Introduction
Chagas disease (CD) is a neglected tropical disease caused by the
intracellular parasite Trypanosoma cruzi (T. cruzi )
transmitted through contact with infected feces of triatomine bugs. CD
affects approximately 6-7 million people worldwide and is a major public
health problem in Latin America1. CD is characterized
by two clinically distinct phases: acute and chronic. Acute infection is
distinguished by parasites in peripheral blood and a non-specific
febrile illness that typically resolves within 4–8
weeks2,3. However, up to 5% of cases develop severe
affections including acute myocarditis, pericardial effusion, and
meningoencephalitis, with an estimated 0.5% risk of
mortality3,4. In chronic phase, most patients remain
asymptomatic throughout life, however, 20-30% of them could develop
chronic chagasic cardiomyopathy (CCC), driving death by heart failure
years to decades after initial infection3,5,6. Current
treatment against CD is restricted by two chemotherapeutic agents,
benznidazole (BNZ) and nifurtimox, nevertheless, are associated with
toxicity, long regimens and do not halt the progression of cardiac
clinical deterioration7–9. Therefore, several studies
have been consolidated on evaluating alternatives for the treatment or
halt T. cruzi- infection.
Control of T. cruzi- infection is dependent on the adaptive immune
response by MHC class I presentation of cytoplasmic antigens, activating
CD8+ cytotoxic T cells which release cytotoxic
granules (perforin and granzymes)10–12. Perforin is a
pore forming cytolytic protein, that allows the transition of granzymes,
a group of serine proteases, which induce programmed cell death inT. cruzi- infected cells13,14. Pre-clinical
studies in murine models, have showed that a T helper (Th) 1 type immune
response with evidence for IFN-γ and TNF-α are required to reduce
parasite dissemination from acute T.
cruzi -infection12,15–17. However, adaptive immune
responses are characterized by being delayed; hence, innate immune
response plays an important role during early infection by T.
cruzi , besides, some studies have suggested that, limited or deficient
innate effector functions during T. cruzi -infection may cause
parasite dissemination, and in some cases,
morbidity18–20. Innate immune response is triggered
by the recognition of pathogen-associated molecular patterns (PAMP´s)
through receptors, such as Toll-like receptors (TLRs) expressed by
“antigen-presenting cells” (APC´s), including macrophages. Macrophages
are recognized as the major components of the inflammatory and
immunological reactions typically seen in several chronic
diseases21. Macrophages classically activated by LPS
or IFN-γ secrete pro-inflammatory cytokines such as, TNF-α, IL-1, and
IL-6, as well as, reactive oxygen species (ROS) and reactive nitrogen
species, which result in a Th1 response driving parasite elimination or
growth inhibition22–24. All this data suggests that,
an appropriate innate immune response which support the activation of
the adaptive immune response is essential to control T.
cruzi -infection and host survival.
To date, the development and use of vaccines are an attractive option
for the treatment or control T. cruzi- infection. Since DNA
vaccines historically have not been approved for use in humans, several
studies have focused on the recombinant protein antigens, representing a
more reliable vaccine strategy17,25. Our program has
been examining the effects of two major recombinant protein antigens,
together with TLR-4 agonist adjuvants. These antigens include the
trypomastigote surface antigen known as TSA-1 and the flagellar
calcium-binding protein, Tc24. The recombinant TSA-1 have demonstrated
reduced parasitemia, cardiac inflammatory cell density and promote a Th1
type immunity through high levels of TSA-1-specific IFN-γ-producing
cells in acutely infected mice26. The recombinant Tc24
was associated with high production of IgG2a levels,
CD4+ T cell activity, and IFN-γ production during
acute T. cruzi -infection in a murine model27.
Hence, both proteins are being produced under current good manufacturing
practices (cGMP) as potential vaccines, even have been shown to recall
memory responses in HLA-A and HLA-B supertypes among CD patients in
Mexico28. Specific mutations have been made in both
recombinant proteins to facilitate production and increase stability
while maintaining immunogenicity, the proteins resulting were named
TSA-1-C4 and Tc24-C429,30, and even Tc24-C4 has showed
therapeutic efficacy in combination with low doses of BNZ duringT. cruzi -acute phase in BALB/c mice31,32. On
account of both, innate and adaptative immune responses are convenient
to control T. cruzi -infection, is necessary to perform studies
evaluating the combination of the recombinant TSA-1-C4 plus Tc24-C4
antigens focusing on the innate immunity and the cytotoxicity activity.
We hypothesized that the use of TSA-1-C4 and Tc24-C4 employed them as a
bivalent recombinant protein strategy may improve the immune response
for eliciting macrophages, pro-inflammatory cytokine-production, as well
as, stimulate the cytotoxic activity. In this study, we evaluated the
immunomodulatory effect of TSA-1-C4 plus Tc24-C4 recombinant antigen
combination by in-vitro assays, additionally, we evaluated the
cytotoxicity effect of the bivalent recombinant protein strategy in
presence with a TLR-4 agonist adjuvant (E6020-SE), by an acute
experimental T. cruzi -infection model.