Pure paraflagellar rod protein protects mice against Trypanosoma cruzi infection

Abstract

The paraflagellar rod proteins (PAR) purified from Trypanosoma cruzi epimastigotes were shown to protect mice against an otherwise lethal challenge inoculum of 10 3 bloodstream-form trypomastigotes. The injection route used for immunization was shown to have a marked impact on the development of protective immunity. Mice receiving subcutaneous (s.c.) injections of PAR proteins had reduced bloodstream parasitemias and showed 100% survival following challenge. In contrast, mice immunized via the intraperitoneal (i.p.) route developed parasitemia levels equivalent to those of unimmunized controls and did not survive infection. Western blotting (immunoblotting) demonstrated that sera from both i.p. and s.c. immunized mice reacted specifically with PAR proteins; however, the antibody titer of the i.p. immunized mice was approximately 64-fold greater than that of the s.c. immunized mice, suggesting that the protective response in the s.c. immunized mice is cell mediated rather than humoral.Trypanosoma cruzi is a parasitic hemoflagellate that causes Chagas' disease, a major human health problem in Central and South America (4). Chemotherapeutic agents have limited effectiveness against the disease, and no vaccines are available for disease prevention. Experimental vaccines consisting of attenuated forms of the parasite, crude parasite lysates, partially purified proteins, and synthetic peptides derived from the sequence of a 24-kDa excretory protein (ESA) have been tested in laboratory animals with varying degrees of success (reviewed in references 2, 5, 7, 16, and 22). Several of these studies show that susceptible hosts may be partially protected against death by vaccination prior to challenge; however, individual antigens capable of ensuring survival against an otherwise lethal challenge have yet to be identified.In an attempt to identify and purify protective antigens, we focused on proteins present in the flagellum of T. cruzi. The rationale for this approach is based on studies demonstrating that vaccination with crude flagellar extracts are effective in both inducing protective immunity (i.e., 90% survival) in mice after challenge (15,20,21) and reducing tissue lesions in immunized mice compared with nonimmunized mice following a challenge infection (14). In contrast, immunization with a membrane-enriched fraction from the body of the parasite was less effective in inducing a protective response, suggesting that the protective antigen(s) may be unique to the flagellum. A potential candidate for such antigens is the paraflagellar rod, a prominent structure present only in the flagellum of the parasite (5). We have shown that the paraflagellar rod contains two distinct proteins, PAR 1 and PAR 2, and have determined conditions that allow these proteins to be isolated in amounts and purity sufficient for immunization trials (3,18).In this paper, we report the ability of the purified PAR proteins to induce an immune response in mice capable of protecting against an otherwise lethal inoculum of T. cruzi trypomastigote...

Trypanosoma cruzi:Protective Immunity in Mice Immunized with Paraflagellar Rod Proteins Is Associated with a T-Helper Type 1 Response

“…1). Consistent with our previous results (Wrightsman et al 1995), a dramatic difference was observed between the titer in the Freund's (sc) group and that in the Freund's (ip) group, with the latter having a 30-fold higher titer. The titer of the Freund's (sc) group was also conspicuously lower than the titer of the alum, QS21, and Ribi groups by factors of 50, 75, and 125, respectively.…”

“…Measurements of anti-PAR antibody titer were performed by enzymelinked immunosorbent assay (ELISA) precisely as described elsewhere (Wrightsman et al 1995). Antibody isotypes were determined using the same ELISA except that the concentration of PAR antisera used was 1/100 for Freund's (sc), 1/2000 for Freund's (ip), and 1/1500 for alum, RIBI, and QS-21.…”

“…In our previous studies (Wrightsman et al 1995), we have shown that the paraflagellar rod proteins (PAR) present in the flagellum of T. cruzi can induce an immune response in mice capable of protecting against an otherwise lethal inoculum of T. cruzi trypomastigotes. In this paper, the effects of antigen dose, immunization regimen, and different adjuvants on parasite clearance and survival of immunized mice challenged with the parasite are reported.…”

Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection

“…Immunization with the paraflagellar rod proteins has previously been shown to be highly effective at reducing acute parasitemia and providing 100% protection against an otherwise lethal challenge with a highly virulent strain of T. cruzi [1,2]. Although potent immune responses against the previously identified PFR proteins have been observed, it was impossible to entirely rule out the possibility that the protective antigen in this preparation was a minor, unidentified component.…”

“…Our laboratory has previously described successful vaccination of mice with paraflagellar rod proteins (PFR) purified from parasite extracts (pPFR). Vaccination with these proteins either emulsified with Freund's adjuvant [1] or co-adsorbed to alum with recombinant IL-12 (rIL-12) [2] resulted in greater than 90% reduction in circulating parasites and 100% survival of an otherwise lethal infection with T. cruzi. The parasite-derived preparation, pPFR, is composed of four distinct proteins, designated PFR-1 through PFR-4 (encoded by the par1-4 genes) [3,4].…”