Anti-Trypanosoma cruzi antibody profiling in patients with Chagas disease treated with benznidazole assessed by genome phage display.

BACKGROUND: There have been significant improvements in Chagas disease therapy and it is now widely accepted that most patients with chronic disease might benefit from therapy. However, there are challenges to monitor drug efficacy and cure for these patients, which are important impediments for current and future therapies. Trypanosoma cruzi-PCR is highly variable while IgG seroconversion takes decades yielding variable results depending on the antigen(s) used for the assay. METHODS AND RESULTS: We used the genomic phage display (gPhage) platform to perform a pairwise comparison of antigens and epitopes recognized by twenty individual patients with chronic Chagas disease before and after treatment with benznidazole. In total, we mapped 54,473 T. cruzi epitopes recognized by IgG from individual patients (N = 20) before benznidazole treatment. After treatment, the number of epitopes recognized by all patients was significantly smaller (21,254), a reduction consistent with a decrease in anti-T. cruzi antibodies. Most of these epitopes represent distinct fragments from the same protein and could, therefore, be grouped into 80 clusters of antigens. After three years of treatment with benznidazole, we observed a 64% reduction in the number of clusters of antigens recognized by patients (59 clusters before versus 21 clusters after treatment). The most abundant antigenic clusters recognized by patients correspond to the surface antigen CA-2 (B13) followed by the microtubule associated antigen, which highlights the value of these epitopes in Chagas disease diagnosis. Most importantly, quantitative pairwise comparison of gPhage data allowed for the prediction of patient response to treatment based on PCR status. PRINCIPAL FINDING: Here, we compiled a list of antigens and epitopes preferentially recognized by Chagas disease patients before and after benznidazole treatment. Next, we observed that gPhage data correlated with patient PCR-status and could, therefore, predict patient response to treatment. Moreover, gPhage results suggest that overall, independent of PCR status, treatment led to a reduction in the presence of T. cruzi-specific antibody levels and the number of antigens and epitopes recognized by these patients. CONCLUSION: The gPhage platform use of unbiased library of antigens, which is different from conventional serological assays that rely on predetermined antigens, is a contribution for the development of novel diagnostic tools for Chagas disease.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

Matrix Metalloproteinase 2 and 9 Enzymatic Activities are Selectively Increased in the Myocardium of Chronic Chagas Disease Cardiomyopathy Patients: Role of TIMPs.

Chronic Chagas disease (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis compared to other cardiomyopathies. We show the expression and activity of Matrix Metalloproteinases (MMP) and of their inhibitors TIMP (tissue inhibitor of metalloproteinases) in myocardial samples of end stage CCC, idiopathic dilated cardiomyopathy (DCM) patients, and from organ donors. Our results showed significantly increased mRNA expression of several MMPs, several TIMPs and EMMPRIN in CCC and DCM samples. MMP-2 and TIMP-2 protein levels were significantly elevated in both sample groups, while MMP-9 protein level was exclusively increased in CCC. MMPs 2 and 9 activities were also exclusively increased in CCC. Results suggest that the balance between proteins that inhibit the MMP-2 and 9 is shifted toward their activation. Inflammation-induced increases in MMP-2 and 9 activity and expression associated with imbalanced TIMP regulation could be related to a more extensive heart remodeling and poorer prognosis in CCC patients.

Reduced T Cell and Antibody Responses to Inactivated Coronavirus Vaccine Among Individuals Above 55 Years Old.

