Epitope mapping of vaccine antigens Tc24 and TSA1 with antibodies from Trypanosoma cruzi infected patients

Epitope mapping of vaccine antigens Tc24 and TSA1 with antibodies from Trypanosoma cruzi infected patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Epitope mapping of vaccine antigens Tc24 and TSA1 with antibodies from Trypanosoma cruzi infected patients Eric Dumonteil, Claudia Herrera This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7216993/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Genes & Immunity → Version 1 posted 11 You are reading this latest preprint version Abstract Chagas disease is a zoonotic disease caused by Trypanosoma cruzi parasites. Tc24 and TSA1 parasite antigens are leading candidates for a therapeutic vaccine to treat infected patients to stop/delay the progression of chronic cardiomyopathy. As these antigens are nearing clinical trials, we aimed to assess their epitope recognition profile by antibodies from Chagas disease patients to better understand their immunogenicity in humans. Peptide microarrays covering Tc24-C4 and TSA1-C4 vaccine antigens were incubated with IgG from 27 T. cruzi -infected patients from Argentina, Honduras and Mexico. Most patients (20/27, 74%) had a highly similar recognition profile of both vaccine antigens, with the same immunodominant epitopes (three epitopes for Tc24-C4 and four for TSA1-C4). Remaining patients had limited reactivity against these antigens, targeting epitopes that varied among patients. All immunodominant epitopes were well conserved among T. cruzi strains and DTUs and most were accessible on the surface of the proteins. The immunodominant epitope recognition profile was observed independently of patient HLA profile, diagnostic test reactivity or T. cruzi parasite burden. Patients were infected with mixtures of TcI, TcII, TcIV, TcV and TcVI parasites. These results present an important baseline for assessing potential changes in epitope profiles following therapeutic vaccination in future clinical trials. Biological sciences/Immunology/Infectious diseases/Parasitic infection Biological sciences/Immunology/Vaccines/Protein vaccines Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Chagas disease is a major parasitic disease in the Americas, affecting over 6 million patients with a large disease burden. Infection with Trypanosoma cruzi , the protozoan parasite responsible for the disease, can lead to chronic cardiomyopathy and/or digestive disease such as megaesophagus or megacolon many years after infection in 30–40% of cases, while the remainer of patients is asymptomatic, despite parasite persistence in all patients 1 , 2 . Current treatments for infected patients are limited to benznidazole and nifurtimox, which are effective during the acute phase following infection, but their efficacy decreases during the chronic phase 3 . In addition, these drugs are associated with multiple side effects that add challenges to treatment completion 4 – 7 . As part of recent efforts at developing a vaccine that may be used as an immunotherapeutic treatment, alone or combined with benznidazole, two parasite antigens have emerged as promising for their inclusion in a vaccine formulation 8 . Tc24 is a flagellar associated calcium binding protein 9 , 10 and TSA1 is part of the trans-sialidase family of multicopy genes encoding major surface proteins of the parasite 11 . Extensive preclinical studies have shown that recombinant Tc24-C4 and TSA1-C4, which underwent cysteine mutagenesis to improve solubility and expression 12 – 14 , can effectively treat infected mice, reducing parasite burden, cardiac inflammation and fibrosis 14 – 19 . Advanced studies in non-human primates 20 – 22 and a first field trial in naturally infected dogs 23 have further evidenced that vaccine treatment can preserve cardiac function, making this vaccine formulation attractive for the development of a human vaccine. Remarkably, T. cruzi infected patients from Mexico were found to have antibodies against Tc24 and TSA1 antigens, and antigen-stimulated PBMCs from these patients were able to mount a recall response, indicating that both antigens may be immunogenic during natural infection in humans 24 . However, T. cruzi is genetically very diverse, being classified into seven major near-clade or discrete-typing units (DTUs) TcI to TcV and TcBat 25 , 26 . While both antigens have been found to be highly conserved across DTUs 27 , 28 , it is still unclear which specific epitopes of these antigens are being targeted by the host antibody response and how these may vary among human populations. As these antigens are nearing clinical trials, it is key to better understand their immunogenicity during natural T. cruzi infection. Thus, our aim was to assess the epitope recognition profile of Tc24-C4 and TSA1-C4 using antibodies from Chagas disease patients from various countries to better understand the immunogenicity of these antigens in humans. We used peptide microarrays to map epitopes from these antigens using plasma samples, and the recognition profile was analyzed in the context of patient characteristics including diagnostic test results, HLA and the infecting T. cruzi parasite strains. Material and Methods Patient samples The present study was approved by Tulane University institutional IRB (No. 2018–2237). De-identified archived samples were derived from a previous study on congenital T. cruzi transmission 29 and consisted in maternal plasma samples collected at birth in Argentina (N = 5), Honduras (N = 5) and Mexico (N = 19), with a well characterized T. cruzi infection status based on Stat-Pak (Chembio Diagnostics) and T-detect (InBios) rapid tests and a recombinant ELISA (Wiener), as well as T. cruzi PCR (Table 1 ). Parasite burden was also measured by qPCR. Ten samples were reactive with 2–3 serological tests, of which 8 were PCR positive. Seventeen samples were serodiscordant (reactive with 0–1 serological tests), but PCR positive for T. cruzi . Two seronegative and PCR negative control samples from Mexico were also included. Positive and serodiscordant samples were equally distributed among the three countries (Table 1 ). IgG was purified from plasma samples using Thermo Scientific™ Melon™ Gel IgG Spin Purification Kit as instructed, and IgG concentration was measured on a Nanodrop2000 spectrophotometer. Table 1 Patient characteristics ID Country Stat-Pak T -detect ELISA ELISA OD $ PCR Parasite burden* P1 Argentina - - - 0.093 + 1.477 P2 Argentina + - - 0 + 4.984 P3 Argentina + + + 2.574 + 14.443 P4 Argentina + - - 0.002 + 2.544 P5 Argentina + + + 2.841 + 4.506 P6 Honduras + + + 2.329 + 5.528 P7 Honduras + + + 1.965 + 8.591 P8 Honduras - - - 0.042 + 5.994 P9 Honduras + - - 0.045 + 3.455 P10 Honduras + - - 0.043 + 6.057 P11 Mexico + + - 0.099 - 0 P12 Mexico + + + 2.644 + 0.973 P13 Mexico + + + 1.556 + 1.469 P14 Mexico + - - 0.026 + 5.233 P15 Mexico - + - 0.03 + 5.288 P16 Mexico + - - 0.029 + 2.789 P17 Mexico - + - 0.037 + 4.154 P18 Mexico - + - 0.039 + 1.157 P19 Mexico + - - 0.041 + 2.207 P20 Mexico - + - 0.037 + 4.101 P21 Mexico + - - 0.034 + 2.837 P22 Mexico + - - 0.043 + 1.34 P23 Mexico + + + 0.777 - 0 P24 Mexico - + - 0.032 + 3.066 P25 Mexico + + + 2.174 + 3.502 P26 Mexico - + - 0.047 + 2.333 P27 Mexico + + + 2.561 + 1.47 N1 Mexico - - - 0.035 - ND N2 Mexico - - - 0.035 - ND $ optical density. * parasite equivalent/ml of blood. ND: not done. Peptide microarrays The sequence from Tc24-C4 and TSA1-C4 (REFS) were used to generate overlapping 15-mers peptide, with an overlap of 13 amino acids covering these antigens. The Herpes envelope epitope SHRANETIYNTTLKY sequence was included as a positive control. Each unique peptide was synthesized in duplicate on a C-terminal — βAla — Asp spacer on glass slides by Schafer-N (Denmark) and Cy3 blank spots were also included as negative controls. Microarray slides were deprotected in TFA EDT H 2 0 for 3h at room temperature and blocked overnight in 0.1% BSA, 0.1% Tween-20 in PBS. After blocking, slides were incubated for 1h at room temperature with purified IgG from patients (100 µg/ml IgG in 0. 1% BSA, 0.1% Tween-20 in PBS), washed 3 x 20 min with 0.1% BSA, 0.1% Tween-20 in PBS and incubated for 1h at room temperature with Cy3- goat anti-Hu IgG (1 µg/ml in 0.1% B SA, 0.1% Tween-20 in PBS). Slides were washed 3 x 20 min with 0.1% BSA in PBS, dried, and scanned using a laser scanner with 1 µm resolution to measure fluorescent signal intensity 30 . IgG binding intensity to duplicate peptides was averaged to assess binding profile along antigen sequences. 3D modeling of epitopes Epitopes were localized on the 3D structure of Tc24 (PDB ID 3CS1) and TSA1 AlphaFold prediction (Uniprot Q26971_TRYCR), to assess potential exposure to IgG in native antigens. Structures and epitopes were visualized in UCSF ChimeraX 31 . HLA typing and T. cruzi genotyping HLA typing of patients was performed by CD Genomics for Class I alleles of HLA-A, -B and -C and Class II alleles of HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, and -DR345 loci, based on sequencing of the respective genes. Allele frequencies from the HLA genes were compared between groups of patients using C 2 tests. Principal component analysis (PCA) was also performed to assess potential differences in HLA profile using the complete Class I and Class II gene set, or the Class II genes only, since these are thought to be more relevant for antibody responses 32 . Genotyping of T. cruzi was performed by deep sequencing of the mini-exon marker, that was PCR amplified as before 33 , 34 . Epitope conservation among T. cruzi strains Tc24 and TSA1 epitope conservation among T. cruzi strains and DTUs was assessed by BLASTp searches on a custom database from 32 T. cruzi genomes covering TcI (n = 12); TcII (n = 4); TcIII (n = 3); TcIV (n = 5); TcV (n = 3) and TcVI (n = 5)(Supplementary Table 1). Sequences from the epitopes identified in these genomes were aligned and visualized with WebLogo 35 . The proportion of genomes from each DTU in which epitopes were identified was also calculated. Results Epitope mapping for Tc24-C4 and TSA1-C4 vaccine antigens was performed using overlapping peptide microarrays with IgG from 27 T. cruzi infected patients and two negative controls. As expected, IgG from negative controls showed no binding to these two antigens. On the other hand, IgG from most T. cruzi infected patients showed some recognition of both antigens, although some variability was detected among individuals (Fig. 1 ). For Tc24-C4, a strongly recognized region with several overlapping epitopes was observed in the middle of the protein for 20/27 patients (E1 109–137 ), including patients from Argentina, Honduras and Mexico. Two weak but consistent epitopes were also observed on both sides of this immunodominant region, epitopes E2 69–83 and E3 179–193 . The seven remaining patients presented weak or no IgG binding to the immunodominant epitopes, but some recognition of several other epitopes that mostly differed among individuals. One patient was from Honduras and six were from Mexico (Fig. 1 ). Two patients (P22 and P27) showed negligible reactivity to Tc24-C4. For TSA1-C4, four dominant epitopes were identified for the same 20/27 patients strongly reacting to Tc24-C4, and TSA1-C4 