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Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis
Veterinapy Parasitology, 37 (1990) 273-284 Elsevier Science Publishers B.V., Amsterdam
273
Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis H. Wedrychowicz and B. Bezubik Department of Parasitology, Zoological Institute, University of Warszawa, Krakowskie Przedmie~cie 26/28, 00-927 Warsaw (Poland) (Accepted for publication 23 May 1990)
ABSTRACT Wedrychowicz, H. and Bezubik, B., 1990. Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis. Vet. Parasitol., 37: 273284. Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 pg of infective larval somatic proteins (L3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10 000 L 3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L 3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L 3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using ~25I-L3SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.
INTRODUCTION
The recent development of genetic engineering has provided convenient methods for manufacturing antigens from organisms which would otherwise be impossible to culture in sufficient quantities, and has opened the way for vaccines containing purified parasite antigens able to generate protective immunity in the host. In the host-parasite systems where humoral antibodies confer protective immunity, idiotype-derived vaccines might also be produced (Kieber-Emmons et al., 1987; Bomford, 1989 ). However, purified antigens are usually much less immunogenic than when they comprise part of a 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
274
H. WEDRYCHOWICZ AND B. BEZUB1K
living parasite and the application of adjuvants is necessary. Adjuvants potentiate the specific immune response, some preferentially activate cell-mediated immunity and others humoral immunity (Bomford, 1989). Beryllium hydroxide has been reported as a powerful immunological adjuvant capable of inducing high levels of specific, high-affinity antibodies in sera and bile against parenterally injected antigens, even in athymic animals (Hall, 1984; Hall and Spencer, 1984 ). More recent investigations suggest that it is possible to use soluble and relatively non-toxic beryllium preparations and thus dissociate the adjuvant and immunomodulatory properties from the toxic and granulomatous reactions (Hall, 1988). Freund's complete adjuvant (FCA) is known to potentiate mainly cell-mediated immunity and is widely used in experimental protozoa or helminth vaccines. However, FCA causes chronic inflammation at the site of injection and occasionally autoimmune complications (Bomford, 1989 ). Vaccination of sheep and laboratory animals against Trichostrongylus colubriformis infection has demonstrated that a very high degree of protective immunity can be induced using normal or irradiated larvae (Gregg et al., 1978; Maclean et al., 1986; Douch, 1988; Bezubik et al., 1988). Attempts to isolate protective antigens operating during vaccination have been carried out in the guinea pig model and it has been found that the fourth stage larvae somatic and excretory-secretory proteins stimulate protective immunity to subsequent challenge infection (Rothwell and Love, 1974; Rothwell and Merritt, 1975 ). Aluminium hydroxide gel, but not Bordetella pertussis or levamisole, improved protection when mixed with soluble extract of the fourth stage larvae and injected intraperitoneally. However, there are some doubts about whether antigens involved in the induction of protective immunity in guinea pigs and the natural host (sheep) are the same (O'Donnell et al., 1985, 1989). The aim of the present study was to assess the capacity of FCA and beryllium hydroxide to enhance protective immunity in rabbits induced against T. colubnformis infection by soluble antigens extracted from infective larvae of the nematode. Rabbits vaccinated orally with normal larvae, which were allowed to develop in the host to the late fourth stage, served as immunization controls. MATERIALS AND METHODS
Experimental design Thirty adult New Zealand rabbits in six experimental groups were used (Table 1 ). Groups I-IV were vaccinated at 2-week intervals, as indicated in Table 1. The immunizing infections in Groups I and II were terminated on Day 7 after the administration of each infection (0.18 mg kg- J body weight
275
ADJUVANT INFLUENCEON IMMUNITY IN L3SP-VACCINATEDRABBITS TABLE 1 Experimental design Group
Antigen
Immunization route
Adjuvant
Percentage of protection J
I II III IV V VI
3 X 2000 L3 3 × 5000 L3 3 × 100/tg L3SP 3 × 100 #g L3SP Challenge controls Parasite-free controls
Oral Oral Subcutaneous Subcutaneous
FCA 2 Be(OH)2
87.6 99.8 29.5 94.8
Percentage of protection =
No. of worms in Group V - N o . of worms in immunized Group× 100 No. of worms in Group V
2FCA = Freund's complete adjuvant.
of ivermectin orally). All vaccinated animals and also five naive (Group V) rabbits were challenged orally with 10 000 infective larvae (L3) 14 days after the administration of the third immunizing dose. Five rabbits remained intact and served as parasite-free controls (Group VI). All experimental animals were killed and dissected on Day 21 after challenge. At necropsy, the number of worms in the intestines was estimated and samples of bile and small intestinal mucosa were collected for antibody measurement.
Parasitological techniques Infective larvae of T. colubriformis were cultured in twist-offjars from faeces of sheep infected with a pure strain of the nematode. The infective larvae, after being purified by decantation and filtration through a bolting cloth, were counted by a dilution method, then used for infection or antigen preparation. During dissections, the small intestines were removed and the intestinal contents were washed out with phosphate-buffered saline (PBS). The total worm count was then estimated on the luminal contents by a 10% aliquot technique. The mucous membrane of the intestine was scraped, weighed and divided into two equal parts. One half was used for the extraction of small intestinal mucosal proteins and the other was placed in digestion fluid ( 13 g pepsin and 10.8 ml concentrated HC1 in 1000 ml distilled water) for 12 h at 37°C and afterwards examined for the nematodes. The number of worms recovered was then multiplied by two to give the number of worms that lodged in the mucosa a n d / o r on the wall of the small intestine. The sum of the worm counts from the small intestinal fluid and the mucosa was considered to give the total number of worms recovered.
