Trypanosoma cruzi Culture Used as Vaccine to Prevent Chronic Chagas' Disease in Mice

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1 INFECTION AND IMMUNITY, Apr. 1982, p /82/ $02.00/0 Vol. 36, No. 1 Trypanosoma cruzi Culture Used as Vaccine to Prevent Chronic Chagas' Disease in Mice MIGUEL A. BASOMBRIOt* AND SANTIAGO BESUSCHIO Instituto de Investigaciones Hematol6gicas, Academia Nacional de Medicina, 1425 Buenos Aires, Argentina Received 8 September 1981/Accepted 8 December 1981 The development of chronic pathology in mice at 2 to 10 months after inoculation of 102 T. cruzi trypomastigotes can be prevented by preimmunization with live, attenuated culture parasites (strain TCC). Swiss mice received one or three immunizing inoculations of 106 TCC organisms and were challenged with 102 Tulahudn blood trypomastigotes. Control groups received only the immunizing or the challenge inoculations. Immunized groups as compared with nonimmunized controls had lower mortality rates at 2 months postchallenge (9% versus 23%; P = 0.059), lower early peaks of parasitemia, lower percentages of positive xenodiagnoses at 5.5 months (40 versus 80%; P = 0.061), and lower incidences of tissue lesions in the skeletal muscle (P < 0.005) at 2, 6, and 10 months postchallenge. Tissue lesions in the heart and smooth muscle were also reduced, reaching statistical significance after 10 months (P < 0.02). Chronic pathology parameters were never enhanced in preimmunized groups. In spite of the putative role that autoimmunity may play in the development of chronic chagasic lesions, the preventive effect of vaccination is readily exerted upon the chronic murine model of Chagas' disease. Almost all of the experimental work on immunization against American trypanosomiasis (Chagas' disease) deals with prevention of the acute, lethal infection which follows inoculation of a relatively high number of parasites (5, 9, 11). Particular attention should be paid, however, to the possible effects of immunization upon chronic T. cruzi infection, since this is the course of disease most often observed in human or animal reservoirs. Protection during the acute phase of the disease may not extend to the chronic phase, since parasites which survive host immunity during the acute period may later cause chronic lesions. On the other hand, the immune response is often regarded as a contributor rather than as a defense against the development of chronic lesions in Chagas' disease (for review, see reference 4). A chronic T. cruzi infection which closely resembles human Chagas' disease can be experimentally induced in Swiss mice (6, 8). The purpose of the work reported here was to determine whether preimmunization with a T. cruzi culture strain (TCC) affects the development of lesions which result from chronic infection with T. cruzi trypomastigotes. t Present address: Laboratorio de Patologia Experimental, Departamento de Ciencias de la Salud, Universidad Nacional de Salta, Calle Buenos Aires 177, 4400 Salta, Argentina. 351 MATERIAL AND METHODS Animals. Swiss outbred mice of both sexes, raised in our laboratory, were used. They were 1 to 2 months old and weighed 28 ± 3.5 g (average ± standard deviation) when the experiments were started. Parasites. A culture T. cruzi strain, designated TCC, was used in the immunizing inoculations. This line is a derivate from the Tulahuen strain and underwent an attenuation in virulence and pathogenicity after long in vitro passage (2). It was cultured in biphasic liver-heart infusion medium containing 20%o rabbit blood (3). Inoculations of 106 TCC organisms did not induce death or significant lesions in adult mice (2). A mouse passage strain (Tulahuen) was used to challenge immunized mice. It was propagated by intraperitoneal inoculation of blood containing 104 parasites every 10 to 13 days. Doses of more than 103 parasites were 100% lethal in 30 days. When doses of 102 organisms or less were injected, a high number of mice survived and presented chronic pathology (6). Fresh blood mounts. Blood obtained by sectioning tail tips of mice was collected in a 0.01-ml capillary pipette and placed between a slide and a cover slip (20 by 20 mm). Mobile parasites were counted in mm-diameter microscope fields. Xenodiagnosis. Groups of 10 second- or third-instar Triatoma infestans which had been fasted for a month were allowed to feed on each sodium pentobarbitalanesthesized mouse for 45 min in the dark. This feeding was repeated on two occasions that were 20 days apart. At 10 and 30 days after the last meal, fresh mounts of the pooled bug feces were diluted in a small drop of culture medium and examined under a microscope for 15 min or until parasites were found. Histology. Tissue samples from hearts, quadriceps

