The impact of vaccine-linked chemotherapy on liver health in a mouse model of chronic Trypanosoma cruzi infection

Background Chagas disease, chronic infection with Trypanosoma cruzi, mainly manifests as cardiac disease. However, the liver is important for both controlling parasite burdens and metabolizing drugs. Notably, high doses of anti-parasitic drug benznidazole (BNZ) causes liver damage. We previously showed that combining low dose BNZ with a prototype therapeutic vaccine is a dose sparing strategy that effectively reduced T. cruzi induced cardiac damage. However, the impact of this treatment on liver health is unknown. Therefore, we evaluated several markers of liver health after treatment with low dose BNZ plus the vaccine therapy in comparison to a curative dose of BNZ. Methodology Female BALB/c mice were infected with a bioluminescent T. cruzi H1 clone for approximately 70 days, then randomly divided into groups of 15 mice each. Mice were treated with a 25mg/kg BNZ, 25μg Tc24-C4 protein/5μg E6020-SE (Vaccine), 25mg/kg BNZ followed by vaccine, or 100mg/kg BNZ (curative dose). At study endpoints we evaluated hepatomegaly, parasite burden by quantitative PCR, cellular infiltration by histology, and expression of B-cell translocation gene 2(BTG2) and Peroxisome proliferator-activated receptor alpha (PPARα) by RT-PCR. Levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were quantified from serum. Results Curative BNZ treatment significantly reduced hepatomegaly, liver parasite burdens, and the quantity of cellular infiltrate, but significantly elevated serum levels of ALT, AST, and LDH. Low BNZ plus vaccine did not significantly affect hepatomegaly, parasite burdens or the quantity of cellular infiltrate, but only elevated ALT and AST. Low dose BNZ significantly decreased expression of both BTG2 and PPARα, and curative BNZ reduced expression of BTG2 while low BNZ plus vaccine had no impact. Conclusions These data confirm toxicity associated with curative doses of BNZ and suggest that the dose sparing low BNZ plus vaccine treatment better preserves liver health.


