RIG-I–Adjuvanted Immunogen Elicited T-Cell Immunity Controls Trypanosoma cruzi, Chagasic Cardiomyopathy, and Left Ventricular Dysfunction

Visual Abstract

Highlights

• •New therapies to prevent cardiomyopathy and heart failure are urgently needed for CD.
• •Bicistronic immunotherapies (BCV, BCVR), designed in regulatory compliance, elicited relevant, functional adaptive immunity against Trypanosoma cruzi infection.
• •BCV and BCVR were safe and highly effective in controlling tissue parasite burden, myocardial inflammation, and fibrosis.
• •Left ventricular function, highly compromised in chronically infected mice, was preserved by treatment (BCVR>BCV) prior to challenge.
• •These findings highlight a nontoxic immunotherapy to mitigate cardiogenic risks of Chagas disease.

Summary

BCVR and BCV (bicistronic immunogen with and without RIG-I adjuvant) immunogens were designed in compliance with Food and Drug Administration guidelines. These immunogens were nontoxic, and highly effective in controlling the replication and persistence of Trypanosoma cruzi. Importantly, BCV/BCVR blocked the myocarditis, cardiac fibrosis, and left ventricular dysfunction that otherwise contribute to heart failure and sudden death in Chagas disease.

Trypanosoma cruzi (Tc) is a parasitic protozoan that is the causative agent of Chagas disease (CD). Tc is endemic in the Americas, ranging from southern United States to Argentina, due to continuous transmission of the parasite in triatomines and wildlife and domestic reservoir hosts.¹ Owing to migration, CD has also emerged in nonendemic areas.² Most individuals infected by Tc develop nonspecific somatic symptoms that resolve in 2 to 3 months but remain seropositive for life.³ In 10% to 40% of infected persons, clinical symptoms progress from cardiac hypertrophy and cardiomyopathy to heart failure.⁴ Tc infection is estimated to have caused 308,000 to 398,000 new cases of Chagas disease in 2023 and ∼12,000 deaths every year.⁵ A comprehensive cost analysis concluded that CD results in an economic burden of >US$10 billion annually.⁶

Currently available antiparasitic therapies (benznidazole and nifurtimox) are highly effective against Tc infection in children (<15 years of age).⁷ However, these drugs are not recommended for the treatment of infected adults and pregnant women because of therapeutic failure and adverse health effects.^8,9 Moreover, several Tc isolates of clinical relevance exhibit resistance to these drugs.¹⁰ No options are available to prevent the risk of Tc infection in adults traveling to endemic countries or achieve parasitological cure in infected adults to halt the CD development. Because of the compelling clinical unmet need for therapies to control CD, Tc infection is recognized by National Institutes of Health, Centers for Disease Control and Prevention, and other public health organizations as a neglected economic and ethical emergency. A potential therapy that is safe to use and can prevent Tc infection and/or treat the infected adults is urgently needed.

Numerous studies in experimental models depleted of specific immune components and in human patients experiencing various stages and severity of CD have informed the key immune factors associated with susceptibility vs resistance to Tc infection. Production of IFNγ and IL-12 by macrophages and natural killer cells was found to be essential for activation of type 1 CD4⁺T cells¹¹ that, in turn, were crucial for parasite control through amplification of phagocytic activity of macrophages, and stimulation of B cells and CD8⁺T cells.^12,13 Antibodies to the Galα(1,3)Galβ(1,4)GlcNAc epitope of surface expressed glycoproteins facilitated opsonization, phagocytosis, and complement-mediated killing of the parasite.¹⁴ Abundantly expressed, glycolipid-anchored surface antigens that were released during intracellular differentiation of infective and intracellular forms of Tc¹⁵ elicited antigen-specific CD8⁺T cells contributing to cytolysis of infected cells and secretion of Th1 cytokines that induce trypanocidal activity.¹⁶ Overall, a protective response to Tc required balanced, efficient, and combined activities of phagocytes, T helper cells, and cytotoxic T lymphocytes. A subpar or delayed activation of either of these components can result in parasite replication, dissemination, and persistence, and overactivation of immune system can lead to myocarditis, hypertrophy, and heart failure in CD.

Despite committed research efforts for the last two decades, none of the newly developed antiparasite drugs have reached the marketplace due to their limited efficacy against Tc in clinical trials.¹⁷ Recently, investigators have paid attention to the development of efficacious immunotherapies against Tc. The premise of these studies has been to identify candidate immunogens that enhance the protective immune response, reduce the parasite load, and thereby decrease the risk of clinically symptomatic CD development. Indeed, several surface antigens of Tc (eg, glycoprotein 90, Tc52 member of thiol-disulfide oxidoreductase family, complement regulatory protein, amastigote surface protein 2) that were recognized by B and T cells in infected host were found to elicit various degrees of protective immunity and control Tc infection in small animal models.¹⁸

To prevent the investigator bias, we developed a bioinformatics-biological screening approach for identifying the novel candidate immunogens (Figure 1A). Briefly, a computational algorithm was employed to screen the Tc sequence database that ignored the multigene families because of their role in immune evasion,¹⁹ and selected genes that were conserved in Tc but lacked similarity in genome of other eukaryotes.²⁰ The selected candidates were parsed for additional characteristics, including presence of human major histocompatibility complex class I (MHCI)/major histocompatibility complex class II (MHCII) epitopes, expression in infective and intracellular stages of Tc, and released into the host cell cytoplasm during parasite differentiation. Of the 11 candidates that were selected, 3 (TcG1, TcG2, and TcG4) were recognized by IgG antibodies and type 1 CD8⁺T cells in multiple infected hosts.^20,21 TcG2 and TcG4 exhibited superior immunogen attributes as these were also conserved (92%-99% homology) in parasite isolates from all lineages (I-VI), thus providing confidence that TcG2/TcG4-based immunotherapy will be effective against diverse Tc isolates circulating in the Americas. After testing several regimens (doses, dosages, timeline), adjuvants (IL-12, GM-CSF, integrin activator, etc.), and homologous and heterologous prime-boost schemes, we found that DNA delivery of TcG2 and TcG4 (without adjuvants) was sufficient to generate resistance to Tc infection.22, 23, 24, 25, 26, 27 Mice injected with TcG2− and −TcG4 expression plasmids exhibited Th1 IgGs, Tc-specific polyfunctional CD8⁺ T cell response, and >85% reduction in parasitemia and cardiac inflammation.^25,26,28 In dogs, TcG2 and TcG4 delivery led to 70% to 85% decline in tissue parasites than was noted in untreated/infected dogs.^29,30

Figure 1.

Immunogen and Study Design

(A) Selection of candidate genes. (B-E) Bicistronic immunogen (BCV) synthesis. (B) Mouse codon–optimized sequence for TcG2 (666bp, tangerine) and TcG4 (276bp, blue), separated by P2A-linker (66bp, green). The bicistronic sequence was cloned in NTC9385R or NTC9385R-eRNA41H-CpG (expresses RIG-I agonist) to generate (C) BCV and (D) bicistronic immunogen with RIG-I adjuvant (BCVR), respectively. The production pipeline included synthesis, amplification, and cloning of bicistronic sequence in NTC plasmids; confirmation of sequence accuracy and directional cloning; and bulk production by antibiotic-free technology. (F) Experimental model. C57BL/6 adult female mice (8 weeks old) were injected with BCV or BCVR at a 21-day interval and challenged (Trypanosoma cruzi SylvioX10, 10,000 parasites per mouse) at 21 days and 100 days after booster to monitor short- and long-term efficacy, respectively. Mice were euthanized at 21 days and >150 days postinfection to evaluate efficacy against acute parasitemic phase and chronic disease phase, respectively. Tissue and blood samples were subjected to various assessments. Ab = antibody; CMV = cytomegalovirus; DTU = discrete typing units; PCR = polymerase chain reaction; SkM = skeletal muscle.

