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Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics
Journalfor Parisatology Vol.
Internolionol Primed in tireof Bnlbh.
12, No. 4, pp. 251-256,
1982
0020-7519/82/040251+6 $03.00/O Pergomon Press Lfd. Soc~ery for Parosifology
0 1982 Austmlion
RESISTANCE OF MICE IMMUNIZED WITH IRRADIATED AND LYOPHILIZED STAGES OF TRYPANOSOMA CRUZI TO INFECTIONS WITH METACYCLICS EMMANUEL 0.
OKANLA, JERRY L. STUMPF and
DONALD G. DUSANIC
Department of Life Sciences, Indiana State University, Terre Haute, IN 47809, U.S.A. (Received 1 June 1981) AbSfraCt-DKANLA E. O., STUMPFJ. L. & DUSANICD. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Tr~panosoma cruzi to infections with metacyclics. Infernational Journalfor Parasitology12: 251-256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosomacruzi in three weekly injections of 1 x lo8 trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund’s complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestuns. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestuns included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and bloodstream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infesrans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector. INDEX KEY WORDS: T. cruzi; epimastigote; metacyclic; bloodstream vaccines; immunization; resistance; mice.
INTRODUCTION ATTEMPTSto immunize animals against experimental Trypanosoma cruzi infections have utilized trypanosomes from cultures or from the blood of infected
animals (Goble, 1970). Kierszenbaum & Budzko (1975) demonstrated partial protection through a reduction in parasitemia and decreased mortality in animals immunized with sodium perchlorate-treated culture forms of T. cruzi. Partial protection in mice was also demonstrated with frozen and thawed and sonicated vaccines against challenge with bloodstream forms or metacyclics from the vector Rhodnius prolixus (McHardy, 1977; McHardy & Neal, 1979). CFI mice immunized with bloodstream forms of T. cruzi that had been attenuated with 1.5 kGy gamma irradiation, displayed reduced parasitemias when challenged with bloodstream forms (Hanson, Chien, Chapman & Roberson, 1974). In studies by Scott & Gary (1979) that utilized T. cruzi cell surface glycoproteins as a vaccine, the glycoprotein alone did not elicit protection in the
form; parasites; cultivation;
immunized mice, while glycoprotein emulsified in adjuvant enhanced resistance. All of these studies utilized culture forms (predominately epimastigotes) or bloodstream forms as vaccines. There are no reports on the use of metacyclic forms of T. cruzi as vaccines. Since this stage initiates natural infections, a vaccine made from the metacyclics may be effective in preventing infections. Experiments were designed to compare the effectiveness of vaccines made from irradiated or lyophilized epimastigotes, metacyclics or bloodstream forms of T. cruzi against challenge infections by metacyclics obtained from culture. Similar studies were conducted with animals which were injected with irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics isolated from the vector Triatoma infestans. MATERIALS AND METHODS Parasites and animals. The Tulahutn strain of T. cruzi was isolated from Chilean T. infesfuns and sent to our
251
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EMMANUEL0. OKANLA, JERRY L. STUMPF and DONALD G. DUSANIC
laboratory by Dr. Ramiro Martinez-Silva, University of Puerto Rico. This strain has been maintained in our laboratory by syringe injection of 1 x lo5 organisms intraperitoneally every 9-10 days in C3H/Anf mice (Cumberland Farms, Clinton, TN). Female C3H/Anf mice and BALB/c mice (Harland Industries, Indianopolis, IN) weighing 18 and 22 g were used for these studies. Holtzman -rats (Charles River Breeding Laboratories. Wilmington. MA) _. weighing bet -en 100 and 120 g were used for the production of bloostream form trypanosomes. The mice were maintained in plastic cages with 5 animals in each cage and the rats were housed 2 animals to a cage. The animals were provided food and water ad libitum. T. infestans, originally from Chile, were acquired from Dr. Robert Taylor (Center for Disease Control, Atlanta, GA). The T. infestans were housed in covered one gallon glass jars in an environmental chamber (Warren-Sherer Division of Kysor Industries, Marshall, MI) at 27°C and 80% relative humidity. They were fed on the blood of white New Zealand rabbits once a week. The triatomes were infected with T. cruzi by feeding them on infected mice anesthetized with Nembutal (HaverLockhart Laboratories, Kansas City, MO). The abdomens of t‘rese mice were shaved and they were placed on bug containers (Ryckman, 1954) and the bugs were allowed to feed for 1 h in the dark. The bugs were fed on uninfected mice once every week thereafter. Cultivation of parasites. Epimastigotes and metacyclics were cultivated in the LMC medium as described by Dusanic (1980). Epimastigotes were harvested after 10 days, while metacyclics were harvested after 14 days of cultivation. The harvested trypanosomes were centrifuged at 1500 g for 20 min in a Sorvall RC 2-B centrifuge maintained at 4°C. The supernatant fluids were discarded. Metacyclic rich preparations were purified by passage through DEAE-Sephacel (Pharmacia Fine Chemicals, Piscataway, N.J.) using the method of Kreier, Al-Abbassy & Seed (1977). These purified metacyclics preparations contained more than 95% metacyclics. All preparations were then washed in 200 times their volume with 0.2 M phosphate buffered saline pH 7.2 (PBS) by centrifugation as described above. Bloodstream form trypanosomes were obtained from rats that had been exposed 18-24 days previously to 5.8 Gv whole body irradiation from a 6oCo gamma irradiation source (Radiation Laboratory, Department of Physics, Indiana State University) and immediately infected by intraperitoneal injection with 5 x lo6 T. cruzi. The trypanosomes were separated from the rat blood by passage through DEAE-Sephacel (Gutteridge, Cover & Gaborak, 1978) and washed several times by centrifugation with PBS at 1500 g at 4°C for 20 min.