276
H. W E D R Y C H O W I C Z AND B. BEZUBIK
Sampling methods Blood for serum preparation was collected from infected and control rabbits by puncture of lateral ear veins at weekly intervals. Sera of individual animals were stored at - 2 0 ° C before serological analysis. Small intestinal mucosal proteins were extracted from scrapings of mucous membrane of the small intestines. Two-gram samples were homogenized in 6 ml PBS containing 0.1 M ethylenediaminetetraacetic acid, olisodium salt (EDTA) and protease inhibitors: 0.5 mM of phenylmethylsulphonyl fluoride (PMSF) and 0.01% soybean trypsin inhibitor (Sigma Chemical Company, Poole, Gt. Britain ). The homogenates were centrifuged for 30 min at 14 000 × g and supernatants used for serological analysis.
Parasite antigens Somatic antigens of infective larvae were obtained by homogenization and extraction of the carefully cleaned worms in PBS containing protease inhibitors: L- 1-tosylamide-2-phenylethylchoromethylketone (TPCK), 50/tg ml- 1; N-a-p-tosyl-L-lysine chloromethylketone HC1 (TLCK), 25/lg ml-1; PMSF, 174/~g m l - l (Sigma Chemical Company, Poole, Gt. Britain), at pH 7.6. The extracts were cleared by centrifugation for 1 h at 15 000 × g and then concentrated to 3 mg of protein m l - 1 using Aquacide (Calbiochem, San Diego, CA, U.S.A.).
Adjuvants The adjuvants were administered only with the first and the second antigen injections. Beryllium hydroxide was freshly precipitated from BeSO4 solution according to Hall (1984). The equivalent of 20 mg of BeSO4 was injected subcutaneously a few seconds prior to antigen. FCA (Difco Labs. Ltd., Detroit, MI ) was emulsified with an equal volume of antigen solution and 1 ml of the FCAantigen mixture was injected subcutaneously.
Antibody measurements Gel double diffusion tests were performed using infective larval somatic extracts (L3SP) as the antigenic source, against sera, bile and mucosal extracts of vaccinated animals, in 1% agarose. Radioimmunoprecipitation (RIP) tests were carried out using L3SP labelled with 125I by the chloramine T method (Greenwood et al., 1963). Approximately 105 counts per minute (c.p.m.) ( 5-10 pl) of the labelled antigen were added to 100/tl of PBS, pH 7.6, which contained some 150 pg of normal
ADJUVANT
INFLUENCE
ON IMMUNITY
IN L3SP-VACCINATED
RABBITS
277
serum proteins and ~ 150/tg of bile, mucosal or serum proteins from infected rabbits. Incubation for 13 h at room temperature and for I h at 37°C was followed by the addition of 50 ~1 of goat anti-rabbit IgA or IgG monospecific antiserum (Nordic Immunology Laboratory Ltd., Amsterdam, The Netherlands), washing three times in cold PBS and transfer into flesh tubes. The radioactivity in the precipitates was measured using a gamma-counter. All samples were assayed in duplicate• Variations around the means were expressed as the standard error (SE). Student's t-test was used to assess the statistical significance of differences between groups of rabbits. RESULTS
Immunity to infection The mean worm counts are presented in Fig. 1. Worm burdens found after challenge in Groups I, II and IV differ significantly ( P < 0.001 ) from those in 4000
m
3000
q~
2000
i. o
1000
__T__ •
I
T
II
III
IV
V
G r o u p s
Fig. 1. Burdens of adult T. colubriformis f o u n d in the intestines of immunized and naive rabbits after challenge with 10 000 larvae. The histograms represent the means of five animals. I = rabbits vaccinated with three doses of 2000 L3; II=rabbits vaccinated with three doses of 5000 L3; III =animals immunized with infective larval somatic proteins and Freund's complete adjuvant; IV=animals vaccinated with the same antigen as Group Ill and beryllium hydroxide; V = challenge controls.
278
H. W E D R Y C H O W 1 C Z A N D B. B E Z U B I K
challenge controls. The highest degree of protection (99.8%) was observed in Group II, vaccinated with three doses of 5000 larvae, and also in rabbits immunized with L3SP and beryllium hydroxide as the adjuvant (94.8%). Interestingly, vaccination with the same close of L3SP emulsified in FCA produced very poor immunity (29.5%); the number of worms found in Group III showed no statistically significant differences in comparison with worm burdens observed in naive challenge control rabbits.
Local antibody response The intestinal and bile IgA, and also intestinal mucosa IgG responses to T.
colubriforrnis antigens were measured by RIP using 125I radioisotop~-i,lz~lled L3SP. The results are presented in Fig. 2. The binding of worm proteins by intestinal mucosa IgG was the highest in challenge control rabbits and the lowest in Group I, although statistically significant differences also occurred between Groups V and IV ( P < 0.05 ). Intestinal IgA reacting with 125I-L3SP showed the highest level in Group III and the percentage of radiolabelled parasite proteins bound in this group was ~ 1.7 times higher than in Group I ( P < 0 . 0 1 ) , 1.3 times higher than in Group II ( P < 0 . 0 1 ) , 1.5 times higher than in Group IV ( P < 0 . 0 0 1 ) , and > 2.6 times higher than in the challenge control ( P < 0.001 ). The amounts of 125I-L3SP bound by bile IgA were much lower than those measured in the intestinal mucosa. The highest binding ac-
I
~ = mucosal IgG []
mucosal
][1[[[I]
bile
IgA
50
iii!i!
lgA
40
30 :: I
,,o
T
-L 20
.7
l
I1
II1
IV
V
VI
Groups
Fig. 2. Bile and mucosal antibodies against infective larvae somatic antigens. (For explanation of groups see Fig. 1. )
279
A D J U V A N T I N F L U E N C E ON I M M U N I T Y IN L3SP-VACCINATED RABBITS
tivity was observed with biles of Group IV and the percentage of antigen radioactivity bound in this group was 2.3 times higher than in Group I ( P < 0 . 0 0 1 ) , 1.9 times higher than in Group II ( P < 0 . 0 0 1 ) , 1.23 times more than in Group III ( P < 0 . 0 1 ) , and 1.7 times higher than in the challenge controls ( P < 0.002).
