Chagas disease is caused by infection with the protozoal parasite Trypanosoma cruzi, affecting approximately 6-7 million people worldwide (Lidani et al., 2019). Infection is mainly spread by contact with feces from an infected hematophagous insect of the Triatomine family, but can also occur through congenital infection, blood transfusion and organ transplant (Rassi et al., 2010). Upon entry into the host, the parasite is capable of invading any nucleated cell where it replicates, then ruptures the cell releasing parasites that circulate in the vasculature and invade cells in any tissues (Machado et al., 2012). Cell damage, as well as pathogen associated molecular patterns from T. cruzi, incite the host inflammatory immune response to control parasite levels (Oliveira et al., 2004). However, the host inflammatory immune response is only able to significantly reduce parasite levels, not eliminate all parasites (Añez et al., 1999; Zhang and Tarleton, 1999). Thereafter, persistence of parasites in tissues leads to chronic inflammation, progressive fibrosis, edema, and hypertrophy (Bonney and Engman, 2008; Bonney et al., 2019). Clinically, infected individuals may develop a self-limiting illness with nonspecific flu-like symptoms during the first 1-2 months after infection, but resolution of symptoms without treatment coincides with significant reduction of parasite levels by the inflammatory immune response (Machado et al., 2012). Subsequently, individuals enter the chronic infection phase where they may remain clinically asymptomatic for years to decades (Machado et al., 2012). Approximately 60-70% of chronically infected individuals remain asymptomatic for life and are considered indeterminate (Machado et al., 2012). The remaining 30-40% develop clinical disease, with approximately 75% of those individuals developing cardiac symptoms and the remaining 25% developing gastrointestinal disease (Rassi et al., 2010). Chronic Chagasic cardiomyopathy (CCC) is the most severe manifestation of chronic T. cruzi infection, resulting in 2.3 million disability adjusted life years (DALYs) and can incur an annual cost of $11,000 per patient for treatment ([[NoAuthor]]; Lee et al., 2012). Thus, there is an urgent need for treatment strategies that can alleviate disease burdens.

Clinical manifestations of CCC encompass a spectrum of symptoms, including mild to severe arrhythmias, apical aneurysms, ventricular dilation, heart failure and sudden death (Ribeiro et al., 2012). Symptoms arise due to the T. cruzi induced inflammatory and fibrotic tissue damage which leads to cardiac remodeling (Cruz et al., 2017). Histologic studies of cardiac tissue from CCC patients have shown that CD8+ and CD4+ T lymphocytes are the predominant infiltrating leukocyte population in cardiac tissue, and increased levels of pro-inflammatory cytokines IFN-γ, TNF-α, and IL-6 are also present (Higuchi Mde et al., 1993; Higuchi et al., 1997; Reis et al., 1997). Additionally, progression of cardiac fibrosis correlates with progression of disease severity (Noya-Rabelo et al., 2018; Senra et al., 2018). Thus, the central problem to addressing cardiac disease due to chronic T. cruzi infection is the fact that specific treatment is limited to only two anti-parasitic drugs, benznidazole (BNZ) and nifurtimox, which act to directly kill parasites peripherally but may not significantly reduce the parasite burden in the heart, nor ameliorate host tissue damage (Viotti et al., 2006; Jackson et al., 2010; Urbina, 2010). CCC can ensue due to the progression of host inflammation and fibrosis. Thus, while both drugs have high efficacy during acute infection (Viotti et al., 2006), a large, multi-center clinical trial demonstrated that in patients with established cardiac disease, BNZ failed to prevent the progression of CCC and ultimately, cardiac death (Morillo et al., 2015). Further complicating the limited efficacy of BNZ is the fact that intolerable side effects occur in 40% of patients, resulting in early termination of treatment (Viotti et al., 2009; Molina et al., 2015). Thus, alternative therapies with improved efficacy, reduced adverse effects, and targeting the deleterious host responses are urgently needed.