epitopes were mostly localized in the C-terminus side of the protein (E1 611–627 ; E2 629–643 ; E3 505–523 ; and E4 281–297 ). Several minor epitopes also seemed to be consistently but weakly recognized by IgG from several of the patients. IgG from the other seven patients showed very weak recognition of TSA1-C4 with a few alternative epitopes in some individual samples, but three patients (P10, P25 and P26) also showed negligible recognition of this antigen (Fig. 1 ). Together, these results suggested that there were two main types of antigen recognition profiles: most patients (20/27, 74%, consisting of P1-P9, P11-P21) had a highly similar recognition profile of both vaccine antigens, with the same immunodominant epitopes. On the other hand, IgGs from a minority of patients (7/27, 26%, consisting of P10, P22-P27) presented a low/absent recognition of these immunodominant epitopes, but most recognized alternative epitopes that varied among individuals. Next, we assessed epitope sequence conservation among T. cruzi strains and DTUs. Both the immunodominant region and secondary Tc24-C4 epitopes were highly conserved with no/negligible amino acid substitutions, except in position 71 of epitope E2 69–83 (Fig. 2 ). Tc24-C4 epitopes were also present in all parasite DTUs analyzed. For TSA1-C4, the four immunodominant epitopes were somewhat less conserved, except for epitope E4 281–297 (Fig. 2 ). Nonetheless, the immunodominant sequences for these epitopes were the most frequent among parasite strains, and these were also detected in most strains and DTUs. The lack of some of the epitopes in one of the TcI strains likely reflected artefacts in genome sequencing/annotation rather than a true absence. Mapping of the epitopes on Tc24-C4 3D structure indicated that all three epitopes from this antigen were accessible on the surface of the proteins, except the N-terminus part of epitope E1 109–137 domain, which corresponded to part of a a-helix inside the protein (Fig. 3 A). While the epitope E1 109–137 contained several overlapping epitopes, this N-terminus part appeared to be less strongly recognized by antibodies than the rest of the E1 109–137 domain (Fig. 1 ). For TSA1-C4, the immunodominant epitopes E1 611–627 and E2 629–643 were well accessible on the surface of the proteins, while epitope E3 505–523 was only partially exposed in a groove of the protein, and only the two loops flanking b-sheets from Epitope E4 281–297 protruded on the surface of the protein (Fig. 3 B). These data suggested that IgG may bind to most of these epitopes within native proteins, except for epitopes E3 505–523 and E4 281–297 from TSA1-C4, for which binding may be more constrained. Because Chagas disease patients seemed to present two main types of antigen recognition profiles, we next assessed patient characteristics that may explain such differences. We first tested for potential differences in T. cruzi diagnostics (Table 1 ). There was no difference in reactivity for Stat-Pak (C 2 = 0.005; d.f.=1; P = 0.94), T-detect (C 2 = 0.59; d.f.=1; P = 0.44), ELISA (C 2 = 0.38; d.f.=1; P = 0.54) tests, PCR assay (C 2 = 0.58; d.f.=1; P = 0.45) reactivity or overall serodiscordance (C 2 = 0.14; d.f.=1; P = 0.71) between the two groups of patients with different antigen recognition profiles (Supplementary Table 2). Similarly, optical density (OD) reading of the ELISA assays and blood parasite burden were not significantly different between the two patient groups (t = 0.2, P = 0.86 and t = 1.5, P = 0.24, respectively) (Supplementary Table 2). Thus, patient response to the various T. cruzi diagnostic assays had no influence on their vaccine epitope recognition profiles. Since HLA is a major component of the immune response, we typed both Class I and Class II HLA from these patients and obtained reliable typing for 17 patients recognizing the immunodominant epitopes and 6 patients recognizing alternative epitopes. Comparison of HLA allele frequencies for individual genes indicated that there were no significant differences between the two groups of patients for any of the genes (Supplementary Table 3). Further, PCA analysis of individual HLA profiles also indicated a similar profile between the two groups (Fig. 4 A), even when only Class II genes were analyzed (Fig. 4 B). Thus, patient HLA profile did not explain the differences in their epitope recognition profiles of Tc24-C4 and TSA1-C4. Finally, we analyzed the contribution of the infecting T. cruzi strains. However, we were only able to genotype parasites from nine patients, seven presenting the immunodominant epitope profile, and two the alternative epitope profile (Fig. 4 C). All patients were infected with mixtures of parasite DTUs including TcI, TcII, TcIV, TcV and TcVI in variable proportions, with no clear pattern suggesting potential differences in parasite composition among the two patient groups, possibly due to the very low sample size. Remarkably, even patients with the same immunodominant epitope profile were infected with a broad diversity of parasite DTUs in different proportions, although TcV predominated. Discussion T. cruzi vaccine antigens Tc24-C4 and TSA1-C4 have been proposed as promising antigens for the development of an immunotherapeutic vaccine to stop or at least delay Chagas disease progression in infected humans 8 , 36 , 37 . In the context of forthcoming clinical trials, it is critical to better understand the immune response to these antigens during natural infections in diverse patient populations. We analyzed here the epitope recognition profile of these antigens by IgGs from patients from Argentina, Honduras and Mexico using overlapping peptide microarrays. A first key observation was that most (74%) of patients presented the same epitope recognition profile for both Tc24-C4 and TSA1-C4, with 3–4 consistently reactive epitopes in each antigen. This recognition profile was observed independently of patient diagnostic tests results, parasite burden, HLA profile or infecting parasite DTUs. Indeed, patients presenting this antigen recognition profile were infected with diverse mixtures of TcI, TcII, TcIV, TcV and TcVI in variable proportions. Together, these results suggest a strong immunodominant epitope recognition profile resulting from natural infections with highly variable mixtures of T. cruzi parasite strains, across broad human populations. We recently proposed the term “cruziome” to refer to the multiple strains co-infecting a host, which we argued may be central to driving the host immune response and Chagas disease progression 38 , as observed in naturally infected macaques 39 , 40 . It is thus striking that such diverse infections in different human populations produce such a consistent epitope recognition pattern of the two vaccine antigens. One factor that may have contributed to this immunodominant epitope profile among most patients was the high level of sequence conservation of these epitopes across all parasite DTUs. Even epitopes from TSA1-C4, which were somewhat less conserved that those from Tc24, still presented limited variability of selected amino acid that may have allowed for sufficient cross-reactivity. Indeed, antibody binding intensity to the epitopes resulted variable among patients, suggesting individual differences in affinity or antibody levels targeting these epitopes. As TSA1 is part of the large family of trans-sialidase multicopy genes, many of which may be simultaneously expressed during infection 41 , 42 , epitopes from different proteins but with sufficient sequence similarity may be targeted by cross-reactive antibodies. Analysis of the 3D structure of the antigens indicated that epitope E1 109–137 from Tc24 covers most of the second EF hand calcium binding domain (EF-2) and E3 179–193 coincides with most of EF-4 43 , which can explain their high sequence conservation. Furthermore, all three Tc24-C4 epitopes regions have been found to be under purifying selection, and epitopes E1 109–137 and E2 69–83 overlap with several HLA class I epitopes 27 . For TSA1-C4 antigen, epitope E2 629–643 includes the trans-sialidase VTVxNVxLYNR signature motif 44 , 45 . Epitope E1 611–627 has some similarity with a trans-sialidase immunodominant epitope (cluster 32 − 3) identified with phage-display library screening with patient sera 46 , and a murine immunodominant and partially protective CD8 + epitope overlaps with E3 505–523 47, 48 . On the other hand, all IgG epitopes identified here differed from HLA class I epitopes from TSA1 28 , as well as from previously identified 49 , 50 or predicted 51 epitopes from other members of the trans-sialidase family. Except for epitopes E3 505–523 and E4 281–297 from TSA1-C4, for which binding may be more constrained, all other epitopes appeared to be readily accessible on the surface of the 3D structure on the antigens, suggesting that IgGs may be able to bind to native proteins. This is also encouraging as vaccination with the recombinant antigens may also target these epitopes 21 , 22 , although this remains to be confirmed in future studies. Despite the consistent epitope recognition profile in most patients, a minority of patients (26%) showed a different profile, with limited/no recognition of the immunodominant epitopes, and some recognition of alternative epitopes that varied among individuals, with IgGs from a few individuals showing no recognition of either antigen. This agrees with previous studies indicating that most but not all patients have antibodies against Tc24 and TSA1 9, 24 . Similarly in dogs, about only about 80–93% of T. cruzi infected dogs have antibodies against these antigens 52 , 53 . However, it is unclear why these few patients presented such a different antibody profile against the vaccine antigens, as this did not appear to be associated with their overall immune response to infection, as assessed by the different serological diagnostic tests, nor with the parasite burden, nor their HLA profile. Our data are too limited to completely rule out parasite strains, but all patients for which parasite genotyping was successful harbored very diverse “cruziomes” as mentioned above. In conclusion, we identified major epitopes from TC24-C4 and TSA1-C4 vaccine antigens recognized by IgGs from T. cruzi infected patients following natural infections with mixtures of parasite strains from TcI, TcII, TcIV, TcV and TcVI DTUs. Most patients presented an immunodominant epitope recognition profile of both antigens, independently of their HLA profile, diagnostic test reactivity or T. cruzi parasite burden. These epitopes are conserved among the six DTUs frequently infecting humans. These results present an important baseline for assessing changes in epitope profiles following therapeutic vaccination in future clinical trials. Declarations CONFLICT OF INTEREST The authors declare no conflict of interest AUTHOR CONTRIBUTION ED: Conceptualization and design, Methodology, Formal analysis and investigation, Writing original draft, Reviewing and editing, Approval of the final manuscript. CH: Conceptualization and design, Methodology, Formal analysis and investigation, Reviewing and editing, Approval of the final manuscript. ACKNOWLEDGEMENTS This work was supported by grant R01HD94955 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development to CH, and grant R01AI162907 from the National Institute of Allergy and Infectious Diseases to ED. