Peptide Microarrays for Flavivirus Diagnosis.

Flavivirus are the most alarming prevalent viruses worldwide due to its vast impact on public health. Most early symptoms of diseases caused by Flavivirus are similar among each other and to other febrile illnesses making the clinical differential diagnosis challenging. In addition, due to cross-reactivity and a relatively limited persistence of viral RNA in infected individuals, the current available diagnosis strategies fail to efficiently provide a differential viral identification. In this context, virus-specific tests are essential to improve patient care, as well as to facilitate disease surveillance and the effective control of transmission. Here, we describe the use of protein microarrays as an effective tool for screening peptides differentially recognized by anti-Yellow Fever virus antibodies induced by vaccination or by natural viral infection.

Hypoxanthine guanine phosphoribosyl transferases SmHGPRTases functional roles in Schistosoma mansoni.

Introduction: Extracellular/environmental stimuli trigger cellular responses to allow Schistosoma sp. parasites adaptation and decide development and survival fate. In this context, signal transduction involving eukaryotic protein kinases (ePKs) has an essential role in regulatory mechanisms. Functional studies had shown the importance of MAPK pathway for Schistosoma mansoni development. In addition, early studies demonstrated that Smp38 MAPK regulates the expression of a large set of genes, among them the hypoxanthine-guanine phosphoribosyl transferase 1 (SmHGPRTase 1, Smp_103560), a key enzyme in the purine salvage pathway that is part of a family comprising five different proteins. Methods: First, the regulation of this gene family by the MAPKs pathways was experimentally verified using Smp38-predicted specific inhibitors. In silico analysis showed significant differences in the predicted structure and the domain sequence among the schistosomal HGPRTase family and their orthologs in humans. In order to interrogate the HGPRTases (Smp_103560, Smp_148820, Smp_168500, Smp_312580 and Smp_332640, henceforth SmHGPRTase -1, -2, -3, -4, -5) functional roles, schistosomula, sporocysts, and adult worms were knocked-down using specific dsRNAs. Results: Our results suggest that SmHGPRTases activity has an essential role in sporocysts and schistosomula development since significant differences in viability, size, and/ or shape were observed after the in vitro knockdown. Also, the knockdown of SmHGPRTases in schistosomula influenced the ovary development and egg maturation in female adult worms during mammalian infection. We also observed alterations in the movement of female adult worms knocked-down in vitro. Most of these results were shown when all gene family members were knocked-down simultaneously, suggesting a redundant function among them. Discussion: Thus, this study helps to elucidate the functional roles of the SmHGPRTase gene family in the S. mansoni life cycle and provides knowledge for future studies required for schistosomiasis treatment and control.

Mapping and Validation of Peptides Differentially Recognized by Antibodies from the Serum of Yellow Fever Virus-Infected or 17DD-Vaccinated Patients.

Yellow Fever disease is caused by the Yellow Fever virus (YFV), an arbovirus from the Flaviviridae family. The re-emergence of Yellow Fever (YF) was facilitated by the increasing urbanization of sylvatic areas, the wide distribution of the mosquito vector, and the low percentage of people immunized in the Americas, which caused severe outbreaks in recent years, with a high mortality rate. Therefore, serological approaches capable of discerning antibodies generated from the wild-type (YFV-WT) strain between the vaccinal strain (YFV-17DD) could facilitate vaccine coverage surveillance, enabling the development of strategies to avoid new outbreaks. In this study, peptides were designed and subjected to microarray procedures with sera collected from individuals infected by WT-YFV and 17DD-YFV of YFV during the Brazilian outbreak of YFV in 2017/2018. From 222 screened peptides, around ten could potentially integrate serological approaches aiming to differentiate vaccinated individuals from naturally infected individuals. Among those peptides, one was synthesized and validated through ELISA.

Docking-Based Virtual Screening Enables Prioritizing Protein Kinase Inhibitors With In Vitro Phenotypic Activity Against Schistosoma mansoni.

Schistosomiasis is a parasitic neglected disease with praziquantel (PZQ) utilized as the main drug for treatment, despite its low effectiveness against early stages of the worm. To aid in the search for new drugs to tackle schistosomiasis, computer-aided drug design has been proved a helpful tool to enhance the search and initial identification of schistosomicidal compounds, allowing fast and cost-efficient progress in drug discovery. The combination of high-throughput in silico data followed by in vitro phenotypic screening assays allows the assessment of a vast library of compounds with the potential to inhibit a single or even several biological targets in a more time- and cost-saving manner. Here, we describe the molecular docking for in silico screening of predicted homology models of five protein kinases (JNK, p38, ERK1, ERK2, and FES) of Schistosoma mansoni against approximately 85,000 molecules from the Managed Chemical Compounds Collection (MCCC) of the University of Nottingham (UK). We selected 169 molecules predicted to bind to SmERK1, SmERK2, SmFES, SmJNK, and/or Smp38 for in vitro screening assays using schistosomula and adult worms. In total, 89 (52.6%) molecules were considered active in at least one of the assays. This approach shows a much higher efficiency when compared to using only traditional high-throughput in vitro screening assays, where initial positive hits are retrieved from testing thousands of molecules. Additionally, when we focused on compound promiscuity over selectivity, we were able to efficiently detect active compounds that are predicted to target all kinases at the same time. This approach reinforces the concept of polypharmacology aiming for "one drug-multiple targets". Moreover, at least 17 active compounds presented satisfactory drug-like properties score when compared to PZQ, which allows for optimization before further in vivo screening assays. In conclusion, our data support the use of computer-aided drug design methodologies in conjunction with high-throughput screening approach.

Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors.

The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.

Design and production of dengue virus chimeric proteins useful for developing tetravalent vaccines.

Dengue virus (DENV) is a Flavivirus estimated to cause 390 million infections/year. Currently, there is no anti-viral specific treatment for dengue, and efficient DENV vector control is still unfeasible. Here, we designed and produced chimeric proteins containing potential immunogenic epitopes from the four DENV serotypes in an attempt to further compose safer, balanced tetravalent dengue vaccines. For this, South American DENV isolate sequences were downloaded from the NCBI/Virus Variation/Dengue virus databases and intraserotype-aligned to generate four consensuses. Four homologous DENV sequences were retrieved using BLAST and then interserotype-aligned. In parallel, sequences were subjected to linear B epitope prediction analysis. Regions of the envelope and NS1 proteins that are highly homologous among the four DENV serotypes, non-conserved antigenic regions and the most antigenic epitopes found in the C, prM, E and NS1 DENV proteins were used to construct 11 chimeric peptides. Genes encoding the chimeric proteins were commercially synthesized, and proteins were expressed, purified by affinity chromatography and further subjected to ELISA assays using sera from individuals infected with DENVs 1, 2, 3 or 4. As a proof-of-concept, the chimeric EnvEpII protein was selected to immunize BALB/c and C57BL/6 mice strains. The immunization with EnvEpII protein associated with aluminum induced an increased number of T CD4+ and CD8+ cells, high production of IgG1 and IgG2 antibodies, and increased levels of IL-2 and IL-17 cytokines, in both mouse strains. Because the EnvEpII protein associated with aluminum induced an efficient cellular response by stimulating the production of IL-2, IL-4, IL-17 and induced a robust humoral response in mice, we conclude that it resembles an efficient specific response against DENV infection. Although further experiments are required, our results indicate that epitope selection by bioinformatic tools is efficient to create recombinant proteins that can be used as candidates for the development of vaccines against infectious diseases.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Desenho e produção de proteínas quiméricas potencialmente aplicáveis no desenvolvimento de testes diagnóstico e/ou vacinas para a febre Dengue

A dengue é uma doença viral transmitida de mosquitos para humanos sendo a arbovirose mais prevalente em países tropicais e subtropicais, atingindo milhões de pessoas em diferentes regiões do mundo. A dengue é causada pelo Dengue virus (DENV), membro da família Flaviviridae, que possuem 4 sorotipos geneticamente distintos conhecidos como DENV 1-4. Uma vacina eficiente necessita de gerar resposta imune tetravalente balanceada. Para alcançarmos essa imunidade tetravalente balanceada, trabalhamos com a hipótese de que proteínas quiméricas expressando epítopos imunogênicos dos quatro sorotipos do DENV possam ser utilizadas no desenvolvimento de uma vacina segura e/ou um sistema de diagnóstico eficiente. Para este estudo onze proteínas quiméricas foram desenhadas contendo regiões de proteínas com potencial imunogênico derivado do envelope, capsídeo, membrana e/ou da proteína não estrutural NS1, dos quatro sorotipos do DENV. Tais regiões foram selecionadas in silico utilizando-se o algoritmo BepiPred. Regiões com alta homologia entre os quatro sorotipos do DENV foram preferencialmente incluídas, mas regiões antigênicas de um ou mais sorotipos do DENV também foram utilizados. As proteínas quiméricas foram construídas pela adição de resíduos de aminoácidos entre as sequências selecionadas, chamados de espaçadores, para que a estrutura dos epítopos expressos fosse mantida.

A sequência final de aminoácidos foi traduzida e a sequência de nucleotídeos foi otimizada utilizando o algoritmo LETO 1.0 (Entelechon). Todas as onze proteínas quiméricas foram produzidas utilizando os vetores de expressão pET 28 TEV, pQE-9 ou pET-21a, transformados em E. coli BL21 ou M15 e foram purificadas por cromatografia de afinidade utilizando resina de níquel para realização de ensaios de Western blot e ELISA para testar a reatividade das proteínas com soros de indivíduos já infectados pelo DENV e indivíduos nunca infectados e testes de imunogenicidade através da imunização de camundongos das linhagens BALB/c e C57BL/6. Nossos resultados mostraram um reconhecimento específico de cinco proteínas quiméricas com soros de pacientes sabidamente infectados pelo DENV-1, DENV-2 ou DENV-3. Além disso, a imunização de camundongos com a proteína quimérica EnvEpII, mostrou que esta proteína foi capaz de estimular uma produção robusta de anticorpos IgG1, IgG2a e IgG2c nas duas linhagens de camundongos testadas e de anticorpos neutralizantes em C57BL/6. Além disso, foi observada a ativação de células T CD4+ e CD8+ em BALB/c e o aumento dos níveis das citocinas IL-2, IL-4, IL-17 e IFNγ, quando camundongos foram imunizados com a proteína EnvEpII. Nossos resultados demonstram que desenhar, sintetizar, expressar e purificar proteínas quiméricas em sistemas bacterianos é viável e, dessa forma, proteínas artificiais podem ser estudadas como candidatas para o desenvolvimento de vacinas e/ou sistemas de diagnóstico contra doenças infecciosas.