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Trypanosoma equiperdum: Active immunization of rabbits with actinomycin D-inactivated parasites
EXPERIMENTAL
46, 323-329
PARASITOLOGY
Tryponosoma
( 1978)
Equiperdum: Actinomycin
Active Immunization of Rabbits with D-Inactivated Parasites
M. H. V. VAN REGENMORTEL,~ K. GILL, AND G. FAZAKERLEY Department
of Microbiology, (Accepted
Unizjersity
for publication
of Cape Town, South Africa 4 October
1978)
VAN REGENMORTEL, M. H. V., GILL, K., AND FAZAKERLEY, G. 1978. TTypanosomn equiperdum: Active immunization of rabbits with actinomycin D-inactivated parasites. Ezpetimental Parasitology 46, 323-329. Trypanosoma equiperdum was inactivated with actinomycin D. Rabbits inoculated with inactivated parasites were completely protected from a challenge inoculation of viable T. equiperdum of the same stabilate. Immune serum from the inoculated rabbits, as well as the 7 S and 19 S serum fractions, were tested for their ability to neutralize trypanosomes. During the first 6 weeks of immunization the neutralizing activity of the 19 S fraction was stronger than that of the 7 S fraction. After 9 weeks, the situation was reversed. INDEX DESCRIPTORS: Trypanosoma equiperdum; Protozoa, parasitic; Rabbit; Immunization; Actinomycin D inactivation; Neutralization; IgM; IgG.
Protection against T. cruzi, the agent of American trypanosomiasis, has been Many attempts have been made to im- achieved in mice by inoculation with acmunize rodents, bovines, and primates tinomycin D-treated parasites (Fernandes against trypanosomiasis. Active immuniza- et al. 1965), with chagastoxin (Seneca et tion against infection with Trypanosomu ~7. 1966) as well as by nonspecific means congolense, T. hue&, T. lewisi or T. rho( Ortiz-Ortiz et al. 1975). Passive transfer desiense has been achieved by inoculating of immunity h’as also been achieved by inanimals with killed parasites (Johnson et jecting mice with immune sera (Krettli and al. 1963) as well as with organisms inacti- Brener 1976). vated by formalin, /?-propiolactone, or y Passive immunity to T. gambiense has irradiation (Soltys 1964, 1967; Sanders and been obtained by immunization with spleen Wallace 1966; Duxbury and Sadun 1969; cells from immune donors (Takayanagi Duxbury et al. 1972; Campbell and Phillips and Nakatake 1975) and with immune sera 1976). Attempts to use attenuated T. equi( Seed and Gam 1966). perdum were unsuccessful, however, as no However, in spite of some limited SUCprotection was found when mice were in- cess, a high level of general immunity oculated with irradiated organisms (Stubbs against trypanosomes has not yet been et al. 1958) . achieved, probably because of the instability of trypanosomal antigens and the vari~Present address: Institut de Biologie Molecuant-specific nature of protective anti-trylaire et Cellulaire, 15, rue Descartes, 67084 Strasbourg Ckdex, France. panosome antibodies. 323 0014-4894/78/0462-0323$02.00~0 All
Copyright 6 1918 by Academic Press, Inc. rights of reproduction in any form reserved.
324
VAN
REGENMORTEL,
GILL,
The present investigation arose during a study of the macroglobulinemia induced in rabbits by infection with 7’. equiperdum (Ross and van Regemnortel 1977). In an attempt to produce increased serum IgM levels, actinomycin D-inactivated trypanosomes were injected into rabbits. It was found that the rabbits became immune to a challenge injection of viable T. equiperdum and that mice were resistant to parasites treated in vitro with the 19 S and 7 S fractions of rabbit immune serum. MATERIALS
AND
AND
TABLE Immunization D-Treated
No. of rabbits
METHODS
Trypanosom43s. Trypanosonw equiperrlum was obtained from Dr. R. D. Bigalke of the Onderstepoort Veterinary Research Institute, Pretoria, and was maintained in liquid nitrogen (Herbert et ~2. 1968) and by peritoneal passage in mice. The blood trypanosomes were counted in a hemocytometer chamber and were harvested and purified by the method of Lanham (1968) using preswollen DE-52 cellulose ( Whatman, Maidstone, Kent). An,imals. Swiss white mice injected with 10” to lo3 organisms developed a high level of parasitemia in 3 to 5 days. Rabbits weighing between 3 and 6 kg and injected intramuscularly with 10’ to log organisms, developed typical dourine symptoms (Haig and Lund 1948; Hoare 1972) after 2 weeks and died ‘after 4 to 5 weeks. Inactivation of trypanosomes. Trypanosomes were inactivated by incubation with actinomycin D (Fernandes et al. 1965). Purified trypanosomes were resuspended in TC 199 (Difco, Detroit, Mich. ) and were incubated ‘at a concentration of 5 x lo7 organisms/ml with 200 yg actinomycin D/ml (Sigma, St. Louis, MO.) for 45 min at 37 C. They were washed twice in phosphatebuffered saline ‘containing 1% glucose and were resuspended to give the final concentrations for immunization indicated in Table I. Rabbits received three intramuscuI~Y injections of the immunizing dose in 0.6 ml at weekly intervals.