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Data Availability Statement All data generated or analysed during this study are included in this published article and its supplementary information files. References Rassi A, Jr., Rassi A, Marin-Neto JA. Chagas disease. Lancet 2010; 375(9723): 1388–402. Rassi A, Jr., Marin JAN, Rassi A. Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the BENznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial. Memorias do Instituto Oswaldo Cruz 2017; 112(3): 224–235. Pecoul B, Batista C, Stobbaerts E, Ribeiro I, Vilasanjuan R, Gascon J et al. The BENEFIT Trial: Where Do We Go from Here? PLoS neglected tropical diseases 2016; 10(2): e0004343. Viotti R, Vigliano C, Lococo B, Alvarez MG, Petti M, Bertocchi G et al. Side effects of benznidazole as treatment in chronic Chagas disease: fears and realities. Expert review of anti-infective therapy 2009; 7(2): 157–63. Pinazo MJ, Munoz J, Posada E, Lopez-Chejade P, Gallego M, Ayala E et al. Tolerance of benznidazole in treatment of Chagas' disease in adults. Antimicrobial agents and chemotherapy 2010; 54(11): 4896–9. Antinori S, Grande R, Bianco R, Traversi L, Cogliati C, Torzillo D et al. High frequency of adverse reactions and discontinuation with benznidazole treatment for chronic Chagas disease in Milano, Italy. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2015; 60(12): 1873–5. Forsyth CJ, Hernandez S, Olmedo W, Abuhamidah A, Traina MI, Sanchez DR et al. Safety Profile of Nifurtimox for Treatment of Chagas Disease in the United States. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2016. Dumonteil E, Bottazzi ME, Zhan B, Heffernan MJ, Jones K, Valenzuela JG et al. Accelerating the development of a therapeutic vaccine for human Chagas disease: rationale and prospects. Expert review of vaccines 2012; 11(9): 1043–55. Krautz GM, Galvao LM, Cancado JR, Guevara Espinoza A, Ouaissi A, Krettli AU. Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease. Journal of clinical microbiology 1995; 33: 2086–90. Ouaissi A, Aguirre T, Plumas Marty B, Piras M, Schoneck R, Gras Masse H et al. Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody. Biol Cell 1992; 75: 11–17. Wrightsman RA, Dawson BD, Fouts DL, Manning JE. Identification of immunodominant epitopes in Trypanosoma cruzi t rypomastigote surface antigen-1 protein that mask protective epitopes. J Immunol 1994; 153: 3148–3154. Seid CA, Jones KM, Pollet J, Keegan B, Hudspeth E, Hammond M et al. Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease. Human vaccines & immunotherapeutics 2017; 13(3): 621–633. Biter AB, Weltje S, Hudspeth EM, Seid CA, McAtee CP, Chen WH et al. Characterization and Stability of Trypanosoma cruzi 24-C4 (Tc24-C4), a Candidate Antigen for a Therapeutic Vaccine Against Chagas Disease. J Pharm Sci 2018; 107(5): 1468–1473. Dzul-Huchim VM, Ramirez-Sierra MJ, Martinez-Vega PP, Rosado-Vallado ME, Arana-Argaez VE, Ortega-Lopez J et al. Vaccine-linked chemotherapy with a low dose of benznidazole plus a bivalent recombinant protein vaccine prevents the development of cardiac fibrosis caused by Trypanosoma cruzi i n chronically-infected BALB/c mice. PLoS neglected tropical diseases 2022; 16(9): e0010258. Barry MA, Wang Q, Jones KM, Heffernan MJ, Buhaya MH, Beaumier CM et al. A therapeutic nanoparticle vaccine against Trypanosoma cruzi in a BALB/c mouse model of Chagas disease. Human vaccines & immunotherapeutics 2016; 12(4): 976–87. de la Cruz JJ, Villanueva-Lizama L, Dzul-Huchim V, Ramírez-Sierra MJ, Martinez-Vega P, Rosado-Vallado M et al. Production of recombinant TSA-1 and evaluation of its potential for the immuno-therapeutic control of Trypanosoma cruzi infection in mice. Human vaccines & immunotherapeutics 2019; 15(1): 210–219. Martinez-Campos V, Martinez-Vega P, Ramirez-Sierra MJ, Rosado-Vallado M, Seid CA, Hudspeth EM et al. Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against Trypanosoma cruzi infection in mice. Vaccine 2015; 33(36): 4505–12. Cruz-Chan JV, Villanueva-Lizama LE, Versteeg L, Damania A, Villar MJ, Gonzalez-Lopez C et al. Vaccine-linked chemotherapy induces IL-17 production and reduces cardiac pathology during acute Trypanosoma cruzi infection. Sci Rep 2021; 11(1): 3222. Jones K, Versteeg L, Damania A, Keegan B, Kendricks A, Pollet J et al. Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease. Infection and immunity 2018; 86(4). Dumonteil E, Herrera C, Marx PA. Safety and preservation of cardiac function following therapeutic vaccination against Trypanosoma cruzi in rhesus macaques. J Microbiol Immunol Infect 2023; 56(2): 400–407. Dumonteil E, Herrera C, Tu W, Goff K, Fahlberg M, Haupt E et al. Safety and immunogenicity of a recombinant vaccine against Trypanosoma cruzi in Rhesus macaques. Vaccine 2020; 38(29): 4584–4591. Dumonteil E, Tu W, Desale H, Goff K, Marx P, Ortega-Lopez J et al. Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naive rhesus macaques. J Biomed Sci 2024; 31(1): 58. Calderon-Quintal JA, Teh-Poot CF, Pech Piste LM, Martinez-Vega PP, Dzul-Huchim V, Torres-Acosta F et al. Randomized field trial of a therapeutic vaccine against Trypanosoma cruzi natural infection in dogs and correlates for efficacy. bioRxiv 2025. Villanueva-Lizama L, Cruz-Chan JV, Cetina-Aguilar AC, Herrera-Sanchez LF, Rodriguez-Perez JM, Rosado-Vallado ME et al. Trypanosoma cruzi vaccine candidate antigens Tc24 and TSA-1 recall memory immune response associated with HLA-A and –B supertypes in Chagasic chronic patients from Mexico. PLoS neglected tropical diseases 2018; 12(1): e0006240. Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Memorias do Instituto Oswaldo Cruz 2009; 104(7): 1051–4. Zingales B, Miles MA, Moraes CB, Luquetti A, Guhl F, Schijman AG et al. Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity. Memorias do Instituto Oswaldo Cruz 2014; 109(6): 828–33. Arnal A, Villanueva-Lizama L, Teh-Poot C, Herrera C, Dumonteil E. Extent of polymorphism and selection pressure on the Trypanosoma cruzi vaccine candidate antigen Tc24. Evol Applications 2020; 13: 2663–2672. Knight JM, Zingales B, Bottazzi ME, Hotez P, Zhan B. Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1. Parasite immunology 2014; 36(12): 708–12. Buekens P, Cafferata ML, Alger J, Althabe F, Belizan JM, Bustamante N et al. Congenital Transmission of Trypanosoma cruzi in Argentina, Honduras, and Mexico: An Observational Prospective Study. The American journal of tropical medicine and hygiene 2018; 98(2): 478–485. Majeau A, Dumonteil E, Herrera C. Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay. Emerg Microbes Infect 2024; 13(1): 2315964. Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH et al. UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 2023; 32(11): e4792. Khan T, Rahman M, Ahmed I, Al Ali F, Jithesh PV, Marr N. Human leukocyte antigen class II gene diversity tunes antibody repertoires to common pathogens. Front Immunol 2022; 13: 856497. Villanueva-Lizama L, Teh-Poot C, Majeau A, Herrera C, Dumonteil E. Molecular genotyping of Trypanosoma cruzi by next-generation sequencing of the mini-exon gene reveals infections with multiple parasite DTUs in Chagasic patients from Yucatan, Mexico. J Inf Dis 2019; 219(12): 1980–1988. Majeau A, Herrera C, Dumonteil E. An improved approach to Trypanosoma cruzi molecular genotyping by next-generation sequencing of the mini-exon gene. Methods Mol Biol 2019; 1955: 47–60. Crooks GE, Hon G, Chandonia J-M, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004; 14: 1188–1190. Dumonteil E, Herrera C. The Case for the Development of a Chagas Disease Vaccine: Why? How? When? Trop Med Infect Dis 2021; 6(1). Dumonteil E, Herrera C. Vaccines against chronic Trypanosoma cruzi infection: progress, challenges and future directions. 2025: submitted. Carlier Y, Dumonteil E, Herrera C, Waleckx E, Tibayrenc M, Buekens P et al. Coinfection by multiple Trypanosoma cruzi clones: a new perspective on host-parasite relationship with consequences for pathogenesis and management of Chagas disease. Microbiol Mol Biol Rev 2025: e0024224. Desale H, Tu W, Goff K, Marx PA, Herrera C, Dumonteil E. PBMC transcriptomic signatures reflect Trypanosoma cruzi strain diversity and trained immunity in chronically infected macaques. JCI Insight 2025; 10(4): e186003. Dumonteil E, Desale H, Tu W, Hernández-Cuevas N, Shroyer M, Goff K et al. Intra-host Trypanosoma cruzi strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis. Microbiol Spectr 2023; 11(5): e0423622. Jager AV, Muia RP, Campetella O. Stage-specific expression of Trypanosoma cruzi trans-sialidase involves highly conserved 3' untranslated regions. FEMS microbiology letters 2008; 283(2): 182–8. Freitas LM, dos Santos SL, Rodrigues-Luiz GF, Mendes TA, Rodrigues TS, Gazzinelli RT et al. Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity. PloS one 2011; 6(10): e25914. Maldonado RA, Mirzoeva S, Godsel LM, Lukas TJ, Goldenberg S, Watterson DM et al. Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein. Molecular and biochemical parasitology 1999; 101(1–2): 61–70. Colli W. Trans-sialidase: a unique enzyme activity discovered in the protozoan Trypanosoma cruzi. FASEB J 1993; 7(13): 1257–64. Alves MJ, Colli W. Role of the gp85/trans-sialidase superfamily of glycoproteins in the interaction of Trypanosoma cruzi with host structures. Subcell Biochem 2008; 47: 58–69. Teixeira AAR, Carnero LR, Kuramoto A, Tang FHF, Gomes CH, Pereira NB et al. A refined genome phage display methodology delineates the human antibody response in patients with Chagas disease. iScience 2021; 24(6): 102540. Wizel B, Nunes M, Tarleton RL. Identification of Trypanosoma cruzi trans-sialidase family members as targets of protective CD8 + TC1 responses. J Immunol 1997; 159(12): 6120–6130. Martin DL, Weatherly DB, Laucella SA, Cabinian MA, Crim MT, Sullivan S et al. CD8 + T-Cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes. PLoS pathogens 2006; 2(8): e77. Pitcovsky TA, Mucci J, Alvarez P, Leguizamon MS, Burrone O, Alzari PM et al. Epitope mapping of trans-sialidase from Trypanosoma cruzi reveals the presence of several cross-reactive determinants. Infection and immunity 2001; 69(3): 1869–75. Pitcovsky TA, Buscaglia CA, Mucci J, Campetella O. A functional network of intramolecular cross-reacting epitopes delays the elicitation of neutralizing antibodies to Trypanosoma cruzi trans-sialidase. J Infect Dis 2002; 186(3): 397–404. Pacini MF, Perdomini A, Bulfoni Balbi C, Dinatale B, Herrera FE, Perez AR et al. The high identity of the Trypanosoma cruzi Group-I of trans-sialidases points them as promising vaccine immunogens. Proteins 2023; 91(10): 1444–1460. Mejia R, Verocai GG, Mosley IA, Zhan B, Vongthavaravat L, Busselman RE et al. Evaluation of a novel Tc-24 recombinant antigen ELISA for serologic testing for Trypanosoma cruzi in dogs. bioRxiv 2024. Calderón-Quintal JA, Escalante-Talavera MJ, Teh-Poot C, Carrera-Campellone MN, Martinez-Vega PP, Dzul-Huchim V et al. Natural infection of Trypanosoma cruzi in client-owned-dogs from rural Yucatan, Mexico. Sci Rep 2025; 15(1): 10263. Additional Declarations There is NO conflict of interest to disclose. The authors declare no conflict of