Produção de anticorpos monoclonais contra um fragmento recombinante da proteína estrutural do capsídeo do vírus da mionecrose infecciosa de camarões

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.O vírus da mionecrose infecciosa de camarões (IMNV) é um patógeno que determina elevadas perdas econômicas nas fazendas de cultivo no Brasil. Os principais sinais da enfermidade são necrose e opacidade dos músculos, além de letargia dos camarões infectados. O agente etiológico da enfermidade, o IMNV, é um vírus icosaédrico e não-envelopado, sendo a proteína estrutural que constitui o capsídeo viral tem aproximadamente 100 kDa e o genoma é constituído por RNA dupla fita que possui 7.560 pb. Métodos moleculares e histológicos de diagnóstico já foram estabelecidos, porém testes rápidos de diagnóstico como testes imunocromatográficos, que possam ser utilizados em campo, ainda não estão disponíveis. O presente trabalho teve como objetivo a clonagem e expressão heteróloga de um fragmento da proteína estrutural do IMNV e sua posterior utilização para a produção de anticorpos monoclonais. O RNA total do músculo esquelético de camarões Litopenaeus vanammei naturalmente infectados foi obtido e o cDNA foi produzido através de RTPCR, sendo posteriormente amplificado por PCR. Iniciadores específicos foram capazes de amplificar, através de PCR, um segmento de cDNA de 600 pb, correspondente a um fragmento da proteína estrutural do capsídeo do IMNV. O fragmento obtido foi clonado, seqüenciado e, posteriormente, sub-clonado no vetor de expressão pET-14b. Este plasmídeo de expressão foi utilizado para tranformação de bactérias E. coli BL21(DE3), com as quais foram realizados testes de expressão que possibilitaram a obtenção de uma banda protéica de 25 kDa. A confirmação da identidade da proteína heteróloga expressa foi verificada por SDS-PAGE e Western-blot. Após a purificação da proteína recombinante, esta foi utilizada na imunização de camundongos Balb/c, cujos esplenócitos foram utilizados na fusão com células de mieloma, para produção de hibridomas. A triagem dos hibridomas foi realizada através de ELISA indireto. Foram obtidos seis linhagens monoclonais produtoras de anticorpos anti-IMNV, sendo quatro anticorpos IgM (11D, 33G, 39G e 54H) e dois anticorpos IgG1 (13H, 46C). A reatividade dos anticorpos monoclonais contra extratos de tecido muscular de camarões infectados por IMNV foi avaliada através de Western blot, confirmando assim a especificidade dos anticorpos contra a proteína estrutural do IMNV. Estes anticorpos poderão ser utilizados futuramente para a produção de um kit de diagnóstico imunocromatográfico, que será útil para testes em campo, por ser uma estratégia rápida, específica e factível de ser utilizado em fazendas de cultivo de camarões