In this study, we aimed to incorporate TcG2/TcG4 immunogenicity in designing a bicistronic immunogen (BCV) in compliance with the regulatory guidance for immunotherapies against infectious agents provided by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services. A mouse codon–optimized TcG2-TcG4 sequence, individual cistrons separated by a P2A linker, was synthesized and cloned into a nanoplasmid. An agonist of RIG-I was added to BCV (bicistronic immunogen with RIG-I adjuvant [BCVR]) to enhance the RIG-I–inducible adaptive immunity. We examined the safety as well as short- and long-term efficacy of BCV and BCVR in providing protection from Tc infection, chronic cardiomyopathy, and left ventricular (LV) dysfunction, and monitored the immune components (antibody response, CD4⁺, CD8⁺, and IL-17⁺ T cell response) associated with Tc infection vs immunogen-associated protection from CD in adult mice.

Methods

Parasite

Tc (SylvioX10, 50823) and C2C12 myoblasts (CRL-1772) were purchased from the American Type Culture Collection resource center. Cells were cultured at 37 °C, 5% CO₂ in RPMI 1640 medium containing 300 mg/L L-glutamine, 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% sodium pyruvate, and infected with Tc trypomastigotes (1:3 cell:parasite ratio). After 5 to 7 days, supernatants containing trypomastigotes released from infected cells were sequentially centrifuged at 300 g for 2 minutes and 3,000 g for 8 minutes, and pelleted parasites were resuspended in phosphate-buffered saline (PBS).

Mice

Adult female mice (C57BL/6, 8 weeks old) were obtained from the Jackson Laboratory. The National Institutes of Health guidelines were followed for housing and care of laboratory animals. All animal experiments were conducted in accordance with the protocol approved by the Institutional Animal Welfare Office (PHS assurance: D16-00202/A3314-01, Protocol: 08-05-029). Mice were housed in micro-isolation cages (maximum 5 mice per cage) to avoid transfer of airborne pathogens and maintained at 12-hour day/night cycle, at the animal facility at University of Texas Medical Branch. The veterinary care of mice was provided by full-time veterinarians and their staff. All animals had ad libitum access to water and standard chow.

Immunogens

GenBank contains the Tc (CL/Brenner) sequences for TcG2 and TcG4 (AY727915 and AY727917, respectively). TcG2 (220 amino acids) and TcG4 (92 amino acids) are relatively small peptides that were selected because of their immunogenicity in eliciting relevant and functional Tc-specific antibody and T cell responses in multiple hosts (Figure 1A).

Self-cleaving 2A peptides (18-22 amino acids) share a core motif (DXEXNPGP) and induce ribosomal skipping between proline and glycine to allow cleavage of polypeptides during translation.³¹ The 2A peptides facilitate the expression and post-translational cleavage of polycistronic gene products and used for expression of multiple genes from a single vector to reduce the cost of production.

Next-generation nanoplasmids are designed to be compliant with FDA regulatory guidance for antigen delivery vectors.³² In NTC series of nanoplasmids, sequences nonessential for plasmid replication in prokaryotes and target gene expression in eukaryotes are eliminated. Instead, NTC vectors employ: 1) synthetic nonhuman eukaryotic mRNA leaders and terminators to minimize gene integration into the host genome; 2) RNA-OUT technology to eliminate antibiotics-based selection and amplification of recombinant DNA; and 3) R6K-derived mini-origin and SV40-CMV-HTLV-1R chimeric promoter-intron to achieve sustained and elevated expression of the target genes in eukaryotic cells.³³ NTC nanoplasmids express encoded protein in cytoplasm making it more accessible for MHCI/MHCII presentation, can be efficiently manufactured in g/L without antibiotic selection, and offer an efficient, cost-effective gene delivery.

Using the previous features, we designed the BCV encoding the two candidate antigens. Briefly, mouse-codon optimized sequence for TcG2 and TcG4 (separated by P2A linker ATNFSLLKQAGDVEENPGP) was synthesized (GenScript Biotech) (Figure 1B). The bicistronic construct was directionally cloned at SalI/BglII restriction sites in NTC9385R and NTC9385R-eRNA41H (contains RIG-I adjuvant) from Nature Technology to generate BCV and BCVR, respectively (Figures 1C and 1D). Recombinant constructs were confirmed by sequencing, and 50 mg of each were purified using the HyperGRO fermentation process (Figure 1E).³⁴

Immunization, challenge, and sample collection

A schematic overview of study design, including sample and data collection, is presented in Figure 1F. Briefly, mice were randomly distributed in study groups. BCV or BCVR (2.5 μg, 5 μg, or 10 μg, intramuscular, twice) were administered at 21-day intervals, and mice were challenged with Tc (10,000-trypomastigotes in 50 μL PBS per mouse, intraperitoneal) at 21 days or 100 days after booster dose to monitor short- and long-term protection, respectively. Mice were euthanized 21 days after booster or challenge to monitor toxicity, immunogenicity, and efficacy of BCV/BCVR against acute Tc infection. Additional mice were euthanized at 150 days postchallenge to monitor BCV/BCVR efficacy against chronic CD. Mice given empty vector or vehicle only or Tc only were used as controls.

Blood, serum, and plasma samples were stored at 4 °C or −20 °C. Tissues were stored at −80 °C for DNA/RNA purification or fixed for histology. Freshly isolated splenocytes were used for flow cytometry. Mice were monitored for heart function during chronic phase. All experiments were conducted by trained personnel in an ABSL2/BSL2-approved laboratory.

Physical and clinical exam

After each treatment, mice were monitored daily for 1 week and on alternate days for 2 weeks. Physical/clinical well-being was scored as 1 (healthy), 2 (ruffled fur, lethargic), 3 (hunched posture, orbital tightening, >15% weight loss), and 4 (reluctance to move when stimulated, >20% weight loss).

Lesion size (<10, 11-15, >16 mm); erythema, induration, and swelling (<20, 21-50, >51 mm); and local reactogenicity lasting for <3, 4 to 7, or >8 days were scored as 1, 2, and 3, respectively. Acute pathophysiological changes were monitored by complete blood count panel and 27-parameter comprehensive chemistry 2 panel analysis of serum samples (IDEXX Reference Laboratory). Peripheral blood smears were analyzed by IDEXX trained personnel.

Parasite burden

Tissues (25 mg each) were homogenized for 2 cycles (1,500 g, 30 seconds) by BeadBug homogenizer (Benchmark Scientific). Total DNA (50 ng) was isolated using the DNeasy Blood & Tissue Kit (Qiagen) and examined for quality (optical density at 260 nm/optical density at 280 nm:1.8–2.0) and quantity (optical density of 1.0 at 260 nm = 50 μg/mL) using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reaction mix (10 μL) consisting 12.5 ng DNA, 300 to 500 nM each oligonucleotide (Tc18SrDNA-F: 5′-TTTTGGGCAACAGCAGGTCT-3′, Tc18SrDNA-R: 5′- CTGCGCCTACGAGACATTCC-3′, Gapdh-F: 5′-AACTTTGGCATTGTGGAAGG-3′, Gapdh-R: 5′-ACACATTGGGGGTAGGAACA-3), and SYBR Green Supermix (172–5271; Bio-Rad) was subjected to quantitative polymerase chain reaction for 40 cycles (95 °C/10 seconds, 63 °C/30 seconds) on an iCycler thermal cycler (Bio-Rad). Each sample was analyzed in duplicate, and relative parasite burden was calculated by 2^−ΔΔCT, where ΔC_T = C_T (Tc18SrDNA) − C_T (Gapdh) and ΔΔC_T = ΔC_T (sample) − ΔC_T (control).