Preparation of vaccines. The epimastigote, metacyclic or bloodstream form trypanosomes to be irradiated were suspended in PBS at a concentration of 5 x lo8 parasites/ml. The tubes containing the parasites were kept in an ice bath and subjected to 3.0 kGy gamma irradiation from a ‘%o gamma irradiation source. These vaccines were used immediately after irradiation. The suspension of trypanosomes to be lyophilized were dispensed into 13 x 100 mm disposable culture tubes with I x lo9 trypanosomes/tube. The tubes were frozen in a mixture of acetone and ethanol (SO/SO) at -76°C. The trypanosomes were then lyophilized with a Vertis lyophilizer (Vertis Co., Gardiner, NY). Rat blood and 0.1% LMC were also irradiated or lyophilized as described for the trypanosomes. In order to test the infectivity of the vaccines, groups of 5 C3H/Anf mice were inoculated subcutaneously with either 1 x 10s irradiated or reconstituted lyophilized preparations of trypanosomes. Blood collected from the tail vein of each animal was
I.J.P. VOL. 12. 1982
examined at 5 day intervals for 15 days. The mice were then decapitated and 1 ml of blood from each mouse was collected and subinoculated into C3H/Anf mice that had been irradiated with 6.0 Gy gamma irradiation. Blood from the tail vein of the mice was sampled after 7 and 14 days. The mice were killed and tissue imprints were made of the heart, spleen, and liver of each animal. The imprints were fixed in 100% methanol, stained with Giemsa and examined microscopically for trypanosomes. Immunization and challenge. Mice were randomly divided in groups of 10 animals each. These animals were injected intramuscularly with 0.2 ml volumes of the vaccines or control preparations. Three injections were administered at weekly intervals. The first injection was made in the muscle of the right thigh, the second in the left thigh and the third in the right thigh. All animals were challenged with T. cruzi 2 weeks after the last injection. Mice were immunized with 1 x 10’ irradiated epimastigotes, metacyclics, or bloodstream forms suspended in PBS in each injection. Three control groups were injected with equivalent volumes of LMC medium, rat blood or PBS. A fourth group of untreated mice was also included. All animals were challenged on the same day with 1 x lo4 culture metacyclics from the same pool of parasites by subcutaneous injection between the scapulae. Three panels of mice were inoculated with lyophilized epimastigotes, metacyclics or bloodstream trypomastigotes suspended in PBS and emulsified in Freund’s complete adjuvant (l:l). Three control groups were injected with LMC medium, rat blood or PBS emulsified with Freund’s complete adjuvant (l:l). The untreated mice also served as a control group for this experiment, since the mice were challenged with 1 x lo4 culture metacyclics from the same pool and via the same route as indicated above. Two groups of mice were injected with 1 x 10s irradiated T. cruzi epimastigotes or metacyclics suspended in PBS for each injection. Groups of control mice were injected with identical volumes of LMC medium or PBS. One group of mice which were not injected served as an additional control. All animals were challenged with 1 x lo2 metacyclics from T. infestans injected subcutaneously in the scapular region. Groups of mice were immunized with lyophilized epimastigotes-or metacyclics in PBS emulsified with Freund’s complete adiuvant (1:l). Two control groups consisted of mice injected with LMC medium or PBS emulsified with the Freund’s complete adjuvant (1:l). The untreated mice described above also served as controls for this experiment, since these mice were also challenged subcutaneously with 1 x IO* metacyclics T. infestans from the same pooled preparation of parasites. The mice were sampled at 3 day intervals for 30 days by microscopic examinations of Giemsa stained thick blood films. Ten microliters of tail vein blood were spread evenly over an 18 x 18 mm area of microscope slides and then allowed to dry overnight. The slides were stained with Giemsa for 20 min and washed with distilled water. Fifty fields of an ocular grid of known dimensions that had been calibrated with a stage micrometer were counted using oil immersion optics (1000 x magnification). The number of trypanosomes counted was then converted to trypanosomes/ml of blood (Roberson, Chapman & Hanson, 1973). Experiments were terminated 30 days after the challenge inoculations. RESULTS
Parasites were not detected in the blood of C3H/Anf mice inoculated subcutaneously to test the infectivity of the vaccines with 1 x lo* irradiated or
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Immunization
against
T. cruzi
253
lyophilized epimastigotes, metacyclics or bloodstream forms and sampled during a 15-day period. When these animals were decapitated and their blood injected intraperitoneally into irradiated mice, parasitemias were not detected in the recipient mice during a 2-week period. Amastigotes were not found in the tissue imprints of the hearts, livers, or spleens of these animals. Animals immunized with irradiated metacyclics, epimastigotes, or bloodstream forms which were challenged with culture metacyclics had significantly fewer (P
3 DAY FIG. 2. Mean parasitemias of groups of BALB/c mice challenged with 1 x IO4T. cruzi metacyclics from culture. The mice received either Freund’s complete adjuvant emulsified with lyophilized rat blood (A-A), lyophilized bloodstream forms (n--n), no treatment (m---W), lyophilized culture metacyclics (O--O), lyophilized LMC medium ( l 0) or lyophilized culture epimastigotes (O--O) of T. cruzi. Parasitemias of control mice injected with PBS (not shown) are similar to those of the untreated animals.