Introduction 69
Chagas disease is a bloodborne parasitic protozoal disease caused by Trypanosoma 70 cruzi, which through human migration, is found in all parts of the world(1). Considered a neglected 71 tropical disease, it is mainly found in lower socioeconomic areas of Latin America, where an 72 estimated 6-7 million people are affected by this disease(2). Being a bloodborne infection, the 73 disease can be disseminated through blood transmission, organ transplant, or congenital 74 transmission from mother to fetus(3). However, the most common path of infections is through 75 contact with the feces of infected Triatomine insect vectors, which are only found in the 76 Americas (4). 77 Most often, people are not even aware of their infection status. In the initial acute phase 78 of infection, the infected individual may be asymptomatic or experience nonspecific, generalized 79 flu-like symptoms such as fever, fatigue, body aches, vomiting, and diarrhea(1,5). During this 80 time, high levels of motile trypomastigotes circulate throughout the individual's bloodstream(6). 81 Left untreated, the disease progresses to a chronic phase, circulating parasite levels in the blood 82 drop to low levels, and detection can be difficult without sensitive PCR or culture methods (7,8). 83 There are also long-term deleterious health effects associated with the chronic phase. 30% of 84 people develop cardiac complications, which is the most significant disease manifestation and 85 may include heart enlargement, conduction disturbances, or even cardiac arrest(9,10). In 10% of 86 people, gastrointestinal complications may occur, causing disorders like megaesophagus or 87 megacolon(11). 88 Though cardiac disease, and to a lesser extent gastrointestinal disease, have been 89 extensively explored, the impact of T. cruzi on the liver need further elucidation. Studies have 90 shown that the liver is important in clearing parasites from the blood during both the acute and 91 chronic phases of infection(12). The release of amastigote nest and immediate phagocytosis by 92 resident immune cells produce nitric oxide and oxygen radicals that kill the parasite but can lead 93 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint to organ damage(13). Once the disease enters the chronic phase, it is characterized by low 94 parasitemia and low-grade tissue inflammation(7). Reactive oxygen species (ROS) can directly 95 stimulate hepatic stellate cells, which are known to produce extracellular matrix proteins that lead 96 to hepatic fibrosis(14). Over time, ROS and chronic low-grade inflammation lead to fibrosis and 97 organ dysfunction (14). 98 The current treatment for Chagas disease is administering the antiparasitic drug 99 benznidazole (BNZ)(10). While BNZ is effective at curing when treatment is initiated during the 100 acute phase, cure rates significantly decline when treatment is initiated during the chronic 101 phase(15). Importantly, a large multicenter trial treating patients with established cardiac disease 102 showed that treatment did not prevent disease progression or cardiac death(16). Another 103 important limitation of benznidazole therapy is the toxic profile. The side effects of BNZ include 104 hypersensitivity, neuropathy, and bone marrow disorders, which can result in individuals 105 discontinuing treatment(17). Benznidazole is metabolized by the cytochrome p450 enzyme, which 106 is found throughout the body but primarily in liver cells (18). The liver has also been shown to 107 efficiently elicit a robust immune response with superior levels of inflammation and IFN-y 108 production(19). These elevated levels of inflammation can also damage the liver and may be 109 associated with immune allergic hepatitis(20 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint Mice were infected with 5000 trypomastigotes of a bioluminescent clone of the T. cruzi H1 strain, 160 generated in our laboratory, by intraperitoneal injection. Naïve age-matched mice were left 161 uninfected as controls. Blood was collected by tail vein microsampling from all mice at 162 approximately 28 days post-infection (DPI) to confirm parasitemia by quantitative PCR. 163 Approximately 70 DPI mice were randomly assigned to treatment groups, with 15 mice per group 164 as described in Table 2. Benznidazole treatments were administered once daily by oral gavage, 165 and vaccinations were administered by subcutaneous injection according to the timeline in Figure  166 1. Mice were monitored daily for morbidity and any mice that reached humane endpoints were 167 humanely euthanized. Cohorts of mice were euthanized at multiple time points after treatments 168 were completed to evaluate the treatment's short-and long-term effects on liver health. At the 169 study endpoints, all mice were weighed, then humanely euthanized. Whole blood was collected 170 postmortem. Livers were removed, and weighed, then one section was frozen for DNA and RNA 171 analysis, and a second portion was placed in 10% neutral buffered formalin for histopathology 172 analysis. 173 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint Whole blood was allowed to clot at room temperature for 30 minutes, then centrifuged at 10,000 190 rpm for 5 minutes at room temperature to separate serum. Serum was analyzed for aspartate 191 aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), and 192 lactate dehydrogenase (LDH) using a Beckman Coulter AU480 Chemistry Analyzer (Clinical 193 Pathology Laboratory, Baylor College of Medicine). 194

Histopathology Analysis: 195
Sections of the liver were fixed in 10% neutral buffered formalin, dehydrated, embedded in 196 paraffin, sectioned to 5µm thickness, and adhered to glass slides. Sections were routinely stained 197 with hematoxylin and eosin (H&E). Images of liver H&E histology slides were taken from 5 198 randomly selected representative fields at 10x magnification using an Amscope ME580 brightfield 199 microscope. Images' color thresholds (hue, saturation, brightness) were adjusted using ImageJ 200 to create uniformity among all images. A count of lymphocyte nuclei was accomplished by setting 201 a particle size limit to exclude larger hepatocyte nuclei and smaller debris from being factored into 202 the count. Lymphocyte counts from all 5 randomly selected fields were averaged to indicate 203 inflammatory cell infiltration in the liver. 204

QRT-PCR for BTG2 and PPARα: 205
According to the manufacturer's guidelines, RNA was isolated from frozen liver tissue (20mg) 206 using RNeasy kit (Qiagen). The concentration of RNA was quantified using Nanodrop with a target 207 concentration of 100ng/uL. cDNA was amplified with RT-PCR master mix (ThermoFisher) and 208 ran in Bio-Rad PCR Thermal Cycler. QRT-PCR was performed using a Quant Studio 3 209 thermocycler (Applied Biosciences). The specific primers were as follows: BTG2 210 All samples were run in duplicate. The relative quantity (RQ) values were calculated according to 213 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint the ΔΔCt method. The infected mice were normalized to the values obtained from non-infected 214 mice (ΔΔCT). Then, the RQ was calculated as RQ = 2-ΔΔCt. 215

Statistical analysis: 216
For each parameter measured, data were plotted using GraphPad Prism 9.4.1 software 217 (GraphPad). Treatments were compared to infected untreated controls using a Kruskal-Wallis 218 one-way ANOVA and Dunn's multiple comparisons tests. When comparing two groups, a Mann-219 Whitney test was used. P values ≤ 0.05 were considered significant. 220