Histology

Heart and skeletal muscle (SkM) tissue sections were fixed in 10% buffered formalin for 24 hours, dehydrated in graded ethyl alcohol, cleared in xylene, and embedded in paraffin. Slides containing 5 μm tissue sections were stained with hematoxylin (stains nuclei blue) and eosin (stains extracellular matrix and cytoplasm pink) or Masson’s trichrome (stains nuclei dark red, cytoplasm red/pink, and collagen blue). Images were acquired using an Olympus IX51 microscope equipped with digital camera and cellSens Imaging software (V4.3). In hematoxylin and eosin images, nuclei and tissue coloration were identified using ilastik (v.1.4.0.post1, released Nov 10, 2023). Pixel classification and ImageJ (v.1.54f, last updated June 05, 2025; National Institutes of Health) were used to quantify pixels with nuclei and tissue colorations, and percent nuclei quantified as [(nuclei pixels / total tissue pixels) × 100]. Each image was scanned in >5 microscopic fields and average % nuclei per tissue section calculated to score the inflammatory infiltrate.

Masson’s trichrome images were scored for percent fibrotic tissue, as previous. Each image was scanned for blue (collagen) and red (fibers) pixels in >5 microscopic fields and average percentage of fibrosis positive area calculated as [(blue pixels / total tissue pixels) × 100].

Antibody levels, agglutination, and trypanolysis

Normal mouse heart tissue (100 mg) or Tc trypomastigotes (1 × 10¹⁰) in 1.5 mL ice-cold RIPA buffer containing protease inhibitors cocktail were subjected to 3 freeze/thaw cycles (−80 °C for 20 minutes and room temperature for 5 minutes), homogenized for 2 cycles (1,500 g, 30 seconds), and sonicated at maximum speed for 5 minutes in Emerson-Branson ultrasonic sonicator. Lysates were centrifuged at 20,000 g for 10 minutes, and protein concentration was evaluated by Bradford assay.

To assess anticardiac antibodies, flat-bottom 96-well plates (Corning) were coated with heart lysate (5 μg/100 μL/well) overnight at 4 °C. Plates were washed with phosphate-buffered saline with 0.05% Tween 20 (PBST), blocked with 1% bovine serum albumin in PBST, and incubated with test sera (1:50 to 1:400 dilutions, 50 μL/well) for 1.5 hours and horseradish peroxidase (HRP) conjugated anti-mouse IgG (1:1,000 dilution; SouthernBiotech) for 1 hour. After adding TMB substrate (100 μL/well; Sigma-Aldrich), the reaction was stopped with 2N H₂SO₄ and change in color recorded at 450 nm by using a Multiskan SkyHigh microplate spectrophotometer (Molecular Devices).

To measure antiparasite antibodies, plates were coated with Tc lysate (5 μg/100 μL/well), washed, and incubated with test sera, as previous. Antibodies were purchased from Abcam unless stated otherwise and diluted in 1% bovine serum albumin–PBST. To measure IgG, plates were coated with goat anti-mouse IgG-HRP (Cell Signaling Technology) at 1:1000 dilution for 1 hour. To measure IgG1 or IgG2a, plates were coated with goat anti-mouse IgG1 or IgG2a (1:500 dilution, 1 hour), and streptavidin-HRP (1:5,000 dilution, 30 minutes). Incubations with primary and secondary antibodies were performed at 37 °C and 25 °C, respectively. Color was developed with TMB substrate and recorded at 450 nm.

To appraise agglutination activity, trypomastigotes (4 × 10⁶/500 μL/well) were distributed in 24-well plates and sera samples (30 μL) added in triplicate at 5%, 0.5%, and 0.05% concentration. After incubation on rotating platform for 1 hour, trypomastigotes agglutination was visualized and imaged by light microscopy (200× magnification). Agglutinate size (#parasites/agglutinate) and number of agglutinates were calculated using ImageJ software.

To examine trypanolytic activity, trypomastigotes (1 × 10⁶/100 μL/well) were incubated in triplicate with 50 μL each of sera samples (5%, 0.5%, or 0.05%, 50 μL/well) for 15 minutes and human complement (Innovative Research) for 1 hour. Motile trypomastigotes were counted by light microscopy, and percent trypanolysis recorded as ([total trypomastigotes added – remaining trypomastigotes] × 100 / total trypomastigotes).

Flow cytometry

Fresh spleen tissues of mice were macerated, passed through 70 μm Falcon cell strainer (22-363-548; Thermo Fisher Scientific) to eliminate clumps and debris, and incubated with red blood cell lysis buffer (00-4333-57; eBioscience) for 5 minutes. After adding 25 mL PBS, cells were centrifuged at 300 g for 5 minutes and processed for flow cytometry. The antibodies/markers and gating strategies for phenotypic and functional characterization of CD4⁺, CD8⁺, and IL-17A⁺ lymphocytes are shown in Supplemental Table 8, Supplemental Figure 2, and Supplemental Figures 3A to 3J. Briefly, splenocytes were incubated for 15 minutes with Fc block (anti-CD16/CD32) in brilliant-stain buffer (BD Biosciences) and washed with staining buffer (00-4222-26; eBioscience). Cells (5 × 10⁴/50 μL) were incubated in the dark for 30 minutes at 4 °C with fluorophore-conjugated antibodies to surface molecules. In some experiments, cells were incubated with fixation-permeabilization solution (BD Biosciences) for 20 minutes, washed, and stained with fluorophore-conjugated antibodies against intracellular molecules for 30 minutes. Cells were washed and resuspended in staining buffer for flow cytometry analysis.

The CD4⁺ T and CD8⁺ T subsets were captured based on surface expression of CD3, CD4, CD8a, CD25, CD27, CD44, CD62L, and CD127. Each subset was further analyzed for perforin (PRF1), granzyme B (GZM), IFNγ, IL2, and TNFα production. The IL-17A–expressing Th17 subsets were captured based on surface expression of CD3, CD4, CD127, Thy1.2, and CD25. All samples were visualized and acquired on a LSRII Fortessa Cell Analyzer (BD Biosciences). Unstained cells, cells incubated with isotype matched IgGs (eBioscience), live/dead stain, and FMO (fluorescence minus one) were included as controls. Data were analyzed using FlowJo software (v.10.9.0; Becton Dickinson). Briefly, an average of 1 × 10⁵ of CD3⁺T live cells from all categorical treatments were selected and combined dataset was subjected to generate common t-distributed stochastic neighbor embedding (tSNE) plots using the following parameters: iteration = 1,000, perplexity = 30, learning rate (eta) = 1400, k-nearest neighbors algorithm = exact, and gradient algorithm = Barnes-Hut, which allows visualization of high-dimensional datasets. Cumulative tSNE maps were applied to individual files to identify treatment-specific T cell subsets.

Echocardiography assessment of cardiac structure and function

Mice were continuously anesthetized by inhalant 1.5% isoflurane/100% O₂ to maintain a light sedation level and placed supine on an electrical heating pad at 37 °C. Electrocardiogram electrodes were attached to mouse paws, and heart rate and respiratory physiology were continuously monitored. Chest area was shaved to apply warmed ultrasound gel. Transthoracic echocardiography was performed using the high-frequency linear array transducer (MS400, 18-38 MHz) on the Vevo2100 ultrasound system (VisualSonics). Images were acquired in B-mode and M-mode to evaluate the heart in diastole and systole. Pulsed wave Doppler imaging was performed to measure diastolic functions. All measurements were obtained in duplicate, and data were analyzed using Vevo2100 software.