2 352 BASOMBRIO AND BESUSCHIO muscles, colons, and urinary bladders of mice were fixed in 10% formaldehyde, and histological, hematoxylin-eosin-stained sections were studied. The lesions found (see Results) were scored blindly as severe, moderate, slight, or absent by one or two observers. After decoding, the significance of the differences found between immunized and control groups was calculated with the Mann-Whitney U test. Experimental protocol. The basic procedure consisted of comparatively studying two groups of mice. The first was immunized with TCC and challenged with a sublethal (102) dose of blood trypomastigotes. The second group received medium only instead of TCC and was challenged with 102 trypomastigotes. A third group, in which mice received the immunizing but not the challenge inoculum, was included in each experiment. In confirmation of previous work (2), all determinations were negative in these animals, which, for the sake of briefness, will seldom be mentioned in Results. Immunizing and challenge inocula were given when the mice were 29 to 59 and 67 to 80 days old, respectively. For immunization, 106 TCC organisms were inoculated intraperitoneally either once or three times (see Results). Controls received culture medium only. The challenge inoculum consisted of 102 blood trypomastigotes injected intraperitoneally. INFECT. IMMUN. Three experiments were performed after this basic procedure. The animals of each experiment were autopsied at 2, 6, and 10 months after challenge, respectively. RESULTS Survival after inoculation of trypomastigotes. Challenge with 102 trypomastigotes killed 23% (12 of 53) of the mice within 2 months. The mortality rate decreased markedly thereafter, reaching only 26% (5 of 19) within 10 months. Preimmunization with TCC slightly decreased the mortality at 2 months; 9% (5 of 54) were dead versus 23% (12 of 53; P = 0.059). The slope of the mortality curve after 2 months was not noticeably affected in immunized animals. Fresh blood mounts. No parasites could be detected in fresh blood mounts from TCC-inoculated mice. Injection of 102 trypomastigotes produced a low peak of parasitemia (average, 2 x 104 trypomastigotes per ml) and, after a month, parasites were seldom detected by this method. Preimmunization with TCC significantly reduced this peak parasitemia. Downloaded from 4,4 -;~~~~~~~~~~~~~~~1 K~~v..&, '~~~~i.n~~~7~~&~4> "F, on August 13, 2018 by guest 1; 't ",A - FIG. 1. Skeletal muscle of a Swiss mouse at 6 months after inoculation of <102 Tulahuen blood trypomastigotes. Interstitial inflammatory mononuclear infiltrates can be seen. 41

3 VOL. 36, 1982 Xenodiagnosis. Triatomae were fed on all mice of experiment 3 (see below) on days 166 and 186. Parasites were recovered from 47% (7 of 15) of the immunized-challenged and from 80% (12 of 15) of the control-challenged mice (P = 0.061). Simultaneous controls which received only epimastigotes did not yield parasites (0 of 4), and additional controls yielded only epimastigotes in 3.5% (2 of 53) of the xenodiagnoses. Histopathology in nonimmunized mice. Since a majority of the animals survived challenge with 102 blood trypomastigotes, the following observations could be made after 2, 6, and 10 months. Skeletal muscle. At 2 months after challenge, most samples had extensive and severe lesions which consisted of interstitial inflammatory mononuclear infiltrates (Fig. 1) and incipient degeneration of muscle fibers. Samples taken at 6 and 10 months showed essentially the same type of infiltration, but fiber degeneration increased and fibrotic scars were often found toward the 10th month. Heart. Lesions on the heart were not as severe and extensive as those in the skeletal muscle. However, most samples presented interstitial 1,~~~~~ ''-4fr4 ~~~~ IMMUNIZATION AGAINST CHRONIC TRYPANOSOMIASIS 353 inflammatory infiltrates, often subendocardial, in the atrial wall. The ventricular tissue occasionally presented widely scattered mononuclear infiltrates which predominated in perivascular areas (Fig. 2). Most lesions were found at 2 months and were infrequent in samples taken at 6 and 10 months. Colon. The muscularis mucosae presented inflammatory mononuclear infiltrates which were often focal and perivascular in 58% of the samples. Again, these lesions were more marked in early (2-month) than in late (6 and 10-month) samples. Urinary bladder. Lesions in the bladder similar to those found in the colon were encountered in samples taken at 6 and 10 months postinoculation. Effect of immunization on histopathology. (i) Experiment 1. A total of 18 mice received a single immunizing TCC inoculation. At 19 days later, they were challenged, together with 17 controls, with trypomastigotes. Autopsies were performed 64 days after challenge. A comparison of lesions in immunized and control mice is shown in Fig. 3A through 5A (2 7-~~~~~~~~~ to", 41~~~~~~ -4,.4 ~~~~~~~~~~~~~~~~~~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~o ~~,.j7.a\, - 4. '-3~~~~~~~~~~~~~~~~ 1', l W-i W f~~~~~445'.i 3'mw'y " K..%-~ -A --W, purn FIG. 2. Heart tissue of a Swiss mouse at 6 months after inoculation of 102 Tulahuen blood trypomastigotes. Widely scattered mononuclear infiltrates are seen predominating in perivascular areas.