”
6
12
18
24
J 30
DAY FIG. 1. Mean parasitemias of groups of BALB/c mice challenged with 1 x lo4 T. cruzi metacyclics from culture. The mice received either irradiated rat blood (A-A), irradiated bloodstream forms (n--n), no treatment (U-m), irradiated culture metacyclics (O--O), irradiated LMC medium (0-O) or irradiated culture epimastigotes (0 - - 0) of T. cruzi.
injected animals. No mortalities were observed in the mice vaccinated with the irradiated metacyclics, epimastigotes, or bloodstream forms, while among the controls, only one of the animals of the unimmunized groups died during the course of the experiment. Mice inoculated with lyophilized metacyclics, epimastigotes, or bloodstream forms emulsified with adjuvant which were challenged with metacyclics from culture showed parasitemias similar to those presented by the mice of the control groups (Fig. 2). These mice and the animals which had been injected with lyophilized LMC medium, rat blood or PBS emulsified with adjuvant survived the infection during the course of the study. Only the single animals of the unimmunized control group indicated above died. During the first 12 days after challenge with metacyclics obtained from T. infestam, there were no differences detected between the parasitemias of animals immunized with irradiated epimastigotes or
EMMANUEL 0. OKANLA,JERRYL. STUMPFand DONALDG. DUSANIC
254
metacyclics and those of the control with irradiated medium, PBS or the
I.J.P. VOL. 12. 1982
mice injected
untreated mice (Fig. 3). The parasitemias of the vaccinated animals were lower on day 15 than those of the controls (P
16
t 12
t
DAY FIG. 4. Mean parasitemias of groups of BALB/c mice challenged with 1 x lo2 T. cruzi metacyclics from T. infestuns. The mice were either untreated (m-m) or injected with Freund’s complete adjuvant emulsified with either lyophilized culture metacyclics (O--O), lyophilized LMC medium ( l 0) or lyophilized culture epimastigotes (O--O) of T. cruzi. Parasitemias of mice injected with a PBS-Freund’s complete adjuvant emulsion (not shown) are similar to those of the untreated animals.
8
DAY FIG. 3. Mean parasitemias of groups of BALB/c mice challenged with I x lo* T. cruzi metacyclics from T. infest&s. The mice were either untreated- (m-m) or injected with irradiated culture metacyclics (O--O), irradiated LMC medium (0-O) or irradiated culture epimastigotes (O--O) of T. cruzi.
than those of the control animals injected with lyophilized medium or PBS emulsified in adjuvant or the untreated animals (X0.05). However, unlike animals immunized with the irradiated trypanosomes, parasites were still detected in the blood of the mice inoculated with lyophilized epimastigotes and metacyclics 30 days after inoculation of the infective metacyclics. No deaths were observed among the mice immunized with lyophilized epimastigotes or metacyclics, while 2 mice of the groups injected with lyophilized medium died during the study period. As indicated above, all of the untreated animals infected with metacyclics from T. infestuns survived. Metacyclic inocula from culture were 100 times greater than the numbers from triatomes used to challenge the mice of the experimental and control groups. Parasitemias were approximately IO-fold lower and appeared to be of shorter duration in
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animals inoculated with culture metacyclics than in mice challenged with metacyclics from T. infestam.
DISCUSSION
Immunization with living trypanosomes has been attempted using avirulent strains of T. cruzi (Menezes, 1969; Kagan & Norman, 1961) or trypanosomes that had been attenuated by irradiation (Hanson et al., 1974) or chemical means (Fernandes, Halsman & Castellani, 1965, 1966). The danger in using live avirulent or attenuated strains of T. cruzi has been repeatedly discussed (Goble, 1970; Brener, 1980). Trypanosomes killed by chemical or physical means have been used as vaccines. Chemical methods included treatment with merthiolate (Muniz, Nobrega, & Cunha, 1946), formalin (Hauschka, Goodwin, Palmquist & Brown, 1950) and sodium perchlorate (Kierszenbaum & Budzko, 1975). Physical methods included sonication, freeze-thawing and lyophilization (McHardy & Neal, 1979; GonzalezCappa, Schmunis, Traversa, Yanowsky & Parodi, 1968; Neal & Johnson, 1977). In addition, nonspecific immunization has been attempted (Ortiz-Ortiz, Gonzalez-Mendoza & Lamayi, 1975). The challenge inocula have included trypanosomes obtained from infected blood, culture, or the vector R. prolixus (Marr & Pike, 1967; Hanson et al., 1974; Kierszenbaum & Budzko, 1975; McHardy, 1977; McHardy & Neal, 1979). Although protection as measured by reduction in the parasitemias, duration of parasitemias, or mortality of hosts was accomplished in many of these experiments, complete protection has not been demonstrated. Since metacyclic trypomastigotes are the developmental