Caracterização funcional de mutantes da proteína NS3hel do vírus da dengue e expressão do domínio RdRp da proteína NS5 para estudos de interação

Orientador: Daisy Maria StrottmannCoorientadores: Claudia N. D. dos Santos e Silvio Marques ZanataMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : A dengue atualmente constitui um dos principais problemas de saúde pública nas regiões tropicais e subtropicais. Apesar dos grandes avanços dos últimos anos para o entendimento da biologia do vírus da dengue (DENV), a patogenia da doença continua sendo um grande desafio. Estudos de infecção em modelo murino identificaram variantes de alta virulência em dengue vírus tipo 1 (DENV-1) que apresentam mutações nas proteínas E e NS3. As mutações na proteína NS3 encontram-se nos resíduos Val209Ile, Leu435Ser e Leu480Ser do domínio helicase. Recentemente, estudos do nosso grupo demonstraram que as mutações Leu435Ser e Leu480Ser aumentam a capacidade replicativa do DENV-1 in vivo, in vitro e ex vivo, enquanto a mutação Val209Ile parece não alterar a atividade biológica do vírus. Os mecanismos pelos quais as mutações modulam a virulência do vírus ainda não foram completamente compreendidos. A proteína NS3 é uma proteína multifuncional que, juntamente com a proteína NS5, desempenha papel crítico durante a replicação do genoma viral. Buscando um melhor entendimento dos mecanismos funcionais relacionados às mutações, o presente estudo visou expressar e purificar as proteínas NS3hel parental do DENV-1 e suas variantes a fim de avaliar o efeito das mutações pontuais na atividade de ATPase da proteína. Além disso, procuramos expressar o domínio NS5RdRp da proteína NS5 para estudos futuros de interação com a proteína NS3hel. Nossos dados revelam que as mutações em estudo conferem maior afinidade da proteína pelo substrato e maior eficiência catalítica quando comparadas com a proteína parental. Os resultados sugerem ainda que a atividade helicase da proteína NS3hel é estimulada pela molécula de dupla fita de RNA. Pelos métodos empregados neste estudo, não foi possível expressar de forma funcional a proteína recombinante NS5RdRp. No entanto, sob condições desnaturantes a proteína foi purificada e poderá ser utilizada para o desenvolvimento de anticorpos monoclonais, que contribuirão para estudos futuros. Os dados obtidos sobre a atividade ATPase da proteína NS3hel são indicativos de que mutações pontuais podem modular a eficiência catalítica da enzima e modificar a aptidão viral para suportar a infecção do DENV-1 em diferentes modelos de estudo. Tais informações são importantes para o entendimento dos mecanismos moleculares envolvidos na patogênese da dengue, os quais são fundamentais no contexto de produção de vacinas efetivas e delineamento de estratégias antivirai

