As doenças respiratórias agudas (DRAs) são as causas mais comuns de morbidez e mortalidade infantil mundial, podendo ser causadas por uma grande variedade de microorganismos. A fim de se detectar os vírus respiratórios mais comumente associados às infecções agudas do trato respiratório e traçar seu perfil epidemiológico, utilizamos um protocolo de GS RT-PCR (GeneScan Transcrição Reversa-Reação em Cadeia da Polimerase) para a rápida detecção simultânea do, vírus influenza A e B, parainfluenzavirus tipo 1, 2 e 3, picornavirus, metapneumovirus e o adenovírus. As amostras clínicas foram colhidas de crianças menores de cinco anos de idade, apresentando sintomas respiratórios, no Hospital Universitário (HU) da Universidade de São Paulo (USP), durante o ano de 2003. O GS RT-PCR se mostrou uma metodologia sensível e específica, capaz de detectar uma diversidade maior de agentes infecciosos do trato respiratório em relação à Imunofluorescência Indireta (IFI), reduzindo neste estudo a porcentagem de amostras negativas de 69,9% (235 amostras) para 22% (74 amostras). / A reverse transcription polymerase chain reaction (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was used to detect nine common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV), human parainfluenza viruses 1, 2, and 3, influenza A and B viruses, human metapneumovirus, adenovirus and picornavirus were incorporated into a screening PCR standard assay format. The optimized assay panel was used to test 336 respiratory specimens obtained from children hospitalized with acute respiratory illness (ARI) that had been previously tested by viral culture and indirect immunofluorescence staining (IIF). GS RT-PCR showed be a sensitive and specific methodology, able to detect a larger diversity of respiratory viruses regarding IFI, reducing in this study the percentage of negative samples of 69,9% (235 samples) to 22% (74 samples).

Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated. Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived fromStaphylococcus aureusprotein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of thePlasmodium falciparumblood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies forin vivoorin vitrolipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived fromToxoplasma gondii. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation. Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product. Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy. We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against theP. falciparumPf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein. <b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,Staphylococcus aureusprotein A, Subunit vaccine,Toxoplasma gondii

<p>Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.</p><p>Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived from<i>Staphylococcus aureus</i>protein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of the<i>Plasmodium falciparum</i>blood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies for<i>in vivo</i>or<i>in vitro</i>lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived from<i>Toxoplasma gondii</i>. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.</p><p>Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.</p><p>Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.</p><p>We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against the<i>P. falciparum</i>Pf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.</p><p><b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,<i>Staphylococcus aureus</i>protein A, Subunit vaccine,<i>Toxoplasma gondii</i></p>

In dieser Arbeit wurden Vektorimpfstoffe auf der Basis rekombinanter Vacciniaviren hinsichtlich ihrer Eignung zur Immunisierung gegen Infektionen mit Respiratorischen Synzytialviren (RSV) untersucht. Hierfür standen genetisches Material und Viruspräparationen des Respiratorischen Synzytialvirus des Rindes (BRSV, Stamm Odijk) sowie des Respiratorischen Synzytialvirus des Menschen (HRSV, Subtyp A2) sowie rekombinante Vacciniaviren MVA-HRSV-F bzw. MVA-HRSV-G zur Verfügung. Rekombinante MVA-Viren, welche die Gene der BRSV-Oberflächenproteine G und F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu), sowie Viren in welchen die Fremdgensequenzen durch Deletion wieder entfernt sind (Revertante Viren MVA-∆BRSV-F und MVA-∆BRSV-G), wurden gentechnologisch hergestellt. Alle rekombinanten MVA-Viren wurden molekular-virologisch charakterisiert und dienten zur Gewinnung und Prüfung von Testimpfstoffen im Tiermodell. Die Untersuchungen zeigen: 1. Alle neu konstruierten rekombinanten MVA-BRSV-Viren produzierten nach Infektion von Zellkulturen die erwünschten Zielantigene, die BRSV-Glykoproteine F und G. Für das durch MVA-Expression hergestellte BRSV-F-Glykoprotein konnte außerdem die biologische Funktionalität in einem Fusionstest in infizierten HeLa-Zellen nachgewiesen werden. 2. Die Charakterisierung der Genome aller MVA-BRSV- sowie MVA-HRSV-Vektorviren bestätigte die exakte Insertion der Fremdgensequenzen im anvisierten Genombereich und zeigte die genetische Stabilität der Virusisolate nach Passagierung. 