Curative Benznidazole treatment reduces T. cruzi induced hepatomegaly. 222
In Liu et al, 2023, we previously reported that combination treatment with low BNZ + vaccine 223 better restored T. cruzi induced metabolic perturbances in several sections of heart compared to 224 curative BNZ treatment (30). However, that work did not evaluate the impact of treatments on 225 liver health. Hepatomegaly is a consistent finding in human cases of Chagas disease as well as 226 experimental animal models(31). To determine if hepatomegaly was also present in our model of 227 chronic T. cruzi infection, the liver weight/ body weight ratio was calculated at study endpoints. 228 The liver weight/ body weight ratio was significantly increased by infection at 90 and 142 DPI 229 ( Figure 2C, red and purple symbols, respectively) compared to naïve controls, indicating 230 hepatomegaly was evident in our model. By 142 DPI, curative BNZ significantly reduced liver 231 weight/body weight ratio compared to infected control mice (Fig 2C maroon symbol), suggesting 232 that curative BNZ treatment ameliorates infection-induced hepatomegaly. However, low BNZ + 233 vaccine had no apparent effect on hepatomegaly. Additionally, infection alone did not induce 234 significant changes in overall body weight compared to naïve controls, but low BNZ + vaccine 235 resulted in overall lower body weight compared to controls (Figure 2A). Overall liver weight was 236 significantly increased only at 90 DPI when compared to naïve controls, but liver weight was not 237 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint increased at other timepoints or with any treatments. Together, these data confirm hepatomegaly 238 is evident in our model and that only curative BNZ ameliorates this finding. We have previously demonstrated that vaccine-linked chemotherapy significantly reduces cardiac 246 parasite burdens in acutely infected mice(24,25) and curative BNZ significantly reduces cardiac 247 parasite burdens and reduces cellular infiltration in chronically infected mice immediately after 248 treatment(32). Therefore, we evaluated the impact of treatments on parasite levels and cellular 249 infiltration in the liver. Curative BNZ effectively decreased parasite burden in the liver to below 250 the limits of quantitation for the assay (Fig 3A dark purple symbol), while low BNZ + vaccine had 251 no effect on parasite burdens (Fig 3A blue symbols). Additionally, when evaluating parasite levels 252 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint 13 in infected untreated mice over time, parasite levels decreased from 90 to 120 and 142dpi (Fig  253   3A red, green and purple symbols, respectively). Evaluation of H&E stained liver sections 254 revealed that treatment with low BNZ significantly increased inflammatory infiltrate at 120 DPI 255 compared to infected untreated mice. (Fig 3B orange symbol Fig 4E). However, curative BNZ 256 significantly decreased inflammatory infiltrate compared to infected untreated mice (Fig 3B dark  257 purple symbol, Fig 4I), while low BNZ + vaccine did not affect the number of inflammatory cells 258 (Fig 3B dark blue symbol, Fig 4H). was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint and AST (Fig 5C, navy and maroon symbols, respectively) and AST when compared to naïve 275 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint mice. Importantly, only curative BNZ induced significant elevation to LDH (Fig 5D, maroon  276 symbols) compared to naïve mice. Infection alone and single treatments did not cause significant 277 elevations to AST, ALT and LDH. Further, no differences in ALP were observed for any groups. 278 Together, these data suggest that our vaccine-linked chemotherapy strategy causes less liver 279 and tissue damage compared to curative BNZ alone.

Benznidazole treatment reduces the expression of liver damage markers 287
To begin to define potential mechanism of liver damage in our model, we evaluated expression 288 of BTG2, a marker of oxidative damage, and PPARα, a regulator of inflammation(33-35). BTG2 289 expression was elevated by 120 DPI in infected mice compared to 90 DPI and 142DPI (Fig 6A  290 green symbols), but this expression was significantly decreased by low BNZ treatment (Fig 6A  291 orange symbols). Similarly, by 142 DPI, infection induced BTG2 expression was significantly 292 reduced by curative BNZ treatment (Fig 6A maroon symbols). Expression of PPARα was elevated 293 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint by 142 DPI in infected mice compared to 90DPI and 120DPI (Fig 6B purple symbols). Only low 294 BNZ significantly decreased PPARα expression by 120DPI (Fig 6B orange symbols). Together, 295 these data suggest that BNZ treatment can affect oxidative damage and regulation of 296 inflammation in the liver, but low BNZ + vaccine has no apparent effect. were measured in liver tissue. **P < 0.01; ****P < 0.0001 301