FAZAKERLEY
I
of Rabbits
with
Trypanosoma
No. of trypanosonles~~
No. of doses”
Actinomycin
E&per&m
Challcttgittg doscc
~~~-~ --- 103 103 10’
0 100 0
104 10” 103
0 100 100
4 x 103 1 x lo”
100 0 INI
3 3 3 3
1 1 I 1
1 x 10” 1 x 108 1X10”
:; 3 3
1 x 2 x
1 x 107
3
2 5 2 2
6 1 1 2
1 2 5
4 2
x x x x
106 107 107 108
Percentage survivald
x x x x
.-
* Trypattosomes (5 X 1O7 organisms/ml) ww inactivated with 200 fig/ml actinomycin D for 45 min at 37 C, washed twice in phosphate-buffered saline and resuspended to provide the ntmibcr of organisms indicated. * Three intramttscular injections of the immunizittg dose of trypanosomcs in 0.F ml were given at weekly intervals. c The attitnals were challenged with one injection uf viable trypanosontes of the same stabilate as the one used for immuttizatiott. d Survival recorded 4 weeks after the challenge injection. No further deaths occttrred in the stthsequent 2 months.
Separation of 7 S and 19 S fractions of sera. Immunized and control rabbits were bled at weekly intervals over a period of 3 months. The 19 S and 7 S components of rabbit sera were separated by density gradient centrifugation on 0 to 40% sucrose gradients in 19-ml cellulose nitrate tubes of a Beckman SW 27.1 rotor. A volume of 0.1 ml serum was placed on the gradients and the tubes were centrifuged at 27,000 rpm for 26 hr in a Beckman Model L3-50 ultracentrifuge. Fractions of 1 ml were collected by upward displacement of the contents of the tubes and were monitored by measuring the absorbance at 280 nm in an ISCO Model 640 fractionator (ISCO, Lincoln, Nebr.). Pooled fractions of the 7 S and 19 S components were concentrated to 0.1 ml by ultrafiltration on Amicon XM lm A membranes (Amicon, Lexington, Mass. ).
T. equiperdum:
IMMUNIZATION
1
I
OF
325
RABBITS
by the method of Herbert (1976).
and Lumsden
RESULTS
\
6
I
I
20
I
1 34
Active Immunization
.-.
I
I 48
I
I
61
I
77
day3
FIG. 1. Anti-trypanosome complement fixation titers of antisera obtained from rabbits immunized with inactivated Trypanosoma equiperdum. Individual results obtained with three rabbits are shown. Immunizing doses (10’ inactivated parasites) were given on Days 1, 6, and 13 and a single challenge dose with viable trypanosomes on Day 20. Rabbit 1 (A) received a challenge dose of 4 X lo3 trypanosomes, rabbit 2 ( l ) received a challenge of 1 X 10’ trypanosomes, and rabbit 3 ( W ) received an immunizing dose of 10' inactivated parasites and no challenge. Immune serum from the same three animals was used to obtain the data of Table 2.
MicrocompZement fixation. Complement fixation tests were carried out by the method of Bradstreet and Taylor (1962) using Microtitre plates (Cooke Instruments, Zurich) and were used to detect the presence of anti-trypanosomal antibodies in rabbit serum and in 19 S and 7 S serum fractions. Suspensions of washed trypanosomes (1 x lo6 organisms/ml) were used as antigen. In vitro neutralization of typanosomes. A volume of 0.5 ml of lo3 trypanosomes/ml in the AB pH 7.4 phosphate buffer of Lumsden et al. (1965) was mixed at 4 C with 0.5 ml of various dilutions of rabbit serum, 7 S, or 19 S serum fraction. Mixing was started 25 min after removing the stabilate from liquid nitrogen (Lumsden et ~1. 1968) and after a 30-min incubation the suspension was injected into mice. Trypanosomes incubated with normal rabbit serum were injected in control mice. Mice were 0bserve.d for parasitemia and, in initial experiments, estimates of the number of organisms per milliliter of blood were made
of Rabbits
Rabbits were inoculated with 5 x lo8 to 2 x lo8 inactivated trypanosomes according to the schedules indicated in Table I. After 20 days, the animals were challenged intramuscularly with lo3 to lo4 viable organisms of the same stabilate as the one used for immunization. Three injections of lo7 inactivated organisms produced complete protection when the challenging dose was lo3 viable organisms but had no effect when the animals received a challenge of lo4 organisms (Table I). When three immunizing doses of IO6 inactivated organisms were used, the animals were protected against a challenge of 4 x lo3 viable organisms. Neutrakation of Trypanosomes with Serum Components from Immunized Rabbits Groups of five mice were injected with lo3 trypanosomes which had been incubated with various dilutions of either whole serum, 7 S, or 19 S serum components obtained from rabbits previously inoculated with lo8 inactivated organisms. Serum was obtained from these rabbits at weekly intervals ‘during the entire experiment. Antitrypanosome complement fixation titer of antisera obtained from three rabbits immunized with inactivated parasites are illustrated in Fig. 1. Maximum activity was demonstrated on Days 20 to 34 following the first immunization. It has been shown previously, by indirect immunofluorescence and complement fixation tests (Ross ‘and Van Regenmortel 1977), that maximum anti-trypanosome activity appears in inoculated rabbits 13 days after infection with a lethal dose of viable trypanosomes. It was also found that anti-trypanosome IgM antibodies decreased after Day 13, but that the level of IgG antibodies could either in-
326
VAN
REGENMORTEL,
GILL,
TABLE Neutralization
of Trypanosoma
Dilution
equip&urn
AND
II
with Serum Components
Serum
Rabbit lb l/20 l/80 l/320 l/1280 l/5120 l/20480