3. Bei der Untersuchung des Wachstumsverhaltens von MVA-BRSV-F und MVA-BRSV-G zeigte sich die eingeschränkte Vermehrungsfähigkeit des Virus MVA-BRSV-G. Die Konstruktion und Untersuchung der revertanten Viren MVA-∆BRSV-F und MVA-∆BRSV-G belegte die Koproduktion des G-Proteins als Ursache des verminderten Replikationsvermögens. Dieser für ein mögliches Impfvirus erhebliche Nachteil konnte durch die Verwendung eines moderateren Vacciniavirus-Promotors zur Fremdgenexpression (rekombinantes Virus MVA-BRSV-Gneu) behoben werden. 4. Die Prüfung von Testimpfstoffen auf der Grundlage der rekombinanten MVA-HRSV-Viren in einem Maus-HRSV-Infektionsmodell zeigte, dass MVA-HRSV-Impfstoffe, im Gegensatz zu Impfstoffen aus mit Formalin-inaktiviertem HRSV, Immunantworten mit einem ausgewogenen TH1/TH2-assoziierten Zytokinprofil induzierten. Eine infolge von Immunisierung verstärkte Einwanderung eosinophiler Zellen (Marker für Immunpathogenese) in die Lungen HRSV-infizierter Tiere, konnte nach MVA-Impfung nicht beziehungsweise in nur sehr geringem Ausmaß festgestellt werden (OLSZEWSKA et al. 2004). 5. Wichtige erste Daten hinsichtlich der Verträglichkeit, Immunogenität und Schutzwirkung rekombinanter Impfstoffe auf der Basis von MVA-BRSV-F und MVA-BRSV-G konnten in einem Kälber BRSV-Infektionsmodell erhoben werden. Die zweimalige Immunisierung mit MVA-Impfstoff verlief bei allen Tieren ohne feststellbare Nebenwirkungen und die Anregung Vaccinia- bzw. BRSV-F-spezifischer Antikörper bestätigte die Immunogenität der Vektorvakzinen. Schließlich belegten klinische Daten, insbesondere die fehlende Fieberreaktion bei Impflingen nach BRSV-Belastungsinfektion, die Schutzwirkung der MVA-BRSV-Impfstoffe. Insgesamt unterstützen die erzielten Ergebnisse dieser Arbeiten die weitere präklinische und klinische Untersuchung von MVA-Vektorimpfstoffen zur wirksameren und sichereren Bekämpfung von Infektionen mit Respiratorischen Synzytialviren. / This study investigated vector vaccines based on recombinant vaccinia virus MVA for their suitability to immunize against infections with respiratory syncytial viruses. Genetic material and virus stocks of bovine respiratory syncytial virus (BRSV, Strain Odijk) and human respiratory syncytial virus (HRSV, Strain A2) and recombinant vaccinia viruses MVA-HRSV-F and MVA-HRSV-G were provided and used in this study. The project work included the genetical engineering of recombinant MVA expressing gene sequences encoding the BRSV surface proteins G and F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu) and the secondary generation of mutant viruses in which recombinant gene sequences have been removed (revertant viruses MVA-∆BRSV-F, MVA-∆BRSV-G). All recombinant MVA were carefully characterized in in vitro experiments and served for generation of vaccine preparations being tested in animal model systems. The investigations demonstrate: 1. All recombinant MVA-BRSV viruses produced the target antigens (BRSV-F and -G proteins) upon tissue culture infections. Functional activity of BRSV-F protein was demonstrated in a cell fusion assay using virus-infected HeLa cells. 2. The characterization of the genomes of all MVA recombinant viruses confirmed the correct insertion of foreign gene sequences into the target site of the MVA genome and demonstrated the genetic stability of the vector viruses upon tissue culture passage. 3. In vitro studies on virus growth revealed a reduced replicative capacity of the recombinant virus MVA-BRSV-G. Construction and growth analysis of revertant viruses MVA-∆BRSV-F and MVA-∆BRSV-G demonstrated that over expression of BRSV-G protein caused this replication deficiency which could be avoided by using a more moderate vaccinia virus promoter for transcriptional control of recombinant gene expression (recombinant virus MVA-BRSV-Gneu). 4. Upon characterization in a mouse-HRSV challenge model candidate vaccines based on recombinant MVA-HRSV viruses, in contrast to formalin inactivated HRSV, and induced a well balanced TH1 and TH2 cytokine profile. In addition, none of the MVA-HRSV-F vaccinated animals and only two of the MVA-HRSV-G immunized mice showed low-level eosinophilia in the lungs after HRSV challenge infection (OLSZEWSKA et al. 2004). 5. Vaccination experiments in the calf-BRSV challenge model generated first relevant data on safety, immunogenicity and protective capacity of MVA-BRSV recombinant vaccines. The repeated application of MVA vaccine was well tolerated by all vaccinated animals and the induction of vaccinia- and BRSV-F-specific antibody responses confirmed the immunogenicity of the MVA vector vaccines. Moreover, clinical data (lack of fever response in vaccines) suggested the protective capacity of MVA-BRSV immunization upon BRSV challenge. The obtained results from these studies clearly support further preclinical and clinical evaluation of recombinant MVA candidate vaccines to immunize against disease caused by RSV infections in cattle and humans.

