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.

Figure 1. TcCRT+/– inoculated mice showed significant reduction of specific anti T. cruzi antibody levels.

BALB/c mice were inoculated with 5×10⁵ metacyclic trypomastigotes. Results are expressed as the ratio of the absorbance of each serum sample at 490-nm. Dotted lines: Cut-off value adopted for positivity calculated as the mean of the values obtained for the negative controls (inoculated with PBS) plus three times the standard deviation. Serum samples were taken at the intervals indicated. TcCRT+/– inoculated mice showed significant reduction of antibody levels compared to those inoculated with wild type and TcCRT+ clones (p = 0.01 for both cases). Positive controls: serum from mice infected with the virulent Tulahuén T. cruzi strain.

Figure 2. TcCRT+/– mutant parasites are stable after prolonged infection in mice.

(A) Schematic representation of the TcCRT genomic locus in TcCRT+/– parasites (B) PCR analysis carried out from genomic DNA of TcCRT+/– and wild type parasites recovered from hemocultures of chronically infected mice. Primers: Pair CRT1-CRT2 and H1-H2 amplify the TcCRT (1.2 KB) and HYG CDS (0.96 kb), respectively. Pair CRT7-H4 amplifies the 5' UTR of TcCRT together with a fragment of the HYG CDS (1.45 kb). Pair CRT93-H6 amplifies the 3' UTR of TcCRT together with a fragment of the HYG CDS (1.4 kb). The sizes of the fragments correspond to those predicted for the replacement of TcCRT by the HYG gene.

Figure 3. Inoculation of TcCRT+/– mutant parasites protects mice against a virulent T. cruzi challenge.

(A) Balb/c mice were primed and boosted with metacyclic trypomastigotes TCC wild type, TcCRT+, TcCRT+/– or PBS. On day 120, all mice were challenged with 10⁴ bloodstream forms of a virulent T. cruzi TcVI isolate. Note the protection (p = 0.0001) in TcCRT+/– and wild type-preinoculated mice. (B) Dispersion diagrams of specific anti-T. cruzi antibody levels elicited in either naive mice (non- immunized) and those immunized and boosted with 5×10⁵ metacyclic trypomastigotes TcCRT+/–, wild type or TcCRT+ clone. The results are expressed as the ratio of the absorbance of each serum sample at 490-nm. Dotted lines: Cut-off value adopted for positivity calculated as the mean of values obtained for the negative controls plus three standard deviations. Serum samples were taken at day 30 post-priming. TcCRT+/– immunized mice showed undetectable antibody levels as compared to mice inoculated with wild type and TcCRT+ clones (p = 0.006 for both cases). Data (mean ± SD) presented are representative of three independent experiments (n = 6/group/experiment).

Figure 4. TcCRT+/– immunization decreases tissue inflammatory response and spleen indexes in challenged mice.

Autopsies were performed on mice 4 months post-priming and 2 months after virulent challenge. Dispersion diagrams of histopathological alterations in hearth muscle (A) and skeletal tissue (B). The inflammatory responses were graded as absent (–), slight (+), moderate (++), and severe (+++). Each dot represents a mouse. Representative images of H&E staining (blue: nuclear, pink: muscle/cytoplasm/keratin) of heart tissue (C and D) and skeletal muscle sections (E and F) from non-immunized and TcCRT+/– mice respectively (magnification, 25X). Li, Lymphocytic infiltrates. (G) Spleen indexes on day 60 post-challenge. Mice inoculated with TcCRT+/– parasites present lower spleen indexes compared with non-immunized controls (p = 0.02) and TcCRT+ parasites (p = 0.004).