4 354 BASOMBRIO AND BESUSCHIO + ++ SEVERE PC4 INFECT. IMMUN. + MWOERATE SLIGHT : : * 0 ABSO#T IMMU- NIZED A CON TROLS ::* i1 IU- CON- NIZED B TROtS WWMM- NZE D C FIG. 3. Evaluation of tissue damage found in skeletal muscles of Swiss mice. Lesions consisted of myositis (interstitial mononuclear infiltrates and edema), followed in later stages by fiber degeneration and partial replacement by fibrous tissue. Control mice and mice immunized with culture epimastigotes (TCC) were challenged with 102 trypomastigotes and autopsied at 2 months (A), 6 months (B), and 10 months (C) postchallenge. months). A marked prevention of tissue lesions was observed in the skeletal muscle (P < 0.002). In heart and colon tissue, the differences were nonsignificant, although lesions were slightly inhibited in preimmunized animals. (ii) Experiment 2. A total of 18 mice were immunized with 106 TCC organisms on three occasions that were 15 days apart. At 20 days later, they were challenged, together with 17 controls, with 102 trypomastigotes. Autopsies were performed 180 days later. A comparison of tissue lesions in immunized and control mice is shown in Fig. 3B through SB (6 months). A sharp protective effect against lesions in the skeletal muscle was detected (P < SEVERE +4. MODERATE N S. a* HEART MUSCLJE N I I P<Io. SLIGHT LIE :::: *- - P I*- CON- TROLS IUmi- CON- NIZED TROLS A 0.001). In contrast, the muscles of hearts, colons, and urinary bladders in the same animals were either normal or presented slight or moderate lesions. No significant protection was observed in these samples. (iii) Experiment 3. A total of 18 mice were immunized with 106 TCC organisms on three occasions that were 11 and 17 days apart. At 23 days later, they were challenged, together with 19 controls, with 102 blood trypomastigotes. Autopsies were performed 304 days after challenge. A comparison of tissue lesions in immunized and control mice is shown in Fig. 3C through SC (10 months). Significant prevention of late lelu NIZED I- B CMN- TROLS ILU NIZED TROLS C FIG. 4. Evaluation of tissue damage found in myocardium of Swiss mice. Lesions consisted of intense perivascular or moderate interstitial mononuclear infiltrates, either focal or diffuse. Control mice and mice immunized with culture epimastigotes (TCC) were challenged with 102 trypomastigotes and autopsied at 2 months (A), 6 months (B), and 10 months (C) postchallenge. N.S., Not significant.