Made available in DSpace on 2015-07-22T20:50:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-25 / O Vírus Sincicial Respiratório (VSR) é o principal agente em infecções agudas do trato respiratório inferior (IATRI) antes de dois anos, com altas taxas de internação e óbito em crianças de alto risco para infecção grave pelo VSR. Objetivo: Identificar os vírus envolvidos nos quadros de infecções agudas de trato respiratório (IATR) e analisar taxas de internação e óbito em crianças submetidas à profilaxia com palivizumabe. Métodos: Coorte prospectiva com 198 crianças menores de um ano de idade nascidas antes de 29 semanas de idade gestacional e crianças menores de 2 anos de idade com cardiopatia hemodinamicamente instável ou doença pulmonar crônica que receberam palivizumabe para profilaxia contra infecções graves pelo VSR em 2008. Nesse período, em cada episódio de IATR foi coletado aspirado de nasofaringe (NPA) para identificação de VSR, adenovírus, parainfluenza 1, 2 e 3, influenza A e B por imunofluorescência direta, e rinovírus e metapneumovírus por RT-PCR. Foram monitoradas internações e óbitos nesse grupo. Resultados: Das 198 crianças acompanhadas, 117 (59,1%) apresentaram IATR, totalizando 175 episódios. Das 76 NAPs coletadas, 37 foram positivas, encontrando-se, rinovírus em 75,7% dessas amostras, VSR (18,9%), parainfluenza (28,1%), adenovírus (2,7%), metapneumovírus (2,7%) e co-infecção em três amostras. Das 48 internações, 18 ocorreram por causa respiratória, sendo apenas 1 por VSR. Em nenhuma das duas crianças que evoluíram para óbito detectou-se o VSR. Conclusão: Na vigência de profilaxia com palivizumabe, a frequência de isolamento de VSR em crianças de alto risco com IATR e naquelas que necessitaram de internação hospitalar foi baixa. / Respiratory Syncytial Virus (RSV) is the most important etiologic agent in acute lower respiratory tract infections (ALRTIs) in children under two years, with high rates of hospitalization and death in high risk children for severe RSV infection. Objective: To identify the virus present in acute respiratory tract infections (ARTIs) and to analyze rates of hospitalization and death in children who received palivizumab prophylaxis. Methods: Prospective cohort of 198 infants up to 1 year old born before 29 weeks gestation and infants less than two years of age with hemodynamically instable cardiopathy or chronic pulmonary disease, who received prophylactic palivizumab against severe RSV infections in 2008. During this period, a nasopharyngeal aspirate (NPA) was collected in each episode of ARTI for identification of RSV, adenovirus, parainfluenza 1, 2 and 3, influenza A and B by direct immunofluorescence, and rhinovirus and metapneumovirus for RT-PCR. Data regarding hospitalization and death were collected. Results: Of the 198 infants included, 117 (59,1%) presented ARTIs, with a total of 175 episodes. Of 76 NPAs collected, 35 were positive, being rhinovirus (75.7%), RSV (18.9%), parainfluenza (8.1%), adenovirus 2 (2.7%), metapneumovirus (2.7%) and 3 samples presented multiple agents. Of 48 hospitalizations, 18 presented respiratory causes, but in only one child was found RSV. None of the 2 children who died had RSV. Conclusion: During the palivizumab prophylaxis period, the frequency of RSV detected in high risk children with ARTIs and those who were hospitalized was low. / TEDE / BV UNIFESP: Teses e dissertações