Avaliação da ativação da via UPR (Unfolded Protein Response) em células de indivíduos HIV positivos sob diferentes esquemas terapêuticos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em BiotecnologiaA via UPR (do inglês Unfolded Protein Response) é uma resposta celular ao acúmulo de proteínas desenoveladas no lúmen do retículo endoplasmático, possuindo três braços (PERK, IRE1 e ATF6) que atuam sinergicamente em controles traducionais e transcricionais com o objetivo de restabelecer a homeostase celular. Os vírus, ao induzirem a célula a produzir proteínas virais, aumentam a carga de proteínas que devem ser dobradas no retículo endoplasmático, frequentemente causando a ativação da via UPR. O presente trabalho teve como objetivo estudar o impacto da infecção do HIV sobre a via UPR em células de indivíduos HIV positivos submetidos a diferentes esquemas terapêuticos. Lisados proteicos provenientes de linfócitos B, linfócitos T CD4+ e monócitos de indivíduos sadios e de pacientes HIV positivos virgens de tratamento, sob tratamento antirretroviral sem inibidor de protease ou com inibidor de protease foram avaliados quanto à presença de proteínas relacionadas à via UPR através de Western Blot. BiP teve expressão significantemente maior em linfócitos B de indivíduos HIV positivos. O fator eIF2., relacionado ao braço PERK, foi detectado em pacientes HIV positivos sob terapia antirretroviral; na sua forma não-fosforilada foi encontrado em monócitos enquanto na sua forma fosforilada e inativada, em linfócitos B e linfócitos T CD4+. P-IRE1 foi expresso em linfócitos B e linfócitos T CD4+ de indivíduos HIV negativos e pacientes HIV positivos, porém esta expressão mostrou-se significantemente maior em células de indivíduos sob tratamento antirretroviral. Células de indivíduos HIV positivos apresentaram ainda níveis de clivagem e ativação de ATF6 significantemente maiores quando comparados a indivíduos sadios. O perfil de ativação da via UPR mostrou-se diferente para indivíduos HIV negativos e HIV positivos e dentre os últimos, indivíduos virgens de tratamento ativaram apenas o braço ATF6 da via UPR, enquanto pacientes sob terapia antirretroviral ativaram os três braços. Os diferentes perfis fenotípicos de expressão de proteínas relacionadas à via UPR parecem estar relacionados à infecção pelo HIV e às cargas virais apresentadas pelos indivíduos HIV positivos, estando estas últimas vinculadas à presença ou não de terapia antirretroviral.The Unfolded Protein Response (UPR) is a mechanism initiated whenever protein folding in the endoplasmic reticulum (ER) is compromised. The UPR pathway has three sensors of ER stress (PERK, IRE1, and ATF6), which promotes the cell metabolism back to homeostasis through transcriptional and translational controls. Virus leads infected cells to produce a great amount of new proteins, which increases the number of unfolded proteins in the ER lumen and activates the UPR signal pathways. The aim of this study was to analyze the HIV infection impact in the UPR activation in cells from HIV-positive individuals under different antiretroviral therapy. Protein lysates from B lymphocytes, T CD4+ lymphocytes and monocytes from healthy individuals, treatment-naive HIV-positive patients, and HIV-positive patients under antiretroviral therapy with or without protease inhibitors were evaluated about their expression of UPR related proteins. Amounts of BiP were significantly higher in B lymphocytes from HIV-positive individuals when compared to healthy individuals. The eIF2. factor, related to the ER stress PERK sensor, was detected in cells from HIV-positive individuals under antiretroviral therapy; non-phosphorylated eIF2. was found in monocytes whereas its phosphorylated form was expressed in B lymphocytes and T CD4+ lymphocytes. P-IRE1 was expressed in B lymphocytes and T CD4+ lymphocytes from HIV-negative and HIV-positive individuals, however this expression was significantly higher in cells from treated HIV-positive individuals. Cells from HIV-positive patients also showed higher levels of ATF6 cleavage and activation in relation to healthy individuals. The expression profile of UPR related proteins showed to be different in HIV-negative and HIV-positive individuals. Differences were also detected between the expression protein profiles from treatment-naive and treated HIV-positive individuals. In treatment-naive patients only the ATF6 pathway was activated while in HIV-positive patients under antiretroviral treatment all the three ER stress sensors were activated. These non-equal phenotypes of UPR activation seem to be related to the HIV infection and the viral loads presented by the HIV-positive patients, being the number of infective particles in their plasmas connected to the presence of antiretroviral therapy

NK cells negatively regulate CD8 T cells via natural cytotoxicity receptor (NCR) 1 during LCMV infection

Besides their function in recognizing cancerous and virally infected cells, natural killer (NK) cells have the potential to shape adaptive immune responses. However, the mechanisms employed by NK cells to negatively regulate virus-specific CD8 T cell responses remain to be fully defined. Using activating receptor natural cytotoxicity receptor (NCR) 1 deficient (NCR1gfp/gfp) mice, we found increased numbers of virus-specific CD8 T cells, leading to enhanced virus control during acute LCMV infection. Furthermore, virus-specific CD8 T cells were more activated in the absence of NCR1, resulting in exacerbated immunopathology, documented by weight loss, and superior virus control early during chronic LCMV infection. Transfer experiments of virus-specific CD8 T cells into NCR1 deficient hosts revealed a direct cross talk between NK and CD8 T cells. Studies on the splenic microarchitecture revealed pronounced disorganization of T cells in infected NCR1gfp/gfp mice, resulting in enhanced immunopathology and disruption of the T cell niche upon chronic LCMV infection. Our data show a novel pathway employed by NK cells to regulate antiviral CD8 T cell responses, namely direct recognition and elimination of activated CD8 T cells via NCR1 early during infection to protect the host from an overshooting T cell response

Neutrophilia, lymphopenia and myeloid dysfunction: A living review of the quantitative changes to innate and adaptive immune cells which define COVID-19 pathology

Destabilisation of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils, and eosinophils represent an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response which bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of ICU admission and death; therefore, this parameter is particularly important for clinical decision making. Mild and severe disease differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decision or predict disease outcomes

