Discussion
CD is a public health problem which affects around 6–7 million people worldwide. First-line drugs available to treat it have limited efficacy and are associated with toxicity. Therefore, there has been an increased interest in vaccine candidate development, exploring a variety of antigens, adjuvants and delivery systems (plasmids, adenoviruses, peptides and recombinant proteins)27,31,41–45. The recombinant parasite-antigens TSA-1 and Tc24 have showed to reduce parasite burdens and increase antigen-specific T cells, as well as, survival of mice during experimental acute T. cruzi -infection26,27,43,46. Specific mutations have been made in both recombinant proteins in order to reduce aggregation during production; the proteins resulting were named TSA-1-C4 and Tc24-C430,47. Previously, has been demonstrated the safety and immunogenicity resulting from the bivalent vaccine formulated with recombinant TSA-1-C4 and Tc24-C4 antigens in non-human primates48 and even a recent study performed by our work team, evaluated the therapeutic effect of a vaccine-linked chemotherapy formulated with the combination of TSA-1-C4 and Tc24-C4 antigens during T. cruzi -chronic infection using a murine model49. Nevertheless, increasing focus on innate immunity studies suggest that, T. cruzi -infection may cause morbidity when innate effector functions, are lacking or down-regulated18–20. Therefore, further studies are necessary in order to clarify the effect of the recombinant TSA-1-C4 and Tc24-C4 antigen combination in the innate immune-response as well as its influence on the CD8+ activation. Hence, we have proposed to evaluate the immunomodulatory effect of TSA-1-C4 and Tc24-C4 recombinant protein combination.
Macrophages are recognized as one of the major components in the inflammatory and immunological responses during the early stages of pathogen infections20,21. With the aim to evaluate if stimulation with the bivalent recombinant protein strategy activate macrophages from naïve mice, we measure levels of NO and H2O2 of supernatants from stimulated peritoneal murine macrophages. We demonstrate that macrophages stimulated with recombinant TSA-1-C4 and Tc24-C4 antigen combination induces production of cytotoxic molecules, which support the innate immune-response. Besides, we observed that from 12 µg/mL of Tc24-C4 stimulation there was an increase in the production of NO and H2O2 molecules compared to TSA-1-C4 plus Tc24-C4 stimulation, which makes us suppose that TSA-1-C4 antigen could have a downregulating effect on cytotoxic molecules production by macrophages. The mechanisms that regulate the production of molecules can be numerous (gene expression, distribution, catabolism, etc.)50,51, however, due to the limitations of the study, we could not clarify how the recombinant proteins interact with these mechanisms. On the other hand, some studies, suggest that, ROS and NO molecules are essential for parasite proliferation and growth, due to provide ideal conditions (e.g., iron availability in macrophages) for parasite-replication52,53. In the present study, the amounts of NO and H2O2 molecules reported are similar to those reported in previous studies36,54,55, which peritoneal murine macrophages were stimulated with extracts of plants or microorganisms with antiparasitic activity. According to those data, toxic effects were not detected, therefore, we believe that the amounts of molecules produced by the TSA-1-C4 plus Tc24-C4 bivalent recombinant strategy is appropriate.
We also evaluated the pro and anti-inflammatory cytokine profile of supernatants from TSA-1-C4 plus Tc24-C4 stimulated macrophages. Here, we demonstrated that there is a benefit to use the bivalent recombinant protein strategy, due to increase the production of pro-inflammatory cytokines compared to individual TSA-1-C4 or Tc24-C4 stimulated-macrophages from naïve mice. Our findings suggest a synergistic effect between the recombinant proteins. Moreover, we observed that, from 12 µg/mL of protein-stimulation, the production of TNF-α, IL-1β and IL-6 increase, while IL-10 tends to reduce, therefore, we suggest that macrophages stimulated with the recombinant antigen combination induce a Th1 profile. It has been reported that during the early stages of T. cruzi infection, macrophages release pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which act synergistically and induce the expression of adhesion molecules, causing chemotaxis and migration of T cells, hence are critical for control of parasite-replication and protective immunity 20,22,56. Nevertheless, an excessive pro-inflammatory response has been correlated with the development of tissue damage in clinical and preclinical models of T. cruzi -infection5,57–60, therefore it has been proposed that a balance between Th1 and Th2 responses is necessary in order to elude an exacerbated response mediated by extensive inflammation31,61,62. Further studies will be necessary perform in order to evaluate if immune responses mediated by recombinant TSA-1-C4 plus Tc24-C4 stimulation protect against tissue damage during T. cruzi- infection.
Protective immunity against intracellular parasites as T. cruziis mediated by CD8+ T cells, which release cytotoxic molecules such as, perforin and granzymes from cytotoxic granules63,64. Moreover, CD8+ T cells modulate the immune response through the secretion of cytokines, which are required for activation of APCs, macrophages and T cells or downregulate the extensive inflammatory response10,16. In this study, we measured the major cytokines representing for Th1 and Th2 immune responses of supernatants from co-cultures of TSA-1-C4 and Tc24-C4 stimulated macrophages and CD8+ T cells from naïve mice. According to our data, the bivalent recombinant protein strategy induced both, pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines responses. When we increased the concentration of stimulation, pro-inflammatory cytokines presented a significant increase paralleled to a decrease for the anti-inflammatory response. This data reveals that the macrophages stimulated with recombinant TSA-1-C4 and Tc24-C4 combination can activate CD8+ T cells which are characterized by inducing a Th1-type response mediated by a strong production of IFN-γ and TNF-α. As we hypothesized, we observed that there is a benefit to use the bivalent recombinant protein combination, suggesting that both recombinant antigens are needed to achieve maximal synergistic effects. Some studies suggest that CD8+ T cells producing IFN-γ perform as central mediators of T. cruzi -control and protective immunity, while the cytolytic activity is not necessarily required for the immuno-protective responses65,66. Even though, we believe that both, cytotoxic and inflammatory responses by CD8+ T cells are necessary, pre-clinical studies focused on the vaccine development against T. cruzi have highlighted the importance of the immune response mediated by pro-inflammatory cytokines26,27,32,46,49. On the other hand, previously, has been demonstrated that the IL-17 family of cytokines plays a critical role in host survival by regulating exuberant inflammation and immunopathology during T. cruzi infection, suggesting that IL-17 have protective roles during adaptive immunity32,67,68. For these reasons, further studies, would be relevant to evaluate the Th17 profile induced by the recombinant TSA-1-C4 plus Tc24-C4 antigen combination.
Finally, we measured the functionality of cytotoxic cells to lyseT. cruzi -infected cells at 50 days post-infection, which corresponding to the end of the acute phase for our experimentalT. cruzi -infection model. We found a significantly higher cytotoxicity percentage in infected-treated mice compared to infected non-treated mice. Although, the administration of the recombinant protein combination did not show a significant difference compared to either TSA-1-C4+E6020-SE or Tc24-C4+E6020-SE, the use of the vaccine formulated with recombinant TSA-1-C4 or Tc24-C4 antigens and E6020-SE induces the activation of cytotoxic effector cells. This data indicates that, CD8+ T cells, as well as, other types of cytotoxic cells (for example Natural killer) from TSA-1-C4 and Tc24-C4 in combination with E6020-SE vaccinated mice are persistent and remained competent to 50 days post infection, therefore, we believe that these cells are in constant activation during the acute phase of T. cruzi -infection.