5 VOL. 36, SEVERE IMMUNIZATION AGAINST CHRONIC TRYPANOSOMIASIS SMOOTH MUSCLE * COLON * URINARY BLADDER NS *PC pz ooq 355 * M * *Oaa SUONT & I'll, 0 0 *8SENT * 0* ** - A aj- NIZED B con TRos WA1U- CON NIZED TROLS FIG. 5. Evaluation of tissue damage found in smooth muscles (colons [0] or urinary bladders [U]) of Swiss mice. Lesions consisted of mononuclear infiltrates. Control mice and mice immunized with culture epimastigotes were challenged with 102 trypomastigotes and autopsied at 2 months (A), 6 months (B), and 10 months (C) postchallenge. N.S., Not significant. sions was detected in the skeletal muscle (P < 0.005), heart (P < 0.05), colon (P < 0.025), and urinary bladder (P < 0.02). Weights of bodies and organs. In all autopsied animals, the weights of bodies, spleens, hearts, and urinary bladders were determined. The average spleen weight increased (x2.45) in both immunized and control mice challenged with trypomastigotes but not in mice which received only TCC. The weights of bodies, hearts and urinary bladders were normal in all groups. DISCUSSION Since most experiments on immunoprotection against T. cruzi deal with the acute, lethal infection which follows the inoculation of high doses of parasites, little attention has been paid to the effects of preimmunization on the development of chronic chagasic lesions. The study of these effects showed that it is possible to prevent several chronic consequences of T. cruzi infection by preimmunization with live, attenuated parasites. Although the effect of immunity on tissue lesions was always in the direction of improvement, the differences were not always significant, owing to two factors. One was the intensity of the background lesions found in challenged controls; when they were intense (skeletal muscle), the preventive effect was more noticeable than when they were moderate (colon). The other factor was the type of tissue alteration; when it was inflammatory (early samples), the preventive effect seemed less remarkable than when it was degenerative or fibrotic (late sam- C ples). Preservation of the inflammatory process and reduction of fibrotic scars would, moreover, seem to be logical consequences of strengthening the host response through immunization. In vaccination experiments involving live immunogens against a chronic T. cruzi infection, it may become difficult to distinguish between the vaccinated and the chronically infected animal. Although on clinical and histological grounds one is apparently healthy (2) and the other is sick (6), the usual diagnostic procedures may fail to draw a sharp distinction between them. Serological reactions may become positive in immunized as well as in chronic animals (11). Fresh blood mounts, although always negative after TCC vaccination, lack diagnostic sensitivity for chronic infection. Hemoculture is sensitive enough to detect less than 10 parasites per blood samples (1) but will also pick up, perhaps selectively, the immunizing, culture-adapted parasites (2). The best practical way to demonstrate the protective effect of attenuated parasites within an animal population potentially infected with wild or virulent T. cruzi strains seems to be xenodiagnosis. The frequency of positive xenodiagnoses among adult mice immunized with TCC is low (2). Instead, the method has a rather good diagnostic sensitivity in clinical practice toward wild or virulent strains of T. cruzi (10). As shown here, xenodiagnosis was able to detect chronic infection with a virulent strain in 80% of the mice and pointed to a difference (P = 0.061) between TCC-immunized and control mice at 5.5 months after they were subjected to

6 356 BASOMBRIO AND BESUSCHIO INFECT. IMMUN. challenge with a virulent strain. The specificity of xenodiagnosis to distinguish between immunizing and pathogenic infections could be increasd by subjecting the feces of positive triatomae to pathogenicity tests in newborn mice. Previously reported immunization-challenge experiments with T. cruzi provided data related to the effects of preimmunization on chronic lesions up to 4 months postchallenge. Taratuto et al. (12) studied histopathological alterations in mice immunized with pressure-killed parasites and subjected to a lethal T. cruzi dose which killed controls within a month. Their study did not allow comparison of chronic lesions in immunized and nonimmunized mice. However, since the immunized group survived, the histopathological alterations of the heart, liver, and spleen could be studied for up to 4 months after challenge. The heart sections showed a progressive healing of lesions which was demonstrable between 2 and 4 months. This finding