41
4 5 :‘, 2
6 4 5 2 2 0
ltabbit 2’ 1po I /so l/320 l/1280 l/5120 1/204SO
i 3 2 I 0
5 3 :: 1 0 0
Rabbit 3” l/20 l/80 l/320 l/1280 l/5120 l/20450
4 3 2 0 0 0
5 3 3 3 0 0
I-
Rabbits
i S Fraction I ):iy
Day
20
1 (1
from Immunized
19 S Fraction ~.-.__-
Day0 6
FAZAKERLEY
61
--
-
6
20
41
61
3
5
5
5
5
4 4 I 0 0
1 0 0 0 0
3 3 0 0 0
3 2 1 0 0
4 1 0 0 0
6
20
41
61
:: 0
3 3
4 3
4
1
I
1
3 2
0
0
1
0
0
0
0 0
0 0
0 0
0 0 0 0 0 0
2 3 2 0 0 0
5 ‘I ; 0 0 0
4 4 4 0 0 0
e Day after initial immunizing injection of the rabbits. b Rabbit 1 received immunizing doses of lo8 inactivated trypanosomes on Days 1, 6, and 13 and a challenge dose of 4 X 10” viable parasites on Day 20. Complement fixation titers of serum from rabbits 1, 2, and 3 are shown in Fig. 1. c Number of mice siuviving 6 days after receiving an injection of lo3 viable trypanosomes preincubated with various dilutions of rabbit immune serum. Five mice per group. No mice survived when the parasites were incubated with nonimmune rabbit serum. d Rabbit 2 received the same immunizing injections as rabbit 1 and a challenge dose of 1 X lo3 viable trypanosomes on Day 20. e Rabbit 3 received an immunizing injection of lo7 inactivated parasit,es but no challenge.
crease and decrease during this period. In an attempt to determine whether the protection demonstrated in Table I was caused by humoral components of the immune response, parasites of the same stabilate as the one used for immunization were preincubated with immune sera from rabbits and the mixtures injected into mice. The 7 S and 19 S serum fractions from immunized animals were also tested for their ability to neutralize trypanosomes. In vitro neutralization results obtained with three rabbits
are presented in Table II. The same animals were used to obtain the complement fixation data shown in Fig. 1. The neutralization data (Table II) showed that the serum from immunized rabbits could inactivate trypanosomes up to a dilution of l/5120. The protective activity of the antisera at Days 6 and 20 after the start of immunization was due mainly to the 19 S serum fraction. At Day 61, however, more neutralizing activity was found in the 7 S than in the 19 S serum fraction,
T. equiperdum:
IMMUNIZATION
DISCUSSION
Fernandes et al. (1965) have shown that when mice are injected with actinomycin D-treate’d Trypanosoma cruxi, the animals are protected against a subsequent exposure to the viruIent parasites, The resu1t.s presented in Table I show that complete protection against a lethal infection with T. equiperdum can also be produced in rabbits by injections of actinomycin D-treated parasites. The animals were fully protected against a challenging dose of 1 x 10” to 4 X lo3 viable organisms but not against a more severe challenge of 104 organisms. Previous work has established that it is possible to obtain complete protection against trypanosomiasis by the passive tr.ansfer of antiserum against trypanosomal antigens (Seed and Gam 1966; Krettli and Brener 1976; Kierszenbaum and Howard 1976). The participation of compIement in host resistance has also been studied extensively (see Jarvinen and Dahnasso 1976) and there is evidence that phagocytosis of trypanosomes by macrophages is enhanced by specific antiserum against the parasites (Patton 1972; Takayanagi et al. 1974). In the case of T. gambiense infections in mice, Takayanagi and Enriquez (1973) showed that the IgM fraction of immune serum is more effective than IgG in protecting mice against infection. The results presented in Fig. 1 and Table II show that with T. equiperdum infected rabbits there is a reasonable correlation between antitrypanosome complement fixation titers and protective activity of the immune serum. During the first few weeks of immunization, the protective activity of the 19 S fraction is stronger than that of the 7 S, but the situation is reversed ‘after 9 weeks. Although it was found previously (Ross and Van Regenmortel 1977) that the anti-trypanosome activity of the 19 S fraction of infected rabbits had decreased considerably 4 weeks after infection, the present data
327
OF RABBITS
(Table II) show that the neutralization potency of the 19 S fraction was still unchanged 6 weeks after the start of immunization. The importance of the protective effects that are observed suggests that the present system might be a useful model for studying the mechanism by which immune protection occurs. Although actinomycin D-treated trypanosomes lose the ability to multiply and are totally noninfectious, their immunogenicity appears to be unimpaired. Numerous workers have shown that when normal division of trypanosomes is suppressed but the organisms are not killed, the parasites produce excretory and secretory antigens which are particularly active in inducing protection (Thillet and Chandler 1957; Yasuda and Dusanic 1969; Duxbury et al. 1974). The immunity induced with inactivated T. equiperdum is likely to be strain and variant-specific (see Wellde et al. 1975; Viens et al. 1975). The recent demonstration of extensive antigenic variation of T. equiperdum in infected rabbits (Capbern et al. 1977) suggests that the present system could provide an interesting approach to the study of variant antigens of this organism. ACKNOWLEDGMENT This work was supported by a grant South African Medical Research Council.
