Genetic diversity and immunogenicity analysis of MAEBL and RON2 of Plasmodium vivax

Orientadores: Fabio Trindade Maranhão Costa, Letusa AlbrechtDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: No último ano foram estimados 212 milhões de casos de malária no mundo. Plasmodium vivax é responsável por 41% dos casos da doença fora do continente africano. Ao contrário do Plasmodium falciparum, as infecções causadas por P. vivax são raramente letais. No entanto, P. vivax tem um impacto significativo sobre a produtividade das populações locais. O surgimento de cepas resistentes às drogas complica ainda mais a infecção por P. vivax o que leva à intensificação de investigações sobre métodos de controle alternativos, como o desenvolvimento de vacinas. Diversas proteínas vêm sendo estudadas a fim de encontrar um bom candidato vacinal. MAEBL é uma molécula quimérica expressa em eritrócitos infectados. Possui dois domínios (M1 e M2) envolvidos no processo de invasão ao eritrócito, sendo o domínio M2 o principal envolvido na invasão do merozoíta, além de exibir alta capacidade adesiva. MAEBL também é expresso em esporozoítas das glândulas salivares e em hepatócitos infectados. Além disso, o gene maebl foi identificado em diferentes espécies de plasmódios. RON2 é uma proteína conservada no filo Apicomplexa, expressa em esquizontes tardios, secretada por organelas denominadas roptrias no merozoíta e também está envolvida no processo de invasão do eritrócito, quando AMA1 junto com proteínas RON estabelecem a estrutura "moving junction" que faz uma conexão entre o merozoíta e o eritrócito. As características de MAEBL e RON2 abrem perspectivas para o desenvolvimento de uma vacina experimental. Visando um bom candidato vacinal, torna-se importante caracterizar o padrão de diversidade genética das proteínas em estudo, bem como avaliar a imunogenicidade das mesmas. Para isso, a partir de amostras de sangue de indivíduos infectados por malária, foi realizado PCR de regiões dos genes maebl e ron2 de Plasmodium vivax. Posteriormente foram sequenciados e avaliados quanto a sua diversidade. As proteínas MAEBL e RON2 foram expressas e testadas por ELISA, a fim de avaliar a sua imunogenicidade frente ao plasma de indivíduos infectados. Foi evidenciado, para ambos os genes, uma baixa diversidade genética. Pvron2 não apresentou mutações não-sinônimas e Pvmaebl teve sete mutações não sinônimas. A prevalência de anticorpos IgM e IgG naturalmente adquiridos contra MAEBL foi de 4,54% para IgM e 55,6% para IgG total. Anticorpos IgG naturalmente adquiridos contra RON2 estiveram presentes em 8,33% dos indivíduos analisados. Portanto, as proteínas MAEBL e RON2 se mostraram conservadas e imunogênicas, reafirmando estas proteínas como potenciais candidatos vacinaisAbstract: Last year, 212 million malaria cases were estimated worldwide. Plasmodium vivax accounts for 41% of the cases of the disease outside the African continent. Unlike Plasmodium falciparum, infections caused by P. vivax are rarely lethal. However, P. vivax has a significant impact on the productivity of local populations. The emergence of drug-resistant strains and complications on P. vivax infection make it extremely necessary to intensify research on definitive control methods such as the development of vaccines. Several proteins have been studied aiming to find a good vaccine candidate. MAEBL is a chimeric molecule expressed on infected erythrocytes that presents two domains involved in the erythrocyte invasion process (M1 and M2). M2 domain is the most important one for merozoite invasion, and it exhibits higher adhesiveness. Recently, it has been shown that MAEBL is also expressed in the salivary sporozoite gland and infected hepatocytes. Moreover, the gene that codifies MAEBL was identified in different Plasmodium species, including P. vivax. RON2 is a conserved protein that belongs to Apicomplexa phylum. It is expressed in late schizonts, secreted by organelles called roptries in the merozoite and it is also involved in the erythrocyte invasion process. In this process AMA-1 together with RON proteins establish the moving junction structure, which makes a connection between the merozoite and the erythrocyte. The characteristics of MAEBL and RON2 open perspectives for the development of an experimental vaccine. It is important to characterize the pattern of genetic diversity of these proteins, as well as to evaluate the immunogenicity of them in order to find a good vaccine candidate. For it to be possible, PCR and sequence analysis were performed. The MAEBL and RON2 proteins were expressed and tested by ELISA to evaluate their immunogenicity against the plasma of infected individuals. A low genetic diversity was evidenced for both genes. Pvron2 did not present non-synonymous mutations and Pvmaebl presented 7 non-synonymous mutations. The reactivity indices of naturally acquired IgM and IgG antibodies against MAEBL were 4.54% for IgM and 55.6% for total IgG. IgG antibodies naturally acquired against RON2 were present in 8.33% of the individuals analyzed. Therefore, MAEBL and RON2 proteins are conserved and immunogenic, thus reaffirming them as potential vaccine candidatesMestradoImunologiaMestra em Genética e Biologia Molecular2015/02808-0FAPES