Vaccines are an attractive option to address the need for improved therapies for Chagas disease. Studies comparing parasite specific immune responses between indeterminate and CCC patients have revealed that a balanced T_H1/T_H2/T_H17 cytokine profile, with increased levels of IFN-γ, IL-10 and IL-17A, correlate with preserved cardiac function, while patients with CCC have predominantly a Th1 response with higher levels of IFN-γ, IL-6, and TNF-α and little IL-10 and IL-17A (Laucella et al., 2004; Magalhaes et al., 2013; Sousa et al., 2014). Building on this knowledge, researchers have developed numerous vaccine candidates that target specific parasite antigens and modulate the host immune responses (Rios et al., 2019; Bivona et al., 2020). One promising vaccine candidate is the T. cruzi flagellar calcium binding protein designated Tc24 (Dumonteil et al., 2012). This antigen is located along the inner leaflet of the flagellar membrane and is highly expressed in the extracellular trypomastigote form as well as in the non-dividing intracellular amastigote form of T. cruzi (Versteeg et al., 2021). Several studies have demonstrated that recombinant protein based vaccines containing Tc24 antigens induce strong immune responses, with increased production of antigen specific CD8+ T cells as well as production of IFN-γ, IL-10 and IL-17A (Barry et al., 2016; Seid et al., 2016; Jones et al., 2018; Cruz-Chan et al., 2021). Importantly, Tc24 based vaccines can reduce parasite burdens and tissue damage when used therapeutically in both acute and chronic mouse models of infection (Barry et al., 2019; Cruz-Chan et al., 2021). Additionally, we have constructed a mutated form of Tc24, designated Tc24-C4, with improved stability suitable for large scale expression and purification processes necessary to develop vaccines for clinical use (Seid et al., 2017; Biter et al., 2018). Therefore, Tc24-C4 based vaccines are a promising candidate to address clinical disease.

Vaccine-linked chemotherapy has been proposed as an improved treatment strategy for parasitic diseases to both reduce infection and reduce morbidity (Bergquist et al., 2005; von Seidlein et al., 2020). We were the first to demonstrate in mouse models of acute infection that vaccine-linked chemotherapy using a low dose of benznidazole and a low dose of vaccine, consisting of a stable squalene emulsion containing recombinant Tc24-C4 protein and a TLR4 agonist adjuvant, either E6020 or glucopyranosyl lipid A (GLA), which leads to greater reductions in parasite burdens, cardiac inflammation, and cardiac fibrosis when compared to BNZ or vaccine only treatment (Jones et al., 2018; Cruz-Chan et al., 2021). Further, the vaccine induced a balanced T_H1/T_H2/T_H17 immune response (Cruz-Chan et al., 2021). More recently, vaccine-linked chemotherapy strategies using recombinant trans-sialidase protein (TSA-1) alone, or TSA1-C4 combined with Tc24-C4 protein, have been tested in mouse models of chronic infection and shown to also reduce tissue pathology (Dzul-Huchim et al., 2022; Prochetto et al., 2022). What we had not yet tested and proved was whether our vaccine-linked chemotherapy strategy improved cardiac function during chronic T. cruzi infection. We previously showed in acute infection models that both sequential and concurrent treatment with a low dose of BNZ combined with the Tc24-C4/E6020 SE significantly reduced cardiac pathology and parasite burdens. Therefore, in the study reported here we tested both sequential and concurrent vaccine-linked chemotherapy strategies and we evaluated left ventricular wall thickness, chamber size and volume over time in an effort to better characterize the changes in cardiac structure induced by chronic T. cruzi infection