from
the
REFERENCES BRADSTREET, C. M. P., AND TAYLOR, C. E. D. 1982. Technique of complement fixation test applicable to the diagnosis of virus disease. Monthly Bulletin of the Ministry of Health and the Public Health Laboratory Service 21,98-104. CAMPBELL, G. H., AND PHILLIPS, S. M. 1976. Adoptive transfer of variant-specific resistance to Trypanosoma Thodesiense with B lymphocytes and serum. Infection and Immunity 14, 11441150. CAPBERN, A., GIROUD, C., BALTZ, T., and MATTERN, P. 1977. Trypanosoma equiperdum: Etude
328
VAN
REGENMORTEL,
GILL,
des variations antigeniques au tours de la tryexpkrimentale du lapin. Experipanosomose mental Parasitology 42, 6-13. DUXBURY, R. E., ANDERSON,J. S., WELLDE, B. T., SADUN, E. H., AND MURIITHI, I. E. 1972. Trypanosoma congolensc: Immunization of mice, dogs and cattle with gamma-irradiated parasites. Experimental Parasitology 32, 527-533. D~XBURY, R. E., AND SADUN, E. H. 1969. Resistance produced in mice and rats by inoculation with irradiated Trypanosoma rhodesiense. Journal of Parasitology 55, 859-865. DUXBUR~, R. E., SADUN, E. H., SCHOENBECHLER, M. J., AND STROUPE,D. A. 1974. Trypanosoma rAodesiense: Protection in mice by inoculations of homologous parasite products. Experimental Parasitology 36, 70-76. FERNANDES,J. F., HALSMAN, M., AND CASTELLANI, 0. 1985. Effect of actinomycin 0 on the infectivity of Typanosoma cruzi. Nature (London) 207,1004-1005. HAIG, D. A., AND LUND, A. S. 1948. Transmission of the South African strain of dourine to laboratory animals. Onderstepoort Journal of Veterinary Science 23, 59-61. HERBERT, W. J., AND LUMSDEN, W. H. R. 1976. “matching” A rapid Trypanosoma brucei: method for estimating the host’s parasitemia. Experimental Parasitology 40, 427-431. HERBERT,W.J., LUMSDEN,W. H. R., AND FREXCH, A. M. 1968. Survival of trypanosomes after rapid cooling and storage at -196°C. Transactions of the Royal Society for Tropical Medicine and Hygiene 62, 209-212. HOARE, C. A. 1972. “The Trypanosomes of Mammals.” Blackwell, Oxford. JARVINEN, J. A., AXD DALMASSO,A. P. 1976. Complement in experimental Trypanosoma lewisi infections of rats. Infection and Zmmunity 14, 894-902. JOHNSON,P., NEAL, R. A., AND GALL, D. 1963. Protective effect of killed trypanosome vaccines with incorporated adjuvants. Nature (London) 200, 83. KIERSZENBAUM, F., AND HOWAHD, J. G. 1976. Mechanisms of resistance against experimental Trypanosoma cruzi infection: The importance of antibodies and antibody-forming capacity in the Biozzi high and low responder mice. ZournaZ of Immunology 116, 1208-1211. KRETTLI, A. U., AND BRENER, Z. 1976. Protective effects of specific antibodies in Trypanosoma cruzi infections. Journal of Immunology 116, 755-760.
AND
FAZAKERLEY
LANHAM, S. M. 1968. Separation
of trypanosomes from the blood of infected rats and mice by anion exchangers. Nature (London) 218, 12731274. LUMSDEN, W. H. R., CUXNINGHA~I, M. P., WEBBER, W. A. F., VAX HOEVE, K., KNIGHT, R. II., AND SIMMONS, V. 1965. Some effects of hydrogen ion concentration on Trypanosome numbers and infectivity. Experimental Parasitology 16, 8-17. LU~ISDEN, W. H. R., CITATIIA, S. Ii., AXD LUTZ, W. 1968. Factors influencing the infectivity of a Trypanosoma brucei stabilate for mice. Journal of ProtozooZogy 15, 129-131.