Pediatric Hospitalizations Associated with 2009 Pandemic Influenza A (H1N1) in Argentina

Fil: Libster, Romina. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Bugna, Jimena. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Coviello, Silvina. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Hijano, Diego R. Hospital De Niños Sor María Ludovica, La Plata; Argentina.Fil: Dunaiewsky, Mariana. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Reynoso, Natalia. Hospital Municipal Materno Infantil de San Isidro; Argentina.Fil: Cavalieri, Maria L. Hospital Eva Perón, Benito Juárez, Buenos Aires; ArgentinaFil: Guglielmo, Maria C. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Areso, M. Soledad. Hospital Eva Perón, Benito Juárez, Buenos Aires; ArgentinaFil: Gilligan, Tomas. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Santucho, Fernanda. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Cabral, Graciela. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Gregorio, Gabriela L. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Moreno, Rina. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Lutz, Maria I. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Panigasi, Alicia L. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Saligari, Liliana. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Caballero, Mauricio T. Hospital De Niños Sor María Ludovica, La Plata; Argentina.Fil: Egües Almeida, Rodrigo M. Hospital De Niños Sor María Ludovica, La Plata; Argentina.Fil: Gutierrez Meyer, Maria E. Hospital De Niños Sor María Ludovica, La Plata; Argentina.Fil: Neder, Maria D. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Davenport, Maria C. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Del Valle, Maria P. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Santidrian, Valeria S. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Mosca, Guillermina. Ministerio de Ciencia, Técnica e Innovación. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Alvarez, Liliana. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Landa, Patricia. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Pota, Ana. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Boloñati, Norma. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Dalamon, Ricardo. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Sanchez Mercol, Victoria I. Hospital Eva Perón, Benito Juárez, Buenos Aires; Argentina.Fil: Espinoza, Marco. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Peuchot, Juan Carlos. Hospital Eva Perón, Benito Juárez, Buenos Aires; Argentina.Fil: Karolinski, Ariel. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Bruno, Miriam. Hospital General de Agudos Carlos G. Durand, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Borsa, Ana. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Ferrero, Fernando. Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Bonina, Angel. Hospital De Niños Sor María Ludovica, La Plata; Argentina.Fil: Ramonet, Margarita. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Albano, Lidia C. Hospital Nacional Profesor Alejandro Posadas, El Palomar, Buenos Aires; Argentina.Fil: Luedicke, Nora. Ministerio de Ciencia, Técnica e Innovación. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Alterman, Elias. Fundación Infant, Ciudad Autónoma de Buenos Aires; Argentina.Fil: Savy, Vilma L. ANLIS Dr.C.G.Malbrán. Instituto de Enfermedades Infecciosas; Argentina.Fil: Baumeister, Elsa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología. Servicio de Virosis Respiratoria; Argentina.Fil: Chappell, James D. Vanderbilt University. Pathology, Nashville, Tennessee; Estados Unidos.Fil: Edwards, Kathryn M. Vanderbilt University. Departments of Pediatrics, Nashville, Tennessee; Estados Unidos.Fil: Melendi, Guillermina A. Vanderbilt University. Departments of Pediatrics, Nashville, Tennessee; Estados Unidos.Fil: Polack, Fernando P. Vanderbilt University. Departments of Pediatrics, Nashville, Tennessee; Estados Unidos.Background: While the Northern Hemisphere experiences the effects of the 2009 pandemic influenza A (H1N1) virus, data from the recent influenza season in the Southern Hemisphere can provide important information on the burden of disease in children. Methods: We conducted a retrospective case series involving children with acute infection of the lower respiratory tract or fever in whom 2009 H1N1 influenza was diagnosed on reverse-transcriptase polymerase-chain-reaction assay and who were admitted to one of six pediatric hospitals serving a catchment area of 1.2 million children. We compared rates of admission and death with those among age-matched children who had been infected with seasonal influenza strains in previous years. Results: Between May and July 2009, a total of 251 children were hospitalized with 2009 H1N1 influenza. Rates of hospitalization were double those for seasonal influenza in 2008. Of the children who were hospitalized, 47 (19%) were admitted to an intensive care unit, 42 (17%) required mechanical ventilation, and 13 (5%) died. The overall rate of death was 1.1 per 100,000 children, as compared with 0.1 per 100,000 children for seasonal influenza in 2007. (No pediatric deaths associated with seasonal influenza were reported in 2008.) Most deaths were caused by refractory hypoxemia in infants under 1 year of age (death rate, 7.6 per 100,000). Conclusions: Pandemic 2009 H1N1 influenza was associated with pediatric death rates that were 10 times the rates for seasonal influenza in previous years