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease

Evaluation of the ATP-adenosine axis in SARS-CoV-2 infection

Adenosina trifosfato (ATP) é uma molécula predominantemente intracelular que pode ser liberada para o meio extracelular durante ativação, estresse tecidual ou dano celular. O ATP extracelular pode ser rapidamente convertido em adenosina pela ação de ectonucleotidases, como CD39 e CD73 que são expressas na membrana de algumas células. A adenosina, por sua vez, pode induzir respostas anti-inflamatórias, participando ativamente da regulação de respostas imunes. O objetivo desse trabalho foi avaliar a participação do eixo ATP-adenosina na patologia da infecção pelo SARS-CoV-2, causador da COVID-19. Essa infecção pode induzir uma série de alterações imunológicas, incluindo o aumento da produção de citocinas pró-inflamatórias, fenômeno conhecido como cytokine storm. Foram analisadas amostras de 88 pacientes com COVID-19 não vacinados e hospitalizados no Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), sendo 44 com sintomas moderados e 44 com doença grave. O grupo controle, composto por indivíduos saudáveis, não vacinados e sem sintomatologia da infecção pulmonar, foi utilizado para comparação. A análise dos achados laboratoriais indicou que marcadores inflamatórios sanguíneos como, proteína C reativa e D-dímero, bem como, a razão N/L estão elevados em pacientes com COVID-19 grave. Também foi verificado que a expressão gênica das nucleotidases ENPP1, ENPP2, ENPP3 e NT5E (CD73), que participam da conversão de ATP em adenosina, está diminuída no sangue periférico de pacientes com COVID-19 grave. Interessantemente a menor expressão dessas enzimas está negativamente correlacionada aos marcadores inflamatórios da doença. Sustentando esses achados, pacientes com COVID-19 mostram maiores concentrações plasmáticas de ATP e menores de adenosina comparados com controles saudáveis. Análises de populações celulares específicas revelaram maior frequência de células T CD39+ em pacientes graves, e redução de células T CD4+ e CD8+ que expressam CD73. A frequência de células B CD39+CD73+ também está diminuída na infecção aguda. Interessantemente, células B de pacientes com COVID-19 foram menos eficazes em hidrolisar ATP em adenosina. Adicionalmente, há uma menor expressão de receptores de adenosina e um comprometimento da ativação da via de sinalização em pacientes infectados. A adição de adenosina in vitro, porém, suprimiu respostas inflamatórias em células de pacientes. Em resumo, esses achados sustentam a hipótese de que alterações no metabolismo de purinas extracelulares contribui com a desregulação imune durante a COVID-19, possivelmente favorecendo a gravidade da doença. A modulação de vias envolvendo a sinalização de adenosina pode representar uma estratégia terapêutica para a infecção aguda por SARS-CoV-2.Adenosine triphosphate (ATP) is a mostly intracellular molecule that can be released to the extracellular medium upon cellular activation, tissue stress, and cellular damage. Extracellular ATP can be converted into adenosine by ectonucleotidases, such as CD39 and CD73 expressed in the cellular membrane. Adenosine, on the other side, can drive anti-inflammatory responses, actively participating in immunoregulation processes. The aim of this study was to evaluate the contribution of the ATP-adenosine axis in the pathogenesis of SARS-CoV-2 infection that causes COVID-19. This infection can trigger alterations in the immune response, including excessive production of pro-inflammatory cytokines, known as cytokine storm. We analyzed samples from 88 COVID-19 patients unvaccinated and hospitalized at the Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), of which 44 had moderate symptoms and 44 had the severe form of the disease. The control group consisted of healthy, unvaccinated donors with no lung infection-associated symptoms. Analysis of laboratory findings indicates that inflammatory markers in the blood, such as C-reactive protein and D-dimer, as well as the N/L ratio are increased in patients with severe COVID-19. It was also verified that the gene expression of the nucleotidases ENPP1, ENPP2, ENPP3 and NT5E (CD73), involved in the hydrolysis of ATP into adenosine is reduced in the whole blood of patients with severe COVID-19. Interestingly, the lower expression of these enzymes is negatively correlated to inflammatory markers of the disease. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of adenosine when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into adenosine. Furthermore, impaired expression of adenosine receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of adenosine in vitro, suppressed inflammatory responses triggered in patients cells. In summary, our findings support the hypothesis that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity. The modulation of adenosine-related signaling pathways might be a therapeutic approach for acute infection with SARS-CoV-2

Immunomodulation of the antiviral response of macrophages of newborns by type I interferon adjuvants