OISTIZ-ONTIZ, I,., GONZALES-MENDOZA, A., AND LAMOYI, E. 1975. A vaccination procedure against Trypanosomu cruzi infection in mice by nonspecific immunization. Journal of Zmmunology114,1424-1425. PATTON, C. L. 1972. Trypanosomu lewisi: Influence of sera and peritoneal exudate cells. Experimental Parasitology 31, 370377. Ross, J. hi., AND VAN REGENMORTEL,M. H. V. 1977. Autoimmunity and absence of rheumatoid factors in experimental Trypanosoma (Trypanozoon) equiperdum infections in the rabbit. Annales d’Zmmunologie (Znstitut Pasteur) 128C, 817-832. SAXDERS,A., AND WALLACE, F. G. 1966. Immunization of rats with irradiated Trypanosoma lewisi. Experimental Parasitology 18, 301-304. SEED, J. R., AND GAM, A. A. 1966. Passive immunity to experimental trypanosomiasis. Journal of Parasitology 52, 1134-1140. SENECA, H., PEER, P., AXD HAMPAR, B. 1966. Active immunization of mice with chagastoxin. Nature (London) 209, 309-310. SOLTYS,hf. A. 1964. Immunity in trypanosomiasis: V, Immunization of animals with dead trypanosomes. Parasitology 54, 585-591. SOLTYS, M. A. 1967. Comparative studies of immunogenic properties of Trypanosoma brucei inactivated with P-propiolactone and with some other inactivating agents. Canadiun Journal of Microbiology 13, 743-747. STUBSS,R. K., BOBALIK, G., AND EHCOLI, N. 1958. Effect of X-ray radiation on Trypanosoma equiper&m in vivo and in vitro. Journal of Znfectious Diseases 102, 3543. TAKAYANAGI, T., AND ENRIQUEZ, G. L. 1973. Effects of the IgG and IgM immunoglobulins in Trypanosoma gambiense infections in mice. JournuZ of Parasitology 59, 644-647. TAKAYANACI, T., AND NAKATAKE, k'. 1975. Trypanosoma gamhiense: Enhancement of agglu-
T.
f3&WdUm:
IMMUNIZATION
tinin and protection in subpopulations by immune spleen cells. Experimental Parasitology 38, 233-239. TAKAYANAGI, T., NAKATAKE, Y., AND ENFUQUEZ, G. L. 1974. Typanosoma gambiense: Phagocytosis in vitro. Experimental Parasitology 36, 106113. THILLET, C. J., JR., AND CHANDLER, A. C. 1957. Immunization against Trypanosoma lewisi in rats by infections of metabolic products. Science 125346-347. VIENS, P., TARGETT, G. A. T., AND LUMSDEN, W. H. R. 1975. The immunological response of CBA
OF
RABBITS
329
mice to Trypanosomu musculi: Mechanisms of protective immunity. International bUTfad fOT Parasitology 5,235-239. WELLDE, B. T., SCHOENBECHLER, M. J., DIGGS, C. L., LANGBEHN, H. R., AND SADIJN, E. H. 1975. Trypanosoma rhodesiense: Variant specificity of immunity induced by irradiated parasites. Experimental Parasitology 37, 12%129. YASUDA, S., AND DUSANIC, D. G. 1969. Comparative immunological studies on somatic antigens and secretion-excretion products of blood stream and culture forms of Trypanosoma lewisi. Journal of Infectious Diseases 119, 562-568.
46, 323-329
PARASITOLOGY
Tryponosoma
( 1978)
Equiperdum: Actinomycin
Active Immunization of Rabbits with D-Inactivated Parasites
M. H. V. VAN REGENMORTEL,~ K. GILL, AND G. FAZAKERLEY Department
of Microbiology, (Accepted
Unizjersity
for publication
of Cape Town, South Africa 4 October
1978)
VAN REGENMORTEL, M. H. V., GILL, K., AND FAZAKERLEY, G. 1978. TTypanosomn equiperdum: Active immunization of rabbits with actinomycin D-inactivated parasites. Ezpetimental Parasitology 46, 323-329. Trypanosoma equiperdum was inactivated with actinomycin D. Rabbits inoculated with inactivated parasites were completely protected from a challenge inoculation of viable T. equiperdum of the same stabilate. Immune serum from the inoculated rabbits, as well as the 7 S and 19 S serum fractions, were tested for their ability to neutralize trypanosomes. During the first 6 weeks of immunization the neutralizing activity of the 19 S fraction was stronger than that of the 7 S fraction. After 9 weeks, the situation was reversed. INDEX DESCRIPTORS: Trypanosoma equiperdum; Protozoa, parasitic; Rabbit; Immunization; Actinomycin D inactivation; Neutralization; IgM; IgG.