Chronic T. cruzi infection induces persistent inflammation, hypertrophy, and progressive fibrosis which leads to significant changes in cardiac structure and function, ultimately manifesting clinically as CCC (Rossi et al., 2003; Alvarez et al., 2014). Thus far, antiparasitic treatments alone are only able to eliminate, or significantly reduce, parasite burdens peripherally and do not prevent progression of clinical cardiac disease due to parasites in the cardiac tissue and resultant inflammation (Morillo et al., 2015). Here we have shown that our vaccine-linked chemotherapy (VLC) treatment regimens, combining a low dose BNZ treatment with our Tc24-C4 protein/TLR-4 agonist (E6020) SE adjuvant vaccine, significantly improves cardiac structure and function in a mouse model of chronic T. cruzi infection. This strategy also reduced cardiac pathology, as evidenced by significantly reduced cellular infiltration and reduced fibrosis. The vaccine induced a durable T_H1/T_H2 cytokine response at low doses, with modest IL-17A production, when administered alone or with BNZ treatment in both sequential (Supplemental Figure 3A) and concurrent (Supplemental Figure 3B) treatment schemes, as well as the high dose vaccine given after low BNZ treatment (Supplemental Figure 3C). These data suggest that modulation of the host immune response effectively ameliorates the deleterious host responses to T. cruzi infection that contribute to tissue pathology and clinical disease. The fact that these effects were observed with a low dose of BNZ combined with a low dose of vaccine, indicates that vaccine-linked chemotherapy is an effective dose-sparing strategy. These data support our prior findings in mouse models of acute infection which demonstrated that vaccine-linked chemotherapy showed improved efficacy compared to monotherapy with BNZ or the Tc24-C4/E6020 SE vaccine alone (Jones et al., 2018; Cruz-Chan et al., 2021). Importantly, our results agree with recently published results demonstrating efficacy of vaccine-linked chemotherapy in mouse models of chronic T. cruzi infection. Prochetto et al. showed that vaccination with three doses of a recombinant Trans-sialidase protein combined with ISPA adjuvant, followed by a curative course of BNZ led to reduced cardiac arrhythmias and fibrosis in chronically infected mice (Prochetto et al., 2022). Dzul-Huchim et al. observed significantly reduced cardiac fibrosis in chronically infected mice that were vaccinated with two doses of a vaccine containing both recombinant TSA-1 protein and Tc24-C4 protein combined with E6020 SE adjuvant concurrently with a 7 day course of low dose BNZ treatment (Dzul-Huchim et al., 2022). Altogether, these data provide proof-of-concept that combining BNZ with vaccines can effectively targeting both the parasite and the deleterious host immune responses and improve cardiac function. Additionally, in both the Dzul-Huchim et al. study and our study, we showed that combining multiple treatments can allow dose sparing of both BNZ and the vaccine, which will likely result in reduced incidence of adverse effects and improve tolerability of treatment.

The clinical manifestations of CCC are due to T. cruzi induced cardiac remodeling (Rossi et al., 2003; Alvarez et al., 2014; Noya-Rabelo et al., 2018; Senra et al., 2018). For example, dilated cardiomyopathy is a severe manifestation of end stage CCC characterized by significant thinning of ventricular walls, left ventricular enlargement, and significantly reduced ejection fraction (Nunes et al., 2013). We previously demonstrated in our mouse model that chronic infection leads to significant compromise of cardiac function as evidenced by significantly increased cardiac circumferential strain on MRI and echocardiography (Hoffman et al., 2019). However, in that study we did not objectively evaluate changes in cardiac structure with imaging modalities as infection progressed. In the study reported here, we evaluated left ventricular wall thickness, chamber size and volume over time in an effort to better characterize the changes in cardiac structure induced by chronic T. cruzi infection. Interestingly, we observed significantly increased LVPW thickness as early as 28 DPI, representing the acute stage of infection. While this wall thickening did naturally regress over time, by approximately 70 DPI it had not yet reached the thickness measured in age matched controls (Figure 2A). Further, LVPW thickness in infected untreated mice did not regress to levels similar to naïve age matched controls until approximately 208 DPI (Figures 3A, B). The increased LVPW thickness induced by T. cruzi infection was accompanied by reductions in left ventricular diameter (Figure 4) and volume (Figure 5) at the end of both systole and diastole, but an increase in ejection fraction (Figure 6A). This suggests that T. cruzi infection can lead to reduced filling of the left ventricle, which may compromise effective tissue perfusion thus the increase in ejection fraction may be a compensatory mechanism to preserve cardiac output. In our previous study characterizing cardiac health and fibrosis during chronic infection, we observed that while all infected mice had increased cardiac strain, only a portion of mice concurrently had reduced ejection fraction while the remaining mice had preserved ejection fraction (Hoffman et al., 2019). This suggests that in our model the mice are developing heart failure with preserved ejection fraction (HFpEF). In HFpEF, it is proposed that systemic inflammation causes microvascular inflammation leading to cardiomyocyte hypertrophy and interstitial fibrosis, resulting in left ventricular stiffness and heart failure (Paulus and Tschöpe, 2013). Further, in HFpEF thickening of the left ventricular wall is accompanied by increased wall stiffness and more forceful contraction, resulting in higher than normal EF in order to preserve cardiac output (Dori et al., 2012). CCC is an inflammatory cardiomyopathy due to parasite persistence, and has a worse prognosis than non-inflammatory cardiomyopathies (Higuchi et al., 1987; Bestetti and Muccillo, 1997). Considering this, we propose that in our model T. cruzi induced inflammation resulted in HFpEF with increased EF and LVPW thickness measured on echocardiography,and increased cardiac fibrosis on histopathology. Additionally, a balanced T_H1/T_H2/T_H17 immune response is associated with better cardiac function in individuals in the indeterminate stage of Chagas disease (Sousa et al., 2014). Here we report that the Tc24-C4/E6020 SE vaccine induced increased antigen specific release of IL-6, IFNγ, IL-4, IL-5, IL-10, and IL-17A. Taking these findings into account, we propose that efficacy of our VLC strategy is due in part to induction of a balanced antigen specific T_H1/T_H2/T_H17 immune response by the vaccine which results in reduced cardiac inflammation and fibrosis, leading to reduced left ventricular posterior wall thickness and reduced EF, and ultimately preservation of cardiac function.