Listeriosis in the far South of Brazil: neglected infection?

Listeriosis is an under-diagnosed and under-reported infection; however, listeriosis is not a compulsorily notifiable disease in Brazil. We provide an overview of the rates of listeriosis in the United States of America (USA), Europe, Latin America, and Brazil during the past decade. We also report a case of miscarriage caused by listeriosis in which there was no suspicion of this infection. This overview and the case we report serve as reminders of the often-neglected threat of listeriosis and its potential to cause miscarriage while highlighting the necessity of recognizing listeriosis as a compulsorily notifiable disease in Brazil

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate

Submitted by Manoel Barata ([email protected]) on 2019-12-04T13:20:20Z No. of bitstreams: 1 s12936-017-2144-ok.pdf: 1645911 bytes, checksum: 0ed5b598ff7fef498aef8fc5b929105c (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-12-26T18:47:27Z (GMT) No. of bitstreams: 1 s12936-017-2144-ok.pdf: 1645911 bytes, checksum: 0ed5b598ff7fef498aef8fc5b929105c (MD5)Made available in DSpace on 2019-12-26T18:47:27Z (GMT). No. of bitstreams: 1 s12936-017-2144-ok.pdf: 1645911 bytes, checksum: 0ed5b598ff7fef498aef8fc5b929105c (MD5) Previous issue date: 2018Universidade Nova de Lisboa. Instituto de Higiene e Medicina Tropical. Centro de Medicina Tropical e Saúde Global. Lisboa, Portugal / Universidade Federal de Goiás. Instituto de Patologia Tropícal e Saúde Pública. GenoBio. Goiânia, GO, Brasil / Centro Universitário de Anápolis. PPG-SOMA. Anápolis, GO, Brasil.Universidade Federal de Goiás. Instituto de Patologia Tropical e Saúde Pública. GenoBio. Goiânia, GO, Brasil.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil.Universidade Federal de Goiás. Instituto de Patologia Tropícal e Saúde Pública. GenoBio. Goiânia, GO, Brasil.Singapore Immunology Network. Agency for Science, Technology and Research. Singapore, Singapore.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil. / Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil / Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. Gerência de Malária. Manaus, AM, Brasil.Universidade de São Paulo. Departamento de Parasitologia. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Ciências Farmaceuticas. Departamento de Análises Clínicas e Toxicológicas. São Paulo, SP, Brasil.Singapore Immunology Network. Agency for Science, Technology and Research. Singapore, Singapore.Universidade de São Paulo. Departamento de Parasitologia. São Paulo, SP, Brasil.Shoklo Malaria Research Unit. Mahidol Oxford Tropical Medicine Research Unit. Faculty of Tropical Medicine. Mahidol University. Mae Sot, Thailand.Department of Microbiology and Immunology. University of Otago. Dunedin, New Zealand.Singapore Immunology Network. Agency for Science, Technology and Research. Singapore, Singapore.Universidade Estadual de Campinas. Departamento de Genética, Evolução, Microbiologia e Imunologia. Laboratório de Doenças Tropicais Prof. Dr. Jacintho da Silva. Campinas, SP, Brasil.Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. Detailed epitope mapping of MAEBL through immunoinformatics identifed several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specifc immunity. Therefore, can concluded that the MAEBL antigen is promising candidate towards the development of a malaria vaccine