Protection against T. cruzi, the agent of American trypanosomiasis, has been Many attempts have been made to im- achieved in mice by inoculation with acmunize rodents, bovines, and primates tinomycin D-treated parasites (Fernandes against trypanosomiasis. Active immuniza- et al. 1965), with chagastoxin (Seneca et tion against infection with Trypanosomu ~7. 1966) as well as by nonspecific means congolense, T. hue&, T. lewisi or T. rho( Ortiz-Ortiz et al. 1975). Passive transfer desiense has been achieved by inoculating of immunity h’as also been achieved by inanimals with killed parasites (Johnson et jecting mice with immune sera (Krettli and al. 1963) as well as with organisms inacti- Brener 1976). vated by formalin, /?-propiolactone, or y Passive immunity to T. gambiense has irradiation (Soltys 1964, 1967; Sanders and been obtained by immunization with spleen Wallace 1966; Duxbury and Sadun 1969; cells from immune donors (Takayanagi Duxbury et al. 1972; Campbell and Phillips and Nakatake 1975) and with immune sera 1976). Attempts to use attenuated T. equi( Seed and Gam 1966). perdum were unsuccessful, however, as no However, in spite of some limited SUCprotection was found when mice were in- cess, a high level of general immunity oculated with irradiated organisms (Stubbs against trypanosomes has not yet been et al. 1958) . achieved, probably because of the instability of trypanosomal antigens and the vari~Present address: Institut de Biologie Molecuant-specific nature of protective anti-trylaire et Cellulaire, 15, rue Descartes, 67084 Strasbourg Ckdex, France. panosome antibodies. 323 0014-4894/78/0462-0323$02.00~0 All
Copyright 6 1918 by Academic Press, Inc. rights of reproduction in any form reserved.
324
VAN
REGENMORTEL,
GILL,
The present investigation arose during a study of the macroglobulinemia induced in rabbits by infection with 7’. equiperdum (Ross and van Regemnortel 1977). In an attempt to produce increased serum IgM levels, actinomycin D-inactivated trypanosomes were injected into rabbits. It was found that the rabbits became immune to a challenge injection of viable T. equiperdum and that mice were resistant to parasites treated in vitro with the 19 S and 7 S fractions of rabbit immune serum. MATERIALS
AND
AND
TABLE Immunization D-Treated
No. of rabbits
METHODS
Trypanosom43s. Trypanosonw equiperrlum was obtained from Dr. R. D. Bigalke of the Onderstepoort Veterinary Research Institute, Pretoria, and was maintained in liquid nitrogen (Herbert et ~2. 1968) and by peritoneal passage in mice. The blood trypanosomes were counted in a hemocytometer chamber and were harvested and purified by the method of Lanham (1968) using preswollen DE-52 cellulose ( Whatman, Maidstone, Kent). An,imals. Swiss white mice injected with 10” to lo3 organisms developed a high level of parasitemia in 3 to 5 days. Rabbits weighing between 3 and 6 kg and injected intramuscularly with 10’ to log organisms, developed typical dourine symptoms (Haig and Lund 1948; Hoare 1972) after 2 weeks and died ‘after 4 to 5 weeks. Inactivation of trypanosomes. Trypanosomes were inactivated by incubation with actinomycin D (Fernandes et al. 1965). Purified trypanosomes were resuspended in TC 199 (Difco, Detroit, Mich. ) and were incubated ‘at a concentration of 5 x lo7 organisms/ml with 200 yg actinomycin D/ml (Sigma, St. Louis, MO.) for 45 min at 37 C. They were washed twice in phosphatebuffered saline ‘containing 1% glucose and were resuspended to give the final concentrations for immunization indicated in Table I. Rabbits received three intramuscuI~Y injections of the immunizing dose in 0.6 ml at weekly intervals.
FAZAKERLEY
I
of Rabbits
with
Trypanosoma
No. of trypanosonles~~
No. of doses”
Actinomycin
E&per&m
Challcttgittg doscc
~~~-~ --- 103 103 10’
0 100 0
104 10” 103
0 100 100
4 x 103 1 x lo”
100 0 INI
3 3 3 3
1 1 I 1
1 x 10” 1 x 108 1X10”
:; 3 3
1 x 2 x
1 x 107
3
2 5 2 2
6 1 1 2
1 2 5
4 2
x x x x
106 107 107 108
Percentage survivald
x x x x
.-
* Trypattosomes (5 X 1O7 organisms/ml) ww inactivated with 200 fig/ml actinomycin D for 45 min at 37 C, washed twice in phosphate-buffered saline and resuspended to provide the ntmibcr of organisms indicated. * Three intramttscular injections of the immunizittg dose of trypanosomcs in 0.F ml were given at weekly intervals. c The attitnals were challenged with one injection uf viable trypanosontes of the same stabilate as the one used for immuttizatiott. d Survival recorded 4 weeks after the challenge injection. No further deaths occttrred in the stthsequent 2 months.