Este estudo visou observar os efeitos plásticos induzidos pelo exercício a curto prazo em regiões motoras do encéfalo de ratos. Observou-se a expressão das subunidades GluR1 e GluR2/3. Os animais foram divididos em grupos de: 3 dias(COR3), 7 dias(COR7) e 15 dias(COR15); e um grupo controle(CONT). Empregaram-se as técnicas de imuno-histoquímica e immunoblotting. A expressão de GluR1 no cerebelo, demonstrou um decréscimo em COR3. No hipocampo houve uma queda na expressão em COR3(40%), retornando aos níveis basais em COR7. No córtex cerebral observou-se uma queda da expressão com máxima queda em COR7(52%), retornando à expressão basal em COR15. O estriado não sofreu alterações na expressão de GluR1 ao longo dos primeiros 7 dias, tendo um aumento em COR15(90%). A expressão de GluR2/3 não foi alterada, exceto no cerebelo, onde houve um decréscimo em dois momentos distintos, COR3(55%) e COR15(25%), retornando à expressão basal em COR7. Os nossos dados revelam que o exercício físico a curto prazo foi capaz de promover alterações plásticas ao longo do treinamento. / This study aimed at analyzing the plastic effects of the short-term exercise upon the rat motor area. We check the expression of GluR1 and GluR2/3. We divided into 3 experimental groups based on duration of exercise: 3 days(COR3), 7 days(COR7), and 15 days(COR15); and a control group(CONT). The experimental animals were subjected to a treadmill exercise protocol. The brains were subjected to the techniques of immunohistochemistry and immunoblotting. In the cerebellum, there was a decrease for COR3(17%). In the hippocampus, there was a decrease of the GluR1 expression for COR3 (40%). In the cerebral cortex there was a drop of GluR1 expression for COR3 and COR7(52%). In the striatum, there was no change of GluR1 expression during the first seven days, with a increase for COR15(90%). The GluR2/3 expression did not change in any brain structure analyzed, except in the cerebellum, where there was a significant decrease for two distinct groups, COR3(55%) and COR15(25%). Our data show that short-term physical exercise was able to promote plastic changes during training.

As doenças respiratórias agudas (DRAs) são as causas mais comuns de morbidez e mortalidade infantil mundial, podendo ser causadas por uma grande variedade de microorganismos. A fim de se detectar os vírus respiratórios mais comumente associados às infecções agudas do trato respiratório e traçar seu perfil epidemiológico, utilizamos um protocolo de GS RT-PCR (GeneScan Transcrição Reversa-Reação em Cadeia da Polimerase) para a rápida detecção simultânea do, vírus influenza A e B, parainfluenzavirus tipo 1, 2 e 3, picornavirus, metapneumovirus e o adenovírus. As amostras clínicas foram colhidas de crianças menores de cinco anos de idade, apresentando sintomas respiratórios, no Hospital Universitário (HU) da Universidade de São Paulo (USP), durante o ano de 2003. O GS RT-PCR se mostrou uma metodologia sensível e específica, capaz de detectar uma diversidade maior de agentes infecciosos do trato respiratório em relação à Imunofluorescência Indireta (IFI), reduzindo neste estudo a porcentagem de amostras negativas de 69,9% (235 amostras) para 22% (74 amostras). / A reverse transcription polymerase chain reaction (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was used to detect nine common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV), human parainfluenza viruses 1, 2, and 3, influenza A and B viruses, human metapneumovirus, adenovirus and picornavirus were incorporated into a screening PCR standard assay format. The optimized assay panel was used to test 336 respiratory specimens obtained from children hospitalized with acute respiratory illness (ARI) that had been previously tested by viral culture and indirect immunofluorescence staining (IIF). GS RT-PCR showed be a sensitive and specific methodology, able to detect a larger diversity of respiratory viruses regarding IFI, reducing in this study the percentage of negative samples of 69,9% (235 samples) to 22% (74 samples).