Serum antibody responses In gel double diffusion tests, antibodies reacting with L3SP were detected only in Groups III and IV. The earliest precipitin reaction was observed in one rabbit of Group III; a single precipitation arc developed in the serum collected on Day 17 of the experiment (DE). All rabbits of this group reacted positively and formed 1-4 precipitation bands 2 weeks later. At the time of • o
I II IV
[ 60
50
i.
40
30
0
e~ 20 \
2
"l
10
I
I
I
I
I
I
0
10
20
30
40
SO
Days
Fig. 3. Serum IgG antibodies against
L3
L3SP and FCA (III) or beryllium (IV).
of
J 60
experiment
in rabbits vaccinated with living larvae (I and II) or
280
~
e,,
H. WEDRYCHOWICZ
60
-
50
-
•
I
0
II
AND
B. B E Z U B I K
40
"0
o
e~
30
¢0
0 e~
20
e~
2
10 _
_
0
I
I
0
10
Days
of
[
I,
I
I
I
20
30
40
50
60
experiment
Fig. 4. Serum IgA antibodies against L 3 in rabbits vaccinated with hivinglarvae (I and 11) o r L3SP and FCA (III) or beryllium (IV).
challenge infection (46 DE), sera of all rabbits from Group III developed four precipitation lines and at the end of the experiment 3 out of 5 rabbits gave positive reactions of 3-4 precipitation arcs. Sera of four rabbits of Group IV developed 1-2 arcs on Day 25. A similar reaction was observed on Day 32 and just before challenge infection only two rabbits reacted positively (two arcs). No precipitation lines were produced by sera of Group IV collected 2 weeks after challenge. Serum IgG antibody levels increased during immunization to the highest degree in Group I, but at the time of challenge the activity of these antibodies was similar in all vaccinated groups (Fig. 3). The percentage of 125I-L3SP bound by IgG of Group V increased slowly after infection and at the end of the experiment reached a value which was not significantly higher than that in Groups III and IV (results not shown). IgA antibodies reacting with 125I-L3SP increased significantly after admin-
ADJUVANT INFLUENCE ON IMMUNITY IN L3SP-VACCINATED RABBITS
281
istration of the third immunizing dose, and reached maxima before challenge in Groups I, II and IV (Fig. 4). Two weeks after challenge, binding of 125IL3SP by serum IgA was very low in these groups, while in susceptible rabbits of Group III it reached the maximum (Fig. 4). No specific reaction was measured in serum IgA of Group V. DISCUSSION
Rabbits vaccinated orally with three doses of 5000 infective larvae of
T. colubriformis demonstrated very high protective immunity; only single worms were found in the intestines of Group II after challenge with 10 000 larvae. Lower (2000 L3 ) vaccination doses induced slightly poorer protection (87.6%), but still at a level comparable with that induced in sheep by vaccination with two doses of 20 000 irradiated larvae (Dineen et al., 1977 ). Parasites coming from subsequent vaccinations were removed on Day 7 after administration, i.e. when the larvae had developed to the late fourth stage (Rapson et al., 1985); thus the rabbit immune system was stimulated only with antigens of parasitic L3 and fourth stage larvae. Animals vaccinated with infective larval somatic proteins and beryllium hydroxide showed a higher protection level than the rabbits immunized orally with three doses of 2000 L3. However, the same antigenic preparation, when introduced to the host with FCA, induced a very low degree of protection. The mean number of adult nematodes found in the intestines of rabbits treated with FCA was lower than in challenge control rabbits, but the difference was not statistically significant. Similar dependence of the protective ability of nematode antigens on the immunomodulatory action of various adjuvants has been recently reported by Monroy et al. ( 1989 ). To our knowledge, beryllium has not been used in immunoparasitological investigations and, although beryllium has been known as a powerful immunological adjuvant for > 20 years, the mechanism of its adjuvant action is not entirely clear. It has been suggested that the immunological actions of beryllium are caused primarily by the physical effects of microcrystalline beryllium oxides and hydroxides on macrophages, and that such affected macrophages possess an increased ability to present antigens. However, Denham and Hall ( 1988 ) showed that the adjuvant properties of beryllium were not confined to the damaging effects of microcrystalline salts of beryllium, but could be expressed by soluble and relatively non-toxic complexes of beryllium and plasma proteins. Such complexes may induce rapid lymphoproliferative responses in the regional lymph nodes and the appearance of huge numbers of immunoblasts in the efferent lymph (Hall, 1988). In the present experiment, only serum IgA antibody during vaccination and bile IgA on Day 21 after challenge showed clearly higher levels in the beryllium-treated group than in other experimental animals. Sera of rabbits immunized with L3SP and FCA
282
H. WEDRYCHOW1CZ AND B. BEZUBIK