is in agreement with our results, which in addition suggested further healing 10 months postchallenge in preimmunized mice. The fact that, in this work, samples taken early and late after challenge were from mice with different immunization protocols may nevertheless bias the comparison of lesions found at 2 and 10 months. Laguens et al. (7) studied the effect of preimmunization with subcellular T. cruzi fractions on a chronic murine model of chagasic infection similar to the one used in this work. Animals were studied for 3 months postchallenge. A reduction of tissue damage was detected in the skeletal muscle but not in the heart. Remarkably, however, electrocardiographic alterations were significantly prevented by preimmunization, and the immunogen itself induced some lesions in adult mice (E. L. Segura, P. Cabeza Meckert, M. Esteva, R. Gelpi, A. R. Compannini, E. Subias, and R. P. Laguens, Medicina (Buenos Aires) 40:807, 1981), a possibility excluded in the case of TCC (2). The applicability of protective immunization against Chagas' disease has been questioned on the grounds that the trypanosomes have the ability to modulate their surface antigens, thereby being able to sneak through into immune hosts. Previous work showing that immunization with live attenuated parasites prevented the acute lethal infection by homologous virulent parasites seemed to be of little relevance, since Chagas' disease may have a very insidious onset and the very immune response itself seemed to play a role in the causation of chronic lesions. This work, however, conveys the reassurance that immunization of adult mice with live attenuated parasites does not induce or enhance chronic lesions but, on the contrary, can prevent them. It follows, therefore, that if the host is adequately primed, it may successfully cope with the parasite at the port of entry, thus abrogating both the acute and chronic consequences of an otherwise pathogenic or lethal infection. ACKNOWLEDGMENTS We are grateful to C. D. Pasqualini for unrelenting support, E. D. de Isola for providing the original seed of the TCC subline, R. Laguens and P. Cabeza Meckert for the evaluation of tissue specimens in experiment 1, A. Bellouard and B. Basombrfo for technical assistance, and J. C. Corradi and G. Martfnez for providing triatomae for xenodiagnosis. This investigation was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas. LITERATURE CITED 1. Abramo Orrego, L., J. C. Lansetti, J. P. Bozzini, and G. Winne de Martini Hemocultivo como metodo de diagn6stico en la enfermedad de Chagas. Medicina (Buenos Aires), Suppl. 1 40: Basombrlo, M. A., S. Besuschio, and P. M. Cossio. Side effects of imunization with live attenuated Trypanosoma cruzi in mice and rabbits. Infect. Immun. 36: Bronia, D. I., E. E. Montamat, E. E. Aeberhard, and E. L. Segura Composici6n lipidica del T. cruzi. Medicina (Buenos Aires) 36: Cossio, P. M., C. Diez, R. P. Laguens, and R. Arana Inmunopatologia de la enfermedad de Chagas. Hechos y perspectivas. Medicina (Buenos Aires), Suppl. 1 40: Kagan, I. G., and L. Norman Immunologic studies on T. cruzi. III. Duration of acquired immunity in mice initially infected with a North American strain of T. cruzi. J. Infect. Dis. 108: Laguens, R. P., P. Cabeza Meckert, M. A. Basombrio, G. J. Chambo, P. Cossio, R. M. Arana, and R. Gelpi Infecci6n cr6nica del rat6n con Trypanosoma cruzi. Modelo experimental de enfermedad de Chagas. Medicina (Buenos Aires), Suppl. 1 40: Laguens, R. P., P. Cabeza Meckert, M. Esteva, A. D. Campanini, R. Gelpi, E. Subias, C. E. del Prado, and E. L. Segura Acci6n de las fracciones subcelulares de Trypanosoma cruzi sobre la enfermedad de Chagas cr6nica en el rat6n. II. Efecto sobre la evoluci6n de la infecci6n. Medicina (Buenos Aires) 87: Laguens, R. P., P. Cabeza Meckert, and R. J. Gelpi Chronic Chagas disease in the mouse. I. Electrocardiographic and morphological patterns of cardiopathy. Medicina (Buenos Aires) 41: Pizzi, T., and R. Prager Inmunidad a la sobreinfecci6n inducida mediante cultivos de Trypanosoma cruzi de virulencia atenuada. Bol. Inf. Parasit. Chil. 7: Schenone, H., E. Alfaro, H. Reyes, and E. Tancher Valor del xenodiagn6stico en la infecci6n chagasica cr6nica. Bol. Chil. Parasitol. 23: Szarfman, A., G. A. Schmufis, N. H. Vattuone, J. F. Yanovsky, P. Cossio, and R. M. Arana Protection against challenge of mice experimentally infected with T. cruzi. Tropenmed. Parasitol. 28: Taratuto, A. L., J. F. Yanovsky, G. A. Schmunis, 0. G. Traversa, S. M. Gonzalez Cappa, and A. S. Parodi Histopathology in Rockland mice immunized against American trypanosomiasis (Chagas disease). Am. J. Trop. Med. 17: