Accessing the Variability of Multicopy Genes in Complex Genomes using Unassembled Next-Generation Sequencing Reads: The Case of Trypanosoma cruzi Multigene Families

Reis-Cunha, João Luís; Coqueiro-dos-Santos, Anderson; Pimenta-Carvalho, Samuel Alexandre; Marques, Larissa Pinheiro; Rodrigues-Luiz, Gabriela F.; Baptista, Rodrigo P.; Almeida, Laila Viana de; Honorato, Nathan Ravi Medeiros; Lobo, Francisco Pereira; Fraga, Vanêssa Gomes; Galvão, Lúcia Maria da Cunha; Bueno, Lilian Lacerda; Fujiwara, Ricardo Toshio; Cardoso, Mariana Santos; Cerqueira, Gustavo C.; Bartholomeu, Daniella Castanheira

doi:10.1128/mbio.02319-22

Cited by 7 publications

(6 citation statements)

References 51 publications

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“…In order to find immunodominant epitopes, the frequency in which the same peptide appeared in the top score for different HLA-ABC alleles was analyzed. Because T. cruzi has a high genetic diversity between strains, especially in the TS multigene family 42 , we also looked for conserved immunogenic epitopes among different discrete typing units (DTUs) (Supplementary Tables 1 , 2 ). Using these strategies, it was possible to detect potentially immunogenic regions in both proteins.…”

Section: Resultsmentioning

confidence: 99%

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

et al. 2023

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Immunization with the Amastigote Surface Protein-2 (ASP-2) and Trans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages of Trypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge with T. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels of T. cruzi-specific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+ T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+ T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+ T cell response. Immunization with either vaccine protected dogs against challenge with T. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.

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Section: Resultsmentioning

confidence: 99%

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

et al. 2023

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“…It was previously observed that for the same amount of parasite DNA, TcI strains had higher Ct values than TcII, which would correspond to a lower number of satDNA copies of the former DTU [ 31 , 39 , 51 , 52 ]. In fact, recent findings have described that the size of the T. cruzi mitochondrial and nuclear genome and the content of chromosomes and multigene families vary in sequence and content among the parasite’s strains, even those classified into the same DTU or between different DTUs [ 53 , 54 , 55 ].…”

Section: Discussionmentioning

confidence: 99%

Validation of the NAT Chagas IVD Kit for the Detection and Quantification of Trypanosoma cruzi in Blood Samples of Patients with Chagas Disease

Moreira

Fernandes

Gomes

et al. 2023

Life

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In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs—TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.

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“…The spot intensity of each peptide was quantitatively measured by densitometry scanning analysis using the software Image J–Protein Array Analyzer plug-in ( http://image.bio.methods.free.fr/ImageJ/?Protein-Array-Analyzer-for-ImageJ.html ) as described elsewhere ( Reis-Cunha et al, 2022 ). Briefly, densitometry values were normalized and the mean densitometric value of the four less reactive peptides from each experiment was subtracted from the densitometric value of each spot of the same membrane.…”

Section: Methodsmentioning

confidence: 99%

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure

Marzano-Miranda,

Pereira Cardoso-Oliveira,

Carla de Oliveira

et al. 2024

PeerJ

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Background The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

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Accessing the Variability of Multicopy Genes in Complex Genomes using Unassembled Next-Generation Sequencing Reads: The Case of Trypanosoma cruzi Multigene Families

Cited by 7 publications

References 51 publications

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

Validation of the NAT Chagas IVD Kit for the Detection and Quantification of Trypanosoma cruzi in Blood Samples of Patients with Chagas Disease

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure

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