demonstrated much higher activity in gel double diffusion tests than the beryllium group, and also the highest IgA level after challenge infection. The role of serum and intestinal antibodies in conferring protective immunity of the host against T. colubriformis is still obscure. Bottjer et al. ( 1985 ) found that pre-exposure of adult nematodes to serum of goats immune to T. colubriformis decreased subsequent feeding activity of the worms, while Bone and Klesius (1986) reported that IgG antibodies from immune hosts reduced the production of eggs by females of this nematode. Findings regarding the IgA response and its role in immunity against T. colubriformis are also conflicting. Increased levels of Secretory IgA (SIgA) and/or the presence of nematode-specific IgA antibodies were observed in the intestines of sheep and rabbits (Cripps and Rothwell, 1978; Wedrychowicz and Bezubik, 1988 ), but attempts to transfer immunity against T. colubriformis with lymph containing anti-nematode IgA antibodies have failed (Adams et al., 1980). Also, in the present experiment local and systemic antibody levels showed little correlation with the degree of protection. Perhaps, the mechanism of resistance of rabbits against T. colubriformis infection is cell mediated and specific antigenic determinants of such responses do not provoke antibody responses. Rabbits immunized orally with living larvae showed a lower bile IgA antibody level than those vaccinated parenterally with L3SP. The active transfer of rabbit polymeric IgA into bile occurs via the same hepatic secretory component (SC)-mediated transport process as that described for the rat (Delacroix et al., 1982 ). Suggested roles for this transfer include the elimination of 'blocking' p-IgA antibodies which could modulate the effect of other immunoglobulin classes on antigens, and of polymeric IgA (p-IgA) immune complexes from blood, and a reinforcement of intestinal secretory IgA immunity (Tomasi and Plaut, 1985 ). This study has demonstrated that the somatic proteins of infective larvae of T. colubriformis may be as good a source of protective immunogens as living parasites when injected subcutaneously with beryllium hydroxide as the adjuvant. Further investigations are necessary to explain the exact role of local and systemic humoral antibodies during T. colubriformis infection, and also to find out whether the antigens involved in inducing the antibody responses possess protective properties.
ACKNOWLEDGEMENTS
The authors are grateful to Professor Peter H. Holmes of the Department of Veterinary Physiology, University of Glasgow Veterinary School, for his critical reading of the manuscript and helpful advice.
ADJUVANT INFLUENCE ON IMMUNITY IN L3SP-VACCINATED RABBITS
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REFERENCES Adams, D.B., Merritt, C.C. and Cripps, A.W., 1980. Intestinal lymph and the local antibody and immunoglobulin response to infection by Trichostrongylus colubriformis in sheep. Aust. J. Exp. Biol. Med. Sci., 58: 167-177. Bezubik, B., Wedrychowicz, H. and Wojciechowska, A., 1988. Trichostrongylus colubriformis in rabbits: some quantitative aspects and pathogenesis of single and multiple infections. Acta Parasitol. Pol., 33:131-142. Bomford, R., 1989. Adjuvants for anti-parasite vaccines. Parasitol. Today, 5: 41-46. Bone, L.W. and Klesius, P.H., 1986. Effect of host serum on in vitro oviposition by Trichostrongylus colubriformis (Nematoda). Int. J. Invert. Reprod. Dev., 10: 27-32. Bottjer, K.P., Klesius, P.H. and Bone, L.W., 1985. Effect of host serum on feeding by Trichostrongylus colubriformis (Nematoda). Parasitol. Immunol., 7: 1-9. Cripps, A.W. and Rothwell, T.L.W., 1978. Immune response of sheep to the parasite nematode Trichostrongylus colubr(/'ormis: infections in Thiry-Vella loops. Aust. J. Exp. Biol. Med. Sci., 56: 99-106. Delacroix, D.L., Denef, A.M., Acosta, G.A., Montgomery, P.C. and Vaerman, J.P., 1982. Immunoglobulins in rabbit hepatic bile: Selective secretion of IgA and IgM and active plasmato-bile transfer of polymeric IgA. Scand. J. Immunol., 16: 347-350. Denham, S. and Hall, J.G., 1988. Studies on the adjuvant action of beryllium. IlI. The activity in the plasma of lymph efferent from nodes stimulated with beryllium. Immunology, 64: 341-344. Dineen, J.K., Gregg, P., Windon, R.G., Donald, A.D. and Kelly, J.D., 1977. The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes. Int. J. Parasitol., 7:211-215. Douch, P.G.C., 1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. Int. J. Parasitol., 18: 1035-1038. Greenwood, F.C., Hunter, W.M. and Glover, J.S., 1963. The preparation of ~3~I-labelled human growth hormone of high specific radioactivity. Biochem. J., 89:114-123. Gregg, P., Dineen, J.K., Rothwell, T.L.W. and Kelly, J.D., 1978. The effect of age on the response of sheep vaccination with irradiated Trichostrongylus colubriformis larvae. Vet. Parasitol., 4: 35-48. Hall, J.G., 1984. Studies on the adjuvant action of beryllium. I. Effects on individual lymph nodes. Immunology, 53:105-113. Hall, J.G., 1988. Studies on the adjuvant action of beryllium. IV. The preparation of beryllium containing macromolecules that induce immunoblasts responses in vivo. Immunology, 64: 345-351. Hall, J.G. and Spencer, J., 1984. Studies on the adjuvant action of beryllium. II. Systemic effects with particular reference to secretory immunity. Immunology, 53:115-120. Kieber-Emmons, T., McNamara, M., Ward, R.E. and Kohler, H., 1987. Structural considerations in idiotype vaccine design. Monogr. Allergy, 22:126-133. Maclean, J.M., Abebe, M., Wedrychowicz, H. and Holmes, P.H., 1986. Local and systemic antibody responses in gerbils following vaccination with irradiated or non-irradiated Trichostrongylus colubriformis larvae. J. Helminthol., 60: 263-278. Monroy, F.G., Adams, J.H., Dobson, C. and East, I.J., 1989. Nematospiroides dubius: Influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms. Exp. Parasitol., 68: 67-73. O'Donnell, I.J., Dineen, J.K., Rothwell, T.L.W. and Marshall, R.C., 1985. Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pigs. lnt. J. Parasitol., 15: 129-136.