Statistical analysis

Data are presented using actual data points with the mean ± SEM values. The Kolmogorov-Smirnov test was used to check data normality. Two groups were compared using an unpaired 2-tailed t test or nonparametric Mann-Whitney U test. Normally distributed data from multiple groups (eg, control, Tc, and BCV.Tc or BCVR.Tc) were compared using 1-way analysis of variance with Tukey's post hoc test. Kruskal-Wallis test with Dunn's post hoc test was performed for the data that were not in normal distribution. Repeated measures (ie, antibody response at different sera dilutions) were analyzed using repeated measures 2-way analysis of variance with Tukey's post hoc test. All datasets were managed in Microsoft Excel (v.16.101.3) and analyzed using GraphPad Prism 10.1.1 (GraphPad Software). A P value of <0.05 was considered statistically significant.

Results

Immunogens, CD model, and study design

The schematics for selection and synthesis of the immunogen and study design are presented in Figure 1. Briefly, a computational/biological screen was employed to select the dominant surface antigens that were conserved in all parasite lineages and recognized by Tc-specific type 1 CD8⁺ T cell response in multiple infected hosts. TcG2 and TcG4 were further chosen because of their superior immunogenicity that offered >85% reduction of parasites in mice and 70% to 85% control of parasites in dogs. To increase the expression and stability of the encoded antigens, and decrease the cost of production, sequence for TcG2 and TcG4 were mouse codon-optimized and synthesized with insertion of a P2A linker (Figure 1B). Synthetic, bicistronic construct was cloned into a nanoplasmid without and with RIG-I agonist to generate BCV and BCVR, respectively (Figures 1C and 1D). The NTC series of nanoplasmids are compliant with FDA guidelines for human use. The RIG-I agonist was added as it is known to increase innate/adaptive immunity to infectious agents. Schematics of the BCV/BCVR production pipeline is presented in Figure 1E.

We established a mouse model to capture human phases of CD progression. C57BL/6 mice infected with Tc (SylvioX10) develop acute parasitemia for 21 to 40 days; indeterminate phase of low-grade parasites, diffused inflammation, and oxidative stress for 6 months; and cardiomyopathy and LV dysfunction at 6 to 9 months.³⁵ This experimental model was utilized to evaluate BCV and BCVR immunogenicity and efficacy in controlling Tc infection and CD in adults (Figure 1F).

Evaluation of BCV and BCVR safety

We examined local reactogenicity and systemic toxicity of BCV and BCVR following the World Health Organization guidelines on nonclinical evaluation of immunotherapies in animals.³⁶ Physical/clinical well-being was scored as 1 to 4 (1 = healthy). BCV/BCVR-immunized mice exhibited slight changes in activity during first week but no changes in food/water uptake and weight. After challenge, all groups of mice exhibited mild physical symptoms of being lethargic (score of 1 of 2) (Supplemental Figure 1A). Local reactogenicity was graded as 1 to 3 based on lesion size and duration at injection site. Mice responded to delivery of vehicle, immunogens, and Tc with mild to no erythema or swelling lasting for 3 to 5 days (Supplemental Figure 1B).

Hematological analysis was performed to measure anemia and nonspecific immune suppression or inflammation in all groups. Clinical chemistry (albumin, calcium, phosphates, glucose, cholesterol, etc.) and biochemistry (bilirubin, blood urea nitrogen, amylase, lipase, alanine aminotransferase, alkaline phosphatase, etc.) analyses were performed to document physiological changes and effects on liver, pancreas and kidney function or glucose/lipid metabolism. BCV elicited proliferation of white blood cells, including monocytes, neutrophils, and lymphocytes, but no alterations in red blood cells. No changes in general parameters of blood hematology in mice injected with vehicle only and no disparities in blood chemistry/biochemistry were observed in all groups of mice (Supplemental Table 1). After challenge, blood platelets were increased in infected mice, while blood monocytes were increased in the BCVR.Tc group. Red blood cell–related parameters and blood chemistry/biochemistry, indicative of acute organ injuries, were not significantly changed in all infected groups (Supplemental Table 1). Microscopic examination of blood smears showed mild polychromasia and anisocytosis of red blood cells and adequate distribution of platelets in all infected groups. Poikilocytes, Heinz bodies, band cells, promyelocyte and blast cells, indicative of excessive immature cells, were not seen in blood smear of all groups of mice.

Tc is a eukaryotic parasite that may exhibit antigenic similarity to self-antigens. Therefore, we titrated anticardiac IgG response by an enzyme-linked immunosorbent assay (Supplemental Table 2). A slight increase in IgGs recognizing cardiac soluble antigens was noted in sera samples (1:50 to 1:100 dilution) of BCV- and BCVR-immunized mice (Supplemental Figure 1C). A similar anticardiac IgG profile was observed in acutely and chronically infected mice, irrespective of immunization status (Supplemental Figures 1D and 1E).

Together, these results demonstrate that BCV and BCVR are safe to use, and do not elicit significant local or systemic toxicity in presence or absence of Tc infection.

Titration of immunogen dosage for controlling Tc infection

We have shown that delivery of TcG2 and TcG4 in pCDNA3 (25 μg each) elicited significant control of Tc infection in mice.²⁵ NTC9385R is >50% smaller (1,753 bp vs 5,546 bp), and >5-fold efficacious in gene expression than pCDNA3. Therefore, we titrated BCV and BCVR at 2.5, 5, and 10 μg concentrations. Mice were challenged at 21 and 100 days after booster dose to evaluate short- and long-term efficacy, respectively (Figure 2, Supplemental Table 3). Tissue parasite burden, monitored at 21 days postinfection, occurred at a very high level in heart and SkM tissues of infected mice (Figures 2A to 2H). In comparison, BCV.Tc and BCVR.Tc (vs Tc only) groups controlled the parasite burden by 83% to 86% and 84% to 92% in heart (Figures 2A and 2B) and 41% to 80% and 37% to 76% in SkM (Figures 2C and 2D) tissues, respectively. When challenged at 100 days, BCV.Tc and BCVR.Tc (vs Tc only) groups exhibited 63% to 82% and 75% to 99% control of cardiac (Figures 2E and 2F) and 58% to 74% and 91% to 98% control of SkM (Figures 2G and 2H) parasite burden, respectively.

Figure 2.

Titration of Immunogens for Controlling Acute Trypanosoma cruzi Infection

Mice were immunized with 2.5, 5, and 10 μg BCV or BCVR at a 21-day interval; challenged with Trypanosoma cruzi (Tc) at (A to D, I-R) 21 days or (E to H) 100 days after booster; and euthanized at 21 days postinfection. Parasite burden. (A, B, E, F) Heart and (C, D, G, H) SkM tissues were analyzed in duplicate for Tc18SrDNA level by quantitative PCR. Data were normalized to GAPDH (n ≥ 10 mice/group). Histology. Hematoxylin eosin–stained 5 μm (I to L) heart and (N to Q) SkM tissue sections were imaged (scale bar =50 μm) and (N, R) inflammatory infiltrate score calculated (n = 5-10 mice per group, 2 tissue slides per mouse, >5-microscopic fields per slide). In bar graphs, the data point for each mouse and mean ± SEM are plotted. Significance was calculated by 1-way analysis of variance with Tukey’s post hoc test or Kruskal-Wallis with Dunn’s post hoc test, and P values are plotted as ∗^,^P < 0.05, ∗∗^,^^P < 0.01, and ∗∗∗^,^^^P < 0.001 (∗control vs Tc, ^Tc vs BCV.Tc or BCVR.Tc). Abbreviations as in Figure 1.

