Immunogenicity of Trypanosoma cruzi mutants: characterization of biochemical markers and models of protection against disease

A. BARRIO, M. CIACCIO, C. PEREZ BRANDÁN , M.P. ZAGO, R.M CORRALES, M.PADILLA & M.A. BASOMBRIO

Tallin, Estonia

Otro; Meeting of HHMI International Research Scholars; 2004

Howard Hughes MedicalInstitute

Partial immunization can be achieved by experimental vaccines, often consisting of parasite antigens plus adjuvants. However, this protection is not long lasting and has not convincingly prevented tissue lesions or alterations in heart function. Working with animal models, we were able to establish in recent years that vaccination can indeed induce solid and long-lasting resistance against infection and disease. But the ability of a vaccine to do so seems to depend on two attributes that raise safety issues: that live parasites be present in the immunogen and that they retain, at least temporarily, some degree of infectivity. Much of this evidence was obtained using the long-term, culture-attenuated TCC strain. Recently, we started using two gene targeted mutants of T. cruzi, derived by homologous recombination, as tools. A gp72-null strain retains the ability to grow in vitro, in spite of a 1745-bp deletion of coding sequence from both alleles of the gene. After eight years of laboratory propagation in the absence of antibiotics, the stability of the mutation was studied. Specific primer annealing sites and length of the construct replacing the gene were analyzed. The deleted gene was still absent; it was replaced by an antibiotic-resistance construct. In mice, the mutated parasites were unable to sustain infection, but the animals were protected against virulent T. cruzi. However, after one year, the protection by gp72-null was no longer detectable, in contrast with the persistently protective TCC strain. A second T. cruzi mutant was analyzed, in which one allele of the cub gene was deleted after homologous recombination with a neomycin-resistance construct. This mutant displayed a remarkable, though incomplete, reduction in virulence and the ability to protect against virulent challenge. The possibility of knocking out one or more genes from the TCC strain while retaining its capacity to provide long-term protection is being explored in our laboratory.