stage encountered by the vertebrate host in natural infections and specific antigens appear to be present in different stages of T. cruzi (Kloetzel, Camargo & Giovannini, 1975), vaccines produced from metacyclics may improve protection when the method of challenge mimics a natural infection (subcutaneous injection of metacyclics). The mice immunized with irradiated epimastigotes, metacyclics, or bloodstream forms and challenged with these metacyclics from culture showed significant, but not complete protection. The parasitemias presented by these mice were lower than those of the control animals. Differences were apparent among the mice immunized with the irradiated trypanosomes of each of the three stages. The most pronounced protection was found in the group immunized with the irradiated metacyclics. None of the mice inoculated with lyophilized vaccines were protected against challenge infections by culture metacyclics. In the groups of mice challenged with metacyclics from T. infestans immunization with the trypanosomes attenuated by irradiation and the lyophilized trypanosomes gave similar patterns of the protection. Differences between the patterns of the parasitemias
255
among the mice challenged by the metacyclics from culture and T. infestans may reflect differences between the numbers of metacyclics in the challenge inocula, their virulence or antigenic compositions. McHardy & Neal (1979) observed differences in the course of infections in mice challenged by bloodstream forms and metacyclics from R. profths. Although Ortiz-Ortiz et al. (1975) detected a nonspecific immune response in mice injected with BCG against T. cruzi, no such effect was observed in animals after multiple injections of LMC medium, rat blood or Freund’s complete adjuvant during this study. Mice injected with these preparations developed parasitemias equivalent to those presented by the untreated controls. As with previous attempts to immunize against T. cruzi, complete protection was not attained in this study and the inability to demonstrate complete protection continues to be an obstacle to the immunization of humans in endemic areas. The survival of the African trypanosomes has been explained in terms of antigenic variability of the surface coat (Cross, Holder, Allen & Boothroyd, 1980). However, antigenic variation has not been observed with T. cruzi. @nary, 1980). Other mechanisms may be employed by the parasites to avoid the host’s immune system and permit them to persist in the immunized host (Miles, 1979). The persistence of the parasites may be in part the result of a variety of factors including the intracellular location of the amastigote stages which isolate them from the host’s immune responses, release of surface antigens from the trypanosomes, mimicking of the host’s antigens by the bloodstream forms and/or adsorption of host proteins on the surfaces of the circulating parasites. Miles (1979) suggested that autoimmune responses observed in T. cruzi infected hosts may be the result of these latter two mechanisms and warns that the absence of immunopathogenesis should be an essential criterion for any vaccine. It is possible that through further studies with stage specific vaccines, improved protection may be achieved without the development of responses toxic to the recipients. Acknowledgements-This Grant AI 14642.
study was supported
by NIH
REFERENCES 1980. Immunity to liypunosoma cruzi. In: Advances in Parasitology (Edited by LUMSDENW. H. R., MULLER R. & BAKER J. R.), Vol. 18. Academic Press, New York. CROSS G. A. M., HOLDER A. A., ALLEN G. & BOO~HROYD J. C. 1980. An introduction to antigenic variation in trypanosomes. American Journal of Tropical Medicine and Hygiene 29: 1027-1032. DUSANIC~?I. G. 1980. In vrtro production of metacyclic trypomastigotes of Trypanosoma cruzi. Journal of Parasirology 66: 1046-1049. FERNANDES J. F., HALSMAN M. & CASTELLANI 0. 1965. Effect of actinomycin D on the infectivity of Trypanosoma cruzi. Nature 207: 1004-1005.
BRENER 2.
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FERNANDES J. F., HALSMAN M. & CASTELLANI 0. 1966. Effect of mitomycin C, actinomycin D and pyrimidine analogs on the growth rate, protein and nucleic acid synthesis and on the viability of Trypanosoma cruzi. Experimental Parasitology 18: 203-210. GOBLE F. C. 1970. South American trypanosomes. In: Immunity to Parasitic Animals (Edited by JACKSON G. J , HERMAN R. & SINGER I.), Vol. 1. Appleton-CenturyCrofts, New York. GONZALEZ-CAPPA S. M., SCHMUNISG. A., TRAVERSA0. C., YANOWSKY J. F. & PARODI A. S. 1968. Complementfixation tests, skin test and experimental immunization with antigens of Trypanosoma cruzi prepared under pressure. American Journal of Tropical Medicine and Hygiene 17: 709-7 15. GUTTERIDGE W. E., COVER B. & GABORAK M. 1978. Isolation of blood and intracellular forms of Trypunosoma cruzi from rats and other