Histological examination was performed in mice immunized with 10 μg of immunogens, challenged 21 days later, and euthanized in acute phase. Representative hematoxylin and eosin–stained images along with semi-quantitative scores of myocardial (Figures 2I to 2M) and SkM (Figures 2N to 2R) tissues are shown. Acutely infected mice exhibited widespread infiltration of inflammatory infiltrate in heart and SkM (histology score of 27.5 and 38.5, respectively). Extensive coalescing inflammatory foci or disseminated inflammation with loss of tissue integrity were predominant in these tissues of infected mice. In comparison, the BCV.Tc and BCVR.Tc groups exhibited a significant decline in heart and SkM inflammatory infiltrate, and coalescing sheets of inflammation were replaced by diffused inflammatory infiltrate in alignment with the control of tissue parasites. Tc and inflammatory infiltrate were undetectable in control mice.

Together, these results suggest that BCV and BCVR dosage at the 10 μg level was maximally effective in controlling the tissue parasites in mice. Both BCV and BCVR enhanced the host’s ability to control myotropic parasite replication and associated inflammatory infiltrate that are known pathological hallmarks of acute CD. BCVR exhibited slightly better short-term and long-term efficacy than BCV in controlling parasites and associate inflammatory pathology in mice.

BCV and BCVR improved the Tc-specific T cell maturation and function

Using a T cells specific gating strategy (Supplemental Figure 2), we examined if protection from acute Tc infection was associated with maturation and functional activation of T cell subsets in BCV- or BCVR-immunized mice. Group-specific tSNE plots of CD4⁺T meta-clusters markers are presented in Figure 3A, and detailed analysis of CD4⁺T subsets from a representative experiment is presented in Figures 3B to 3K and Supplemental Table 4. Acutely infected (vs control) mice exhibited 74.8% increase in CD4⁺ effector T cell population that was mirrored in a 42%, 228%, 33%, and 93% increase in effector T cells of intermediate phenotype (Teff-I), effector T cells of late phenotype (Teff-L), effector memory T cells of early phenotype (Tem-E) and effector memory T cells of late phenotype (Tem-L), respectively (Figures 3B to 3E). The BCV.Tc and BCVR.Tc groups exhibited 19% to 54% further increase in the frequencies of effector T cells and Teff-L and Tem-L subsets (Figures 3B to 3E), and maximal increase in mature CD4⁺ T cell subsets was observed in BCVR.Tc group. All CD4⁺Teff subsets exhibited functional response, though CD4⁺Teff-L subset was the major source of cytotoxic molecules postinfection (Figures 3F to 3K). Though detected at <1% level, frequencies of GZM-, PRF1-, and TNFα-producing CD4⁺ Teff-L subsets were observed in the order of BCVR.Tc ≥ BCV.Tc > Tc, while IFNγ- and IL2-producing CD4⁺ Teff-L subsets were expanded in infected mice only (Figure 3H). Low frequencies of CD4⁺T memory subsets (<0.05%) exhibiting proinflammatory/cytotoxic response and IL-2 production were detectable in infected mice only.

Figure 3.

Maturation and Functional Activation of CD4⁺T cells in Tc-Infected Mice (±BCV/BCVR)

Mice were immunized with BCV or BCVR (2 doses, 10 μg), infected with Tc, and euthanized at 21-day intervals. Splenocytes were labeled with fluorescent-conjugated antibodies and analyzed by flow cytometry (see gating strategy in Supplemental Figure 2). (A) Phenotypic profiling. The t-distributed stochastic neighbor embedding (tSNE) plots, visualizing color-coded CD4⁺T cell subsets in 2-dimensional space in all groups. Percentage frequencies of (B, C) CD4⁺ effector T cells (Teff) (total, effector T cells of early phenotype [Teff-E], effector T cells of intermediate phenotype [Teff-I], effector T cells of late phenotype [Teff-L]) subsets and (D, E) CD4⁺ memory T cells (Tmem) (total, central memory T cells [Tcm], effector memory T cells of early phenotype [Tem-E], effector memory T cells of late phenotype [Tem-L]) subsets are shown. (F-K) Functional profiling. Percentage frequencies of GZM-, PRF1-, IFNγ-, TNF⍺-, and IL-2–producing CD4⁺ subsets. Datapoint for each mouse and mean ± SEM (n = 5 or 6 per group, duplicate analysis per mouse) are plotted. Significance comparing 3 groups (ie, control, Tc, and BCV.Tc or BCVR.Tc) was calculated by 1-way analysis of variance with Tukey’s post hoc test or Kruskal-Wallis with Dunn’s post hoc test. Significance comparing 2 groups was calculated by unpaired t test or Mann-Whitney U test. The P values of ∗P < 0.05, ∗P < 0.01, and ∗∗∗P < 0.001 comparing control vs infected groups are plotted. Abbreviations as in Figures 1 and 2.

A detailed characterization of CD8⁺T cells in enrolled groups is shown in Figure 4 and Supplemental Table 5. Group-specific tSNE plots of CD8⁺T meta-clusters are presented in Figure 4A. Infected (vs control) mice exhibited potent (331%) increase in CD8⁺Teff cells leading to 214%, 632%, and 100% increase in Teff-I, Teff-L, and central memory T cell subsets, respectively (Figures 4B to 4E). A further increase of 48% to 82% and 104% to 184% in the frequencies of CD8⁺ Teff and Teff-L subsets, respectively, was noted in the BCV.Tc and BCVR.Tc groups (Figures 4B to 4E). Functionally active CD8⁺ T cell subsets (0.01%-3%) producing GZM, PRF1, IFNγ, TNFα, or IL-2 were also primarily increased in infected mice when compared with that noted in the BCV.Tc and BCVR.Tc groups (Figures 4F to 4K).

Figure 4.

Maturation and Activation of CD8⁺ T Cells in Tc–Infected Mice (±BCV/BCVR)

Mice were immunized with BCV/BCVR (2 doses, 10 μg), infected with Tc, and euthanized at 21-day intervals. Splenocytes were labeled with fluorescent-conjugated antibodies and analyzed by flow cytometry (gating strategy in Supplemental Figure 2). (A) Phenotypic profiling. The tSNE plots, visualizing color-coded CD8⁺T cell subsets in all groups. Percentage frequencies of (B, C) CD8⁺ Teff (total, Teff-E, Teff-I, Teff-L) subsets and (D, E) CD8⁺ Tmem (total, Tcm, Tem-E, Tem-L) subsets are shown. (F-K) Functional profiling. Percentage frequencies of GZM, PRF1, IFNγ, TNF⍺, and IL2 producing CD8⁺ subsets. Datapoint for each mouse and mean ± SEM (n = 5 or 6 per group, duplicate analysis per mouse) are plotted. Significance comparing 3 groups (ie, control, Tc, and BCV.Tc or BCVR.Tc) was calculated by 1-way analysis of variance with Tukey’s post hoc test or Kruskal Wallis with Dunn’s post hoc test. Significance comparing 2 groups was calculated by unpaired t test or Mann-Whitney U test. The P values are plotted as ∗^,^P < 0.05, ∗∗^,^^P < 0.01, and ∗∗∗^,^^^P < 0.001 (∗control vs infected, ^Tc vs BCV.Tc or BCVR.Tc). Abbreviations as in Figures 1, 2, and 3.

IL-17A contributes to control of infectious agents by inducing neutrophils recruitment and secretion of inflammatory mediators. In context to Tc infection, an IL-17 protective function was attributed to recruitment of IL-10⁺ neutrophils that limited IFNγ production and tissue damage.³⁷ The gating strategy to capture IL-17A⁺ splenic subsets and changes in IL-17A⁺ subsets in various groups is shown in Supplemental Figures 3A to 3J. The IL-17A⁺ cells constituted <2% of total CD3⁺ and CD3⁻ lymphocytes in normal mice, and these were increased by 53% to 200% in infected (vs control) groups (Supplemental Figures 3K and 3L). Further characterization of CD3⁻IL-17⁺ cells identified low frequencies of ILC1-like and ILC2-like phenotype, of which IL-17⁺ ILC2-like cells were elevated in infected (vs control) mice (Supplemental Figures 3M and 3N). The IL-17A⁺CD3⁺ subset remained elevated, and CD3⁻IL-17A⁺ subset was further increased by 47% in BCV.Tc (vs infected) mice (Supplemental Figures 2K and 2L).