Separation of 7 S and 19 S fractions of sera. Immunized and control rabbits were bled at weekly intervals over a period of 3 months. The 19 S and 7 S components of rabbit sera were separated by density gradient centrifugation on 0 to 40% sucrose gradients in 19-ml cellulose nitrate tubes of a Beckman SW 27.1 rotor. A volume of 0.1 ml serum was placed on the gradients and the tubes were centrifuged at 27,000 rpm for 26 hr in a Beckman Model L3-50 ultracentrifuge. Fractions of 1 ml were collected by upward displacement of the contents of the tubes and were monitored by measuring the absorbance at 280 nm in an ISCO Model 640 fractionator (ISCO, Lincoln, Nebr.). Pooled fractions of the 7 S and 19 S components were concentrated to 0.1 ml by ultrafiltration on Amicon XM lm A membranes (Amicon, Lexington, Mass. ).
T. equiperdum:
IMMUNIZATION
1
I
OF
325
RABBITS
by the method of Herbert (1976).
and Lumsden
RESULTS
\
6
I
I
20
I
1 34
Active Immunization
.-.
I
I 48
I
I
61
I
77
day3
FIG. 1. Anti-trypanosome complement fixation titers of antisera obtained from rabbits immunized with inactivated Trypanosoma equiperdum. Individual results obtained with three rabbits are shown. Immunizing doses (10’ inactivated parasites) were given on Days 1, 6, and 13 and a single challenge dose with viable trypanosomes on Day 20. Rabbit 1 (A) received a challenge dose of 4 X lo3 trypanosomes, rabbit 2 ( l ) received a challenge of 1 X 10’ trypanosomes, and rabbit 3 ( W ) received an immunizing dose of 10' inactivated parasites and no challenge. Immune serum from the same three animals was used to obtain the data of Table 2.
MicrocompZement fixation. Complement fixation tests were carried out by the method of Bradstreet and Taylor (1962) using Microtitre plates (Cooke Instruments, Zurich) and were used to detect the presence of anti-trypanosomal antibodies in rabbit serum and in 19 S and 7 S serum fractions. Suspensions of washed trypanosomes (1 x lo6 organisms/ml) were used as antigen. In vitro neutralization of typanosomes. A volume of 0.5 ml of lo3 trypanosomes/ml in the AB pH 7.4 phosphate buffer of Lumsden et al. (1965) was mixed at 4 C with 0.5 ml of various dilutions of rabbit serum, 7 S, or 19 S serum fraction. Mixing was started 25 min after removing the stabilate from liquid nitrogen (Lumsden et ~1. 1968) and after a 30-min incubation the suspension was injected into mice. Trypanosomes incubated with normal rabbit serum were injected in control mice. Mice were 0bserve.d for parasitemia and, in initial experiments, estimates of the number of organisms per milliliter of blood were made
of Rabbits
Rabbits were inoculated with 5 x lo8 to 2 x lo8 inactivated trypanosomes according to the schedules indicated in Table I. After 20 days, the animals were challenged intramuscularly with lo3 to lo4 viable organisms of the same stabilate as the one used for immunization. Three injections of lo7 inactivated organisms produced complete protection when the challenging dose was lo3 viable organisms but had no effect when the animals received a challenge of lo4 organisms (Table I). When three immunizing doses of IO6 inactivated organisms were used, the animals were protected against a challenge of 4 x lo3 viable organisms. Neutrakation of Trypanosomes with Serum Components from Immunized Rabbits Groups of five mice were injected with lo3 trypanosomes which had been incubated with various dilutions of either whole serum, 7 S, or 19 S serum components obtained from rabbits previously inoculated with lo8 inactivated organisms. Serum was obtained from these rabbits at weekly intervals ‘during the entire experiment. Antitrypanosome complement fixation titer of antisera obtained from three rabbits immunized with inactivated parasites are illustrated in Fig. 1. Maximum activity was demonstrated on Days 20 to 34 following the first immunization. It has been shown previously, by indirect immunofluorescence and complement fixation tests (Ross ‘and Van Regenmortel 1977), that maximum anti-trypanosome activity appears in inoculated rabbits 13 days after infection with a lethal dose of viable trypanosomes. It was also found that anti-trypanosome IgM antibodies decreased after Day 13, but that the level of IgG antibodies could either in-
326
VAN
REGENMORTEL,
GILL,
TABLE Neutralization
of Trypanosoma
Dilution
equip&urn
AND
II
with Serum Components
Serum
Rabbit lb l/20 l/80 l/320 l/1280 l/5120 l/20480
41
4 5 :‘, 2
6 4 5 2 2 0
ltabbit 2’ 1po I /so l/320 l/1280 l/5120 1/204SO
i 3 2 I 0
5 3 :: 1 0 0
Rabbit 3” l/20 l/80 l/320 l/1280 l/5120 l/20450
4 3 2 0 0 0
5 3 3 3 0 0
I-
Rabbits
i S Fraction I ):iy
Day
20
1 (1
from Immunized
19 S Fraction ~.-.__-
Day0 6
FAZAKERLEY
61
--
-
6
20
41
61
3
5
5
5
5
4 4 I 0 0
1 0 0 0 0
3 3 0 0 0
3 2 1 0 0
4 1 0 0 0
6
20
41
61
:: 0
3 3
4 3
4
1
I
1
3 2
0
0
1
0
0
0
0 0
0 0
0 0
0 0 0 0 0 0
2 3 2 0 0 0
5 ‘I ; 0 0 0
4 4 4 0 0 0
e Day after initial immunizing injection of the rabbits. b Rabbit 1 received immunizing doses of lo8 inactivated trypanosomes on Days 1, 6, and 13 and a challenge dose of 4 X 10” viable parasites on Day 20. Complement fixation titers of serum from rabbits 1, 2, and 3 are shown in Fig. 1. c Number of mice siuviving 6 days after receiving an injection of lo3 viable trypanosomes preincubated with various dilutions of rabbit immune serum. Five mice per group. No mice survived when the parasites were incubated with nonimmune rabbit serum. d Rabbit 2 received the same immunizing injections as rabbit 1 and a challenge dose of 1 X lo3 viable trypanosomes on Day 20. e Rabbit 3 received an immunizing injection of lo7 inactivated parasit,es but no challenge.