rodents and preliminary studies of their metabolism. Parasitology 76: 159-l 76. HANSON W. L., CHIEN J. J., CHAPMAN W. L. & ROBERSON E. L. 1974. Current status of vaccination for Chagas’ disease: use of irradiated parasites. In: Parasitic Zoonoses: Clinical and Experimental Studies (Edited by SOULSBY E. J. L.). Academic Press, New York. HAUSCHKAT. S., Goo~wr~ M. B., PALMQUISTJ. & BROWN E. 1950. Immunological relationship between seven strains of Trypanosoma cruzi and its application in the diagnosis of Chagas’ disease. American Journal of Tropical Medicine and Hygiene 30: I-16. KACAN I. G. & NORMAN L. 1961. Immunological studies of Trypanosoma cruzi. III. Duration of acquired immunity in mice initially infected with a North American strain of T. cruzi. Journal of Infectious Diseases 107: 165-167. KIERSZENBAUMF. & BUDZKO D. G. 1975. Immunization against experimental Chagas’ disease by using culture forms of Trypanosoma cruzi killed with a solution of sodium perchlorate. Infection and Immunity 23: 117-120. KLOETZEL J., CAMARGO M. E. & GIOVANNINI V. C. 1975. Antigenic differences among epimastigotes, amastigotes and trypomastigotes of Trypanosoma cruzi. Journal of Protozoology 22: 259-261. KREIER J. P., AL-ABBASSY S. N. & SEED T. M. 1977. Trypanosoma cruzi: surface charge characteristics of cultured epimastigotes. trypomastigotes and amastigotes. Revista do Instituto de Medicina Tropical de Scio Paul0 19: 10-20. MCHARDY N. 1977. Immunization of mice against
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Trypanosoma cruzi. The effect of size of dose and route of injection of immunizing and challenge inocula. Tropenmedizin und Parasitologie 28: 1 l-l 6. MCHARDY N. & NEAL R. A. 1979. A comparison of challenge with Trypanosoma cruzi bloodstream trypoand metacyclic trypomastigotes from mastigotes Rhodnius prolixus in mice immunized with killed antigens. Transactions of the Royal Society of Tropical Medicine and Hygiene 13: 409-414. MARR J. S. & PIKE H. 1967. The protection of mice by “Corpus Christi” strain of Trvnanosoma cruzi when __ challenged with “Brash” strain. Journal of Parasitology 53: 657-659. MENEZES H. 1969. Active immunization of mice with the avirulent Y strain of Trypanosoma cruzi against heterologous virulent strains of the same parasite. Revista do Institute de Medicina Tropical Silo Paul0 11: 335-342. MILES M. A. 1979. Transmission cycles and the heterogeneity of Trypanosoma cruzi. In: Biology of Kinetoplastida (Edited by LUMSDEN W. H. R. & EVANS D. A.), Vol. 1. Academic-Press, New York. Mu~rz J., NOBREGAG. & CUNHA A. M. DA. 1946. Ensaios de vacinaca6 preventiva e curativa nos infecbes pelo Schizotrypanum cruzi. Memorias fo Instituto Oswald0 Cruz 44: 529-541. NEAL R. A. & JOHNSON P. 1977. Immunization against Trypanosoma cruzi using killed antigens with saponin as adjuvant. Acta Tropica 34: 87-96. ORTIZ-ORTIZ L., GONZALEZ-MENDOZAA. & LAMAYIE. 1975. A vaccination procedure against Trypanosoma cruzi infection in mice by non-specific immunization. Journal of Immunology 114: 1424-1425. ROBERSON E. L., CHAPMAN W. L. & HANSON W. L. 1973. The effects of total-body X-irradiation on Trypanosoma cruzi infection (Chagas’ disease) in mice and rats. Zeitschrift .ftir Parasitenkunde 41: 83-94. RYCKMANR. E. 1954. A method of collecting large quantities of feces from triatominae infected with Trypanosoma cruzi. Journal of Economic Entomology 229: 170-l 71. SCOTT M. T. & SNARY D. 1979. Protective immunization of mice using cells surface glycoprotein from Trypanosoma cruzi. Nature 282: 73-74. SNARY D. 1980. Trypanosoma cruzi: antigenic invariance of the cell surface glycoprotein. Experimental Purasitology 49: 68-77.
Internolionol Primed in tireof Bnlbh.
12, No. 4, pp. 251-256,
1982
0020-7519/82/040251+6 $03.00/O Pergomon Press Lfd. Soc~ery for Parosifology
0 1982 Austmlion
RESISTANCE OF MICE IMMUNIZED WITH IRRADIATED AND LYOPHILIZED STAGES OF TRYPANOSOMA CRUZI TO INFECTIONS WITH METACYCLICS EMMANUEL 0.
OKANLA, JERRY L. STUMPF and
DONALD G. DUSANIC
Department of Life Sciences, Indiana State University, Terre Haute, IN 47809, U.S.A. (Received 1 June 1981) AbSfraCt-DKANLA E. O., STUMPFJ. L. & DUSANICD. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Tr~panosoma cruzi to infections with metacyclics. Infernational Journalfor Parasitology12: 251-256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosomacruzi in three weekly injections of 1 x lo8 trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund’s complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestuns. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestuns included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and bloodstream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infesrans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector. INDEX KEY WORDS: T. cruzi; epimastigote; metacyclic; bloodstream vaccines; immunization; resistance; mice.