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O'Donnell, I.J., Dineen, J.K., Wagland, B., Letho, S., Werkmeister, J.A. and Ward, C.W., 1989. A novel host-protective antigen from Trichostrongylus colubriformis. Int. J. Parasitol., 19" 327-335. Rapson, E.B., Jenkins, D.C. and Topley, P., 1985. Trichostrongylus colubriformis: in vitro culture of parasitic stages and their use for the evaluation of anthelmintics. Res. Vet. Sci.. 39: 90-94. Rothwell, T.L.W. and Love, R.J., 1974. Vaccination against the nematode Trichostrongylus colubriformis. 1. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance. Int. J. Parasitol., 4: 293-299. Rothwell, T.L.W. and Merritt, G.C., 1975. Vaccination against the nematode Trichostrongylus colubriformis, lI. Attempts to protect guinea-pigs with worm acetylcholinesterase. Int. J. Parasitol., 5: 453-460. Tomasi, T. and Plaut, A.G., 1985. Humoral aspects of mucosal immunity. Adv. Host Defense Mech., 4: 31-61. Wedrychowicz, H. and Bezubik, B., 1988. Systemic and local humoral responses in rabbits following single and multiple infections with Trichostrongylus colubr~forrnis. Acta Parasitol. Pol., 33: 79-89.
273
Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis H. Wedrychowicz and B. Bezubik Department of Parasitology, Zoological Institute, University of Warszawa, Krakowskie Przedmie~cie 26/28, 00-927 Warsaw (Poland) (Accepted for publication 23 May 1990)
ABSTRACT Wedrychowicz, H. and Bezubik, B., 1990. Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis. Vet. Parasitol., 37: 273284. Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 pg of infective larval somatic proteins (L3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10 000 L 3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L 3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L 3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using ~25I-L3SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.
INTRODUCTION
The recent development of genetic engineering has provided convenient methods for manufacturing antigens from organisms which would otherwise be impossible to culture in sufficient quantities, and has opened the way for vaccines containing purified parasite antigens able to generate protective immunity in the host. In the host-parasite systems where humoral antibodies confer protective immunity, idiotype-derived vaccines might also be produced (Kieber-Emmons et al., 1987; Bomford, 1989 ). However, purified antigens are usually much less immunogenic than when they comprise part of a 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
274
H. WEDRYCHOWICZ AND B. BEZUB1K
living parasite and the application of adjuvants is necessary. Adjuvants potentiate the specific immune response, some preferentially activate cell-mediated immunity and others humoral immunity (Bomford, 1989). Beryllium hydroxide has been reported as a powerful immunological adjuvant capable of inducing high levels of specific, high-affinity antibodies in sera and bile against parenterally injected antigens, even in athymic animals (Hall, 1984; Hall and Spencer, 1984 ). More recent investigations suggest that it is possible to use soluble and relatively non-toxic beryllium preparations and thus dissociate the adjuvant and immunomodulatory properties from the toxic and granulomatous reactions (Hall, 1988). Freund's complete adjuvant (FCA) is known to potentiate mainly cell-mediated immunity and is widely used in experimental protozoa or helminth vaccines. However, FCA causes chronic inflammation at the site of injection and occasionally autoimmune complications (Bomford, 1989 ). Vaccination of sheep and laboratory animals against Trichostrongylus colubriformis infection has demonstrated that a very high degree of protective immunity can be induced using normal or irradiated larvae (Gregg et al., 1978; Maclean et al., 1986; Douch, 1988; Bezubik et al., 1988). Attempts to isolate protective antigens operating during vaccination have been carried out in the guinea pig model and it has been found that the fourth stage larvae somatic and excretory-secretory proteins stimulate protective immunity to subsequent challenge infection (Rothwell and Love, 1974; Rothwell and Merritt, 1975 ). Aluminium hydroxide gel, but not Bordetella pertussis or levamisole, improved protection when mixed with soluble extract of the fourth stage larvae and injected intraperitoneally. However, there are some doubts about whether antigens involved in the induction of protective immunity in guinea pigs and the natural host (sheep) are the same (O'Donnell et al., 1985, 1989). The aim of the present study was to assess the capacity of FCA and beryllium hydroxide to enhance protective immunity in rabbits induced against T. colubnformis infection by soluble antigens extracted from infective larvae of the nematode. Rabbits vaccinated orally with normal larvae, which were allowed to develop in the host to the late fourth stage, served as immunization controls. MATERIALS AND METHODS
Experimental design Thirty adult New Zealand rabbits in six experimental groups were used (Table 1 ). Groups I-IV were vaccinated at 2-week intervals, as indicated in Table 1. The immunizing infections in Groups I and II were terminated on Day 7 after the administration of each infection (0.18 mg kg- J body weight
275
ADJUVANT INFLUENCEON IMMUNITY IN L3SP-VACCINATEDRABBITS TABLE 1 Experimental design Group
Antigen
Immunization route
Adjuvant
Percentage of protection J
I II III IV V VI
3 X 2000 L3 3 × 5000 L3 3 × 100/tg L3SP 3 × 100 #g L3SP Challenge controls Parasite-free controls
Oral Oral Subcutaneous Subcutaneous
FCA 2 Be(OH)2
87.6 99.8 29.5 94.8
Percentage of protection =
No. of worms in Group V - N o . of worms in immunized Group× 100 No. of worms in Group V
2FCA = Freund's complete adjuvant.