Genetic sequence characterization and naturally acquired immune response to Plasmodium vivax Rhoptry Neck Protein 2 (PvRON2)

Abstract Background The genetic diversity of malaria antigens often results in allele variant-specific immunity, imposing a great challenge to vaccine development. Rhoptry Neck Protein 2 (PvRON2) is a blood-stage antigen that plays a key role during the erythrocyte invasion of Plasmodium vivax. This study investigates the genetic diversity of PvRON2 and the naturally acquired immune response to P. vivax isolates. Results Here, the genetic diversity of PvRON21828–2080 and the naturally acquired humoral immune response against PvRON21828–2080 in infected and non-infected individuals from a vivax malaria endemic area in Brazil was reported. The diversity analysis of PvRON21828–2080 revealed that the protein is conserved in isolates in Brazil and worldwide. A total of 18 (19%) patients had IgG antibodies to PvRON21828–2080. Additionally, the analysis of the antibody response in individuals who were not acutely infected with malaria, but had been infected with malaria in the past indicated that 32 patients (33%) exhibited an IgG immune response against PvRON2. Conclusions PvRON2 was conserved among the studied isolates. The presence of naturally acquired antibodies to this protein in the absence of the disease suggests that PvRON2 induces a long-term antibody response. These results indicate that PvRON2 is a potential malaria vaccine candidate

Plasmodium vivax AMA1: Implications of distinct haplotypes for immune response.

In Brazil, Plasmodium vivax infection accounts for around 80% of malaria cases. This infection has a substantial impact on the productivity of the local population as the course of the disease is usually prolonged and the development of acquired immunity in endemic areas takes several years. The recent emergence of drug-resistant strains has intensified research on alternative control methods such as vaccines. There is currently no effective available vaccine against malaria; however, numerous candidates have been studied in the past several years. One of the leading candidates is apical membrane antigen 1 (AMA1). This protein is involved in the invasion of Apicomplexa parasites into host cells, participating in the formation of a moving junction. Understanding how the genetic diversity of an antigen influences the immune response is highly important for vaccine development. In this study, we analyzed the diversity of AMA1 from Brazilian P. vivax isolates and 19 haplotypes of P. vivax were found. Among those sequences, 33 nonsynonymous PvAMA1 amino acid sites were identified, whereas 20 of these sites were determined to be located in predicted B-cell epitopes. Nonsynonymous mutations were evaluated for their influence on the immune recognition of these antigens. Two distinct haplotypes, 5 and 16, were expressed and evaluated for reactivity in individuals from northern Brazil. Both PvAMA1 variants were reactive. Moreover, the IgG antibody response to these two PvAMA1 variants was analyzed in an exposed but noninfected population from a P. vivax endemic area. Interestingly, over 40% of this population had antibodies recognizing both variants. These results have implications for the design of a vaccine based on a polymorphic antigen

Gas6 drives Zika virus-induced neurological complications in humans and congenital syndrome in immunocompetent mice

Zika virus (ZIKV) has the ability to cross placental and brain barriers, causing congenital malformations in neonates and neurological disorders in adults. However, the pathogenic mechanisms of ZIKV-induced neurological complications in adults and congenital malformations are still not fully understood. Gas6 is a soluble TAM receptor ligand able to promote flavivirus internalization and downregulation of immune responses. Here we demonstrate that there is a correlation between ZIKV neurological complications with higher Gas6 levels and the downregulation of genes associated with anti-viral response, as type I IFN due to Socs1 upregulation. Also, Gas6 gamma-carboxylation is essential for ZIKV invasion and replication in monocytes, the main source of this protein, which was inhibited by warfarin. Conversely, Gas6 facilitates ZIKV replication in adult immunocompetent mice and enabled susceptibility to transplacental infection. Our data indicate that ZIKV promotes the upregulation of its ligand Gas6, which contributes to viral infectivity and drives the development of severe adverse outcomes during ZIKV infection