CoronaVac is an inactivated SARS-CoV-2 vaccine that has been rolled out in several low and middle-income countries including Brazil, where it was the mainstay of the first wave of immunization of healthcare workers and the elderly population. We aimed to assess the T cell and antibody responses of vaccinated individuals as compared to convalescent patients. We detected IgG against SARS-CoV-2 antigens, neutralizing antibodies against the reference Wuhan SARS-CoV-2 strain and used SARS-CoV-2 peptides to detect IFN-g and IL-2 specific T cell responses in a group of CoronaVac vaccinated individuals (N = 101) and convalescent (N = 72) individuals. The frequency among vaccinated individuals, of whom 96% displayed T cell and/or antibody responses to SARS-CoV-2, is comparable to 98.5% responses of convalescent individuals. We observed that among vaccinated individuals, men and individuals 55 years or older developed significantly lower anti-RBD, anti-NP and neutralization titers against the Wuhan strain and antigen-induced IL-2 production by T cells. Neutralizing antibody responses for Gamma variant were even lower than for the Wuhan strain. Even though some studies indicated CoronaVac helped reduce mortality among elderly people, considering the appearance of novel variants of concern, CoronaVac vaccinated individuals above 55 years old are likely to benefit from a heterologous third dose/booster vaccine to increase immune response and likely protection.

Recurrence of COVID-19 associated with reduced T-cell responses in a monozygotic twin pair.

Recurrence of COVID-19 in recovered patients has been increasingly reported. However, the immune mechanisms behind the recurrence have not been thoroughly investigated. The presence of neutralizing antibodies (nAbs) in recurrence/reinfection cases suggests that other types of immune response are involved in protection against recurrence. Here, we investigated the innate type I/III interferon (IFN) response, binding and nAb assays and T-cell responses to severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) with IFN gamma (IFNγ) enzyme-linked spot assay (ELISPOT) in three pairs of young adult monozygotic (MZ) twins with previous confirmed COVID-19, one of them presenting a severe recurrence four months after the initial infection. Twin studies have been of paramount importance to comprehend the immunogenetics of infectious diseases. Each MZ twin pair was previously exposed to SARS-CoV-2, as seen by clinical reports. The six individuals presented similar overall recovered immune responses except for the recurrence case, who presented a drastically reduced number of recognized SARS-CoV-2 T-cell epitopes on ELISPOT as compared to her twin sister and the other twin pairs. Our results suggest that the lack of a broad T-cell response to initial infection may have led to recurrence, emphasizing that an effective SARS-CoV-2-specific T-cell immune response is key for complete viral control and avoidance of clinical recurrence of COVID-19.

Protocol for design, construction, and selection of genome phage (gPhage) display libraries.

This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).

A refined genome phage display methodology delineates the human antibody response in patients with Chagas disease.

Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens.

Polymorphism in the alpha cardiac muscle actin 1 gene is associated to susceptibility to chronic inflammatory cardiomyopathy.

AIMS: Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America, and may lead to a life-threatening inflammatory dilated, chronic Chagas cardiomyopathy (CCC). One third of T. cruzi-infected individuals progress to CCC while the others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Since mutations in multiple sarcomeric genes, including alpha-cardiac actin (ACTC1) have been involved in hereditary dilated cardiomyopathy, we investigated the involvement of the ACTC1 gene in CCC pathogenesis. METHODS AND RESULTS: We conducted a proteomic and genetic study on a Brazilian study population. The genetic study was done on a main cohort including 118 seropositive asymptomatic subjects and 315 cases and the replication was done on 36 asymptomatic and 102 CCC cases. ACTC1 protein and mRNA levels were lower in myocardial tissue from patients with end-stage CCC than those found in hearts from organ donors. Genotyping a case-control cohort of CCC and ASY subjects for all informative single nucleotide polymorphism (SNP) in the ACTC1 gene identified rs640249 SNP, located at the 5' region, as associated to CCC. Associations are borderline after correction for multiple testing. Correlation and haplotype analysis led to the identification of a susceptibility haplotype. Functional assays have shown that the rs640249A/C polymorphism affects the binding of transcriptional factors in the promoter regions of the ACTC1 gene. Confirmation of the detected association on a larger independent replication cohort will be useful. CONCLUSIONS: Genetic variations at the ACTC1 gene may contribute to progression to chronic Chagas Cardiomyopathy among T. cruzi-infected patients, possibly by modulating transcription factor binding to ACTC1 promoter regions.