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.