L’avènement des multi-thérapies antirétrovirales a permis une réduction importante de la mortalité associée au VIH en induisant notamment la chute de la charge virale à moins de 50 copies/mL et une récupération progressive du nombre de lymphocytes T CD4+ (LT-CD4). Cependant, certains patients définis comme mauvais répondeurs immunologiques (MRI) ne parviennent pas à récupérer un taux de CD4 généralement considéré comme « protecteur » (>500cellules/µL). L’interleukine-7 (IL-7), cytokine essentielle à la thymopoïèse et à l’homéostasie lymphocytaire T, a été utilisée en étude clinique afin de restaurer et maintenir le taux de LT-CD4 chez les patients MRI. La première partie de mon travail de thèse visait à évaluer l’impact d’une telle thérapie sur le réservoir viral circulant. Dans l’essai clinique sur lequel nous avons travaillé (INSPIRE 3, Cytheris), des cycles d’administration d’IL-7 ont induit une augmentation significative du nombre de LT-CD4 et CD8 circulants, avec une expansion majoritaire des populations naïves et centrales mémoires. Nous avons montré qu’un cycle d’injections d’IL-7 induisait une augmentation significative de la quantité de cellules infectées circulantes 28 jours et 3 mois post-injection. Cependant, malgré l’accroissement de la fréquence de LT-CD4 infectés 28 jours post-injection, nous avons observé une diminution significative de la charge virale ADN par million de LT-CD4 chez la majorité des patients 3 mois après l’initiation de la thérapie, suggérant une élimination partielle de cellules infectées. Suite au second cycle d’injections, nous n’avons pas observé d’évolution de la quantité de cellules infectées circulantes ni de la fréquence de LT-CD4 infectés, suggérant un impact différent des 2 cycles d’injections sur la dynamique du réservoir viral périphérique. Enfin, certains patients ayant développé des anticorps neutralisants anti-IL-7 (Nab) suite au second cycle d’injections d’IL-7, nous avons cherché à identifier des facteurs prédictifs de l’apparition de ces anticorps ainsi que leurs conséquences physiologiques in vivo. Le seul paramètre caractérisant ces patients est l’amplitude de la reconstitution T-CD4 au cours du premier cycle d’injections d’IL-7. Il semble donc qu’une meilleure réponse à l’IL-7 ait pour conséquence de faciliter le développement de la réponse immune contre cette cytokine. Cependant, ces anticorps ne sont détectables que de façon transitoire chez les patients. De plus, nous avons observé une diminution significative, mais transitoire, de la prolifération des thymocytes chez les patients présentant des Nab, démontrant un impact fonctionnel de ces anticorps sur l’activité biologique de l’IL-7 endogène. L’injection systémique d’IL-7 induit la migration des cellules circulantes vers différents compartiments tissulaires lymphoïdes et non lymphoïdes. Dans une seconde partie de mon travail de thèse, j’ai étudié le pouvoir adjuvant de cette cytokine administrée localement par pulvérisation à la surface de la muqueuse vaginale. Dans le modèle macaque rhésus, nous avons mis en évidence une augmentation de la production d’un large spectre de chimiokines dans le chorion et l’épithélium vaginal des animaux 48 heures après l’administration vaginale d’IL-7. Cette surexpression de chimiokines s’accompagne d’une migration massive de LT-CD4, CD8, macrophages, cellules dendritiques et cellules NK dans cette muqueuse, suggérant l’augmentation de la vigilance immunologique. L’effet adjuvant de cette cytokine a été confirmé par l’analyse de la réponse humorale muqueuse de macaques vaccinés par pulvérisation vaginale d’antigènes 48h après l’administration du spray d’IL-7. Dans les lavages cervicovaginaux (CVL) des animaux traités à l’IL-7, nous avons mis en évidence des réponses spécifiques de type IgA et IgG plus rapides, plus fortes et plus durables que chez les animaux contrôles, démontrant la capacité de l’IL-7 à préparer la muqueuse vaginale à répondre à une stimulation antigénique locale. / Highly Active Antiretroviral Therapy (HAART) has led to significant reduction of HIV-associated mortality by maintaining an undetectable viral load and inducing progressive CD4-T cell restoration. However, some patients, defined as poor immunological responders (PIR), fail to restore their CD4 counts to 500cells/µL during treatment, a threshold considered as the protective against AIDS related or non AIDS related malignancies, opportunistic infections and cardiovascular events. Interleukin-7 (IL-7), an essential cytokine for thymopoïesis and T cell homeostasis has been used in clinical trials