RAFAEL RIBEIRO ALMEIDA
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina
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- Disease Tolerance and Pathogen Resistance Genes May Underlie Trypanosoma cruzi Persistence and Differential Progression to Chagas Disease Cardiomyopathy(2018) CHEVILLARD, Christophe; NUNES, Joao Paulo Silva; FRADE, Amanda Farage; ALMEIDA, Rafael Ribeiro; PANDEY, Ramendra Pati; NASCIMENTO, Marilda Savoia; KALIL, Jorge; CUNHA-NETO, EdecioChagas disease is caused by infection with the protozoan Trypanosoma cruzi and affects over 8 million people worldwide. In spite of a powerful innate and adaptive immune response in acute infection, the parasite evades eradication, leading to a chronic persistent infection with low parasitism. Chronically infected subjects display differential patterns of disease progression. While 30% develop chronic Chagas disease cardiomyopathy (CCC)-a severe inflammatory dilated cardiomyopathy-decades after infection, 60% of the patients remain disease-free, in the asymptomatic/indeterminate (ASY) form, and 10% develop gastrointestinal disease. Infection of genetically deficient mice provided a map of genes relevant for resistance to T. cruzi infection, leading to the identification of multiple genes linked to survival to infection. These include pathogen resistance genes (PRG) needed for intracellular parasite destruction, and genes involved in disease tolerance (protection against tissue damage and acute phase death-DTG). All identified DTGs were found to directly or indirectly inhibit IFN-gamma production or Th1 differentiation. We hypothesize that the absolute need for DTG to control potentially lethal IFN-gamma PRG activity leads to T. cruzi persistence and establishment of chronic infection. IFN-gamma production is higher in CCC than ASY patients, and is the most highly expressed cytokine in CCC hearts. Key DTGs that downmodulate IFN-gamma, like IL-10, and Ebi3/IL27p28, are higher in ASY patients. Polymorphisms in PRG and DTG are associated with differential disease progression. We thus hypothesize that ASY patients are disease tolerant, while an imbalance of DTG and IFN-gamma PRG activity leads to the inflammatory heart damage of CCC.
- Broad and Cross-Clade CD4(+) T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides(2012) ALMEIDA, Rafael Ribeiro; ROSA, Daniela Santoro; RIBEIRO, Susan Pereira; SANTANA, Vinicius Canato; KALLAS, Esper Georges; SIDNEY, John; SETTE, Alessandro; KALIL, Jorge; CUNHA-NETO, EdecioT-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-gamma secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-gamma and TNF-alpha, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.
- A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4(+) and CD8(+) T Cell Responses(2011) ROSA, Daniela Santoro; RIBEIRO, Susan Pereira; ALMEIDA, Rafael Ribeiro; MAIRENA, Eliane Conti; POSTOL, Edilberto; KALIL, Jorge; CUNHA-NETO, EdecioT-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFN gamma/TNF alpha, IFN gamma/IL-2 or TNF alpha/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFN gamma/TNF alpha/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses-elicited by other HIV immunogens.
- A promiscuous T cell epitope-based HIV vaccine providing redundant population coverage of the HLA class II elicits broad, polyfunctional T cell responses in nonhuman primates(2022) RIBEIRO, Susan Pereira; MATTARAIA, Vania Gomes De Moura; ALMEIDA, Rafael Ribeiro; VALENTINE, Elizabeth Juliana Ghiuro; SALES, Natiely Silva; FERREIRA, Luis Carlos S.; SA-ROCHA, Luiz Carlos; JACINTHO, Lucas Caue; SANTANA, Vinicius Canato; SIDNEY, John; SETTE, Alessandro; ROSA, Daniela Santoro; KALIL, Jorge; CUNHA-NETO, EdecioOver the last few decades, several emerging or reemerging viral diseases with no readily available vaccines have ravaged the world. A platform to fastly generate vaccines inducing potent and durable neutralizing antibody and T cell responses is sorely needed. Bioinformatically identified epitope-based vaccines can focus on immunodominant T cell epitopes and induce more potent immune responses than a whole antigen vaccine and may be deployed more rapidly and less costly than whole-gene vaccines. Increasing evidence has shown the importance of the CD4+ T cell response in protection against HIV and other viral infections. The previously described DNA vaccine HIVBr18 encodes 18 conserved, promiscuous epitopes binding to multiple HLA-DR-binding HIV epitopes amply recognized by HIV-1-infected patients. HIVBr18 elicited broad, polyfunctional, and durable CD4(+) and CD8+ T cell responses in BALB/c and mice transgenic to HLA class II alleles, showing cross-species promiscuity. To fully delineate the promiscuity of the HLA class II vaccine epitopes, we assessed their binding to 34 human class II (HLA-DR, DQ, and -DP) molecules, and immunized nonhuman primates. Results ascertained redundant 100% coverage of the human population for multiple peptides. We then immunized Rhesus macaques with HIVBr18 under in vivo electroporation. The immunization induced strong, predominantly polyfunctional CD4+ T cell responses in all animals to 13 out of the 18 epitopes; T cells from each animal recognized 7-11 epitopes. Our results provide a preliminary proof of concept that immunization with a vaccine encoding epitopes with high and redundant coverage of the human population can elicit potent T cell responses to multiple epitopes, across species and MHC barriers. This approach may facilitate the rapid deployment of immunogens eliciting cellular immunity against emerging infectious diseases, such as COVID-19.