Discussion 302
The current first-line treatment for Chagas disease is oral BNZ, which is effective mainly 303 in the acute phase but has unwanted side effects, including dermatologic and neurologic 304 manifestations(36,37). Liver toxicity from BZN is thought to be caused by the formation of reactive 305 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint metabolites, which can damage liver cells and lead to inflammation and liver injury(36). Symptoms 306 of BZN-induced liver toxicity may include fatigue, abdominal pain, jaundice, and elevated liver 307 enzymes in the blood(20). Specifically, BNZ treatment results in elevations of AST, ALT, and 308 ALP(38). Our group has shown that a vaccine-linked chemotherapy strategy allows the reduction 309 of BNZ dose, while still effectively reducing cardiac inflammation and fibrosis, and improving 310 cardiac function(24-26). While the evidence showed that this dose-sparing strategy improved 311 cardiac health, similar to a curative dose of BNZ, the impact of vaccine-linked chemotherapy on 312 liver health was unknown. Therefore, we conducted this study to compare the effects of curative 313 benznidazole treatment and vaccine-linked chemotherapy on liver health. T. cruzi infection 314 causes inflammation and edema, which can contribute to hepatomegaly(31). Additionally, right-315 sided heart failure can cause congestion of the liver; thus, an increased liver weight can indicate 316 cardiac dysfunction(39,40). We confirmed in our model that curative BNZ treatment was able to 317 ameliorate the infection-induced hepatomegaly, decrease liver parasite burdens, and reduce 318 cellular infiltrate. This agrees with our prior observations in our chronic infection mouse model 319 that curative BNZ significantly reduced cardiac parasite burdens and cardiac cellular infiltration 320 immediately after treatment was completed(32), and reduced cardiac cellular infiltration over 3 321 months after treatment was completed(26). In contrast, low BNZ + vaccine had no apparent effect 322 on T. cruzi induced hepatomegaly, liver parasite burdens, or cellular infiltrate. We have previously 323 shown that our Tc24-C4/E6020 SE vaccine induced increased antigen specific CD8+ cells in the 324 spleen(25), thus it is possible that the cellular infiltrate within the liver of vaccinated mice showed 325 no apparent reduction due to increased infiltration of antigen specific CD8+ cells into tissues, respectively. This suggests low dose BNZ or vaccine do not cause as much tissue damage as 355 curative BNZ treatment. However, the group treated with low BNZ + vaccine sequentially had 356 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint elevated serum levels of ALT and AST at 142dpi, similar to curative BNZ. his suggests that despite 357 the dose sparing effect of this strategy, the combination of treatments does still result in elevation 358 of tissue damage enzymes. Further studies evaluating ALT and AST at later timepoints after 359 treatment would be needed to determine if the elevations in these tissue damage markers is 360