interest. Supplementary Files SupplementaryTable1.docx Supplementary Table 1 SupplementaryTable2.docx Supplementary Table 2 SupplementaryTable3.docx Supplementary Table 3 Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Genes & Immunity → Version 1 posted Editorial decision: revise 04 Sep, 2025 Review # 2 received at journal 18 Aug, 2025 Review # 1 received at journal 13 Aug, 2025 Reviewer # 4 agreed at journal 07 Aug, 2025 Reviewer # 3 agreed at journal 05 Aug, 2025 Reviewer # 2 agreed at journal 04 Aug, 2025 Reviewer # 1 agreed at journal 04 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Submission checks completed at journal 28 Jul, 2025 First submitted to journal 25 Jul, 2025 Editor assigned by journal 25 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7216993","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":495470680,"identity":"ef0322b9-82de-4309-a79a-a9b4b3c880fe","order_by":0,"name":"Eric Dumonteil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYFCCBAjFz8DAeABFhIAWAwbJNgYGErUYHCNWCz97jumGjzv+yBnf7zE48HOHDUjEAK8WyZ43ZjdnnjEwNjvGY3Cw90waSAS/FoMbOWa3edsMErcBtRzgbTsMEsGvxR6qpX5zG9CWv23/QSIEbJGAaEkwYOMxOMzbdgAkgl+LxJlnZTdnthkbzjiWVnBYti2ZByhSgFcLf3vythsf2+Tk+ZsPb3z4ts1ODiiyAa8WDMBDmvJRMApGwSgYBVgBAMZdSPNDCee/AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9376-0209","institution":"Department of Tropical Medicine, Vector-Borne and Infectious Disease Research Center, School of Public Health and Tropical Medicine, Tulane University, New Orleans","correspondingAuthor":true,"prefix":"","firstName":"Eric","middleName":"","lastName":"Dumonteil","suffix":""},{"id":495470681,"identity":"a732b13b-ff62-4cdc-85f3-6cbae0098254","order_by":1,"name":"Claudia Herrera","email":"","orcid":"","institution":"School of Public Health and Tropical Medicine, Tulane University, New Orleans","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Herrera","suffix":""}],"badges":[],"createdAt":"2025-07-25 19:55:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7216993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7216993/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41435-026-00380-8","type":"published","date":"2026-02-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88504893,"identity":"46ef11fe-44a2-43f0-9beb-4d9ccf126232","added_by":"auto","created_at":"2025-08-07 07:12:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":406603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpitope mapping of Tc24-C4 and TSA1-C4 vaccine antigens.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverlapping peptides covering the full length of the primary sequence for Tc24-C4 (left) and TSA1-C4 (right), horizontal axis, were evaluated in microarrays with IgGs from 27 individual Chagas disease patients from the indicated countries (P1-P27) and 2 negative controls (C1 and C2). The main epitopes (E1-E4) are indicated for each antigen.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/f62138e517e056bf0f044a99.png"},{"id":88503692,"identity":"7f54f9cf-4fbf-49ec-b059-342f9cbdee4f","added_by":"auto","created_at":"2025-08-07 07:04:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpitope sequence conservation among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. cruzi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence conservation of the indicated epitopes was visualized using Weblogo. Amino acids are color-coded according to their chemistry: Green= Polar; Purple= Neutral; Blue= Basic; Red= Acidic; Black= Hydrophobic. The bar graphs show the proportion of \u003cem\u003eT. cruzi\u003c/em\u003estrains from DTUs TcI to TcVI in which the epitope was identified, with the full bars indicating strains in which it is present and the empty bars indicating strains in which it is absent.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/5cc94c8b5069cf27ee14d26c.png"},{"id":88503694,"identity":"ef581305-c509-4a5c-bd8c-4b41d672098f","added_by":"auto","created_at":"2025-08-07 07:04:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":585938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization of epitopes on antigen 3D structures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpitopes (E1 to E4) from Tc24-C4 (\u003cstrong\u003eA\u003c/strong\u003e) and TSA1-C4 (\u003cstrong\u003eB\u003c/strong\u003e) are highlighted in colors in ribbons (top) and surface (bottom) renderings. The N terminus (red) and C-terminus (green) of the proteins are indicated.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/46f1e7d3a2eb6e5da5a457e3.png"},{"id":88503661,"identity":"3681084e-78ba-4f58-bc52-e4b3a86e31cf","added_by":"auto","created_at":"2025-08-07 07:04:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePatient HLA profile and infecting \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. cruzi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCA analysis of Class I and Class II (\u003cstrong\u003eA\u003c/strong\u003e) and only Class II HLA alles (\u003cstrong\u003eB\u003c/strong\u003e). Patients are grouped as with an immunodominant epitope profile (P1-P9, P11-P21, N=20), or as with an alternative epitope profile (P10, P22-P27, N=7). (\u003cstrong\u003eC\u003c/strong\u003e) DTU composition of \u003cem\u003eT. cruzi\u003c/em\u003e strains infecting individual patients. The proportion of DTUs is shown, with DTU color-coded as indicated.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/141b669b607e89c903670a9b.png"},{"id":102386999,"identity":"134fb939-70f8-46a0-94e1-2532dfb4b943","added_by":"auto","created_at":"2026-02-11 08:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2447104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/a94526d5-55ae-4ebc-bf51-c3b4f6b608d0.pdf"},{"id":88504892,"identity":"80f23d11-7560-432d-a0c1-55a2cf00d697","added_by":"auto","created_at":"2025-08-07 07:12:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19366,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/c508ac0bf0d4e2b59a4f9862.docx"},{"id":88504889,"identity":"0f9aa2a4-b039-41d5-9912-28c78fe74320","added_by":"auto","created_at":"2025-08-07 07:12:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19084,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/a146228e8709197693b1fc12.docx"},{"id":88503668,"identity":"891663a5-dee5-47cb-a1ba-b4e9648d191d","added_by":"auto","created_at":"2025-08-07 07:04:14","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21308,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"SupplementaryTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7216993/v1/b65b1c0f6af19deb82c81776.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.\nThe authors declare no conflict of interest.","formattedTitle":"Epitope mapping of vaccine antigens Tc24 and TSA1 with antibodies from Trypanosoma cruzi infected patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChagas disease is a major parasitic disease in the Americas, affecting over 6\u0026nbsp;million patients with a large disease burden. Infection with \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e, the protozoan parasite responsible for the disease, can lead to chronic cardiomyopathy and/or digestive disease such as megaesophagus or megacolon many years after infection in 30\u0026ndash;40% of cases, while the remainer of patients is asymptomatic, despite parasite persistence in all patients \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Current treatments for infected patients are limited to benznidazole and nifurtimox, which are effective during the acute phase following infection, but their efficacy decreases during the chronic phase \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition, these drugs are associated with multiple side effects that add challenges to treatment completion \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs part of recent efforts at developing a vaccine that may be used as an immunotherapeutic treatment, alone or combined with benznidazole, two parasite antigens have emerged as promising for their inclusion in a vaccine formulation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Tc24 is a flagellar associated calcium binding protein \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and TSA1 is part of the trans-sialidase family of multicopy genes encoding major surface proteins of the parasite \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Extensive preclinical studies have shown that recombinant Tc24-C4 and TSA1-C4, which underwent cysteine mutagenesis to improve solubility and expression \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, can effectively treat infected mice, reducing parasite burden, cardiac inflammation and fibrosis \u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Advanced studies in non-human primates \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and a first field trial in naturally infected dogs \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e have further evidenced that vaccine treatment can preserve cardiac function, making this vaccine formulation attractive for the development of a human vaccine.\u003c/p\u003e\u003cp\u003eRemarkably, \u003cem\u003eT. cruzi\u003c/em\u003e infected patients from Mexico were found to have antibodies against Tc24 and TSA1 antigens, and antigen-stimulated PBMCs from these patients were able to mount a recall response, indicating that both antigens may be immunogenic during natural infection in humans \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003eT. cruzi\u003c/em\u003e is genetically very diverse, being classified into seven major near-clade or discrete-typing units (DTUs) TcI to TcV and TcBat \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. While both antigens have been found to be highly conserved across DTUs \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, it is still unclear which specific epitopes of these antigens are being targeted by the host antibody response and how these may vary among human populations. As these antigens are nearing clinical trials, it is key to better understand their immunogenicity during natural \u003cem\u003eT. cruzi\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003eThus, our aim was to assess the epitope recognition profile of Tc24-C4 and TSA1-C4 using antibodies from Chagas disease patients from various countries to better understand the immunogenicity of these antigens in humans. We used peptide microarrays to map epitopes from these antigens using plasma samples, and the recognition profile was analyzed in the context of patient characteristics including diagnostic test results, HLA and the infecting \u003cem\u003eT. cruzi\u003c/em\u003e parasite strains.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003ePatient samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe present study was approved by Tulane University institutional IRB (No. 2018\u0026ndash;2237). De-identified archived samples were derived from a previous study on congenital \u003cem\u003eT. cruzi\u003c/em\u003e transmission \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and consisted in maternal plasma samples collected at birth in Argentina (N\u0026thinsp;=\u0026thinsp;5), Honduras (N\u0026thinsp;=\u0026thinsp;5) and Mexico (N\u0026thinsp;=\u0026thinsp;19), with a well characterized \u003cem\u003eT. cruzi\u003c/em\u003e infection status based on Stat-Pak (Chembio Diagnostics) and T-detect (InBios) rapid tests and a recombinant ELISA (Wiener), as well as \u003cem\u003eT. cruzi\u003c/em\u003e PCR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Parasite burden was also measured by qPCR. Ten samples were reactive with 2\u0026ndash;3 serological tests, of which 8 were PCR positive. Seventeen samples were serodiscordant (reactive with 0\u0026ndash;1 serological tests), but PCR positive for \u003cem\u003eT. cruzi\u003c/em\u003e. Two seronegative and PCR negative control samples from Mexico were also included. Positive and serodiscordant samples were equally distributed among the three countries (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). IgG was purified from plasma samples using Thermo Scientific\u0026trade; Melon\u0026trade; Gel IgG Spin Purification Kit as instructed, and IgG concentration was measured on a Nanodrop2000 spectrophotometer.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCountry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStat-Pak\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT -detect\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eELISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eELISA OD\u003csup\u003e$\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePCR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eParasite burden*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArgentina\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.093\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.477\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArgentina\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.984\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArgentina\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.574\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.443\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArgentina\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.544\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArgentina\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.841\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.506\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHonduras\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.528\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHonduras\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8.591\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHonduras\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.994\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHonduras\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.455\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHonduras\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.043\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.057\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.099\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.644\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.973\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.556\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.469\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.233\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.288\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.789\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.154\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.157\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.041\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.207\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.101\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.837\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.043\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.777\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.066\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.502\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMexico\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e$\u003c/sup\u003e optical density. * parasite equivalent/ml of blood. ND: not done.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide microarrays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe sequence from Tc24-C4 and TSA1-C4 (REFS) were used to generate overlapping 15-mers peptide, with an overlap of 13 amino acids covering these antigens. The Herpes envelope epitope SHRANETIYNTTLKY sequence was included as a positive control. Each unique peptide was synthesized in duplicate on a C-terminal \u0026mdash; βAla \u0026mdash; Asp spacer on glass slides by Schafer-N (Denmark) and Cy3 blank spots were also included as negative controls. Microarray slides were deprotected in TFA EDT H\u003csub\u003e2\u003c/sub\u003e0 for 3h at room temperature and blocked overnight in 0.1% BSA, 0.1% Tween-20 in PBS. After blocking, slides were incubated for 1h at room temperature with purified IgG from patients (100 \u0026micro;g/ml IgG in 0. 1% BSA, 0.1% Tween-20 in PBS), washed 3 x 20 min with 0.1% BSA, 0.1% Tween-20 in PBS and incubated for 1h at room temperature with Cy3- goat anti-Hu IgG (1 \u0026micro;g/ml in 0.1% B SA, 0.1% Tween-20 in PBS). Slides were washed 3 x 20 min with 0.1% BSA in PBS, dried, and scanned using a laser scanner with 1 \u0026micro;m resolution to measure fluorescent signal intensity \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. IgG binding intensity to duplicate peptides was averaged to assess binding profile along antigen sequences.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3D modeling of epitopes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEpitopes were localized on the 3D structure of Tc24 (PDB ID 3CS1) and TSA1 AlphaFold prediction (Uniprot Q26971_TRYCR), to assess potential exposure to IgG in native antigens. Structures and epitopes were visualized in UCSF ChimeraX \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHLA typing and\u003c/b\u003e \u003cb\u003eT. cruzi\u003c/b\u003e \u003cb\u003egenotyping\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHLA typing of patients was performed by CD Genomics for Class I alleles of HLA-A, -B and -C and Class II alleles of HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, and -DR345 loci, based on sequencing of the respective genes. Allele frequencies from the HLA genes were compared between groups of patients using C\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e tests. Principal component analysis (PCA) was also performed to assess potential differences in HLA profile using the complete Class I and Class II gene set, or the Class II genes only, since these are thought to be more relevant for antibody responses \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Genotyping of \u003cem\u003eT. cruzi\u003c/em\u003e was performed by deep sequencing of the mini-exon marker, that was PCR amplified as before \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpitope conservation among\u003c/b\u003e \u003cb\u003eT. cruzi\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTc24 and TSA1 epitope conservation among \u003cem\u003eT. cruzi\u003c/em\u003e strains and DTUs was assessed by BLASTp searches on a custom database from 32 \u003cem\u003eT. cruzi\u003c/em\u003e genomes covering TcI (n\u0026thinsp;=\u0026thinsp;12); TcII (n\u0026thinsp;=\u0026thinsp;4); TcIII (n\u0026thinsp;=\u0026thinsp;3); TcIV (n\u0026thinsp;=\u0026thinsp;5); TcV (n\u0026thinsp;=\u0026thinsp;3) and TcVI (n\u0026thinsp;=\u0026thinsp;5)(Supplementary Table\u0026nbsp;1). Sequences from the epitopes identified in these genomes were aligned and visualized with WebLogo \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The proportion of genomes from each DTU in which epitopes were identified was also calculated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEpitope mapping for Tc24-C4 and TSA1-C4 vaccine antigens was performed using overlapping peptide microarrays with IgG from 27 \u003cem\u003eT. cruzi\u003c/em\u003e infected patients and two negative controls. As expected, IgG from negative controls showed no binding to these two antigens. On the other hand, IgG from most \u003cem\u003eT. cruzi\u003c/em\u003e infected patients showed some recognition of both antigens, although some variability was detected among individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For Tc24-C4, a strongly recognized region with several overlapping epitopes was observed in the middle of the protein for 20/27 patients (E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e), including patients from Argentina, Honduras and Mexico. Two weak but consistent epitopes were also observed on both sides of this immunodominant region, epitopes E2\u003csup\u003e69\u0026ndash;83\u003c/sup\u003e and E3\u003csup\u003e179\u0026ndash;193\u003c/sup\u003e. The seven remaining patients presented weak or no IgG binding to the immunodominant epitopes, but some recognition of several other epitopes that mostly differed among individuals. One patient was from Honduras and six were from Mexico (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two patients (P22 and P27) showed negligible reactivity to Tc24-C4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor TSA1-C4, four dominant epitopes were identified for the same 20/27 patients strongly reacting to Tc24-C4, and TSA1-C4 epitopes were mostly localized in the C-terminus side of the protein (E1\u003csup\u003e611\u0026ndash;627\u003c/sup\u003e; E2\u003csup\u003e629\u0026ndash;643\u003c/sup\u003e; E3\u003csup\u003e505\u0026ndash;523\u003c/sup\u003e; and E4\u003csup\u003e281\u0026ndash;297\u003c/sup\u003e). Several minor epitopes also seemed to be consistently but weakly recognized by IgG from several of the patients. IgG from the other seven patients showed very weak recognition of TSA1-C4 with a few alternative epitopes in some individual samples, but three patients (P10, P25 and P26) also showed negligible recognition of this antigen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTogether, these results suggested that there were two main types of antigen recognition profiles: most patients (20/27, 74%, consisting of P1-P9, P11-P21) had a highly similar recognition profile of both vaccine antigens, with the same immunodominant epitopes. On the other hand, IgGs from a minority of patients (7/27, 26%, consisting of P10, P22-P27) presented a low/absent recognition of these immunodominant epitopes, but most recognized alternative epitopes that varied among individuals.\u003c/p\u003e\u003cp\u003eNext, we assessed epitope sequence conservation among \u003cem\u003eT. cruzi\u003c/em\u003e strains and DTUs. Both the immunodominant region and secondary Tc24-C4 epitopes were highly conserved with no/negligible amino acid substitutions, except in position 71 of epitope E2\u003csup\u003e69\u0026ndash;83\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tc24-C4 epitopes were also present in all parasite DTUs analyzed. For TSA1-C4, the four immunodominant epitopes were somewhat less conserved, except for epitope E4\u003csup\u003e281\u0026ndash;297\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nonetheless, the immunodominant sequences for these epitopes were the most frequent among parasite strains, and these were also detected in most strains and DTUs. The lack of some of the epitopes in one of the TcI strains likely reflected artefacts in genome sequencing/annotation rather than a true absence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMapping of the epitopes on Tc24-C4 3D structure indicated that all three epitopes from this antigen were accessible on the surface of the proteins, except the N-terminus part of epitope E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e domain, which corresponded to part of a a-helix inside the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While the epitope E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e contained several overlapping epitopes, this N-terminus part appeared to be less strongly recognized by antibodies than the rest of the E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For TSA1-C4, the immunodominant epitopes E1\u003csup\u003e611\u0026ndash;627\u003c/sup\u003e and E2\u003csup\u003e629\u0026ndash;643\u003c/sup\u003e were well accessible on the surface of the proteins, while epitope E3\u003csup\u003e505\u0026ndash;523\u003c/sup\u003e was only partially exposed in a groove of the protein, and only the two loops flanking b-sheets from Epitope E4\u003csup\u003e281\u0026ndash;297\u003c/sup\u003e protruded on the surface of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These data suggested that IgG may bind to most of these epitopes within native proteins, except for epitopes E3\u003csup\u003e505\u0026ndash;523\u003c/sup\u003e and E4\u003csup\u003e281\u0026ndash;297\u003c/sup\u003e from TSA1-C4, for which binding may be more constrained.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause Chagas disease patients seemed to present two main types of antigen recognition profiles, we next assessed patient characteristics that may explain such differences. We first tested for potential differences in \u003cem\u003eT. cruzi\u003c/em\u003e diagnostics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no difference in reactivity for Stat-Pak (C\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.005; d.f.=1; P\u0026thinsp;=\u0026thinsp;0.94), T-detect (C\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.59; d.f.=1; P\u0026thinsp;=\u0026thinsp;0.44), ELISA (C\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.38; d.f.=1; P\u0026thinsp;=\u0026thinsp;0.54) tests, PCR assay (C\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.58; d.f.=1; P\u0026thinsp;=\u0026thinsp;0.45) reactivity or overall serodiscordance (C\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.14; d.f.=1; P\u0026thinsp;=\u0026thinsp;0.71) between the two groups of patients with different antigen recognition profiles (Supplementary Table\u0026nbsp;2). Similarly, optical density (OD) reading of the ELISA assays and blood parasite burden were not significantly different between the two patient groups (t\u0026thinsp;=\u0026thinsp;0.2, P\u0026thinsp;=\u0026thinsp;0.86 and t\u0026thinsp;=\u0026thinsp;1.5, P\u0026thinsp;=\u0026thinsp;0.24, respectively) (Supplementary Table\u0026nbsp;2). Thus, patient response to the various \u003cem\u003eT. cruzi\u003c/em\u003e diagnostic assays had no influence on their vaccine epitope recognition profiles.\u003c/p\u003e\u003cp\u003eSince HLA is a major component of the immune response, we typed both Class I and Class II HLA from these patients and obtained reliable typing for 17 patients recognizing the immunodominant epitopes and 6 patients recognizing alternative epitopes. Comparison of HLA allele frequencies for individual genes indicated that there were no significant differences between the two groups of patients for any of the genes (Supplementary Table\u0026nbsp;3). Further, PCA analysis of individual HLA profiles also indicated a similar profile between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), even when only Class II genes were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, patient HLA profile did not explain the differences in their epitope recognition profiles of Tc24-C4 and TSA1-C4. Finally, we analyzed the contribution of the infecting \u003cem\u003eT. cruzi\u003c/em\u003e strains. However, we were only able to genotype parasites from nine patients, seven presenting the immunodominant epitope profile, and two the alternative epitope profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). All patients were infected with mixtures of parasite DTUs including TcI, TcII, TcIV, TcV and TcVI in variable proportions, with no clear pattern suggesting potential differences in parasite composition among the two patient groups, possibly due to the very low sample size. Remarkably, even patients with the same immunodominant epitope profile were infected with a broad diversity of parasite DTUs in different proportions, although TcV predominated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eT. cruzi\u003c/em\u003e vaccine antigens Tc24-C4 and TSA1-C4 have been proposed as promising antigens for the development of an immunotherapeutic vaccine to stop or at least delay Chagas disease progression in infected humans \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In the context of forthcoming clinical trials, it is critical to better understand the immune response to these antigens during natural infections in diverse patient populations. We analyzed here the epitope recognition profile of these antigens by IgGs from patients from Argentina, Honduras and Mexico using overlapping peptide microarrays.\u003c/p\u003e\u003cp\u003eA first key observation was that most (74%) of patients presented the same epitope recognition profile for both Tc24-C4 and TSA1-C4, with 3\u0026ndash;4 consistently reactive epitopes in each antigen. This recognition profile was observed independently of patient diagnostic tests results, parasite burden, HLA profile or infecting parasite DTUs. Indeed, patients presenting this antigen recognition profile were infected with diverse mixtures of TcI, TcII, TcIV, TcV and TcVI in variable proportions. Together, these results suggest a strong immunodominant epitope recognition profile resulting from natural infections with highly variable mixtures of \u003cem\u003eT. cruzi\u003c/em\u003e parasite strains, across broad human populations. We recently proposed the term \u0026ldquo;cruziome\u0026rdquo; to refer to the multiple strains co-infecting a host, which we argued may be central to driving the host immune response and Chagas disease progression \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, as observed in naturally infected macaques \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is thus striking that such diverse infections in different human populations produce such a consistent epitope recognition pattern of the two vaccine antigens.\u003c/p\u003e\u003cp\u003eOne factor that may have contributed to this immunodominant epitope profile among most patients was the high level of sequence conservation of these epitopes across all parasite DTUs. Even epitopes from TSA1-C4, which were somewhat less conserved that those from Tc24, still presented limited variability of selected amino acid that may have allowed for sufficient cross-reactivity. Indeed, antibody binding intensity to the epitopes resulted variable among patients, suggesting individual differences in affinity or antibody levels targeting these epitopes. As TSA1 is part of the large family of trans-sialidase multicopy genes, many of which may be simultaneously expressed during infection \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, epitopes from different proteins but with sufficient sequence similarity may be targeted by cross-reactive antibodies.\u003c/p\u003e\u003cp\u003eAnalysis of the 3D structure of the antigens indicated that epitope E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e from Tc24 covers most of the second EF hand calcium binding domain (EF-2) and E3\u003csup\u003e179\u0026ndash;193\u003c/sup\u003e coincides with most of EF-4 \u003csup\u003e43\u003c/sup\u003e, which can explain their high sequence conservation. Furthermore, all three Tc24-C4 epitopes regions have been found to be under purifying selection, and epitopes E1\u003csup\u003e109\u0026ndash;137\u003c/sup\u003e and E2\u003csup\u003e69\u0026ndash;83\u003c/sup\u003e overlap with several HLA class I epitopes \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor TSA1-C4 antigen, epitope E2\u003csup\u003e629\u0026ndash;643\u003c/sup\u003e includes the trans-sialidase VTVxNVxLYNR signature motif \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Epitope E1\u003csup\u003e611\u0026ndash;627\u003c/sup\u003e has some similarity with a trans-sialidase immunodominant epitope (cluster 32\u0026thinsp;\u0026minus;\u0026thinsp;3) identified with phage-display library screening with patient sera \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and a murine immunodominant and partially protective CD8\u003csup\u003e+\u003c/sup\u003e epitope overlaps with E3\u003csup\u003e505\u0026ndash;523 47, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. On the other hand, all IgG epitopes identified here differed from HLA class I epitopes from TSA1 \u003csup\u003e28\u003c/sup\u003e, as well as from previously identified \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e or predicted \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e epitopes from other members of the trans-sialidase family. Except for epitopes E3\u003csup\u003e505\u0026ndash;523\u003c/sup\u003e and E4\u003csup\u003e281\u0026ndash;297\u003c/sup\u003e from TSA1-C4, for which binding may be more constrained, all other epitopes appeared to be readily accessible on the surface of the 3D structure on the antigens, suggesting that IgGs may be able to bind to native proteins. This is also encouraging as vaccination with the recombinant antigens may also target these epitopes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, although this remains to be confirmed in future studies.\u003c/p\u003e\u003cp\u003eDespite the consistent epitope recognition profile in most patients, a minority of patients (26%) showed a different profile, with limited/no recognition of the immunodominant epitopes, and some recognition of alternative epitopes that varied among individuals, with IgGs from a few individuals showing no recognition of either antigen. This agrees with previous studies indicating that most but not all patients have antibodies against Tc24 and TSA1 \u003csup\u003e9, 24\u003c/sup\u003e. Similarly in dogs, about only about 80\u0026ndash;93% of \u003cem\u003eT. cruzi\u003c/em\u003e infected dogs have antibodies against these antigens \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. However, it is unclear why these few patients presented such a different antibody profile against the vaccine antigens, as this did not appear to be associated with their overall immune response to infection, as assessed by the different serological diagnostic tests, nor with the parasite burden, nor their HLA profile. Our data are too limited to completely rule out parasite strains, but all patients for which parasite genotyping was successful harbored very diverse \u0026ldquo;cruziomes\u0026rdquo; as mentioned above.\u003c/p\u003e\u003cp\u003eIn conclusion, we identified major epitopes from TC24-C4 and TSA1-C4 vaccine antigens recognized by IgGs from \u003cem\u003eT. cruzi\u003c/em\u003e infected patients following natural infections with mixtures of parasite strains from TcI, TcII, TcIV, TcV and TcVI DTUs. Most patients presented an immunodominant epitope recognition profile of both antigens, independently of their HLA profile, diagnostic test reactivity or \u003cem\u003eT. cruzi\u003c/em\u003e parasite burden. These epitopes are conserved among the six DTUs frequently infecting humans. These results present an important baseline for assessing changes in epitope profiles following therapeutic vaccination in future clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTION\u003c/h2\u003e\u003cp\u003eED: Conceptualization and design, Methodology, Formal analysis and investigation, Writing original draft, Reviewing and editing, Approval of the final manuscript. \u003cbr\u003eCH: Conceptualization and design, Methodology, Formal analysis and investigation, Reviewing and editing, Approval of the final manuscript. \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eThis work was supported by grant R01HD94955 from the Eunice Kennedy Shriver National Institute of Child Health \u0026amp; Human Development to CH, and grant R01AI162907 from the National Institute of Allergy and Infectious Diseases to ED. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRassi A, Jr., Rassi A, Marin-Neto JA. Chagas disease. \u003cem\u003eLancet\u003c/em\u003e 2010; 375(9723): 1388\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRassi A, Jr., Marin JAN, Rassi A. Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the BENznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial. \u003cem\u003eMemorias do Instituto Oswaldo Cruz\u003c/em\u003e 2017; 112(3): 224\u0026ndash;235.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePecoul B, Batista C, Stobbaerts E, Ribeiro I, Vilasanjuan R, Gascon J \u003cem\u003eet al.\u003c/em\u003e The BENEFIT Trial: Where Do We Go from Here? \u003cem\u003ePLoS neglected tropical diseases\u003c/em\u003e 2016; 10(2): e0004343.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViotti R, Vigliano C, Lococo B, Alvarez MG, Petti M, Bertocchi G \u003cem\u003eet al.\u003c/em\u003e Side effects of benznidazole as treatment in chronic Chagas disease: fears and realities. \u003cem\u003eExpert review of anti-infective therapy\u003c/em\u003e 2009; 7(2): 157\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePinazo MJ, Munoz J, Posada E, Lopez-Chejade P, Gallego M, Ayala E \u003cem\u003eet al.\u003c/em\u003e Tolerance of benznidazole in treatment of Chagas' disease in adults. \u003cem\u003eAntimicrobial agents and chemotherapy\u003c/em\u003e 2010; 54(11): 4896\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntinori S, Grande R, Bianco R, Traversi L, Cogliati C, Torzillo D \u003cem\u003eet al.\u003c/em\u003e High frequency of adverse reactions and discontinuation with benznidazole treatment for chronic Chagas disease in Milano, Italy. \u003cem\u003eClinical infectious diseases: an official publication of the Infectious Diseases Society of America\u003c/em\u003e 2015; 60(12): 1873\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eForsyth CJ, Hernandez S, Olmedo W, Abuhamidah A, Traina MI, Sanchez DR \u003cem\u003eet al.\u003c/em\u003e Safety Profile of Nifurtimox for Treatment of Chagas Disease in the United States. \u003cem\u003eClinical infectious diseases: an official publication of the Infectious Diseases Society of America\u003c/em\u003e 2016.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Bottazzi ME, Zhan B, Heffernan MJ, Jones K, Valenzuela JG \u003cem\u003eet al.\u003c/em\u003e Accelerating the development of a therapeutic vaccine for human Chagas disease: rationale and prospects. \u003cem\u003eExpert review of vaccines\u003c/em\u003e 2012; 11(9): 1043\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrautz GM, Galvao LM, Cancado JR, Guevara Espinoza A, Ouaissi A, Krettli AU. Use of a 24-kilodalton \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e recombinant protein to monitor cure of human Chagas' disease. \u003cem\u003eJournal of clinical microbiology\u003c/em\u003e 1995; 33: 2086\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOuaissi A, Aguirre T, Plumas Marty B, Piras M, Schoneck R, Gras Masse H \u003cem\u003eet al.\u003c/em\u003e Cloning and sequencing of a 24-kDa \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e specific antigen released in association with membrane vesicles and defined by a monoclonal antibody. \u003cem\u003eBiol Cell\u003c/em\u003e 1992; 75: 11\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWrightsman RA, Dawson BD, Fouts DL, Manning JE. Identification of immunodominant epitopes in \u003cem\u003eTrypanosoma cruzi t\u003c/em\u003erypomastigote surface antigen-1 protein that mask protective epitopes. \u003cem\u003eJ Immunol\u003c/em\u003e 1994; 153: 3148\u0026ndash;3154.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeid CA, Jones KM, Pollet J, Keegan B, Hudspeth E, Hammond M \u003cem\u003eet al.\u003c/em\u003e Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease. \u003cem\u003eHuman vaccines \u0026amp; immunotherapeutics\u003c/em\u003e 2017; 13(3): 621\u0026ndash;633.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiter AB, Weltje S, Hudspeth EM, Seid CA, McAtee CP, Chen WH \u003cem\u003eet al.\u003c/em\u003e Characterization and Stability of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e 24-C4 (Tc24-C4), a Candidate Antigen for a Therapeutic Vaccine Against Chagas Disease. \u003cem\u003eJ Pharm Sci\u003c/em\u003e 2018; 107(5): 1468\u0026ndash;1473.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDzul-Huchim VM, Ramirez-Sierra MJ, Martinez-Vega PP, Rosado-Vallado ME, Arana-Argaez VE, Ortega-Lopez J \u003cem\u003eet al.\u003c/em\u003e Vaccine-linked chemotherapy with a low dose of benznidazole plus a bivalent recombinant protein vaccine prevents the development of cardiac fibrosis caused by \u003cem\u003eTrypanosoma cruzi i\u003c/em\u003en chronically-infected BALB/c mice. \u003cem\u003ePLoS neglected tropical diseases\u003c/em\u003e 2022; 16(9): e0010258.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarry MA, Wang Q, Jones KM, Heffernan MJ, Buhaya MH, Beaumier CM \u003cem\u003eet al.\u003c/em\u003e A therapeutic nanoparticle vaccine against \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in a BALB/c mouse model of Chagas disease. \u003cem\u003eHuman vaccines \u0026amp; immunotherapeutics\u003c/em\u003e 2016; 12(4): 976\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede la Cruz JJ, Villanueva-Lizama L, Dzul-Huchim V, Ram\u0026iacute;rez-Sierra MJ, Martinez-Vega P, Rosado-Vallado M \u003cem\u003eet al.\u003c/em\u003e Production of recombinant TSA-1 and evaluation of its potential for the immuno-therapeutic control of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e infection in mice. \u003cem\u003eHuman vaccines \u0026amp; immunotherapeutics\u003c/em\u003e 2019; 15(1): 210\u0026ndash;219.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartinez-Campos V, Martinez-Vega P, Ramirez-Sierra MJ, Rosado-Vallado M, Seid CA, Hudspeth EM \u003cem\u003eet al.\u003c/em\u003e Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e infection in mice. \u003cem\u003eVaccine\u003c/em\u003e 2015; 33(36): 4505\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCruz-Chan JV, Villanueva-Lizama LE, Versteeg L, Damania A, Villar MJ, Gonzalez-Lopez C \u003cem\u003eet al.\u003c/em\u003e Vaccine-linked chemotherapy induces IL-17 production and reduces cardiac pathology during acute \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e infection. \u003cem\u003eSci Rep\u003c/em\u003e 2021; 11(1): 3222.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJones K, Versteeg L, Damania A, Keegan B, Kendricks A, Pollet J \u003cem\u003eet al.\u003c/em\u003e Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease. \u003cem\u003eInfection and immunity\u003c/em\u003e 2018; 86(4).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Herrera C, Marx PA. Safety and preservation of cardiac function following therapeutic vaccination against \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in rhesus macaques. \u003cem\u003eJ Microbiol Immunol Infect\u003c/em\u003e 2023; 56(2): 400\u0026ndash;407.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Herrera C, Tu W, Goff K, Fahlberg M, Haupt E \u003cem\u003eet al.\u003c/em\u003e Safety and immunogenicity of a recombinant vaccine against \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in Rhesus macaques. \u003cem\u003eVaccine\u003c/em\u003e 2020; 38(29): 4584\u0026ndash;4591.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Tu W, Desale H, Goff K, Marx P, Ortega-Lopez J \u003cem\u003eet al.\u003c/em\u003e Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naive rhesus macaques. \u003cem\u003eJ Biomed Sci\u003c/em\u003e 2024; 31(1): 58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCalderon-Quintal JA, Teh-Poot CF, Pech Piste LM, Martinez-Vega PP, Dzul-Huchim V, Torres-Acosta F \u003cem\u003eet al.\u003c/em\u003e Randomized field trial of a therapeutic vaccine against \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e natural infection in dogs and correlates for efficacy. \u003cem\u003ebioRxiv\u003c/em\u003e 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVillanueva-Lizama L, Cruz-Chan JV, Cetina-Aguilar AC, Herrera-Sanchez LF, Rodriguez-Perez JM, Rosado-Vallado ME \u003cem\u003eet al. Trypanosoma cruzi\u003c/em\u003e vaccine candidate antigens Tc24 and TSA-1 recall memory immune response associated with HLA-A and \u0026ndash;B supertypes in Chagasic chronic patients from Mexico. \u003cem\u003ePLoS neglected tropical diseases\u003c/em\u003e 2018; 12(1): e0006240.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O \u003cem\u003eet al.\u003c/em\u003e A new consensus for \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e intraspecific nomenclature: second revision meeting recommends TcI to TcVI. \u003cem\u003eMemorias do Instituto Oswaldo Cruz\u003c/em\u003e 2009; 104(7): 1051\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZingales B, Miles MA, Moraes CB, Luquetti A, Guhl F, Schijman AG \u003cem\u003eet al.\u003c/em\u003e Drug discovery for Chagas disease should consider \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e strain diversity. \u003cem\u003eMemorias do Instituto Oswaldo Cruz\u003c/em\u003e 2014; 109(6): 828\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArnal A, Villanueva-Lizama L, Teh-Poot C, Herrera C, Dumonteil E. Extent of polymorphism and selection pressure on the \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e vaccine candidate antigen Tc24. \u003cem\u003eEvol Applications\u003c/em\u003e 2020; 13: 2663\u0026ndash;2672.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKnight JM, Zingales B, Bottazzi ME, Hotez P, Zhan B. Limited antigenic variation in the \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e candidate vaccine antigen TSA-1. \u003cem\u003eParasite immunology\u003c/em\u003e 2014; 36(12): 708\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuekens P, Cafferata ML, Alger J, Althabe F, Belizan JM, Bustamante N \u003cem\u003eet al.\u003c/em\u003e Congenital Transmission of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in Argentina, Honduras, and Mexico: An Observational Prospective Study. \u003cem\u003eThe American journal of tropical medicine and hygiene\u003c/em\u003e 2018; 98(2): 478\u0026ndash;485.