Recém-natos (RNs) são mais susceptíveis a infecções devido à relativa imaturidade das respostas imunes inata e adaptativa. Nesse cenário, a modulação imunológica tem sido investigada como uma estratégia para aumentar a proteção contra infecções. Os macrófagos atuam tanto na imunidade inata quanto adaptativa, sendo potenciais alvos para estimular a resposta imune neonatal. Na infecção pelo HIV, os macrófagos atuam como reservatórios virais contribuindo com a replicação viral por longos períodos de tempo. Agonistas de receptores do tipo Toll podem controlar a replicação do HIV-1 em macrófagos de adultos in vitro, mas o impacto de tais moléculas em macrófagos de RNs ainda não foi verificado. Assim, o objetivo desse trabalho foi avaliar o efeito imunomodulador e antiviral de adjuvantes indutores de interferon tipo I em macrófagos de neonatos e adultos. Para isso, macrófagos foram gerados a partir de monócitos isolados de sangue de cordão umbilical e sangue periférico de adultos. Foi observado que os macrófagos de RNs possuem um perfil anti-inflamatório e de produção de IL-10. Os achados mostram ainda que os macrófagos neonatais são semelhantes aos macrófagos de adultos quanto à expressão gênica de componentes da imunidade inata. No entanto, as células neonatais mostram maior replicação viral quando infectadas com HIV-1 in vitro. Também verificou-se que o tratamento com os agonistas de TLR7/TLR8 (CL097), STING (cGAMP) e TLR3/RIG-I/MDA-5 (Poly-I:C) induz a expressão de IFN-&#946 e do fator antiviral MxA em macrófagos de RNs e adultos, mas CL097 é mais eficaz em promover a expressão de sensores citosólicos, em especial RIG-I e cGAS, além de inibir a expressão de TREX-1. Esse agonista também promove a indução de citocinas inflamatórias e &#223-quimiocinas, bem como, da citocina reguladora IL-10. Os resultados indicam ainda que CL097 inibe a replicação do HIV-1 em macrófagos de RNs e adultos, e esse efeito não parece ser dependente da ativação de NF-&#967B. Portanto, o agonista CL097 mostra um potencial terapêutico relevante como adjuvante da resposta neonatal, sendo capaz de induzir fatores antivirais que inibem a replicação do HIV-1.Newborns (NBs) are more susceptible to infections due to the relative immaturity of innate and adaptive immune responses. In this scenario, immunological modulation has been investigated as a strategy to increase protection against infections. Macrophages play a role on both innate and adaptive immunity, being potential targets for stimulating the neonatal immune response. In HIV infection, macrophages act as viral reservoirs contributing to viral replication for long periods of time. Toll-like receptor agonists can control HIV-1 replication in adult macrophages in vitro but the impact of such molecules on macrophages of NBs has not yet been verified. Therefore, the aim of this study was to evaluate the immunomodulatory and antiviral effects of type I interferon adjuvants on macrophages of neonates and adults. For this, macrophages were generated from monocytes isolated from umbilical cord blood and peripheral blood from adults. It was observed that the macrophages of NBs have an anti-inflammatory profile with IL-10 production. The findings also show that neonatal macrophages are similar to adult macrophages regarding the gene expression of innate immunity components. However, neonatal cells show increased viral replication when infected with HIV-1 in vitro. It has also been found that the treatment with TLR7/TLR8 (CL097), STING (cGAMP) and TLR3/RIG-I/MDA-5 (Poly-I:C) agonists induces the expression of IFN-&#223 and the antiviral factor MxA in macrophages of NBs and adults, however CL097 is more effective in promoting the expression of cytosolic sensors, especially RIG-I and cGAS, in addition to inhibit the expression of TREX-1. This agonist also promotes the induction of inflammatory cytokines and &#223-chemokines, as well as the regulatory cytokine IL-10. The results further indicate that CL097 inhibits HIV-1 replication in macrophages of NBs and adults, and this effect does not seem to be dependent on NF-&#967B activation. Therefore, CL097 shows a relevant therapeutic potential as adjuvant of the neonatal response, being able to induce antiviral factors that inhibit HIV-1 replication

Role of Histamine in Modulating the Immune Response and Inflammation

Inflammatory mediators, including cytokines, histamine, bradykinin, prostaglandins, and leukotrienes, impact the immune system, usually as proinflammatory factors. Other mediators act as regulatory components to establish homeostasis after injury or prevent the inflammatory process. Histamine, a biogenic vasoactive amine, causes symptoms such as allergies and has a pleiotropic effect that is dependent on its interaction with its four histamine receptors. In this review, we discuss the dualistic effects of histamine: how histamine affects inflammation of the immune system through the activation of intracellular pathways that induce the production of inflammatory mediators and cytokines in different immune cells and how histamine exerts regulatory functions in innate and adaptive immune responses. We also evaluate the interactions between these effects

Contrasting patterns of insecticide resistance and knockdown resistance (kdr) in Aedes aegypti populations from Jacarezinho (Brazil) after a Dengue Outbreak

