A Monoallelic Deletion of the TcCRT Gene Increases the Attenuation of a Cultured Trypanosoma cruzi Strain, Protecting against an In Vivo Virulent Challenge

Abstract

Trypanosoma cruzi calreticulin (TcCRT) is a virulence factor that binds complement C1, thus inhibiting the activation of the classical complement pathway and generating pro-phagocytic signals that increase parasite infectivity. In a previous work, we characterized a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another overexpressing it (TcCRT+), both derived from the attenuated TCC T. cruzi strain. The TcCRT+/- mutant was highly susceptible to killing by the complement machinery and presented a remarkable reduced propagation and differentiation rate both in vitro and in vivo. In this report, we have extended these studies to assess, in a mouse model of disease, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Balb/c mice were inoculated with TcCRT+/- parasites and followed-up during a 6-month period. Mutant parasites were not detected by sensitive techniques, even after mice immune suppression. Total anti-T. cruzi IgG levels were undetectable in TcCRT+/- inoculated mice and the genetic alteration was stable after long-term infection and it did not revert back to wild type form. Most importantly, immunization with TcCRT+/- parasites induces a highly protective response after challenge with a virulent T. cruzi strain, as evidenced by lower parasite density, mortality, spleen index and tissue inflammatory response. TcCRT+/- clones are restricted in two important properties conferred by TcCRT and indirectly by C1q: their ability to evade the host immune response and their virulence. Therefore, deletion of one copy of the TcCRT gene in the attenuated TCC strain generated a safe and irreversibly gene-deleted live attenuated parasite with high immunoprotective properties. Our results also contribute to endorse the important role of TcCRT as a T. cruzi virulence factor.

Author SummaryTrypanosoma cruzi is a protozoan parasite which infects 9 million people in Latin America. Currently there is no vaccine to prevent this disease. Therefore, different approaches or alternatives are urgently needed to identify new protective immunogens. Live vaccines are likely to be most effective in inducing protection; however, safety issues associated with their use have been raised. Hence, we genetically manipulated an attenuated strain of T. cruzi as a safety device to rule out the possibility of reversion to the virulent phenotype. The genetically modified parasites were highly susceptible to killing by the complement machinery and presented a reduced propagation and differentiation rate. We have extended these studies to assess, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Accordingly, we show that genetically modified parasites present attenuated virulence in mice. The genetic alteration was stable and, after long term infection, it did not revert back to wild type form. Furthermore, after challenge with a virulent T. cruzi strain, mutant immunization induces a highly protective response evidenced by significantly lowered parasite density, mortality, spleen weight index and tissue inflammatory response. Our study provides new insights into the host-pathogen interactions and into the use and evaluation of irreversibly gene-deleted live attenuated parasites to protect against Chagas disease.

Keywords

KeyWords Plus:CHAGAS-DISEASE; TARGETED DELETION; HUMAN-COMPLEMENT; APOPTOTIC CELLS; 1ST COMPONENT; GUINEA-PIGS; MICE; INFECTION; CALRETICULIN; VACCINE