Together, these results (Figures 3 and 4, Supplemental Figure 3) suggest that BCV and BCVR enhanced the differentiation of naïve T cells toward mature CD4⁺ T and CD8⁺ Teff-L and Tem-L subsets, that persist in the lymphoid organs for long time. While CD4⁺ T effectors in BCVR.Tc and BCV.Tc mice responded with increased production of cytolytic molecules for parasite killing, CD8⁺ Teff-L and CD4⁺ and CD8⁺ Tem-L subsets displayed protective function with minimal production of cytolytic/inflammatory molecules to limit tissue damage. In untreated/infected mice, CD4⁺T and CD8⁺T subsets exhibited an ongoing proinflammatory response that failed to control parasite and promoted host tissue damage.

BCV and BCVR induced functional, antiparasite antibody response

Next, we monitored if BCV and BCVR protect from Tc by eliciting function antibody response. We, first, titrated the antiparasite antibody levels in sera samples at 1:50 to 1:400 dilutions (Figure 5, Supplemental Table 6). Immunized (vs control) mice exhibited a 67% to 329% increase in anti-Tc IgG, IgG1, and IgG2a levels (BCVR>BCV, 1:50 to 1:100 dilutions) (Figures 5A to 5C). Tc challenge resulted in 11- to 86-fold and 1.8- to 20.4-fold increase in IgG, IgG1, and IgG2a levels during acute (Figures 5D to 5F) and chronic (Figures 5G to 5I) infection (1:50 dilution), respectively. BCV.Tc and BCVR.Tc groups exhibited similar or slightly lower level of antibody response than untreated/infected mice (Figures 5D to 5I). IgG2a antibodies are associated with protection from parasitic infections,³⁸ and therefore the IgG2a/IgG1 ratio was calculated as a measure of protection (Supplemental Table 6). All infected groups exhibited a high IgG2a/IgG1 ratio during acute (range 4.19-14.5, BCVR.Tc=BCV.Tc>Tc at 1:200 to 1:400 dilutions) and chronic (range 2.15-4.23, BCV.Tc>BCVR.Tc>Tc at 1:50 to 1:200 dilutions) phases of infection and disease progression, and the maximal increase was observed in mice given BCV or BCVR.

Figure 5.

BCV/BCVR-Induced Antiparasite Antibody Response (±Tc)

Mice were immunized with BCV or BCVR (2 doses, 10 μg) and infected with Tc at 21-day intervals. Serum samples were obtained at 42 days postimmunization, and 21 days (acute) and 150 days (chronic) postinfection. Parasite-specific total IgGs, IgG1, and IgG2a/b (A to C) after immunization, and (D to F) acute and (G to I) chronic infection were titrated by an enzyme-linked immunosorbent assay. Agglutinate size and agglutinate number show serum IgG capacity to bind trypomastigotes in (J and K) immunized and (M and N) chronically infected mice. (L, O, P) Trypanolytic activity of serum IgGs. Data are presented as mean ± SEM (≥5 mice per group, duplicate analysis per sample). Significance, with consideration of serum dilution, was calculated by repeated measures 2-way analysis of variance with Tukey’s post hoc test, and P values are plotted as ∗^,^P < 0.05, ∗∗^,^^P < 0.01, and ∗∗∗^,^^^P < 0.001 (∗control vs infected, ^Tc vs BCV.Tc or BCVR.Tc). Significance comparing trypanolytic activity between BCV and BCVR groups (5% dilution) is plotted as ⁺⁺⁺P < 0.001, and it was calculated by nonparametric Mann-Whitney U test. Abbreviations as in Figures 1, 2, and 3.

To determine if BCV/BCVR-induced antibodies are functional against Tc, we titrated agglutination (inhibits parasite motility and infectivity) and trypanolytic (ie, direct parasite killing) activities using 5%, 0.5% and 0.05% sera dilutions. Immunized mice exhibited potent agglutination and parasite lysis capacity (BCVR>BCV, 5% to 0.5% serum dilution) (Figures 5J to 5L). Dominance of antibodies to agglutinate parasites was maintained in chronically infected BCV.Tc and BCVR.Tc (vs Tc only) mice (Figures 5M and 5N). A high level of trypanolytic activity of antibodies was observed in all infected groups (Figures 5O and 5P).

We surmise that BCV/BCVR enhanced the antibody switch to protective IgG2a isotype, and these antibodies were functionally active and capable of controlling the parasite motility and survivability. Trypanolytic antibody response, though slightly lower in BCV.Tc and BCVR.Tc (vs Tc) mice, was still associated with a potent decline in tissue parasite load.

Cardiomyopathy and LV dysfunction in chronic Chagas disease (±BCV or BCVR)

Low-grade parasitism, tissue inflammation, and fibrosis leading to LV dysfunction are the hallmarks of chronic CD. Therefore, we examined if BCVs were effective in controlling CD-associated pathologies. Tissue parasites were readily detectable in chronically infected mice (SkM>>heart). In contrast, chronic Tc burden was decreased by 54% to 77% and 86% to 99.5%, respectively, in the heart and SkM of immunized mice, with maximal reduction observed in BCVR.Tc group (Figures 6A and 6B, Supplemental Table 3). Microscopic evaluation of hematoxylin and eosin–stained tissues revealed coalescing inflammatory infiltrate (score 15.8-28) in the heart and SkM of chronically infected mice, whereas diffused inflammatory infiltrate (score 12.3-14.3) was observed in immunized (BCVR.Tc < BCV.Tc) mice (Figures 6C to 6L).

Figure 6.

Cardiac Pathology in Chronically Infected Mice (±BCV/BCVR)

Mice were immunized with BCV or BCVR (2 doses, 10 μg), infected with Tc at 21-day intervals, and euthanized at >150 days postinfection. (A and B) Parasite burden. Tc18SrDNA in heart and SkM tissues was analyzed in duplicate (n ≥ 10 mice per group) by quantitative PCR. See detailed data in Supplemental Table 3. Histology. (C to G, M to Q) Five-micron heart and (H to L, R to V) SkM tissue sections were subjected to (C to L) hematoxylin and eosin (H&E) or (M-V) Masson’s trichrome (MT) staining, and representative images identifying inflammatory infiltrate and fibrosis are shown (scale bar = 50 μm). Histoscore for (G and L) inflammatory infiltrate and (Q and V) fibrosis was measured as described in methods (n = 5-8 mice per group, 2 tissue slides per mouse, ≥5 microscopic fields per slide). In bar graphs, the data point for each mouse and mean ± SEM are plotted. Significance was calculated by 1-way analysis of variance with Tukey’s post hoc test or Kruskal-Wallis with Dunn’s post hoc test, and P values are plotted as ∗^,^P < 0.05, ∗∗^,^^P < 0.01, and ∗∗∗^,^^^P < 0.001 (∗control vs Tc, ^Tc vs BCV.Tc or BCVR.Tc). Abbreviations as in Figures 1, 2, and 3.

Masson's trichrome staining identified extensive collagen deposition (score 74.4-89.9) in heart and SkM fibers (including endomysium and perimysium) of CD (vs control) mice. Cardiac fibrosis was completely controlled (Figure 6M to 6Q) and collagen deposition in SkM fibers was markedly reduced (Figures 6R to 6V) in BCV.Tc and BCVR.Tc groups. These results suggest that BCVs (BCVR>BCV) were highly effective in controlling persistent tissue parasite burden and associated inflammatory infiltrate and fibrosis, which otherwise were pronounced in the heart and SkM of nontreated/CD mice.