T cell phenotypes in COVID-19 - a living review

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation

The role and uses of antibodies in COVID-19 infections: a living review

Coronavirus disease 2019 has generated a rapidly evolving field of research, with the global scientific community striving for solutions to the current pandemic. Characterizing humoral responses towards SARS-CoV-2, as well as closely related strains, will help determine whether antibodies are central to infection control, and aid the design of therapeutics and vaccine candidates. This review outlines the major aspects of SARS-CoV-2-specific antibody research to date, with a focus on the various prophylactic and therapeutic uses of antibodies to alleviate disease in addition to the potential of cross-reactive therapies and the implications of long-term immunity

Impact of asymmetric cell division on T cell differentiation

Successful immune responses rely on diversity. Being equipped with highly variable T cell receptors (TCRs), which convey antigen specificity, CD8+ T cells exhibit an immense repertoire of different naïve cells even before antigen encounter, but for an immune response to be effective, both clonal expansion, and differentiation must take place. Upon cognate antigen activation, one single naïve CD8+ T cell can give rise to different progenies, including effector and memory cells. How this diversity is generated, is still a phenomenon incompletely elucidated, but some mechanisms were reported to be involved in fate determination, such as differential strength of TCR activation, and differential exposure to inflammatory stimuli, leading to differential transcriptional and metabolic profiles, epigenetic control of gene expression, and asymmetric cell division (ACD). Concerning ACD as a means to foster diversification, most studies were limited to describe the mitotic polarization of a variety of components in activated naïve T lymphocytes, leading to the rise of two daughter cells inheriting distinct potential fates. We set out to study whether the ability to undergo ACD is limited to certain CD8+ T cell subsets and developed a strategy to modulate ACD rates. Using the murine Lymphocytic Choriomeningitis virus (LCMV) infection model, we established a correlation between ACD and cellular stemness, as naïve and memory CD8+ T cells (which exhibit stemness in terms of self-maintenance and being able to generate progenies with different fates) were found to divide asymmetrically, while terminally differentiated cells, as short-lived effector and exhausted cells, lacked this ability. ACD modulation was achieved by transient mTOR inhibition, leading to higher ACD rates in naïve and memory cells, and reestablishment of ACD in pre-terminally exhausted PD-1int CD8+ T cells. The ability to undergo ACD correlated with memory potential. Upon adoptive transfer, progenies of mTOR-inhibited LCMV-specific TCR transgenic P14 cells, exhibiting increased ACD rates, showed improved expansion upon acute or chronic LCMV infection, resulting in more efficient viral control. mTOR inhibition also led to higher ACD rates in stimulated naïve and memory human CD8+ T cells. Thus, ACD modulation might be a potential useful tool to enforce memory differentiation in T cells. Memory formation is known to be impaired upon aging as a consequence of profound remodelling of immune responses, in particular CD8+ T cells. This led us to investigate how aging affects the ability of CD8+ T cells from aged individuals to undergo ACD and whether transient mTOR inhibition could reinvigorate memory features that are impaired during immunosenescence. Analysis of CD8+ T cells isolated from young and old naïve P14 mice showed that aging led to overall decreased ACD rates, which could be increased upon transient mTOR inhibition and also led to better memory potential. Being aware of the heterogeneity within CD8+ T cell populations, and that aging leads to an increased CD44hi/CD44lo T cell ratio, we further dissected the ability of CD8+ T cells with high or low CD44 expression levels to undergo ACD. We found that CD44hi cells showed intrinsic higher ACD rates compared to their CD44lo counterparts. Moreover, ACD modulation by transient mTOR inhibition was only effective in CD44lo cells. Phenotypical analysis of CD44hi cells revealed them as virtual memory T cells (TVM). Functionally, TVM showed better expansion potential in adoptive transfer experiments, which is likely explained by their ability to survive better upon limiting availability of homeostatic cytokines when compared to “truly naïve” CD44lo CD8+ T cells. In this perspective, TVM emerge as a possible solution on how the adaptive immune system counteracts immunosenescence by endowing this T cell subset with long-term survival and function during advanced aging. As enhanced ACD proved to correlate with superior long-term survival and function, our results open new perspectives on vaccination and adoptive transfer therapies, of particular relevance in the context of tumours, chronic infections and improvement of immune responses in the elderly