of ivermectin orally). All vaccinated animals and also five naive (Group V) rabbits were challenged orally with 10 000 infective larvae (L3) 14 days after the administration of the third immunizing dose. Five rabbits remained intact and served as parasite-free controls (Group VI). All experimental animals were killed and dissected on Day 21 after challenge. At necropsy, the number of worms in the intestines was estimated and samples of bile and small intestinal mucosa were collected for antibody measurement.
Parasitological techniques Infective larvae of T. colubriformis were cultured in twist-offjars from faeces of sheep infected with a pure strain of the nematode. The infective larvae, after being purified by decantation and filtration through a bolting cloth, were counted by a dilution method, then used for infection or antigen preparation. During dissections, the small intestines were removed and the intestinal contents were washed out with phosphate-buffered saline (PBS). The total worm count was then estimated on the luminal contents by a 10% aliquot technique. The mucous membrane of the intestine was scraped, weighed and divided into two equal parts. One half was used for the extraction of small intestinal mucosal proteins and the other was placed in digestion fluid ( 13 g pepsin and 10.8 ml concentrated HC1 in 1000 ml distilled water) for 12 h at 37°C and afterwards examined for the nematodes. The number of worms recovered was then multiplied by two to give the number of worms that lodged in the mucosa a n d / o r on the wall of the small intestine. The sum of the worm counts from the small intestinal fluid and the mucosa was considered to give the total number of worms recovered.
276
H. W E D R Y C H O W I C Z AND B. BEZUBIK
Sampling methods Blood for serum preparation was collected from infected and control rabbits by puncture of lateral ear veins at weekly intervals. Sera of individual animals were stored at - 2 0 ° C before serological analysis. Small intestinal mucosal proteins were extracted from scrapings of mucous membrane of the small intestines. Two-gram samples were homogenized in 6 ml PBS containing 0.1 M ethylenediaminetetraacetic acid, olisodium salt (EDTA) and protease inhibitors: 0.5 mM of phenylmethylsulphonyl fluoride (PMSF) and 0.01% soybean trypsin inhibitor (Sigma Chemical Company, Poole, Gt. Britain ). The homogenates were centrifuged for 30 min at 14 000 × g and supernatants used for serological analysis.
Parasite antigens Somatic antigens of infective larvae were obtained by homogenization and extraction of the carefully cleaned worms in PBS containing protease inhibitors: L- 1-tosylamide-2-phenylethylchoromethylketone (TPCK), 50/tg ml- 1; N-a-p-tosyl-L-lysine chloromethylketone HC1 (TLCK), 25/lg ml-1; PMSF, 174/~g m l - l (Sigma Chemical Company, Poole, Gt. Britain), at pH 7.6. The extracts were cleared by centrifugation for 1 h at 15 000 × g and then concentrated to 3 mg of protein m l - 1 using Aquacide (Calbiochem, San Diego, CA, U.S.A.).
Adjuvants The adjuvants were administered only with the first and the second antigen injections. Beryllium hydroxide was freshly precipitated from BeSO4 solution according to Hall (1984). The equivalent of 20 mg of BeSO4 was injected subcutaneously a few seconds prior to antigen. FCA (Difco Labs. Ltd., Detroit, MI ) was emulsified with an equal volume of antigen solution and 1 ml of the FCAantigen mixture was injected subcutaneously.
Antibody measurements Gel double diffusion tests were performed using infective larval somatic extracts (L3SP) as the antigenic source, against sera, bile and mucosal extracts of vaccinated animals, in 1% agarose. Radioimmunoprecipitation (RIP) tests were carried out using L3SP labelled with 125I by the chloramine T method (Greenwood et al., 1963). Approximately 105 counts per minute (c.p.m.) ( 5-10 pl) of the labelled antigen were added to 100/tl of PBS, pH 7.6, which contained some 150 pg of normal
ADJUVANT
INFLUENCE
ON IMMUNITY
IN L3SP-VACCINATED
RABBITS
277
serum proteins and ~ 150/tg of bile, mucosal or serum proteins from infected rabbits. Incubation for 13 h at room temperature and for I h at 37°C was followed by the addition of 50 ~1 of goat anti-rabbit IgA or IgG monospecific antiserum (Nordic Immunology Laboratory Ltd., Amsterdam, The Netherlands), washing three times in cold PBS and transfer into flesh tubes. The radioactivity in the precipitates was measured using a gamma-counter. All samples were assayed in duplicate• Variations around the means were expressed as the standard error (SE). Student's t-test was used to assess the statistical significance of differences between groups of rabbits. RESULTS
Immunity to infection The mean worm counts are presented in Fig. 1. Worm burdens found after challenge in Groups I, II and IV differ significantly ( P < 0.001 ) from those in 4000
m
3000
q~
2000
i. o
1000
__T__ •
I
T
II
III
IV
V
G r o u p s
Fig. 1. Burdens of adult T. colubriformis f o u n d in the intestines of immunized and naive rabbits after challenge with 10 000 larvae. The histograms represent the means of five animals. I = rabbits vaccinated with three doses of 2000 L3; II=rabbits vaccinated with three doses of 5000 L3; III =animals immunized with infective larval somatic proteins and Freund's complete adjuvant; IV=animals vaccinated with the same antigen as Group Ill and beryllium hydroxide; V = challenge controls.