Orientador: Elenice Deffune / Banca: Mirthes Ueda / Banca: Rejane Tommasini Grotto / Resumo: O Vírus Sincicial Respiratório humano (VSR) é o principal agente causal das Infecções Respiratórias Agudas (IRAs) em lactantes e pré-escolares. Apresenta dois subgrupos - A e B - sendo o primeiro responsável por quadros clínicos mais graves. Os RNA mensageiros virais codificam 11 proteínas conhecidas, das quais a proteína F é responsável pela fusão viral à célula e se apresenta na forma de F0 com peso molecular de 67kDa, podendo ser clivada em duas unidades: F2 de 20kDa e a F1 de 47kDa. A proteína F recombinante (Fr), expressa em E.coli (BL21A no vetor pET28a), foi cedida por pesquisadores da UNESP de São José do Rio Preto. Com intuito de produzir anticorpos monoclonais contra a proteína Fr para fins de diagnóstico laboratorial, inoculamo-la em camundongos isogênicos Balb/c. Durante o desenvolvimento do monoclonal, foi necessário a eliminação da contaminação de cultura por Mycoplasma ssp. Após ajuste de dose de inoculação e descontaminação do antígeno (hipótese: contaminação com endotoxinas provenientes de E.coli), seis clones secretores de anticorpos monoclonais foram produzidos (5 do tipo IgM e 1 do tipo IgG2a) e testados, simultâneamente, contra toxina bacteriana e proteína Fr (40μg/mL) por técnica de ELISA indireto. Paralelamente, testes por citometria de fluxo foram realizados, incubando-se os clones obtidos com E.coli (bactéria com membrana permeabilizada e in natura). Os clones (VIRSV2-87A74 e VIRSV2-87A80) apresentam um reconhecimento inespecífico de proteínas da E. coli e não foi possível caracterizar os clones obtidos em função da dificuldade de se obter novas amostras de proteína Fr. Para o futuro, os anticorpos obtidos deverão ser marcados com isotiocinato de fluoroceína e comporem um amplo teste epidemiológico em paralelo com o kit disponível em mercado respeitando a sazonalidade da doença. Novos ensaios para caracterização... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Human Respiratory Syncytial Virus (HRSV) is the main cause of Acute Respiratory Insufficiency in suckling child and children below 5 years old. It has 2 subgroups - A and B - being the first one responsible for more dangerous clinical situations. The viral messenger RNA codifies 11 known proteins, which the F protein is responsible for the viral fusion and it is presented as F0 with molecular weight of 67kDa, but once is broke it generates two units: F2 with 20kDa and F1 with 47kDa. The recombinant F (Fr) protein were expressed on E.coli (BL21A, on vector pET28a), and it was given to us by researches from UNESP of São José do Rio Preto. Wishing to produce monoclonal antibodies (Mab) against Fr protein to laboratorial diagnosis, mice (Balb/c) were inoculated with de antigen (Fr protein). During the development of the Mab, it was necessary to eliminate, from the culture, Mycoplasma spp. After we adjusted the necessary amount to inoculate on the mice and eradicated the contamination of the antigen (hypotheses: there were endotoxins from the E.coli), six clones that secrete Mab were produced (5 of the kind IgM, and 1 of the kind IgG2a) and they were tested, simultaneously, against bacterium toxin and Fr protein (40μg/mL) using ELISA technique. Together, flow cytometry test were performed when we incubated the clones with E. coli (bacterium with permeable membrane and in natura). The clones (VIRSV2-87A74 and VIRSV2-87A80) presented an unspecific recognition for the proteins from E.coli and it was not possible to characterize the cells because we couldn't get more samples of Fr protein. For the future, the Mab should be conjugated with FITC and then, they must be part of a wide epidemiologic test side by side from the commercial kit, always respecting the seasonal disease. New assays to characterize antibodies (like Western blotting and imunepreciptation) are already being done... (Complete abstract click electronic access below) / Mestre

Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract disease among infants and young children worldwide, and understanding the immune response to its infection is essential for intervention strategies. The innate-response serum mannose-binding lectin (MBL), which recognizes a broad range of pathogens and subsequently activates the complement system, has an essential role in the early phase of infection contributing to the development of an acquired immune response. In this study, 82 children <5 years old with confirmed acute RSV infection and 70 controls had MBL levels measured by an indirect ELISA and PBMC phenotypes characterized by flow cytometry. Samples were distributed in four groups: serum/case (81), serum/control (40), PBMC/case (33), PBMC/control (58). Thirty eight cases were <6 months old and most of them had been interned (33/38). The MBL concentrations from all children presented a wide range of values, however, the greater percentage of cases, i.e. 67.9% (55/81) had <500 ng/mL (low/intermediate) MBL levels compared