aimed at restoring and maintaining CD4 counts in PIR patients. The first part of my thesis project aimed at assessing the impact of IL-7 therapy on circulating HIV reservoir. In the clinical study we worked on (INSPIRE 3, Cytheris), cycles of IL-7 injections led to a significant increase of the number of circulating CD4 and CD8 T-cells, with a predominance of naïve and central memory T cell expansion. We have shown that one cycle of IL-7 injections induced a significant increase in the number of circulating infected cells 28 days and 3 months post-injections. However, despite a significant increase in the frequency of infected CD4 T-cells 28 days post-injections, we observed a significant decrease of HIV-DNA load in CD4 T-cells in the majority of patients 3 months after the therapy initiation. These data suggest a partial elimination of HIV infected cells. After the second cycle of IL-7 injections, we did not observed any change in the number or frequency of circulating infected cells, suggesting a differential impact of the two IL-7 injection cycles on the dynamics of circulating HIV-reservoir. Finally, considering that some patients developed anti-IL-7 neutralizing antibodies (Nab) after the second cycle of IL-7 injections, we looked for predictive factors of this immunogenicity and analyzed its physiological consequences in vivo. The only parameter that distinguished Nab and non-Nab patients was the extent of CD4 T-cell reconstitution during the first cycle of therapy. This suggests that a better response to IL-7 also facilitates the development of auto-antibodies to the cytokine. However, these antibodies were only transiently detectable after the second cycle of therapy. Moreover, the appearance of Nab was associated with a significant but transient decrease of thymocyte proliferation, suggesting a functional impact of these antibodies on the endogenous IL-7 function. Systemic injection of IL-7 induces circulating T cells homing from the blood into lymphoid and non-lymphoid tissues. In the second part of my thesis project, I evaluated whether this cytokine could be used as an adjuvant when sprayed on the vaginal mucosa. Ten micrograms of IL-7 directly sprayed in the vaginal tract of rhesus monkeys induced, 48h after administration, the production of a large pattern of chemokines in the vaginal chorion and epithelium. This chemokine expression was accompanied by massive homing of CD4 and CD8-T cells, macrophages, dendritic cells and NK cells in the vaginal mucosa, suggesting an increased immunological vigilance. Finally, the adjuvant potential of this cytokine was confirmed by analyzing local humoral immune response after vaginal administration of antigens 48h following IL-7 spray. In cervicovaginal washes (CVL) of treated animals, we observed a faster, stronger and longer-lasting specific IgA and IgG response than in control animals, highlighting the capacity of IL-7 to prepare the vaginal mucosa response to local antigen stimulation.

Die Transplantation von porzinen Organen könnte eine Lösung des akuten Mangels von Allotransplantaten in der Transplantationsmedizin darstellen. Bevor die Xenotransplantation klinische Realität werden kann, sind jedoch zahlreiche Hürden zu überwinden. Insbesondere die mögliche Übertragung porziner endogener Retroviren (PERVs), die integraler Bestandteil des porzinen Genoms sind, stellt ein mikrobiologisches Risiko dar. PERVs können humane Zellen in vitro produktiv infizieren. Mögliche Strategien zur Abwehr von Xenosen sind die Verwendung von PERV-knockout Tieren oder die Entwicklung eines effektiven Impfstoffes, durch den der Transplantatrezipient vor einer möglichen Übertragung geschützt werden kann. Dazu wurden Antiseren gegen die Hauptstrukturproteine von PERV generiert. Es konnte gezeigt werden, dass in Ziegen und Ratten durch Immunisierung mit der rekombinant generiereten Ektodomäne des transmembranen Hüllprotein p15E neutralisierende Antikörper induziert werden konnten. Die Epitopkartierung der induzierten Antikörper zeigt eine Bindung an eine Domäne im N-terminalen, nahe des Fusionspeptids (E1, GPQQLEK) und eine Domäne im C-terminalen, membranproximalen (E2, FEGWFN) Bereich der p15E-Ektodomäne. Diese Sequenzen sind in allen PERVs identisch und innerhalb der Gammaretroviren hochkonserviert. Aus AIDS-Patienten isolierte neutralisierende Antikörper (mAb2F5: ELDKWA, mAb4E10: LWNWFN) binden ebenfalls an den C-terminalen Bereich der Ektodomäne des Transmembranproteins gp41. Der Bindungsmechannismus dieser Antikörper wurde in ELISA-Experimenten und in vitro-Inhibitionsassays