278
H. W E D R Y C H O W 1 C Z A N D B. B E Z U B I K
challenge controls. The highest degree of protection (99.8%) was observed in Group II, vaccinated with three doses of 5000 larvae, and also in rabbits immunized with L3SP and beryllium hydroxide as the adjuvant (94.8%). Interestingly, vaccination with the same close of L3SP emulsified in FCA produced very poor immunity (29.5%); the number of worms found in Group III showed no statistically significant differences in comparison with worm burdens observed in naive challenge control rabbits.
Local antibody response The intestinal and bile IgA, and also intestinal mucosa IgG responses to T.
colubriforrnis antigens were measured by RIP using 125I radioisotop~-i,lz~lled L3SP. The results are presented in Fig. 2. The binding of worm proteins by intestinal mucosa IgG was the highest in challenge control rabbits and the lowest in Group I, although statistically significant differences also occurred between Groups V and IV ( P < 0.05 ). Intestinal IgA reacting with 125I-L3SP showed the highest level in Group III and the percentage of radiolabelled parasite proteins bound in this group was ~ 1.7 times higher than in Group I ( P < 0 . 0 1 ) , 1.3 times higher than in Group II ( P < 0 . 0 1 ) , 1.5 times higher than in Group IV ( P < 0 . 0 0 1 ) , and > 2.6 times higher than in the challenge control ( P < 0.001 ). The amounts of 125I-L3SP bound by bile IgA were much lower than those measured in the intestinal mucosa. The highest binding ac-
I
~ = mucosal IgG []
mucosal
][1[[[I]
bile
IgA
50
iii!i!
lgA
40
30 :: I
,,o
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-L 20
.7
l
I1
II1
IV
V
VI
Groups
Fig. 2. Bile and mucosal antibodies against infective larvae somatic antigens. (For explanation of groups see Fig. 1. )
279
A D J U V A N T I N F L U E N C E ON I M M U N I T Y IN L3SP-VACCINATED RABBITS
tivity was observed with biles of Group IV and the percentage of antigen radioactivity bound in this group was 2.3 times higher than in Group I ( P < 0 . 0 0 1 ) , 1.9 times higher than in Group II ( P < 0 . 0 0 1 ) , 1.23 times more than in Group III ( P < 0 . 0 1 ) , and 1.7 times higher than in the challenge controls ( P < 0.002).
Serum antibody responses In gel double diffusion tests, antibodies reacting with L3SP were detected only in Groups III and IV. The earliest precipitin reaction was observed in one rabbit of Group III; a single precipitation arc developed in the serum collected on Day 17 of the experiment (DE). All rabbits of this group reacted positively and formed 1-4 precipitation bands 2 weeks later. At the time of • o
I II IV
[ 60
50
i.
40
30
0
e~ 20 \
2
"l
10
I
I
I
I
I
I
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10
20
30
40
SO
Days
Fig. 3. Serum IgG antibodies against
L3
L3SP and FCA (III) or beryllium (IV).
of
J 60
experiment
in rabbits vaccinated with living larvae (I and II) or
280
~
e,,
H. WEDRYCHOWICZ
60
-
50
-
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I
0
II
AND
B. B E Z U B I K
40
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o
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I
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experiment
Fig. 4. Serum IgA antibodies against L 3 in rabbits vaccinated with hivinglarvae (I and 11) o r L3SP and FCA (III) or beryllium (IV).
challenge infection (46 DE), sera of all rabbits from Group III developed four precipitation lines and at the end of the experiment 3 out of 5 rabbits gave positive reactions of 3-4 precipitation arcs. Sera of four rabbits of Group IV developed 1-2 arcs on Day 25. A similar reaction was observed on Day 32 and just before challenge infection only two rabbits reacted positively (two arcs). No precipitation lines were produced by sera of Group IV collected 2 weeks after challenge. Serum IgG antibody levels increased during immunization to the highest degree in Group I, but at the time of challenge the activity of these antibodies was similar in all vaccinated groups (Fig. 3). The percentage of 125I-L3SP bound by IgG of Group V increased slowly after infection and at the end of the experiment reached a value which was not significantly higher than that in Groups III and IV (results not shown). IgA antibodies reacting with 125I-L3SP increased significantly after admin-
ADJUVANT INFLUENCE ON IMMUNITY IN L3SP-VACCINATED RABBITS
281
istration of the third immunizing dose, and reached maxima before challenge in Groups I, II and IV (Fig. 4). Two weeks after challenge, binding of 125IL3SP by serum IgA was very low in these groups, while in susceptible rabbits of Group III it reached the maximum (Fig. 4). No specific reaction was measured in serum IgA of Group V. DISCUSSION
Rabbits vaccinated orally with three doses of 5000 infective larvae of