- Recurrence of COVID-19 associated with reduced T-cell responses in a monozygotic twin pair(2022) V, Mateus de Castro; SANTOS, Keity S.; APOSTOLICO, Juliana S.; FERNANDES, Edgar R.; ALMEIDA, Rafael R.; LEVIN, Gabriel; MAGAWA, Jhosiene Y.; NUNES, Joao Paulo S.; BRUNI, Mirian; YAMAMOTO, Marcio M.; LIMA, Ariane C.; SILVA, Monize V. R.; MATOS, Larissa R. B.; CORIA, Vivian R.; CASTELLI, Erick C.; SCLIAR, Marilia O.; KURAMOTO, Andreia; BRUNO, Fernanda R.; JACINTHO, Lucas C.; NUNES, Kelly; WANG, Jaqueline Y. T.; COELHO, Veronica P.; NETO, Miguel Mitne; MACIEL, Rui M. B.; NASLAVSKY, Michel S.; PASSOS-BUENO, Maria Rita; BOSCARDIN, Silvia B.; ROSA, Daniela S.; KALIL, Jorge; ZATZ, Mayana; CUNHA-NETO, EdecioRecurrence 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 (IFN gamma) 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.
- Multiple Approaches for Increasing the Immunogenicity of an Epitope-Based Anti-HIV Vaccine(2015) ROSA, Daniela Santoro; RIBEIRO, Susan Pereira; FONSECA, Simone Goncalves; ALMEIDA, Rafael Ribeiro; SANTANA, Vinicius Canato; APOSTOLICO, Juliana de Souza; KALIL, Jorge; CUNHA-NETO, EdecioThe development of a highly effective vaccine against the human immunodeficiency virus (HIV) will likely be based on rational vaccine design, since traditional vaccine approaches have failed so far. In recent years, an understanding of what type of immune response is protective against infection and/or disease facilitated vaccine design. T cell-based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. In this context, CD4(+) T cells play a direct cytotoxic role and are also important for the generation and maintenance of functional CD8(+) T and B cell responses. The use of MHC-binding algorithms has allowed the identification of novel CD4(+) T cell epitopes that could be used in vaccine design, the so-called epitope-driven vaccine design. Epitope-based vaccines have the ability to focus the immune response on highly antigenic, conserved epitopes that are fully recognized by the target population. We have recently mapped a set of conserved multiple HLA-DR-binding HIV-1 CD4 epitopes and observed interferon (IFN)--producing CD4(+) T cells when we tested these peptides in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals. We then designed multiepitopic DNA vaccines that induced broad and polyfunctional T cell responses in immunized mice. In this review we will focus on alternative strategies to increase the immunogenicity of an epitope-based vaccine against HIV infection.
- Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease(2021) OUARHACHE, Maryem; MARQUET, Sandrine; FRADE, Amanda Farage; FERREIRA, Ariela Mota; IANNI, Barbara; ALMEIDA, Rafael Ribeiro; NUNES, Joao Paulo Silva; FERREIRA, Ludmila Rodrigues Pinto; RIGAUD, Vagner Oliveira-Carvalho; CANDIDO, Darlan; MADY, Charles; ZANIRATTO, Ricardo Costa Fernandes; BUCK, Paula; TORRES, Magali; GALLARDO, Frederic; ANDRIEUX, Pauline; BYDLOWSKY, Sergio; LEVY, Debora; ABEL, Laurent; CARDOSO, Clareci Silva; SANTOS-JUNIOR, Omar Ribeiro; OLIVEIRA, Lea Campos; OLIVEIRA, Claudia Di Lorenzo; NUNES, Maria Do Carmo; COBAT, Aurelie; KALIL, Jorge; RIBEIRO, Antonio Luiz; SABINO, Ester Cerdeira; CUNHA-NETO, Edecio; CHEVILLARD, ChristopheCardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-gamma and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy. Methods We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY. Results We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related - most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-gamma on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (Delta psi M), indicating mitochondrial dysfunction. Conclusion Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-gamma-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.
- miRNAs may play a major role in the control of gene expression in key pathobiological processes in Chagas disease cardiomyopathy(2020) LAUGIER, Laurie; FERREIRA, Ludmila Rodrigues Pinto; FERREIRA, Frederico Moraes; CABANTOUS, Sandrine; FRADE, Amanda Farage; NUNES, Joao Paulo; RIBEIRO, Rafael Almeida; BROCHET, Pauline; TEIXEIRA, Priscila Camillo; SANTOS, Ronaldo Honorato Barros; BOCCHI, Edimar A.; BACAL, Fernando; CANDIDO, Darlan da Silva; MASO, Vanessa Escolano; NAKAYA, Helder I.; KALIL, Jorge; CUNHA-NETO, Edecio; CHEVILLARD, ChristopheChronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFN gamma, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-gamma-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy. Author summary Chronic Chagas disease cardiomyopathy (CCC), an aggressive dilated cardiomyopathy caused by Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Little is known about the molecular mechanisms responsible for its severity. Authors study the possible role of microRNAs in the regulation of gene expression in relevant pathways and pathobiological processes. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) -small RNAs that can regulate gene expression-associated to severe cardiomyopathy development. The inflammatory mediator Interferon-gamma was the most likely inducer of gene expression in CCC, and most genes belonged to the immune response, fibrosis, hypertrophy and mitochondrial metabolism. A discrete number of differentially expressed mRNAs targeted a high number of differentially expressed mRNAs in multiple processes. Moreover, several pathways had multiple targets regulated by microRNAs, suggesting synergic effect. Results suggest that microRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue.
conferenceObject Vaccine Antigen Design to Maximize anti-HIV CD4+T-cell Responses: From Mice to Non-human Primates(2014) CUNHA-NETO, Edecio; RIBEIRO, Susan; SANTORO-ROSA, Daniela; ALMEIDA, Rafael; SANTANA, Vinicius; KALIL, Jorge- Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy(2022) BROCHET, Pauline; IANNI, Barbara Maria; LAUGIER, Laurie; FRADE, Amanda Farage; NUNES, Joao Paulo Silva; TEIXEIRA, Priscila Camillo; MADY, Charles; FERREIRA, Ludmila Rodrigues Pinto; FERRE, Quentin; SANTOS, Ronaldo Honorato Barros; KURAMOTO, Andreia; CABANTOUS, Sandrine; STEFFEN, Samuel; STOLF, Antonio Noedir; POMERANTZEFF, Pablo; FIORELLI, Alfredo Inacio; BOCCHI, Edimar Alcides; PISSETTI, Cristina Wide; SABA, Bruno; CANDIDO, Darlan da Silva; DIAS, Fabricio C.; SAMPAIO, Marcelo Ferraz; GAIOTTO, Fabio Antonio; MARIN-NETO, Jose Antonio; FRAGATA, Abilio; ZANIRATTO, Ricardo Costa Fernandes; SIQUEIRA, Sergio; PEIXOTO, Giselle De Lima; RIGAUD, Vagner Oliveira-Carvalho; BACAL, Fernando; BUCK, Paula; ALMEIDA, Rafael Ribeiro; LIN-WANG, Hui Tzu; SCHMIDT, Andre; MARTINELLI, Martino; HIRATA, Mario Hiroyuki; DONADI, Eduardo Antonio; PEREIRA, Alexandre Costa; RODRIGUES JUNIOR, Virmondes; PUTHIER, Denis; KALIL, Jorge; SPINELLI, Lionel; CUNHA-NETO, Edecio; CHEVILLARD, ChristopheChagas 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.