analysiert. Die Ergebnisse legen die Bindung eines Konformationsepitopes nahe, das aus der E1 und der E2 Domäne gebildet wird. Die Aufklärung des Bindungsmechnismus breit neutralisierender Antikörper gegen Transmembranproteine von Retroviren könnte die Basis für neue Impfstoffansätze darstellen. / Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, goat and rat antisera against the recombinant ectodomain of the transmembrane envelope protein p15E were found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, one near the fusion peptide (E1, GPQQLEK) and the other near the transmembrane domain (E2, FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, neutralizing antibodies isolated from AIDS patients that recognize regions partially homologous with E2 (mAb4E10, LWNWFN) or located in close proximity to E2 (mAb2F5, ELDKWA) are known to neutralize a broad range of HIV-1 strains. The binding mechanisms of these HIV neutralizing antibodies were analyzed in ELISA experiments and in vitro inhibition assays. The results indicate that the two most broadly reactive HIV-1 envelope gp41 human mAbs are specific for a discontinuous epitope composed of the E1 and the E2 domain. If so, these two transmembrane protein domains in different retroviruses act as effective targets for neutralizing antibodies and may provide the basis for effective antiretroviral vaccines.

INTRODUÇÃO: A vacina contra febre amarela é a principal forma de prevenção da doença, e é raramente associada a eventos adversos graves, para os quais pessoas que vivem com o vírus da imunodeficiência humana (HIV) teoricamente possuem risco aumentado. Nessa população, estudos sugerem que a imunogenicidade da vacina é inferior, e fatores associados à resposta vacinal são pouco conhecidos. Neste estudo, avaliamos a imunogenicidade e reatogenicidade da vacina contra febre amarela em pessoas infectadas por HIV e controles, comparando os títulos de anticorpos neutralizantes, ocorrência de viremia pelo vírus vacinal e eventos adversos após a vacinação, e investigamos potenciais preditores da resposta vacinal. Avaliamos ainda o grau de conhecimento a respeito da febre amarela e a adesão às recomendações de vacinação entre pessoas que vivem com HIV. MÉTODOS: No Estudo 1, indivíduos com infecção por HIV e controles com indicação de receber a vacina foram incluídos em uma coorte prospectiva com um ano de acompanhamento, com avaliação periódica de eventos adversos, viremia pelo vírus vacinal e títulos de anticorpos neutralizantes específicos contra febre amarela após a vacinação. No Estudo 2, indivíduos com infecção por HIV sob tratamento antirretroviral e controles com uma única dose da vacina contra febre amarela no passado foram incluídos em um estudo de corte transversal para avaliação dos títulos de anticorpos neutralizantes contra febre amarela. Finalmente, no Estudo 3 pessoas infectadas por HIV foram convidadas a completar um questionário avaliando o grau de conhecimento a respeito da febre amarela e a adesão às recomendações de prevenção. RESULTADOS: Não observamos entre pessoas infectadas por HIV maior risco de viremia pelo vírus vacinal, ocorrência de eventos adversos ou diferença estatisticamente significante nos títulos de anticorpos nos primeiros três meses após a vacinação. Entretanto a persistência de anticorpos foi significantemente inferior entre indivíduos infectados por HIV, e associou-se inversamente à relação CD4+/CD8+, um marcador de ativação imune e inflamação de importância crescente. Nas respostas ao questionário, embora os participantes tenham demonstrado conhecimento a respeito da febre amarela e sua prevenção, a prevalência de discrepância entre as recomendações e o uso da vacina foi de 19%. CONCLUSÕES: Nossos resultados enfatizam a necessidade de novos estudos e intervenções entre pessoas infectadas por HIV a fim de melhorar a adesão às recomendações de uso da vacina, reduzir a ativação imune excessiva associada à pior resposta vacinal, e determinar o intervalo de tempo ideal para administração de reforço vacinal nessa população / INTRODUCTION: The yellow fever vaccine is the main prevention strategy against the disease, and is rarely associated with severe adverse events for which HIV-infected persons present theoretical increased risk. Studies suggest that the immune response to the vaccine is reduced in this population, but predictors of the vaccine immunogenicity are not well known. In this study, we assessed yellow fever vaccine immunogenicity and reactogenicity among HIV-infected persons and controls by comparing yellow fever-specific neutralizing