Cardiac fibrosis is a consistent finding in CCC and a major driver of clinical disease (Marin-Neto et al., 2007; Senra et al., 2018). Even in indeterminate patients, cardiac fibrosis is detectable with advanced imaging techniques (Macedo et al., 2015). In preclinical models of acute and chronic T. cruzi infection, therapies that interrupt key promoters of fibrosis, including TGFβ and pY-STAT3, do effectively reduce cardiac fibrosis (Waghabi et al., 2009; de Oliveira et al., 2012; Hoffman et al., 2021). Additionally, we have previously shown that Tc24 based vaccines when used alone or in a VLC strategy significantly reduce cardiac fibrosis in both acute and chronic mouse models of infection (Barry et al., 2019; Cruz-Chan et al., 2021; Dzul-Huchim et al., 2022). We reported here that while the low dose vaccine alone was able to significantly reduce endpoint fibrosis compared to infected untreated controls, statistically significant reductions in fibrosis were not observed in any groups that received VLC (Figure 9B and Figure 11). This may be due to differences in the vaccination doses, timing of vaccination, or our use of a monovalent vaccine compared to the bivalent vaccine used by Dzul-Hchim et al. This also suggests that while VLC is partially efficacious, additional targeting of the host fibrotic response to T. cruzi is necessary to improve treatment efficacy. We have previously shown that by inhibiting the activity of pY-STAT3 with a targeted inhibitor, TTI-101, we significantly reduced cardiac fibrosis and serum levels of TGFβ and PDGF-D without affecting tissue parasite burdens (Hoffman et al., 2021). However, since pY-STAT3 regulates inflammatory responses, both by blocking IFNγ induced STAT1 and IL-6 induced NFκB (Hovsepian et al., 2013; Yu et al., 2017), inhibiting pYSTAT3 exacerbated cardiac inflammation (Hoffman et al., 2021). In the Hoffman et al. study, as well as in the study reported here, we observed that while treatment with a curative dose of BNZ alone did not significantly reduce cardiac fibrosis, it did reduce cardiac inflammation. Thus, we propose that we could improve treatment efficacy by first using a treatment to significantly reduce cardiac inflammation, then follow with a targeted anti-fibrotic treatment, such as TTI-101. Studies are ongoing to evaluate whether such a sequential, multi-modal treatment strategy would better resolve T. cruzi induced cardiac pathology.