sustained. 361
In addition to ALT, AST, and ALP, we evaluated serum lactate dehydrogenase (LDH) 362 levels, which are also abundantly expressed in the liver, heart, and muscle tissues(45,46). In 363 damaged tissues, LDH leaks out of the tissues and into the serum(47). The elevated LDH levels 364 of curative BNZ-treated mice suggest that the drug has hepatotoxic effects on tissues, which may 365 lead to acute liver failure(48). Furthermore, previous studies have also concluded that LDH is a 366 good prognosticator for death in individuals with acute liver failure(49). Studies in mice acutely 367 infected with T. cruzi show that LDH levels are detectable in both serum and tissues at the early 368 stages of infection, with elevations evident in serum before tissue damage is evident 369 microscopically(45). This suggests that LDH elevations caused by T. cruzi are due to the 370 combined effect of early changes in cell membrane permeability and later structural damage to 371 tissues(45). Our results showed that only treatment with the curative dose of BNZ resulted in 372 significant elevations in serum LDH levels. Importantly, the lack of LDH elevation in mice receiving 373 low BNZ + vaccine suggests that this treatment is less damaging to tissues, potentially in both the 374 liver and heart, than curative BNZ. Considering the know exacerbation of liver damage caused 375 by the combined effects of T. cruzi infection and BNZ(21), a less damaging treatment strategy, 376 such as vaccine-linked chemotherapy, is desirable to preserve overall health. 377 In an effort to further characterize potential mechanisms of liver-specific damage in our 378 model, we evaluated gene expression of B-cell translocation gene 2 (BTG2) and Peroxisome 379 proliferator-activated receptor alpha (PPARα). BTG2 is a protein-coding gene involved in various 380 cellular processes, including cell cycle regulation, apoptosis, differentiation, hepatic 381 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint gluconeogenesis, and lipid homeostasis(50-52). Importantly, BTG2 is induced in response to 382 DNA damage, and upregulation of BTG2 has been shown to protect against oxidative stress in 383 human mammary epithelial cells(33,34). T. cruzi infection leads to DNA damage in mouse 384 models, specifically in heart cells and splenocytes, due to induction of reactive oxygen species 385 (ROS), and BNZ has been shown to reduce that effect(53,54). In our study, treatment with either 386 low BNZ or curative BNZ significantly decreased BTG2 expression levels compared to infected, 387 untreated mice at 120 DPI and 142 DPI, respectively. This suggests that in addition to DNA 388 damage in the heart, T. cruzi also induces DNA damage in the liver leading to elevated BTG2 389 expression, which is ameliorated with BNZ treatment. BTG2 expression is also upregulated in 390 response to inflammatory stimuli, including IL-6 and NFκB(55). In prior studies, we showed that 391 curative BNZ treatment of chronically infected mice did not significantly reduce NFκB, pSTAT3, 392 or IL-6 in cardiac tissue(32). However, specific evaluation of NFκB and IL-6 in the liver would be 393 needed to determine if curative BNZ treatments has a tissue specific impact on those 394 inflammatory stimuli. PPARα is involved in regulating lipid and glucose metabolism, is abundantly 395 expressed in the liver, and is critical for reducing inflammation and protecting against liver 396 injury(35,56,57). In T. cruzi infected macrophages, PPARα ligands drive M1-to-M2 conversion, 397 regulating inflammatory responses(58). We observed that low BNZ significantly reduced 398 expression of PPARα in the liver, similar to the effect on BTG2 expression. While this reduction 399 did not have an apparent effect on liver cellular infiltrate, further studies would be needed to 400 determine any impact on other inflammatory markers specifically in the liver. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint induced hepatotoxicity(59). However, a 10 year longitudinal study of patients with Chagas disease 407 found that male patients developed cardiac disease at a higher rate compared to female 408 patients(60). Further, heart failure can also result in liver damage(61). Thus, in future studies it 409 will be essential to specifically evaluate the impact of T. cruzi infection and vaccine-linked 410 chemotherapy on liver health in male mice and compare to the impact in female mice. Another 411 limitation identified in this study is the limited time points after infection and treatment that were 412 evaluated. In this study we evaluated liver health primarily at 120 DPI and 142 DPI, representing 413 early chronic infection, to evaluate short term effects of our treatments. However, future studies 414 will need to look at later time points to determine the duration of any treatment effects on liver 415 health. Indeed, we observed elevated AST and ALT in mice treated with low BNZ + vaccine at 416 142DPI, representing approximately 36 days after treatment ended. It is possible that at later 417 timepoints, AST and ALT levels would return to normal range indicating that any toxic effects in 418 the liver are transient. Finally, cardiac fibrosis is a consistent finding in chronic Chagasic 419 cardiomyopathy, and is associated with more severe cardiac disease(62,63). Because the 420 relationship between the heart health and liver health has been demonstrated, assessing whether 421 our treatment affects liver fibrosis will also be important. 422 BNZ treatment of patients with Chagas disease is problematic due to prolonged treatment 423 courses and significant toxicity, resulting in up to 40% of patients terminating treatment 424 early(15,37). We developed a vaccine-linked chemotherapy strategy that is dose-sparing, and 425 demonstrated to be efficacious at reducing cardiac pathology in preclinical models(24-26). Here 426 we present data suggesting that in addition to the beneficial cardiac effects, vaccine-linked 427 chemotherapy may be less damaging to the liver compared to curative BNZ treatment. This 428 further supports vaccine-linked chemotherapy as an attractive strategy to bridge the efficacy and 429 tolerability gaps of standard anti-parasitic treatment for patients with Chagas disease, and 430 ultimately improve overall health. 431 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2023. ; https://doi.org/10.1101/2023.07.11.548497 doi: bioRxiv preprint