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMajeau A, Dumonteil E, Herrera C. Identification of highly conserved \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e antigens for the development of a universal serological diagnostic assay. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e 2024; 13(1): 2315964.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH \u003cem\u003eet al.\u003c/em\u003e UCSF ChimeraX: Tools for structure building and analysis. \u003cem\u003eProtein Sci\u003c/em\u003e 2023; 32(11): e4792.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan T, Rahman M, Ahmed I, Al Ali F, Jithesh PV, Marr N. Human leukocyte antigen class II gene diversity tunes antibody repertoires to common pathogens. \u003cem\u003eFront Immunol\u003c/em\u003e 2022; 13: 856497.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVillanueva-Lizama L, Teh-Poot C, Majeau A, Herrera C, Dumonteil E. Molecular genotyping of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e by next-generation sequencing of the mini-exon gene reveals infections with multiple parasite DTUs in Chagasic patients from Yucatan, Mexico. \u003cem\u003eJ Inf Dis\u003c/em\u003e 2019; 219(12): 1980\u0026ndash;1988.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMajeau A, Herrera C, Dumonteil E. An improved approach to \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e molecular genotyping by next-generation sequencing of the mini-exon gene. \u003cem\u003eMethods Mol Biol\u003c/em\u003e 2019; 1955: 47\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrooks GE, Hon G, Chandonia J-M, Brenner SE. WebLogo: a sequence logo generator. \u003cem\u003eGenome Res\u003c/em\u003e 2004; 14: 1188\u0026ndash;1190.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Herrera C. The Case for the Development of a Chagas Disease Vaccine: Why? How? When? \u003cem\u003eTrop Med Infect Dis\u003c/em\u003e 2021; 6(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Herrera C. Vaccines against chronic \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e infection: progress, challenges and future directions. 2025: submitted.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarlier Y, Dumonteil E, Herrera C, Waleckx E, Tibayrenc M, Buekens P \u003cem\u003eet al.\u003c/em\u003e Coinfection by multiple \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e clones: a new perspective on host-parasite relationship with consequences for pathogenesis and management of Chagas disease. \u003cem\u003eMicrobiol Mol Biol Rev\u003c/em\u003e 2025: e0024224.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesale H, Tu W, Goff K, Marx PA, Herrera C, Dumonteil E. PBMC transcriptomic signatures reflect \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e strain diversity and trained immunity in chronically infected macaques. \u003cem\u003eJCI Insight\u003c/em\u003e 2025; 10(4): e186003.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumonteil E, Desale H, Tu W, Hern\u0026aacute;ndez-Cuevas N, Shroyer M, Goff K \u003cem\u003eet al.\u003c/em\u003e Intra-host \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e 2023; 11(5): e0423622.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJager AV, Muia RP, Campetella O. Stage-specific expression of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e trans-sialidase involves highly conserved 3' untranslated regions. \u003cem\u003eFEMS microbiology letters\u003c/em\u003e 2008; 283(2): 182\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFreitas LM, dos Santos SL, Rodrigues-Luiz GF, Mendes TA, Rodrigues TS, Gazzinelli RT \u003cem\u003eet al.\u003c/em\u003e Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e reveal an undetected level of complexity. \u003cem\u003ePloS one\u003c/em\u003e 2011; 6(10): e25914.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaldonado RA, Mirzoeva S, Godsel LM, Lukas TJ, Goldenberg S, Watterson DM \u003cem\u003eet al.\u003c/em\u003e Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein. \u003cem\u003eMolecular and biochemical parasitology\u003c/em\u003e 1999; 101(1\u0026ndash;2): 61\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColli W. Trans-sialidase: a unique enzyme activity discovered in the protozoan Trypanosoma cruzi. \u003cem\u003eFASEB J\u003c/em\u003e 1993; 7(13): 1257\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlves MJ, Colli W. Role of the gp85/trans-sialidase superfamily of glycoproteins in the interaction of Trypanosoma cruzi with host structures. \u003cem\u003eSubcell Biochem\u003c/em\u003e 2008; 47: 58\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeixeira AAR, Carnero LR, Kuramoto A, Tang FHF, Gomes CH, Pereira NB \u003cem\u003eet al.\u003c/em\u003e A refined genome phage display methodology delineates the human antibody response in patients with Chagas disease. \u003cem\u003eiScience\u003c/em\u003e 2021; 24(6): 102540.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWizel B, Nunes M, Tarleton RL. Identification of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e trans-sialidase family members as targets of protective CD8\u0026thinsp;+\u0026thinsp;TC1 responses. \u003cem\u003eJ Immunol\u003c/em\u003e 1997; 159(12): 6120\u0026ndash;6130.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin DL, Weatherly DB, Laucella SA, Cabinian MA, Crim MT, Sullivan S \u003cem\u003eet al.\u003c/em\u003e CD8\u0026thinsp;+\u0026thinsp;T-Cell responses to \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e are highly focused on strain-variant trans-sialidase epitopes. \u003cem\u003ePLoS pathogens\u003c/em\u003e 2006; 2(8): e77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePitcovsky TA, Mucci J, Alvarez P, Leguizamon MS, Burrone O, Alzari PM \u003cem\u003eet al.\u003c/em\u003e Epitope mapping of trans-sialidase from \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e reveals the presence of several cross-reactive determinants. \u003cem\u003eInfection and immunity\u003c/em\u003e 2001; 69(3): 1869\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePitcovsky TA, Buscaglia CA, Mucci J, Campetella O. A functional network of intramolecular cross-reacting epitopes delays the elicitation of neutralizing antibodies to \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e trans-sialidase. \u003cem\u003eJ Infect Dis\u003c/em\u003e 2002; 186(3): 397\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePacini MF, Perdomini A, Bulfoni Balbi C, Dinatale B, Herrera FE, Perez AR \u003cem\u003eet al.\u003c/em\u003e The high identity of the \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e Group-I of trans-sialidases points them as promising vaccine immunogens. \u003cem\u003eProteins\u003c/em\u003e 2023; 91(10): 1444\u0026ndash;1460.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMejia R, Verocai GG, Mosley IA, Zhan B, Vongthavaravat L, Busselman RE \u003cem\u003eet al.\u003c/em\u003e Evaluation of a novel Tc-24 recombinant antigen ELISA for serologic testing for \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in dogs. \u003cem\u003ebioRxiv\u003c/em\u003e 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCalder\u0026oacute;n-Quintal JA, Escalante-Talavera MJ, Teh-Poot C, Carrera-Campellone MN, Martinez-Vega PP, Dzul-Huchim V \u003cem\u003eet al.\u003c/em\u003e Natural infection of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in client-owned-dogs from rural Yucatan, Mexico. \u003cem\u003eSci Rep\u003c/em\u003e 2025; 15(1): 10263.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7216993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7216993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChagas disease is a zoonotic disease caused by \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e parasites. Tc24 and TSA1 parasite antigens are leading candidates for a therapeutic vaccine to treat infected patients to stop/delay the progression of chronic cardiomyopathy. As these antigens are nearing clinical trials, we aimed to assess their epitope recognition profile by antibodies from Chagas disease patients to better understand their immunogenicity in humans. Peptide microarrays covering Tc24-C4 and TSA1-C4 vaccine antigens were incubated with IgG from 27 \u003cem\u003eT. cruzi\u003c/em\u003e-infected patients from Argentina, Honduras and Mexico. Most patients (20/27, 74%) had a highly similar recognition profile of both vaccine antigens, with the same immunodominant epitopes (three epitopes for Tc24-C4 and four for TSA1-C4). Remaining patients had limited reactivity against these antigens, targeting epitopes that varied among patients. All immunodominant epitopes were well conserved among \u003cem\u003eT. cruzi\u003c/em\u003e strains and DTUs and most were accessible on the surface of the proteins. The immunodominant epitope recognition profile was observed independently of patient HLA profile, diagnostic test reactivity or \u003cem\u003eT. cruzi\u003c/em\u003e parasite burden. Patients were infected with mixtures of TcI, TcII, TcIV, TcV and TcVI parasites. These results present an important baseline for assessing potential changes in epitope profiles following therapeutic vaccination in future clinical trials.\u003c/p\u003e","manuscriptTitle":"Epitope mapping of vaccine antigens Tc24 and TSA1 with antibodies from Trypanosoma cruzi infected patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 07:04:06","doi":"10.21203/rs.3.rs-7216993/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-04T15:35:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-18T20:04:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-13T16:21:06+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-08T00:10:11+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-05T14:36:19+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-04T15:27:44+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-04T15:15:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-04T14:31:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-28T12:41:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Immunity","date":"2025-07-25T19:53:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T19:53:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d313b3e3-dab2-4505-8c2d-731fc98c3ad0","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52619116,"name":"Biological sciences/Immunology/Infectious diseases/Parasitic infection"},{"id":52619117,"name":"Biological sciences/Immunology/Vaccines/Protein vaccines"},{"id":52619118,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies"}],"tags":[],"updatedAt":"2026-02-11T08:06:32+00:00","versionOfRecord":{"articleIdentity":"rs-7216993","link":"https://doi.org/10.1038/s41435-026-00380-8","journal":{"identity":"genes-and-immunity","isVorOnly":false,"title":"Genes \u0026 Immunity"},"publishedOn":"2026-02-10 05:00:00","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-08-07 07:04:06","video":"","vorDoi":"10.1038/s41435-026-00380-8","vorDoiUrl":"https://doi.org/10.1038/s41435-026-00380-8","workflowStages":[]},"version":"v1","identity":"rs-7216993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7216993","identity":"rs-7216993","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}