crease and decrease during this period. In an attempt to determine whether the protection demonstrated in Table I was caused by humoral components of the immune response, parasites of the same stabilate as the one used for immunization were preincubated with immune sera from rabbits and the mixtures injected into mice. The 7 S and 19 S serum fractions from immunized animals were also tested for their ability to neutralize trypanosomes. In vitro neutralization results obtained with three rabbits
are presented in Table II. The same animals were used to obtain the complement fixation data shown in Fig. 1. The neutralization data (Table II) showed that the serum from immunized rabbits could inactivate trypanosomes up to a dilution of l/5120. The protective activity of the antisera at Days 6 and 20 after the start of immunization was due mainly to the 19 S serum fraction. At Day 61, however, more neutralizing activity was found in the 7 S than in the 19 S serum fraction,
T. equiperdum:
IMMUNIZATION
DISCUSSION
Fernandes et al. (1965) have shown that when mice are injected with actinomycin D-treate’d Trypanosoma cruxi, the animals are protected against a subsequent exposure to the viruIent parasites, The resu1t.s presented in Table I show that complete protection against a lethal infection with T. equiperdum can also be produced in rabbits by injections of actinomycin D-treated parasites. The animals were fully protected against a challenging dose of 1 x 10” to 4 X lo3 viable organisms but not against a more severe challenge of 104 organisms. Previous work has established that it is possible to obtain complete protection against trypanosomiasis by the passive tr.ansfer of antiserum against trypanosomal antigens (Seed and Gam 1966; Krettli and Brener 1976; Kierszenbaum and Howard 1976). The participation of compIement in host resistance has also been studied extensively (see Jarvinen and Dahnasso 1976) and there is evidence that phagocytosis of trypanosomes by macrophages is enhanced by specific antiserum against the parasites (Patton 1972; Takayanagi et al. 1974). In the case of T. gambiense infections in mice, Takayanagi and Enriquez (1973) showed that the IgM fraction of immune serum is more effective than IgG in protecting mice against infection. The results presented in Fig. 1 and Table II show that with T. equiperdum infected rabbits there is a reasonable correlation between antitrypanosome complement fixation titers and protective activity of the immune serum. During the first few weeks of immunization, the protective activity of the 19 S fraction is stronger than that of the 7 S, but the situation is reversed ‘after 9 weeks. Although it was found previously (Ross and Van Regenmortel 1977) that the anti-trypanosome activity of the 19 S fraction of infected rabbits had decreased considerably 4 weeks after infection, the present data
327
OF RABBITS
(Table II) show that the neutralization potency of the 19 S fraction was still unchanged 6 weeks after the start of immunization. The importance of the protective effects that are observed suggests that the present system might be a useful model for studying the mechanism by which immune protection occurs. Although actinomycin D-treated trypanosomes lose the ability to multiply and are totally noninfectious, their immunogenicity appears to be unimpaired. Numerous workers have shown that when normal division of trypanosomes is suppressed but the organisms are not killed, the parasites produce excretory and secretory antigens which are particularly active in inducing protection (Thillet and Chandler 1957; Yasuda and Dusanic 1969; Duxbury et al. 1974). The immunity induced with inactivated T. equiperdum is likely to be strain and variant-specific (see Wellde et al. 1975; Viens et al. 1975). The recent demonstration of extensive antigenic variation of T. equiperdum in infected rabbits (Capbern et al. 1977) suggests that the present system could provide an interesting approach to the study of variant antigens of this organism. ACKNOWLEDGMENT This work was supported by a grant South African Medical Research Council.
from
the
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f3&WdUm:
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