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Home arrow Economics arrow American Trypanosomiasis Chagas Disease, Second Edition: One Hundred Years of Research


Vaccination with attenuated parasites (premunition)

Basic laboratory studies on premunition against Typanosoma cruzi

In 1952, Pizzi56 published that mice that had been inoculated with a T. cruzi epi- mastigote culture presenting low virulence became resistant to reinoculations of virulent parasites. From 1965 to 1990, Menezes published a series of papers based on the vaccination of mice against virulent T. cruzi infection by means of preinoculations with an avirulent strain (PF), derived from the virulent Y strain.57-59 Animals inoculated with cultures where epimastigote forms predominated, became resistant and survived lethal T. cruzi inoculations.60 The experiments were replicated in dogs where the levels of parasitemia and mortality rates were significantly reduced. Moreover, electrocardiographic determinations and histopathological studies of the myocardium showed a clear prevention of functional and anatomic lesions of the heart.61 Dr. Menezes’ preclinical and Phase I clinical studies were completed with inoculations into Callitrix monkeys and humans, including himself.62 Most inoculations used by Menezes for experimental vaccination contained mostly epi- mastigotes, with a low proportion of trypomastigotes. The term “avirulent” was challenged by Chiari,63 who demonstrated infections by means of hemoculture in mice inoculated with doses as low as 5000 metacyclic trypomastigotes from PF cultures, although direct blood examination was negative. Since Menezes himself had acknowledged exceptional cases of demonstrated infection in PF strain-inoculated animals, the term “attenuated” seemed more adequate to describe T. cruzi strains of very low infectivity.

Some other attenuated T. cruzi strains with protective activity have been described. Cultures of the “Corpus Christi” strain were unable to establish apparent infections when 107 live culture forms were inoculated into C3H(He) mice. Mice preinoculated with this strain developed resistance against a further infection with the virulent Brazil strain. Moreover, this resistance could be also obtained in naive mice by transfer of spleen cells from immunized mice to nonimmunized receptor ones. Depletion of the B cell population but not of the T cell components abrogated the adoptive transfer of resistance.64

The group of Gattas made a series of presentations to the Brazilian Meetings of Basic Research in Chagas Disease reporting the apparent lack of infectivity of a T. cruzi clone derived from the CL strain of T. cruzi and named the clone CL-14. Inoculations of 2 X 106 metacyclic forms into highly susceptible, newborn BALB mice produced no detectable parasitemias and protected against virulent challenge in a time- and dose-dependent manner, as shown by blood parasite counts and detailed histological observations.65 Further studies with more sensitive methods such as hemoculture and xenodiagnosis also failed to reveal infection with metacyclic forms of this clone, whether they were derived from vectors or from axenic cul- tures.66 The specific antibody levels were much lower in mice inoculated with clone CL-14 than in those inoculated with the parental strain CL. Furthermore CL-14 infected mice did not produce the polyclonal response which is characteristic of T. cruzi infection.67

Some partial insights into the mechanisms of attenuation of the CL-14 clone were obtained by the group of Yoshida et al.68 They carried out a series of comparisons between the attenuated CL-14 and the “wild-type” CL strain dominant surface glycoproteins. The ability of this attenuated clone to infect HeLa cells in vitro was reduced four-fold and no intracellular replication was observed. The expression of GP82, a dominant surface glycoprotein known to play an important role in infectivity, was shown to be close to 10-fold lower, as measured by fluorescent antibody-labeled parasites analyzed by flow cytometry. The genomic organization of the GP82 gene family, as shown by the hybridization patterns of GP82-labeled probes that did not show remarkable differences in the attenuated clone when compared to the original CL parental strain. However, chromosomal mapping of the gene family, which displayed a wide distribution across several pulse-field electrophoretically separated chromosomes, revealed some distinct patterns in CL-14 as compared with CL.

Other metacyclic trypomastigote surface molecules known to interact with host cells, such as cruzipain (GP 57-51), trans-sialidase (TS), and the mucin-like GP

35-50, were examined in the CL-14 clone. The expression of cruzipain was higher, a finding consistent with determinations by Paiva et al.69 for clone CL-14. However, in three other attenuated strains (TCC, Tul 0, and Y-null), a clear reduction of cruzipain gelatinolytic activity as compared with three virulent strains was detected.70 Regarding TS, its activity in CL-14 was 1.6-fold higher than in the CL wild-type strain,68 confirming previous studies by Gattas et al.71 Similarly, a remarkable increase in TS activity was detected in the TCC-attenuated strain. The relative increase, in strains of low infectivity, of proteins known to play a role in infection may seem, in principle, paradoxical. However, the existence of multiple isoforms of these proteins, many of which may be inactive or irrelevant for infection, may explain their overexpression or rebound production in parasites of low infectivity.

A long-term culture, named TCC (Trypanosoma cruzi de Cultivo) was shown to be unable to persistently infect mice or rabbits.72 It was cloned twice, in 1980 and 1990, and characterized after 2000 as belonging to T. cruzi I lineage. The attenuated phenotype was stable, since no virulent sublines could be derived by either mouse culture or insect vector passage, tested repeatedly until lines were extinguished. Since strains of very low virulence but unexpectedly displaying high pathogenicity have been described,73 this possibility was examined for the TCC strain. Mice inoculated with cultures were subjected to long-term histopathological studies. This strain was unable to trigger immunopathological responses or to induce histopatho- logical alterations in immunocompetent mice.72 Experiments involving laboratory conditions to enforce infection (high trypomastigote inocula into newborn or immu- nosuppressed animals) allowed the demonstration of infections with extremely low density; however, attempts to propagate these infections indefinitely from mouse to mouse, always failed.

Leguizamon et al.74 examined the process of attenuation by prolonged passage in culture in an originally virulent T. cruzi strain, named RA. They demonstrated a correlation between the attenuation of infectivity and the time the parasites were propagated in axenic culture (up to 2 years). Even though the original line was highly virulent, after 2 years in culture, inocula of 107 epimastigotes were unable to produce detectable parasitemia in immunocompetent, susceptible BALB mice. However, reactivation of virulence was shown by serial passage in athymic mice. Conversely, similar serial passages were attempted with the attenuated TCC strain but inocula of 105 TCC trypomastigotes into nu/nu athymic mice rarely caused detectable blood parasitemia, although parasite tissue nests were found more often in urinary bladder and heart atria. Homogenates of these tissues, where the presence of parasites was verified, were thus used routinely for serial passage and weakly infective TCC sublines could be recovered, but not serially propagated in immuno- deficient mice.

Serological studies in mice75,76 and dogs77 indicated antibody titer regression to negative values in most animals, although some mice remained seropositive after 1 year. Inoculation of live TCC epimastigotes afforded protection, not only against acute infection, but also against late development of chronic pathology induced by either the Tulahuen laboratory strain or also by 17 wild isolates obtained in a broad endemic area of Argentina.78 Electrocardiographic studies in mice showed that

TCC preinoculations before virulent infection significantly reduced functional alterations of the heart, such as sinus bradycardia, supraventricular tachycardia, and atrioventricular blocks.79

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