Limitations of the study reported here have been identified and are under careful consideration to better inform the design of future studies. Here we demonstrated that chronic T. cruzi H1 infection induced significant changes in cardiac structure and function in female BALB/c mice that were ameliorated with VLC. We elected to use female mice only because we have previously demonstrated that T. cruzi H1 infection induces characteristic cardiac pathology and changes in cardiac function in both acutely and chronically infected female BALB/c mice (Jones et al., 2018; Hoffman et al., 2019; Cruz-Chan et al., 2021; Hoffman et al., 2021). Additionally, we demonstrated initial proof-of-concept for therapeutic efficacy of our VLC strategy using acutely infected female mice (Jones et al., 2018; Cruz-Chan et al., 2021). Therefore we elected to use our established female BALB/c mouse model of chronic T. cruzi H1 strain infection to evaluate efficacy of our VLC strategy initiated during chronic infection. However, some studies have shown that male Chagas patients have a higher incidence of developing cardiac disease compared to females (Sabino et al., 2013). Therefore we are planning future studies with both male and female mice to evaluate VLC efficacy in chronically infected male mice, and to determine if efficacy is comparable between the sexes. An additional limitation is that we only evaluated left ventricular wall thickness, diameter, volume, ejection fraction and fractional shortening to determine the effects of VLC in our chronic infection mouse model. Evaluation of circumferential strain, as performed in prior studies, would have allowed us to determine if increased left ventricular wall thickness correlated with increased circumferential strain, further supporting our hypothesis that cardiac disease in our model can be characterized as HFpEF. We only observed significant reductions in fibrosis in the Low Vaccine (sequential) group and a trend toward reduced fibrosis in the Low BNZ + High Vaccine group (p=0.1403). In a prior study where we observed significantly reduced fibrosis in chronically infected mice treated with Tc24/E6020 SE vaccine mice were evaluated at approximately 180 DPI (Barry et al., 2019), compared to approximately 208 DPI as in the study reported here. Thus, in future studies we will evaluate the effect of treatment on cardiac fibrosis at earlier timepoints to determine if treatment efficacy may wane with the current strategy, and additional vaccine doses may be needed to improve durability of the effect. In this study, while we showed that the Tc24-C4/E6020 SE vaccine induced the desired T_H1/T_H2/T_H17 cytokine profile that has been associated with less clinical disease in indeterminate individuals (Sousa et al., 2014), we did not show any significant increases in antigen specific CD8+ cytokine producing T cells with the vaccine. This is consistent with what was observed by Dzul-Huchim et al, who also showed that VLC induced increased CD4+ and CD8+ perforin producing effector T cells (Dzul-Huchim et al., 2022). In the study reported here we did not evaluate functional activity of effector cells, including production of perforin and granzyme, thus it will be important to evaluate effector cell functions in addition to cytokine producing cells in future studies. This may also correlate with the low parasite burden detected. Further, it will be important in future studies to immunophenotype cardiac derived immune cells, and measure cardiac specific cytokine production and expression of inflammatory markers, such as STAT1 and NFκB. This knowledge would better define disease pathogenesis and mechanisms of protection induced by the VLC strategy.

The etiology of Chagas disease was fully described over 100 years ago, but there are as yet only two approved treatments for the disease with limited efficacy during chronic infection and high rates of adverse side effects (Lewinsohn, 1979; Viotti et al., 2009; Jackson et al., 2010). Several studies have focused on identifying new antiparasitic drugs, testing new drug combinations, and developing new dosing strategies in an effort to improve efficacy and tolerability (Perez-Mazliah et al., 2013; Bustamante et al., 2014; García-Huertas and Cardona-Castro, 2021). Additionally, dozens of candidate vaccines have been designed and tested in pre-clinical models, but none have yet reached testing in human clinical trials (Rios et al., 2019; Bivona et al., 2020). Due to the complex nature of disease pathogenesis, therapies that target only the parasite or only the host response are unlikely to have high efficacy. Vaccine-linked chemotherapy has been proposed as an effective strategy to reduce both infection and disease burdens for parasitic diseases, including schistosomiasis and malaria (Bergquist et al., 2005; von Seidlein et al., 2020). Thus, we have designed vaccine-linked chemotherapy strategy to both reduce parasite burdens and ameliorate the deleterious host responses that contribute to clinical cardiac disease. Here we provide important evidence that such a strategy will help bridge the current tolerability and efficacy gaps of the current single drug standard of treatment, and alleviate disease and disability of millions of Chagas patients worldwide.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Baylor College of Medicine’s Institutional Animal Care and Use Committee.

KJ, MB, and PH designed the study. KJ, EM, LEV, JC, LV, BK, and AK acquired the data. JP and FG provided reagents. KJ, CR, LEV, and LV analyzed the data. KJ drafted the manuscript. All authors critically reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.