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.