assistance.
REFERENCES Allison, A.C. and Gregoriadis, G., 1974. Liposomes as immunological adjuvants. Nature, 252: 252. Alwing, C.R. and Richards, R.L., 1990. Liposomes containing lipid-A: A potent nontoxic adjuvant for a human malaria sporozoite vaccine. Immunol. Lett., 25: 1-3. Bangham, A.D., 1980. Development of the liposome concept. In: G. Gregoriadis and A.C. Allison (Editors), Liposomes in Biological Systems. John Wiley, New York, pp. 1-24. Beatty, J.D., Beatty, B.G., Paraskevas, F. and Froese, E., 1984. Liposomes as immune adjuvants: T cell dependence. Surgery, 96: 345-351. Bindseil, E., 1969. Immunity to A s c a r i s s u u m . 1. Immunity induced in mice by means of material from adult worms. Acta Pathol. Microbiol. Scand., 77:218-222. Bindseil, E., 1971. Immunity to A s c a r i s s u u m . 5. The effect of X-radiation and neonatal thymectomy on a primary infection in mice. Acta Pathol. Microbiol. Scand., 79:511-518. Bomford, R., 1989, Adjuvants for anti-parasite vaccines. Parasitol. Today, 5: 41-46. Claassen, E., Kors, N. and van Rooijen, N., 1987. Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphone and surface associated antigen as hapten. Immunology, 60:509-515. Engvall, E. and Perlmann, P., 1979. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry, 8: 871-874. Euzeby, J.P., 1986. Propri6t6s immunostimulantes du Levamisole. 2. Applications en mddicinc vdtdrinaire et effects secondaires. Rev. Med. Vet., 137: 499-520. Gaur, S.N.S. and Dutt, S.C., 1976. Studies on irradiated form of A s c a r i s s u u m : immunizing effect of irradiated A. s u u m eggs. Indian Vet. J., 53: 305-306. Gregoriadis, G. and Allison, A.C., 1974. Entrapment of proteins in liposomes prevents allergy reactions in preimmunized mice. FEBS Lett., 45:71-75. Gregoriadis, G., Davis, D., Garcon, N., Tan, L., Weissig, V. and Xiao, Q., 1989. The immunoadjuvant action of liposomes. In: G. Lopez-Berenstein (Editor), Liposomes in the Therapy of Infectious Diseases and Cancer. UCLA Symposia, Vol. 89, pp. 35-56. Guerrero, J. and Silverman, P.H., 1972. Active immunization against A s c a r i s s u u m minimum lethal dose in mice. Z. Parasitenkd., 39: 339-344. Hermanek, J. and Koudela, B., 1990. Influence of nonspecific immunomodulation with thymic
VACCINATION AGAINST ASCARIS SUUM IN MICE
113
preparations on the result of infection in some experimental parasitoses. In: K.N. Masihi and W. Lange (Editors), Immunotherapeutic Prospects of Infectious Diseases. Springer, Berlin, pp. 71-76. Kennedy, M.W., 1989. Genetic control of the immune repertoire in Nematode Infections. Parasitol. Today, 56: 316-324. Lowry, O.H., Rosebrough, N.R., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Lukes, S., 1982. Immunological methods in experimental migration of larvae of some ascarids in rabbits. Ph.D. Thesis, Institute of Parasitology, CSAV, Czechoslovakia, 266 pp. Lukes, S., 1988. Levamisole - - biological response modifier in Taenia c r a s s i c e p s - mouse experimental system, Folia Parasitol., 35: 37-40. Lukes, S., 1991. Preliminary results of the experiments with vaccines against helminths on basis of immobilized antigens. In: Proceedings of a Conference on Parasitoses of Domestic Animals, 1989, C. Budejovice, (in Czech), pp. 123-126. Renoux, G. and Renoux, M., 1977a. Influence du levamisole sur la production ~ anticorps. Ann. Immunol. Inst. Pasteur, 128c: 273-274. Renoux, G. and Renoux, M., 1977b. MOchanismes d'action du levamisole, stimulant des responses d'immunit6 cellulaire. Ann. Immunol. Inst. Pasteur, 128c: 275-277. Renoux, G. and Renoux, M., 1977c. Thymus-like activities of sulphur derivatives on T-cell differentiation. J. Exp. Med., 145: 466-471. Rhodes, M.B., Baker, P.K., Christensen, D.L. and Anderson, G.A., 1988. Ascaris suum antigens incorporated into liposomes used to stimulate protection to migrating larvae. Vet. Parasitol., 26: 343-349. Richards, R.L., Hayre, M.D., Hockmeyer, W.T. and Alving, C.R., 1988. Liposomes, lipid A, and aluminium hydroxide enhance the immune response to a synthetic malaria sporozoite antigen. Infect. Immun., 56: 682-686. Shek, P.N. and Lukovich, S., 1982. The role of macrophages in promoting the antibody response mediated by liposome-associated protein antigens. Immunol. Lett., 5: 305-309. Stromberg, B.E. and Soulsby, E.J.L., 1977. Ascaris suum: immunization with soluble antigens in the guinea pigs. Int. J. Parasitol., 7: 287-291. Symoens, J., Rosenthal, M., de Brabander, M. and Goldstein, G., 1979. Immunoregulation with levamisol. Springer Semin. Immunopathol., 2: 49-59. Tendler, M., Pinto, R.N., Lima, A.O., Gebara, G. and Katz, N., 1986. Schistosoma mansoni: vaccination with adult worm antigens. Int. J. Parasitol., 16: 347-352. Urban, J.F. and Tromba, F.G., 1984. An ultraviolet-attenuated egg vaccine for swine ascariasis: Parameters affecting the development of protective immunity. Am. J. Vet. Res., 45: 21042108. Van Rooijen, N. and van Nieuwmegen, R., 1983. Use ofliposomes as biodegradable and harmless adjuvants. Methods Enzymol., 93: 83-95. Yasui, H. and Ohwaki, M., 1983. Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen. Microbiol. Immunol., 27:1107-1116.