INTRODUCTION ATTEMPTSto immunize animals against experimental Trypanosoma cruzi infections have utilized trypanosomes from cultures or from the blood of infected
animals (Goble, 1970). Kierszenbaum & Budzko (1975) demonstrated partial protection through a reduction in parasitemia and decreased mortality in animals immunized with sodium perchlorate-treated culture forms of T. cruzi. Partial protection in mice was also demonstrated with frozen and thawed and sonicated vaccines against challenge with bloodstream forms or metacyclics from the vector Rhodnius prolixus (McHardy, 1977; McHardy & Neal, 1979). CFI mice immunized with bloodstream forms of T. cruzi that had been attenuated with 1.5 kGy gamma irradiation, displayed reduced parasitemias when challenged with bloodstream forms (Hanson, Chien, Chapman & Roberson, 1974). In studies by Scott & Gary (1979) that utilized T. cruzi cell surface glycoproteins as a vaccine, the glycoprotein alone did not elicit protection in the
form; parasites; cultivation;
immunized mice, while glycoprotein emulsified in adjuvant enhanced resistance. All of these studies utilized culture forms (predominately epimastigotes) or bloodstream forms as vaccines. There are no reports on the use of metacyclic forms of T. cruzi as vaccines. Since this stage initiates natural infections, a vaccine made from the metacyclics may be effective in preventing infections. Experiments were designed to compare the effectiveness of vaccines made from irradiated or lyophilized epimastigotes, metacyclics or bloodstream forms of T. cruzi against challenge infections by metacyclics obtained from culture. Similar studies were conducted with animals which were injected with irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics isolated from the vector Triatoma infestans. MATERIALS AND METHODS Parasites and animals. The Tulahutn strain of T. cruzi was isolated from Chilean T. infesfuns and sent to our
251
252
EMMANUEL0. OKANLA, JERRY L. STUMPF and DONALD G. DUSANIC
laboratory by Dr. Ramiro Martinez-Silva, University of Puerto Rico. This strain has been maintained in our laboratory by syringe injection of 1 x lo5 organisms intraperitoneally every 9-10 days in C3H/Anf mice (Cumberland Farms, Clinton, TN). Female C3H/Anf mice and BALB/c mice (Harland Industries, Indianopolis, IN) weighing 18 and 22 g were used for these studies. Holtzman -rats (Charles River Breeding Laboratories. Wilmington. MA) _. weighing bet -en 100 and 120 g were used for the production of bloostream form trypanosomes. The mice were maintained in plastic cages with 5 animals in each cage and the rats were housed 2 animals to a cage. The animals were provided food and water ad libitum. T. infestans, originally from Chile, were acquired from Dr. Robert Taylor (Center for Disease Control, Atlanta, GA). The T. infestans were housed in covered one gallon glass jars in an environmental chamber (Warren-Sherer Division of Kysor Industries, Marshall, MI) at 27°C and 80% relative humidity. They were fed on the blood of white New Zealand rabbits once a week. The triatomes were infected with T. cruzi by feeding them on infected mice anesthetized with Nembutal (HaverLockhart Laboratories, Kansas City, MO). The abdomens of t‘rese mice were shaved and they were placed on bug containers (Ryckman, 1954) and the bugs were allowed to feed for 1 h in the dark. The bugs were fed on uninfected mice once every week thereafter. Cultivation of parasites. Epimastigotes and metacyclics were cultivated in the LMC medium as described by Dusanic (1980). Epimastigotes were harvested after 10 days, while metacyclics were harvested after 14 days of cultivation. The harvested trypanosomes were centrifuged at 1500 g for 20 min in a Sorvall RC 2-B centrifuge maintained at 4°C. The supernatant fluids were discarded. Metacyclic rich preparations were purified by passage through DEAE-Sephacel (Pharmacia Fine Chemicals, Piscataway, N.J.) using the method of Kreier, Al-Abbassy & Seed (1977). These purified metacyclics preparations contained more than 95% metacyclics. All preparations were then washed in 200 times their volume with 0.2 M phosphate buffered saline pH 7.2 (PBS) by centrifugation as described above. Bloodstream form trypanosomes were obtained from rats that had been exposed 18-24 days previously to 5.8 Gv whole body irradiation from a 6oCo gamma irradiation source (Radiation Laboratory, Department of Physics, Indiana State University) and immediately infected by intraperitoneal injection with 5 x lo6 T. cruzi. The trypanosomes were separated from the rat blood by passage through DEAE-Sephacel (Gutteridge, Cover & Gaborak, 1978) and washed several times by centrifugation with PBS at 1500 g at 4°C for 20 min.
Preparation of vaccines. The epimastigote, metacyclic or bloodstream form trypanosomes to be irradiated were suspended in PBS at a concentration of 5 x lo8 parasites/ml. The tubes containing the parasites were kept in an ice bath and subjected to 3.0 kGy gamma irradiation from a ‘%o gamma irradiation source. These vaccines were used immediately after irradiation. The suspension of trypanosomes to be lyophilized were dispensed into 13 x 100 mm disposable culture tubes with I x lo9 trypanosomes/tube. The tubes were frozen in a mixture of acetone and ethanol (SO/SO) at -76°C. The trypanosomes were then lyophilized with a Vertis lyophilizer (Vertis Co., Gardiner, NY). Rat blood and 0.1% LMC were also irradiated or lyophilized as described for the trypanosomes. In order to test the infectivity of the vaccines, groups of 5 C3H/Anf mice were inoculated subcutaneously with either 1 x 10s irradiated or reconstituted lyophilized preparations of trypanosomes. Blood collected from the tail vein of each animal was
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examined at 5 day intervals for 15 days. The mice were then decapitated and 1 ml of blood from each mouse was collected and subinoculated into C3H/Anf mice that had been irradiated with 6.0 Gy gamma irradiation. Blood from the tail vein of the mice was sampled after 7 and 14 days. The mice were killed and tissue imprints were made of the heart, spleen, and liver of each animal. The imprints were fixed in 100% methanol, stained with Giemsa and examined microscopically for trypanosomes. Immunization and challenge. Mice were randomly divided in groups of 10 animals each. These animals were injected intramuscularly with 0.2 ml volumes of the vaccines or control preparations. Three injections were administered at weekly intervals. The first injection was made in the muscle of the right thigh, the second in the left thigh and the third in the right thigh. All animals were challenged with T. cruzi 2 weeks after the last injection. Mice were immunized with 1 x 10’ irradiated epimastigotes, metacyclics, or bloodstream forms suspended in PBS in each injection. Three control groups were injected with equivalent volumes of LMC medium, rat blood or PBS. A fourth group of untreated mice was