No abstract

A cross section of a human population (501 individuals)

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.The protozoan parasite Trypanosoma cruzi causes a persistent infection which can lead to Chagas' disease, a major public health concern in Latin America, where an estimated 18 million people are infected (23). The infection results in a generally self-limiting acute parasitemic phase, followed by an indeterminant phase where parasitemia is commonly undetectable and most patients remain asymptomatic. Approximately 30% of individuals in the indeterminant phase progress to a chronic, symptomatic phase involving severe cardiomyopathy or gastrointestinal pathology. Several recent studies have provided information regarding the protective roles of antibodies, Th1-type cytokines, and cytotoxic T cells (CTL) (4,8,20). An increased understanding of the host immune responses to the organism and the pathogenesis of the disease (20) has provided a rationale for the pursuit of vaccine development as a means of control of T. cruzi infections. Recent advances in DNA vaccine technology make this an attractive vehicle for vaccine development against Chagas' disease for immunologic as well as economic reasons.The complement regulatory protein (CRP) of T. cruzi has been described as a virulence factor that enables the parasite to escape lysis by the host complement system (14). The expression of the crp gene is developmentally regulated and is coincident with conversion of the parasite from the complement-sensitive insect stage (epimastigote) to the complementresistant bloodstream stage (trypomastigote) (14, 17). The CRP is a surface glycoprotein that interferes with complement activation via noncovalent binding to the complement proteins C3b and C4b, the central components of the C3 convertase of the alternative and classical complement pathways, respectively (12, 16). Antibodies that block the complement regulatory activity of the CRP promote complement-mediated lysis of the parasites and have been detected in sera from chagasic patients (15). Transfection of the insect-stage epimastigotes with the crp gene resulted in constitutive expression of crp and conversion of the complement-sensitive epimast...

Monoclonal antibodies were raised against a recombinant ribosomal P2β protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti‐P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra‐cellular loop of the human β1‐adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist‐like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart β1‐adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross‐reaction with the human β1‐adrenergic receptor.

Antibodies from patients with Chagas heart disease and monoclonal antibodies (or mAb) to the carboxy-terminal end (B cell epitope R13) of the ribosomal P2beta protein of Trypanosoma cruzi (TcP2beta) cross-react with the beta1 adrenergic receptor (beta1-AR). Two single-chain Fv fragments (scFv) C5 and B7 derived from the variable regions of the anti-R13 mAb 17.2 were expressed. scFv C5 was a dimer and bound to TcP2beta with an affinity of K(d) = 8 nM, whereas scFv B7 was monomeric and had less affinity than scFv C5 for TcP2beta, K(d) = 46 nM. The affinity constant of scFv C5 to the second extracellular loop of the human beta1-AR was of 10 microM. Moreover, scFv C5 induced an increase in cAMP levels of CHO-K cells transfected with the human beta1-AR; scFv B7 had no effect but blocked isoproterenol stimulation. The agonist-like activity of scFv C5 and the antagonist activity of scFv B7 were both confirmed in vivo on heart beating frequency after their passive transfer to mice. Molecular modeling of the variable region of mAb 17.2 indicated which amino acids were likely to be involved in recognizing both peptide EDDDMGFGLF, derived from the R13 epitope of TcP2beta, and peptide ESDEARRCYN from the second extracellular loop of the human beta1-AR. It is plausible that the recently described cross-reaction of mAb 17.2 with rhodopsin can also be explained by this model. The physiological effects of this type of anti-T. cruzi antibodies may increase the liability of patients with Chagas disease.