to 40% (16/40) for control subjects. Case and control MBL levels from children <1 month old were not statistically different, but were substantially lower in cases >24 months old (p=0.034). The CD4+ T cells percentage was lower (p<0.001) in children >6 months old compared to controls, while, the CD8+ T cells percentage in cases and controls was similar except for lower frequency in the 6 12 months old cases (p=0,003). T cell activation (CD3+HLA-II+) was significantly higher in cases compared to controls (p<0,001), in contrast to natural killer cells which were generally decreased (p<0,001). These results suggest that acute RSV infection in these children seems to be associated with low MBL levels, but not interfering in T cell activation. / O virus respiratório sincicial (VRS) é a principal causa de doença do trato respiratório inferior em crianças no mundo todo, principalmente nas menores de seis meses de idade e, compreender a resposta imune contra ele é essencial para desenvolver estratégias de intervenção. A lectina ligante de manose (LLM) do presente no soro, relacionada à resposta imune inata, reconhece uma gama de patógenos, ativa o sistema complemento e tem um papel essencial na fase inicial da infecção, contribuindo para o desenvolvimento de uma resposta adaptativa. Neste estudo, 82 crianças <5 anos de idade com infecção pelo VRS confirmada e 70 controles tiveram os níveis de LLM no soro medidos por um ensaio imuno enzimático indireto e também fenótipos das células mononucleares do sangue periférico (CMSP) caracterizado por citometria de fluxo. As amostras foram distribuidas em quatro grupos: soro/caso (81), soro/controle (40), CMSP/caso (33), CMSP/controle (58). Trinta e oito casos eram <6 meses de idade sendo que a maioria deles foi internada (33/38). As concentrações de LLM em todas as idades tiveram uma ampla distribuição, porém, uma grande porcentagem dos casos, isto é, 67,9% (55/81) tiveram níveis de LLM <500 ng/mL (baixo/intermediário), comparado com 40% (16/40) dos controles. Os nívels de LLM dos casos e controles <1 mês de idade não foram diferentes, mas para as crianças >24 meses de idade, os níveis dos casos foram menores (p=0,034). A porcentagem de células T CD4+ dos casos >6 meses de idade foi menor (p<0.001) do que a dos controles. Ainda, não houve diferença entre casos e controles para as células T CD8+, com excessão da faixa de idade entre 6-12 meses em que os casos apresentaram uma menor freqüência (p=0,003). A ativação de células T (CD3+HLA-II+) foi significativamente maior nos casos do que nos controles (p<0,001), em contraste com as células natural killer que geralmente estiveram diminuidas nos casos (p<0,001). Estes resultados sugerem que a infecção aguda por VRS nessas crianças parece estar associada com baixos nívels de LLM, porém não interferindo na ativação de linfócitos. / Mestre em Imunologia e Parasitologia Aplicada

O vírus respiratório sincicial humano (HRSV) é um dos agentes patogênicos respiratórios de grande importância clínica, tendo em vista que acomete 64 milhões de crianças por ano em todo o mundo. A resposta imune do hospedeiro e a variabilidade genética do HRSV podem interferir na produção de uma vacina eficaz, tal como a presença de quasispecies na população viral. O objetivo deste trabalho foi detectar quasispecies em amostras de HRSV e verificar se soros obtidos da criança na fase convalescente da doença e de sua respectiva mãe selecionam estes mutantes. Uma alteração não sinonímia foi detectada no gene F em um dos clones seqüenciados, enquanto duas alterações sinonímias e duas não sinonímias foram encontradas no gene G do HRSV, sendo as últimas no mesmo nucleotídeo. Um dos clones pré-selecionados com soro humano apresentou a mesma alteração não-sinonímia, encontrada na ausência de anticorpos no gene G. Os resultados sugerem que diferentes sequencias virais presentes em menor quantidade na população podem ser selecionadas pelo sistema imunológico do hospedeiro. / Human respiratory syncytial virus (HRSV) is one of the most important clinical respiratory pathogens, since 64 millions children in the world are infected by this agent every year. Host immunity and viral genetic variability are important factors to a vaccine development, besides quasispecies presence in the viral population. In this work, HRSV quasispecies were detected in clinical samples in absence and presence of human polyclonal serum collected by children in the convalescent phase and mother serum. A non-synonymy variation was found in the F gene in antibodies absence. Four mutations were found at HRSV G2 in polyclonal serum absence. Two were synonymy and two were non-synonymy variation, the last in the same nucleotide. A non-synonymy mutation was found in the G2 region in presence of polyclonal serum collected from child convalescent phase. This alteration was the same of the observed in absence of polyclonal serum so it is possible that host antibodies can selected different viral minority sequences present in the population.