Echocardiography imaging showed LV dysfunction in CD mice (Figure 7, Supplemental Table 7). Structurally, LV mass was increased by >2-fold, while the LV posterior wall was thinned during systole and diastole by 30% to 37% in CD (vs control) mice (Figures 7A to 7C). Up to 36% increase in LV inner diameter at systole and 16% decline in LV volume at diastole indicated increased stiffness that impaired the heart’s ability to pump blood in CD (vs control) mice (Figures 7D and 7E). In comparison, BCV.Tc and BCVR.Tc mice maintained the LV mass, LV posterior wall thickness, and LV internal diameter at systole at normal levels (Figures 7A to 7D). Indices of LV systolic function—stroke volume, cardiac output, and ejection fraction—were decreased by 31.8%, 35%, and 37.2%, respectively, in CD (vs control) mice (Figures 7F to 7H). Fractional shortening, which calculates the LV size reduction during systole, was also compromised by 40.2% in CD (vs control) mice (Figure 7G). In comparison, BCV.Tc and BCVR.Tc groups exhibited partial to full recovery of systolic and diastolic functions, with maximal benefits observed in the BCVR.Tc group. Together, these results (Figures 6 and 7) demonstrate that changes in the LV wall thickness (and thereby contractile capacity) contributed to compromised systolic and diastolic performance of the heart in CD mice. The protective effects of BCV/BCVR in preserving LV hemodynamics and myocardial performance were mediated via control of collagen deposition and reduced cardiac stiffness in CD mice.

Figure 7.

LV Dysfunction in Chagas Disease (± BCV/BCVR)

Mice were immunized with BCV or BCVR and infected with Tc, and transthoracic echocardiography was performed at >180 days postinfection. Shown are (A) left ventricular (LV) mass, (B and C) left ventricular posterior wall thickness at systole (LVPW-s) and left ventricular posterior wall thickness at diastole (LVPW-d), (D) left ventricular inner diameter at systole (LVID-s), (E) left ventricular volume at diastole (LV volume-d), (F) stroke volume, (G) cardiac output, (H) ejection fraction, (I) fractional shortening, and (J) heart rate. Average values for each mouse and mean ± SEM are plotted (n = 8-14 mice per group). Significance was calculated by 1-way analysis of variance with Tukey’s post hoc test or Kruskal-Wallis with Dunn’s post hoc test, and P values are plotted as ∗^,^P < 0.05, ∗∗^,^^P < 0.01, and ∗∗∗^,^^^P < 0.001 (∗control vs Tc, ^Tc vs BCV.Tc or BCVR.Tc). Abbreviations as in Figures 1, 2, and 3.

Discussion

Among the technologies available for immunotherapies development, DNA- and RNA-based approaches are highly promising because of the ability to elicit both humoral and cellular immune responses. Recent outbreaks of life-threatening infectious diseases, such as H5N1 influenza, Ebola, MERS-CoV, and SARS-CoV-2, have also shown that DNA- and RNA-based approaches have the greatest potential for reproducible production of effective immunogens at a faster rate. Along with several COVID messenger RNA vaccines, the first DNA vaccine for human use, the Indian ZyCovD vaccine against SARS-CoV-2, was approved in 2021.³⁹ Continuing research has shown that DNA offers a rapid and feasible approach for screening multiple immunogens in human trials because of relative ease and low cost of manufacture, transport, stability, and storage.⁴⁰ DNA-based immunogens are also found to be relatively safe, well tolerated, and elicit no significant side effects even after multiple administrations. Several clinical trials have almost unanimously concluded that DNA-based delivery of antigens elicit both humoral (facilitated by CD4⁺T cells) and cellular (mostly by CD8⁺T cells) responses that work synergistically to eliminate the target cancer or pathogen. Several strategies to further improve DNA immunogenicity include structural modifications (eg, optimization of the coding and noncoding sequence in plasmids, incorporation of powerful promotor and nuclear import signals and codon optimization to improve encoded antigen expression), route of administration and boosting to facilitate antigen uptake, and adjuvants aimed at enhancing the innate or adaptive immune responses. Several of these approaches are being tested in ongoing clinical trials of DNA cancer vaccines, and merit future investments. Thus, DNA immunization offers promising, widespread application in clinical settings as a game-changing treatment for infectious and noninfectious diseases.

Considering the complexity of multigene families in Tc that promotes immune evasion and the concerns that attenuated parasites can regain virulence in immunocompromised host, current research efforts have led to the selection and testing of candidate immunogens for control of CD. Indeed, several antigens, including complement regulatory protein, cruzipain, GP82, KMP11, LYT1, paraflagellar rod proteins, Tc24, Tc52, and trans-sialidases have been identified as potential immunogens candidates. Though researchers’ preferences contributed to selection bias, almost all of these were recognized by antibodies and IFNγ-producing CD8⁺T cells in experimental models of CD and/or Chagas patients and consequently offered a degree of immune resistance to Tc infection.⁴¹

The TcG2 and TcG4 antigens used in the designing of BCV in this study were carefully selected because of their small size, high level of expression in clinically relevant isolates of all Tc lineages, and immunogenicity. In general, Tc infection elicits delayed and subpar T cell activation and low levels of effector molecules that fail to control tissue dissemination and replication of the parasite and provide no protection against repeat challenge infection. TcG2 and TcG4 primed the splenic and lymph node macrophages and dendritic cells with increased expression of markers of stimulation (CD80), antigen uptake (CD205), and antigen processing and presentation (MHCI/MHCII), and enhanced the frequencies of splenic Th1 CD4⁺ T cells and type 1 CD8⁺T cells capable of responding to in vitro Tc antigenic stimulation with production of several markers of cytolytic activity.²⁵ Further, TcG2/TcG4 promoted early activation and production of IFNγ by CD4⁺T and cytolytic molecules by CD4⁺ T and CD8⁺ T cells immediately after challenge infection that was further expanded during acute phase,^25,26 when parasite replicates and disseminates to tissues. Moreover, TcG2/TcG4-immunized mice responded to rechallenge infection with high levels of effector molecules production by CD4⁺ T and CD8⁺ T cells that offered even better control of tissue parasites than was observed after first infection.²⁶ These studies allow us to surmise excellent immunogenicity of TcG2 and TcG4 that protects against repeat Tc infections.

Along with the extensive research efforts dedicated to defining the appropriate antigenic immunogens, it is also important that adjuvants that can adequately enhance the protective immune response elicited by a given immunogen are also identified and developed further. Several adjuvants including integrin activators, recombinant cytokines (GM-CSF, IL-12), saponins, cyclic dinucleotides, and alum have been tested to boost the antigen-induced immune response against Tc, some of which offered limited to no improvement,^24,25 while others improved protective effects against Tc infection via triggering a Th1-dependent cell-mediated immunity, along with a balanced Th17 response.⁴² RIG-I is a cytoplasmic double-stranded sRNA pattern receptor that induces type 1 interferon and inflammatory cytokines production through activation of IRF3 and nuclear factor κB signaling. Thus, BCVR was designed to combine high-level antigen expression with immunostimulatory RNA-mediated innate immune activation that, in turn, translates into improved adaptive immune response even at low antigen doses.⁴³