ABSTRACT After a dengue outbreak, the knowledge on the extent, distribution and mechanisms of insecticide resistance is essential for successful insecticide-based dengue control interventions. Therefore, we evaluated the potential changes to insecticide resistance in natural Aedes aegypti populations to Organophosphates (OP) and Pyrethroids (PY) after chemical vector control interventions. After a Dengue outbreak in 2010, A. aegypti mosquitoes from the urban area of Jacarezinho (Paraná, Brazil) were collected in 2011 and 2012. Insecticide resistance to OP Temephos was assessed in 2011 and 2012 by dose–response bioassays adopting WHO-based protocols. Additionally, in both sampling, PY resistance was also investigated by the Val1016Ile mutation genotyping. In 2011, a random collection of mosquitoes was carried out; while in 2012, the urban area was divided into four regions where mosquitoes were sampled randomly. Bioassays conducted with larvae in 2011 (82 ± 10%; RR95 = 3.6) and 2012 (95 ± 3%; RR95 = 2.5) indicated an incipient altered susceptibility to Temephos. On the other hand, the Val1016IIe mutation analysis in 2011, presented frequencies of the 1016Ilekdr allele equal to 80%. Nevertheless, in 2012, when the urban area of Jacarezinho was analyzed as a single unit, the frequency of the mutant allele was 70%. Additionally, the distribution analysis of the Val1016Ile mutation in 2012 showed the mutant allele frequencies ≥60% in all regions. These outcomes indicated the necessity of developing alternative strategies such as insecticide rotations for delaying the evolution of resistance

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life

Impact of Inflammatory Immune Dysfunction in Psoriasis Patients at Risk for COVID-19

Psoriasis is an immune-mediated dermatosis usually associated with comorbidities. Treatment varies from topicals to systemic drugs and data on susceptibility to viral infections in psoriatic patients are scarce. The objectives of this study were to analyze psoriatic patients on different therapies who were at risk for COVID-19 for seroprevalence of SARS-COV-2, pro-inflammatory cytokine profile, comorbidities and outcomes in order to unveil the immunological mechanisms involved in the anti-viral response in patients with psoriasis. Seventy-five patients with psoriasis were divided according to treatment: immunobiologics, methotrexate, topicals and acitretin. Twenty healthy controls were included. Plasma samples were collected for: IgG SARS-COV-2 (ELISA); IL-27, IL-29 and IL-18 (ELISA); and IL-1β, IL-17A, IL-6 and TNF (cytometric array). Seropositivity for SARS-COV-2 was detected in 24 out of 75 psoriasis patients and did not relate to COVID-19 symptoms and/or hospitalization, despite associated comorbidities. Psoriasis patients who were asymptomatic for SARS-COV-2 exhibited immune imbalance with high levels of IL-18, IL-17A and IL-6, and low levels of IL-27 compared to healthy controls. Psoriasis groups showed significant increased cytokine levels only in the group with immunobiologics. Despite immune deviations and lower IL-27, which has a potential antiviral impact, psoriatic patients did not exhibit complications related to COVID-19. An understanding of this kind of proinflammatory profile of psoriatic patients and of the lack of severe outcomes for COVID-19 is essential to establish novel therapeutic approaches and preventive measures, including with regard to the concomitance of viral infections

SARS-CoV-2 Infection and CMV Dissemination in Transplant Recipients as a Treatment for Chagas Cardiomyopathy: A Case Report

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has infected over 90 million people worldwide, therefore it is considered a pandemic. SARS-CoV-2 infection can lead to severe pneumonia, acute respiratory distress syndrome (ARDS), septic shock, and/or organ failure. Individuals receiving a heart transplantation (HT) may be at higher risk of adverse outcomes attributable to COVID-19 due to immunosuppressives, as well as concomitant infections that may also influence the prognoses. Herein, we describe the first report of two cases of HT recipients with concomitant infections by SARS-CoV-2, Trypanosoma cruzi, and cytomegalovirus (CMV) dissemination, from the first day of hospitalization due to COVID-19 in the intensive care unit (ICU) until the death of the patients