Antigens of Trypanosoma cruzi: Evidence That the 90-kd Protective Glycoprotein Antigen Is Expressed in Blood-Form Trypomastigotes and May Not Be Functional in Dead Epimastigotes

Abstract

Attempts to induce protection against infection with Trypanosoma cruzi have revealed that immunization with dead parasites of the epimastigote stage is far less effective than immunization with live organisms (Brener, 1980, Adv. Parasitol. 18: 247-292). Recently, Snary (1983, Trans. R. Soc. Trop. Med. Hyg. 77: 126-129) demonstrated that immunization of mice with a glycoprotein of Mr 90 kd isolated from epimastigotes protected against a challenge with blood and metacyclic trypomastigotes, whereas immunization with another, similarly-isolated glycoprotein of Mr 72 kd protected only against a challenge with metacyclic trypomastigotes. Both glycoproteins have been shown to be present in the epimastigote and metacyclic trypomastigotes, but the 72-kd molecule has been reported to be absent from the amastigote and blood-form trypomastigote stages of T. cruzi (Nogueira et al., 1981, J. Exp. Med. 153: 629-639; Snary et al., 1981, Mol. Biochem. Parasitol. 3: 343-356). To define the antigens of the epimastigote stage of T. cruzi (Strain Y) that were recognized by antibodies formed following injection with different stages of the parasite, groups of New Zealand white female adult rabbits were injected with epimastigotes, amastigotes or blood-form trypomastigotes. All animals received three intraperitoneal inoculations 15 days apart. Four rabbits were inoculated with live epimastigotes from cultures in exponential growth in the medium of Warren (1960, J. Parasit. 46: 529-535) (5 x 104 organisms/rabbit/inoculation). No trypomastigotes were noted by microscopy of fresh and stained smears of this preparation. Three rabbits were inoculated with epimastigotes killed with 1% formalin (1 hr at 4 C) (2 x 105 organisms/rabbit/injection); three rabbits were inoculated with amastigotes (2 x 105 organisms/rabbit/injection) from cultures in L929 cells (Araujo and Remington, 1981, J. Immunol. 127: 855859) after formalinization as above; and five rabbits were inoculated with live blood-form trypomastigotes (5 x 104 organisms/rabbit/injection), isolated (Budzko and Kierszenbaum, 1974, J. Parasit. 60: 1037-1038) from blood of acutely infected mice. Seven days following the last inoculation, the animals were bled, and the serum from at least two animals of each group were pooled. Immunofluorescent antibody titers with epimastigote antigens (Araujo and Batista, 1969, Rev. Inst. Med. Trop. Sao Paulo 11: 104-110) were 1: 640 for the pool of sera from rabbits immunized with live trypomastigotes, 1: 320 for the sera of rabbits immunized with live epimastigotes or with killed amastigotes, and 1: 160 for the sera of rabbits immunized with killed epimastigotes. The sera were then reacted with epimastigote antigens following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transfer of the antigens to nitrocellulose paper according to the technique described by Towbin et al. (1979, Proc. Natl. Acad. Sci. USA 76: 4350-4354). The results revealed striking differences (Fig. 1). The two bands between the Mr markers 92 and 66.5 kd (a and b) represent the glycoprotein antigens of Mr 90 and 72 kd which have been described for the epimastigote stage (Snary and Hudson, 1979, FEBS Lett. 100: 166-170; Snary et al., 1981, loc. cit.), because they could be removed from the epimastigote lysate by affinity chromatography with lectins. These antigens were recognized by antibodies in the sera of the rabbits injected with live epimastigotes (Lanes 1) and by antibodies in the sera of rabbits injected with live blood-form trypomastigotes (Lanes 4). Also, only the 72kd antigen was clearly defined by antibodies in the sera of rabbits immunized with killed epimastigotes (Lanes 2). However, only the 90-kd antigen was detected by antibodies in the sera of rabbits immunized with killed amastigotes (Lanes