BCV and BCVR exhibited excellent safety profiles, and both immunogens were highly effective in controlling the acute parasite replication, tissue parasite persistence, and inflammatory infiltrate. Subsequently myocardial fibrosis was decreased, and LV function was preserved in immunized/chronically infected mice, which otherwise were severely compromised in untreated/infected mice. These benefits were offered with expansion of mature T cell subsets (Teff-L and Tem-L) that provide stable, long-term protection from infection. Of these, CD8⁺ subsets displayed balanced immune characteristics geared toward limiting the tissue damage, while CD4⁺T subsets mounted cytolytic function, evidenced by increased GZM, PRF1, and TNFα production, for parasite killing. Such cytolytic function of the CD4⁺T cells has previously been described in experimental colorectal tumor,⁴⁴ and chronic viral infections.⁴⁵ This, to the best of our knowledge, is the first study demonstrating a nontoxic bicistronic immunogen that is effective in controlling the Tc-associated pathologies and preserving the heart function in a CD model. A recent study has documented the trivalent cruzipain-TS-ASP2 immunogen (Traspain) provided promising protection from Tc infection in mice.⁴²

Antigen-presenting cells serve as first responder to infection and play a crucial role in directly killing the pathogen and instructing T cells to develop into type 1 effector CD4⁺ and CD8⁺ cells that exhibit cytotoxic activity to kill extracellular and intracellular pathogens. In context of Tc infection, macrophages produce reactive oxygen and nitrogen species to exert trypanolytic effects through oxidation of cellular components, including DNA, proteins, and lipids of parasites.⁴⁶ However, free radicals, especially when produced in excess, cause collateral damage to bystander host cells.⁴⁷ Transcriptome profiling and other studies have shown that up-regulation of IFNγ-inducible genes and elevated levels of TNFα and lipid hydroperoxides were associated with worsened cardiac function in Chagas patients.^48,49 These and other studies conclude that while proinflammatory cytokine secreting CD4⁺ T and CD8⁺ T cells and cytotoxic T lymphocytes are essential for protection from infection, persistence of these cells is detrimental and associated with tissue pathology and tissue damage in Chagas patients.⁵⁰ Indeed, IL-10⁺ T cells predominantly found in clinically asymptomatic Chagas patients⁵¹ play a vital role in regulation of pathogenic responses. Our findings in this study provide compelling evidence that BCV and BCVR, after achieving parasite control, did not continue to stimulate pathological/inflammatory T cells in CD mice.

In general, T cells differentiate with the expression of T cell receptors and undergo training to prevent self-antigen recognition in the thymus and then migrate to secondary lymphoid organs.⁵² Upon encountering a foreign antigen presented by antigen-presenting cells, T cells rapidly proliferate and differentiate as effector cells, which can migrate to tissue sites of pathogen invasion and mediate cytotoxic killing of infected cells or assist humoral response. Subsequently, some cells transition to memory T cells, which can provide long-term immunity by responding more efficiently upon re-exposure to pathogens.^52,53 This process ensures an effective immune surveillance to combat infections. The assessment of T lymphocytes through 3 stages of differentiation showed that Tc prevents the generation of mature effectors and their conversion to memory T cells, while mature Teff-L and Tem-L subsets of CD4⁺ and CD8⁺ lineage that can provide an effective immune response peaked in immunized (BCVR>BCV) mice. Indeed, mature CD4⁺T subsets of immunized mice responded to challenge infection with type 1 cytotoxic activation, while CD8⁺ cells became resilient memory cells to provide protection from re-exposure. We have found that CD4⁺ and CD8⁺ cells of Tc-infected mice exhibit a continuous dependency on glucose metabolism, which can provide immediate energy for cell proliferation but compromises long-term survival and functional responsiveness and limit their ability to effectively respond to persistent infection.⁵⁴ We propose that CD4⁺ and CD8⁺ effector and memory T cells of immunized mice likely relied on lipid and thiol metabolism that can provide a survival advantage in oxidative stress conditions, as is noted during Tc infection,⁴⁷ and enhance longevity and effectiveness against re-exposures.

Anti-Tc antibodies must promote opsonization, phagocytosis, and complement-mediated killing of the parasite to prevent invasion and intracellular replication, and complement the T cells in controlling systemic Tc infection.⁵⁵ Current literature suggest that antibodies to Galα(1,3)Galβ(1,4)GlcNAc epitope of surface expressed mucin glycoproteins exhibit such antiparasite activity.¹⁴ Our data provide evidence that BCV and BCVR primed high-affinity, high-avidity neutralizing IgG response capable of inhibiting parasite invasion and survival.

Conclusions, Limitations, and Future Directions

This study has established that control of Tc infection and associated pathologies that contribute to heart failure is an achievable goal with the use of a bicistronic immunogen. BCV/BCVR are simple in design and delivery and offer low-cost, safe alternatives to prevent and control infection in adults that has not been uniformly feasible with benznidazole and nifurtimox treatment. The present study did not address gender associations (if any) because it was conducted in female mice only, and safety and efficacy of BCV/BCVR during pregnancy with the aim of decreasing the congenital Chagas disease also remains to be tested. Considering that TcG2/TcG4 are highly conserved across different Tc lineages and expressed in all life-cycle stages of the parasite, we expect that BCV/BCVR will offer broad-range immunity to diverse Tc isolates circulating in the endemic areas. Studies verifying the immunogenicity of BCV/BCVR in outbred experimental models or naturally infected animals will pave the way for the development of this immunotherapy for human use.

A few studies have documented limited efficacy of candidate immunogens in controlling CD postinfection, especially when the indeterminate-to-chronic phase of disease is developing in the infected host. It is speculated that immunogens to achieve a stimulation of adaptive immunity to attack persistent parasites need to be administered with other agents to prevent cardiac damage. Towards this goal, antiparasite therapies have been adjuvanted with antioxidants to prevent mitochondrial dysfunction and oxidative damage and improve LV contractile activity in the Chagas heart.⁵⁶ Delivery of low-dose benznidazole also enhanced the efficacy of a recombinant protein vaccine (TSA1/Tc24) in controlling cardiac fibrosis in chronically infected mice.⁵⁷ The development of next generation of immunotherapies against CD would likely involve efforts focused on enhancing the stability and immunogenicity of immunogens (with or without adjuvants and adjunct therapeutic agents) using liposomes, biopolymers, or virus like particles. Cost-effective, plant-based production of immunogens for oral delivery may also offer promising perspective for continuing the fight against CD.

Perspectives.

COMPETENCY IN MEDICAL KNOWLEDGE: The Tc protozoan parasite is the most common cause of nonischemic cardiomyopathy, known as CD. This chronic condition leads to severe heart failure, arrhythmias, and sudden death, affecting millions in endemic regions and posing a significant public health challenge in the United States. Benznidazole and nifurtimox are FDA approved for the treatment of CD in children. However, therapeutic failure and toxicity involving severe adverse effects often results in treatment discontinuation in adults. Thus, no treatment is available to prevent the risk of CD in adults. Host-targeted therapies emerging as anti-infectives interfere with host cell factors required for pathogen replication, boosting of immune defense, or modulating excessive inflammation, and serve as valuable adjuvant to standard antimicrobial treatments. We have developed regulatory compliant, bicistronic immunogens (BCV, BCVR) that are safe, and low-dosage, 2-dose treatment provided immune protection from parasites in a murine model of CD. BCV and BCVR had a powerful cardioprotective effect, evidenced by control of cardiac inflammation and fibrosis, and preservation of myocardial structure and LV systolic and diastolic function.

TRANSLATIONAL OUTLOOK: BCV/BCVR, once successfully tested for efficacy and safety in clinical trials, would potentially offer low-cost, safe alternatives to prevent and control CD in adults.

Data Availability

All data are provided within the paper.

Funding Support and Author Disclosures

Research reported in this publication was supported by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R44AI172437 to Drs Marathi, Garg, and De and award number R01AI136031 to Dr Garg. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Efforts of Drs Lokugamage and Choudhuri were partially supported by Robert E. Shope, MD Distinguished Chair in Global Health Endowment award to Dr Garg. Funders had no role in data collection or presentation. Dr De holds equity in 7 Hills Pharma. Dr Marathi is an investor and officer and holds equity in 7 Hills Pharma. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.