also included. All animals were challenged on the same day with 1 x lo4 culture metacyclics from the same pool of parasites by subcutaneous injection between the scapulae. Three panels of mice were inoculated with lyophilized epimastigotes, metacyclics or bloodstream trypomastigotes suspended in PBS and emulsified in Freund’s complete adjuvant (l:l). Three control groups were injected with LMC medium, rat blood or PBS emulsified with Freund’s complete adjuvant (l:l). The untreated mice also served as a control group for this experiment, since the mice were challenged with 1 x lo4 culture metacyclics from the same pool and via the same route as indicated above. Two groups of mice were injected with 1 x 10s irradiated T. cruzi epimastigotes or metacyclics suspended in PBS for each injection. Groups of control mice were injected with identical volumes of LMC medium or PBS. One group of mice which were not injected served as an additional control. All animals were challenged with 1 x lo2 metacyclics from T. infestans injected subcutaneously in the scapular region. Groups of mice were immunized with lyophilized epimastigotes-or metacyclics in PBS emulsified with Freund’s complete adiuvant (1:l). Two control groups consisted of mice injected with LMC medium or PBS emulsified with the Freund’s complete adjuvant (1:l). The untreated mice described above also served as controls for this experiment, since these mice were also challenged subcutaneously with 1 x IO* metacyclics T. infestans from the same pooled preparation of parasites. The mice were sampled at 3 day intervals for 30 days by microscopic examinations of Giemsa stained thick blood films. Ten microliters of tail vein blood were spread evenly over an 18 x 18 mm area of microscope slides and then allowed to dry overnight. The slides were stained with Giemsa for 20 min and washed with distilled water. Fifty fields of an ocular grid of known dimensions that had been calibrated with a stage micrometer were counted using oil immersion optics (1000 x magnification). The number of trypanosomes counted was then converted to trypanosomes/ml of blood (Roberson, Chapman & Hanson, 1973). Experiments were terminated 30 days after the challenge inoculations. RESULTS
Parasites were not detected in the blood of C3H/Anf mice inoculated subcutaneously to test the infectivity of the vaccines with 1 x lo* irradiated or
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Immunization
against
T. cruzi
253
lyophilized epimastigotes, metacyclics or bloodstream forms and sampled during a 15-day period. When these animals were decapitated and their blood injected intraperitoneally into irradiated mice, parasitemias were not detected in the recipient mice during a 2-week period. Amastigotes were not found in the tissue imprints of the hearts, livers, or spleens of these animals. Animals immunized with irradiated metacyclics, epimastigotes, or bloodstream forms which were challenged with culture metacyclics had significantly fewer (P
3 DAY FIG. 2. Mean parasitemias of groups of BALB/c mice challenged with 1 x IO4T. cruzi metacyclics from culture. The mice received either Freund’s complete adjuvant emulsified with lyophilized rat blood (A-A), lyophilized bloodstream forms (n--n), no treatment (m---W), lyophilized culture metacyclics (O--O), lyophilized LMC medium ( l 0) or lyophilized culture epimastigotes (O--O) of T. cruzi. Parasitemias of control mice injected with PBS (not shown) are similar to those of the untreated animals.
”
6
12
18
24
J 30
DAY FIG. 1. Mean parasitemias of groups of BALB/c mice challenged with 1 x lo4 T. cruzi metacyclics from culture. The mice received either irradiated rat blood (A-A), irradiated bloodstream forms (n--n), no treatment (U-m), irradiated culture metacyclics (O--O), irradiated LMC medium (0-O) or irradiated culture epimastigotes (0 - - 0) of T. cruzi.
injected animals. No mortalities were observed in the mice vaccinated with the irradiated metacyclics, epimastigotes, or bloodstream forms, while among the controls, only one of the animals of the unimmunized groups died during the course of the experiment. Mice inoculated with lyophilized metacyclics, epimastigotes, or bloodstream forms emulsified with adjuvant which were challenged with metacyclics from culture showed parasitemias similar to those presented by the mice of the control groups (Fig. 2). These mice and the animals which had been injected with lyophilized LMC medium, rat blood or PBS emulsified with adjuvant survived the infection during the course of the study. Only the single animals of the unimmunized control group indicated above died. During the first 12 days after challenge with metacyclics obtained from T. infestam, there were no differences detected between the parasitemias of animals immunized with irradiated epimastigotes or
EMMANUEL 0. OKANLA,JERRYL. STUMPFand DONALDG. DUSANIC
254
metacyclics and those of the control with irradiated medium, PBS or the
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mice injected
untreated mice (Fig. 3). The parasitemias of the vaccinated animals were lower on day 15 than those of the controls (P
16
t 12
t
DAY FIG. 4. Mean parasitemias of groups of BALB/c mice challenged with 1 x lo2 T. cruzi metacyclics from T. infestuns. The mice were either untreated (m-m) or injected with Freund’s complete adjuvant emulsified with either lyophilized culture metacyclics (O--O), lyophilized LMC medium ( l 0) or lyophilized culture epimastigotes (O--O) of T. cruzi. Parasitemias of mice injected with a PBS-Freund’s complete adjuvant emulsion (not shown) are similar to those of the untreated animals.
8
DAY FIG. 3. Mean parasitemias of groups of BALB/c mice challenged with I x lo* T. cruzi metacyclics from T. infest&s. The mice were either untreated- (m-m) or injected with irradiated culture metacyclics (O--O), irradiated LMC medium (0-O) or irradiated culture epimastigotes (O--O) of T. cruzi.
than those of the control animals injected with lyophilized medium or PBS emulsified in adjuvant or the untreated animals (X0.05). However, unlike animals immunized with the irradiated trypanosomes, parasites were still detected in the blood of the mice inoculated with lyophilized epimastigotes and metacyclics 30 days after inoculation of the infective metacyclics. No deaths were observed among the mice immunized with lyophilized epimastigotes or metacyclics, while 2 mice of the groups injected with lyophilized medium died during the study period. As indicated above, all of the untreated animals infected with metacyclics from T. infestuns survived. Metacyclic inocula from culture were 100 times greater than the numbers from triatomes used to challenge the mice of the experimental and control groups. Parasitemias were approximately IO-fold lower and appeared to be of shorter duration in
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animals inoculated with culture metacyclics than in mice challenged with metacyclics from T. infestam.
