**3.2 TcISP2 is immunogenic in mice with chronic infection**

To verify the immunogenicity of the recombinant form of the TcISP2 protein produced in *E. coli*, the indirect ELISA method was initially standardized. Recombinant proteins have different biochemical properties that determine the best conditions for coating ELISA plates with histidine-tagged TcISP2. Three different types of microplates were tested, each with a binding capacity and with different concentrations of recombinant protein. To assess the distribution of the protein in the wells, serial dilutions of mouse-produced anti-histidine antibodies obtained from Invitrogen were also analyzed. The results obtained showed that the medium binding capacity plate with 2 ug of recombinant protein per well presented the best results.

To confirm the infection of mice with the Y strain of *T. cruzi*, after 7 and 15 days, PCR was performed using the Tc121/122 oligos from a fraction of the clot obtained by puncturing the submandibular vein. The four mice infected after 7 days with the Y strain of *T. cruzi* were PCR negative, while the same mice were PCR positive after 15 days (**Figure 6**). After 60 days of infection, nucleic acids extracted from the cardiac lavage of two euthanized mice with infection confirmed by PCR and light microscopy were positive by PCR (**Figure 6**). These results indicate that blood parasites can only be detected 15 days post-infection in Black mice.

Serum from mice infected by *T. cruzi* strain Y, and collected after 7, 15, and 60 days, with results confirmed by PCR and light microscopy, was used in an ELISA experiment to evaluate the immunogenicity of TcISP2r. The results are shown in (**Figure 7**). The identification of TcISP2r was observed in the serum samples of mice

## **Figure 6.**

*Electrophoresis in 2% agarose gel stained with ethidium bromide containing the PCR products of the Trigonoscuta cruzi molecular test with the Tc121/122 oligonucleotides that amplify a 330 bp fragment corresponding to the gene encoding the parasite's kDNA. Blood clots of 4 infected mice after 7 and 15 days and the washing of cardiac tissue from 2 mice euthanized after 60 days of infection, were evaluated as indicated in the wells. C-, corresponds to the negative control of the PCR reaction; C+, corresponds to the positive control with genomic DNA obtained from T. cruzi of the Y strain; M – 100 bp ladder from synapse biotechnology.*


### **Figure 7.**

*Indirect ELISA test with different concentrations of TcISP2r and serum from mice infected with Trigonoscuta cruzi strain Y after 7, 15, and 60 days. A. Experimental design to verify the immunogenicity of TcISP2r in mouse sera collected 7, 15, and 60 days after infection with the Y strain of T. cruzi; negative control serum was obtained from uninfected mice; 2 μg; 5 μg and 10 μg are the amount of TcISP2r per well; B. absorbance results obtained after indirect ELISA test with alkaline phosphatase-labeled second antibody against mouse IgG, developed with P-nitrophenyl phosphate substrate.*

60 days post-infection, suggesting an immune response against ISP2 from *T. cruzi* in the chronic phase of the disease. In ELISA tests, using mouse serum, the best protein concentration for antibody detection was also 2 ug.

The ELISA test revealed antibodies against TcISP2 in serum samples from *T. cruzi*infected mice only after 60 days post-infection, during the chronic phase. The high activity of TcISP2 on neutrophil elastase indicates its inhibiting action on the immune system [44, 50]. In this way, inflammation would occur in infected organs and tissues during the chronic phase, when cardiomyopathy and mega syndromes (megaesophagus and megacolon) develop. Thus, we could infer that the activity of TcISP2 is to inhibit inflammatory activity and thus facilitate the colonization of tissues and organs by the parasite.

According to Faria et al. [33], ISPs are responsible for inhibiting the neutrophil elastase present in macrophages, through the toll-like receptor 4. Thus, cells with phagocytic activity can be used by the protozoan for their proliferation and escape from the immune system. In Chagas' disease, macrophages are infected and carry the parasite into tissues. The phagocytic action of macrophages is more relevant in tissues compared to other professional phagocytic cells, due to their ability to migrate from the bloodstream to different tissues and organs [51]. Thus, the TcISP2 can play an essential role in acute and chronic Chagas' disease.