T. colubriformis demonstrated very high protective immunity; only single worms were found in the intestines of Group II after challenge with 10 000 larvae. Lower (2000 L3 ) vaccination doses induced slightly poorer protection (87.6%), but still at a level comparable with that induced in sheep by vaccination with two doses of 20 000 irradiated larvae (Dineen et al., 1977 ). Parasites coming from subsequent vaccinations were removed on Day 7 after administration, i.e. when the larvae had developed to the late fourth stage (Rapson et al., 1985); thus the rabbit immune system was stimulated only with antigens of parasitic L3 and fourth stage larvae. Animals vaccinated with infective larval somatic proteins and beryllium hydroxide showed a higher protection level than the rabbits immunized orally with three doses of 2000 L3. However, the same antigenic preparation, when introduced to the host with FCA, induced a very low degree of protection. The mean number of adult nematodes found in the intestines of rabbits treated with FCA was lower than in challenge control rabbits, but the difference was not statistically significant. Similar dependence of the protective ability of nematode antigens on the immunomodulatory action of various adjuvants has been recently reported by Monroy et al. ( 1989 ). To our knowledge, beryllium has not been used in immunoparasitological investigations and, although beryllium has been known as a powerful immunological adjuvant for > 20 years, the mechanism of its adjuvant action is not entirely clear. It has been suggested that the immunological actions of beryllium are caused primarily by the physical effects of microcrystalline beryllium oxides and hydroxides on macrophages, and that such affected macrophages possess an increased ability to present antigens. However, Denham and Hall ( 1988 ) showed that the adjuvant properties of beryllium were not confined to the damaging effects of microcrystalline salts of beryllium, but could be expressed by soluble and relatively non-toxic complexes of beryllium and plasma proteins. Such complexes may induce rapid lymphoproliferative responses in the regional lymph nodes and the appearance of huge numbers of immunoblasts in the efferent lymph (Hall, 1988). In the present experiment, only serum IgA antibody during vaccination and bile IgA on Day 21 after challenge showed clearly higher levels in the beryllium-treated group than in other experimental animals. Sera of rabbits immunized with L3SP and FCA
282
H. WEDRYCHOW1CZ AND B. BEZUBIK
demonstrated much higher activity in gel double diffusion tests than the beryllium group, and also the highest IgA level after challenge infection. The role of serum and intestinal antibodies in conferring protective immunity of the host against T. colubriformis is still obscure. Bottjer et al. ( 1985 ) found that pre-exposure of adult nematodes to serum of goats immune to T. colubriformis decreased subsequent feeding activity of the worms, while Bone and Klesius (1986) reported that IgG antibodies from immune hosts reduced the production of eggs by females of this nematode. Findings regarding the IgA response and its role in immunity against T. colubriformis are also conflicting. Increased levels of Secretory IgA (SIgA) and/or the presence of nematode-specific IgA antibodies were observed in the intestines of sheep and rabbits (Cripps and Rothwell, 1978; Wedrychowicz and Bezubik, 1988 ), but attempts to transfer immunity against T. colubriformis with lymph containing anti-nematode IgA antibodies have failed (Adams et al., 1980). Also, in the present experiment local and systemic antibody levels showed little correlation with the degree of protection. Perhaps, the mechanism of resistance of rabbits against T. colubriformis infection is cell mediated and specific antigenic determinants of such responses do not provoke antibody responses. Rabbits immunized orally with living larvae showed a lower bile IgA antibody level than those vaccinated parenterally with L3SP. The active transfer of rabbit polymeric IgA into bile occurs via the same hepatic secretory component (SC)-mediated transport process as that described for the rat (Delacroix et al., 1982 ). Suggested roles for this transfer include the elimination of 'blocking' p-IgA antibodies which could modulate the effect of other immunoglobulin classes on antigens, and of polymeric IgA (p-IgA) immune complexes from blood, and a reinforcement of intestinal secretory IgA immunity (Tomasi and Plaut, 1985 ). This study has demonstrated that the somatic proteins of infective larvae of T. colubriformis may be as good a source of protective immunogens as living parasites when injected subcutaneously with beryllium hydroxide as the adjuvant. Further investigations are necessary to explain the exact role of local and systemic humoral antibodies during T. colubriformis infection, and also to find out whether the antigens involved in inducing the antibody responses possess protective properties.
ACKNOWLEDGEMENTS
The authors are grateful to Professor Peter H. Holmes of the Department of Veterinary Physiology, University of Glasgow Veterinary School, for his critical reading of the manuscript and helpful advice.
ADJUVANT INFLUENCE ON IMMUNITY IN L3SP-VACCINATED RABBITS
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O'Donnell, I.J., Dineen, J.K., Wagland, B., Letho, S., Werkmeister, J.A. and Ward, C.W., 1989. A novel host-protective antigen from Trichostrongylus colubriformis. Int. J. Parasitol., 19" 327-335. Rapson, E.B., Jenkins, D.C. and Topley, P., 1985. Trichostrongylus colubriformis: in vitro culture of parasitic stages and their use for the evaluation of anthelmintics. Res. Vet. Sci.. 39: 90-94. Rothwell, T.L.W. and Love, R.J., 1974. Vaccination against the nematode Trichostrongylus colubriformis. 1. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance. Int. J. Parasitol., 4: 293-299. Rothwell, T.L.W. and Merritt, G.C., 1975. Vaccination against the nematode Trichostrongylus colubriformis, lI. Attempts to protect guinea-pigs with worm acetylcholinesterase. Int. J. Parasitol., 5: 453-460. Tomasi, T. and Plaut, A.G., 1985. Humoral aspects of mucosal immunity. Adv. Host Defense Mech., 4: 31-61. Wedrychowicz, H. and Bezubik, B., 1988. Systemic and local humoral responses in rabbits following single and multiple infections with Trichostrongylus colubr~forrnis. Acta Parasitol. Pol., 33: 79-89.