DISCUSSION
Immunization with living trypanosomes has been attempted using avirulent strains of T. cruzi (Menezes, 1969; Kagan & Norman, 1961) or trypanosomes that had been attenuated by irradiation (Hanson et al., 1974) or chemical means (Fernandes, Halsman & Castellani, 1965, 1966). The danger in using live avirulent or attenuated strains of T. cruzi has been repeatedly discussed (Goble, 1970; Brener, 1980). Trypanosomes killed by chemical or physical means have been used as vaccines. Chemical methods included treatment with merthiolate (Muniz, Nobrega, & Cunha, 1946), formalin (Hauschka, Goodwin, Palmquist & Brown, 1950) and sodium perchlorate (Kierszenbaum & Budzko, 1975). Physical methods included sonication, freeze-thawing and lyophilization (McHardy & Neal, 1979; GonzalezCappa, Schmunis, Traversa, Yanowsky & Parodi, 1968; Neal & Johnson, 1977). In addition, nonspecific immunization has been attempted (Ortiz-Ortiz, Gonzalez-Mendoza & Lamayi, 1975). The challenge inocula have included trypanosomes obtained from infected blood, culture, or the vector R. prolixus (Marr & Pike, 1967; Hanson et al., 1974; Kierszenbaum & Budzko, 1975; McHardy, 1977; McHardy & Neal, 1979). Although protection as measured by reduction in the parasitemias, duration of parasitemias, or mortality of hosts was accomplished in many of these experiments, complete protection has not been demonstrated. Since metacyclic trypomastigotes are the developmental stage encountered by the vertebrate host in natural infections and specific antigens appear to be present in different stages of T. cruzi (Kloetzel, Camargo & Giovannini, 1975), vaccines produced from metacyclics may improve protection when the method of challenge mimics a natural infection (subcutaneous injection of metacyclics). The mice immunized with irradiated epimastigotes, metacyclics, or bloodstream forms and challenged with these metacyclics from culture showed significant, but not complete protection. The parasitemias presented by these mice were lower than those of the control animals. Differences were apparent among the mice immunized with the irradiated trypanosomes of each of the three stages. The most pronounced protection was found in the group immunized with the irradiated metacyclics. None of the mice inoculated with lyophilized vaccines were protected against challenge infections by culture metacyclics. In the groups of mice challenged with metacyclics from T. infestans immunization with the trypanosomes attenuated by irradiation and the lyophilized trypanosomes gave similar patterns of the protection. Differences between the patterns of the parasitemias
255
among the mice challenged by the metacyclics from culture and T. infestans may reflect differences between the numbers of metacyclics in the challenge inocula, their virulence or antigenic compositions. McHardy & Neal (1979) observed differences in the course of infections in mice challenged by bloodstream forms and metacyclics from R. profths. Although Ortiz-Ortiz et al. (1975) detected a nonspecific immune response in mice injected with BCG against T. cruzi, no such effect was observed in animals after multiple injections of LMC medium, rat blood or Freund’s complete adjuvant during this study. Mice injected with these preparations developed parasitemias equivalent to those presented by the untreated controls. As with previous attempts to immunize against T. cruzi, complete protection was not attained in this study and the inability to demonstrate complete protection continues to be an obstacle to the immunization of humans in endemic areas. The survival of the African trypanosomes has been explained in terms of antigenic variability of the surface coat (Cross, Holder, Allen & Boothroyd, 1980). However, antigenic variation has not been observed with T. cruzi. @nary, 1980). Other mechanisms may be employed by the parasites to avoid the host’s immune system and permit them to persist in the immunized host (Miles, 1979). The persistence of the parasites may be in part the result of a variety of factors including the intracellular location of the amastigote stages which isolate them from the host’s immune responses, release of surface antigens from the trypanosomes, mimicking of the host’s antigens by the bloodstream forms and/or adsorption of host proteins on the surfaces of the circulating parasites. Miles (1979) suggested that autoimmune responses observed in T. cruzi infected hosts may be the result of these latter two mechanisms and warns that the absence of immunopathogenesis should be an essential criterion for any vaccine. It is possible that through further studies with stage specific vaccines, improved protection may be achieved without the development of responses toxic to the recipients. Acknowledgements-This Grant AI 14642.
study was supported
by NIH
REFERENCES 1980. Immunity to liypunosoma cruzi. In: Advances in Parasitology (Edited by LUMSDENW. H. R., MULLER R. & BAKER J. R.), Vol. 18. Academic Press, New York. CROSS G. A. M., HOLDER A. A., ALLEN G. & BOO~HROYD J. C. 1980. An introduction to antigenic variation in trypanosomes. American Journal of Tropical Medicine and Hygiene 29: 1027-1032. DUSANIC~?I. G. 1980. In vrtro production of metacyclic trypomastigotes of Trypanosoma cruzi. Journal of Parasirology 66: 1046-1049. FERNANDES J. F., HALSMAN M. & CASTELLANI 0. 1965. Effect of actinomycin D on the infectivity of Trypanosoma cruzi. Nature 207: 1004-1005.
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