antibody titers, detection of viremia by the vaccine virus and adverse events following vaccination. We also investigated potential predictors of vaccine response. Furthermore, we assessed knowledge and perceptions about yellow fever, and adherence to yellow fever vaccine recommendations among HIV-infected individuals. METHODS: In Study 1, HIV-infected participants and controls with indication to receive yellow fever vaccine were enrolled in a prospective cohort study and followed for one year with serial assessments of adverse events, viremia by the vaccine virus and yellow fever-specific neutralizing antibody titers after vaccination. In study 2, HIV-infected individuals under antiretroviral therapy and controls with a history of a single dose of yellow fever vaccine in the past were enrolled in a cross sectional study to evaluate yellow fever-specific neutralizing antibody titers after vaccination. Finally, in Study 3, HIV-infected persons under clinical follow up were invited to complete a survey assessing knowledge and perceptions about yellow fever and adherence to yellow fever prevention recommendations. RESULTS: We found no increased risk for the occurrence of viremia by the vaccine virus or adverse events, and no significant difference in yellow fever-specific antibody titers among HIVinfected participants in the first three months after vaccination. However, the duration of antibody response was reduced in HIV-infected persons, and was inversely associated to CD4+/CD8+ ratio, a biomarker of immune activation and inflammation of increasing importance. In the survey responses, although participants demonstrated awareness about yellow fever and yellow fever prevention, the prevalence of discrepancy between vaccine recommendation and actual compliance was 19%. CONCLUSIONS: Our results reinforce the need for new studies and interventions among HIVinfected persons to improve adherence to yellow fever vaccine recommendations, reduce excessive immune activation associated with impaired vaccine response, and to determine the ideal interval for a booster vaccination in this population

Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste. / Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test

INTRODUÇÃO: A vacina contra febre amarela é a principal forma de prevenção da doença, e é raramente associada a eventos adversos graves, para os quais pessoas que vivem com o vírus da imunodeficiência humana (HIV) teoricamente possuem risco aumentado. Nessa população, estudos sugerem que a imunogenicidade da vacina é inferior, e fatores associados à resposta vacinal são pouco conhecidos. Neste estudo, avaliamos a imunogenicidade e reatogenicidade da vacina contra febre amarela em pessoas infectadas por HIV e controles, comparando os títulos de anticorpos neutralizantes, ocorrência de viremia pelo vírus vacinal e eventos adversos após a vacinação, e investigamos potenciais preditores da resposta vacinal. Avaliamos ainda o grau de conhecimento a respeito da febre amarela e a adesão às recomendações de vacinação entre pessoas que vivem com HIV. MÉTODOS: No Estudo 1, indivíduos com infecção por HIV e controles com indicação de receber a vacina foram incluídos em uma coorte prospectiva com um ano de acompanhamento, com avaliação periódica de eventos adversos, viremia pelo vírus vacinal e títulos de anticorpos neutralizantes específicos contra febre amarela após a vacinação. No Estudo 2, indivíduos com infecção por HIV sob tratamento antirretroviral e controles com uma única dose da vacina contra febre amarela no passado foram incluídos em um estudo de corte transversal para avaliação dos títulos de anticorpos neutralizantes contra febre amarela. Finalmente, no Estudo 3 pessoas infectadas por HIV foram convidadas a completar um questionário avaliando o grau de conhecimento a respeito da febre amarela e a adesão às recomendações de prevenção. RESULTADOS: Não observamos entre pessoas infectadas por HIV maior risco de viremia pelo vírus vacinal, ocorrência de eventos adversos ou diferença estatisticamente significante nos títulos de anticorpos nos primeiros três meses após a vacinação. Entretanto a persistência de anticorpos foi significantemente inferior entre indivíduos infectados por HIV, e associou-se inversamente à relação CD4+/CD8+, um marcador de ativação imune e inflamação de importância crescente. Nas respostas ao questionário, embora os participantes tenham demonstrado conhecimento a respeito da febre amarela e sua prevenção, a prevalência de discrepância entre as recomendações e o uso da vacina foi de 19%. CONCLUSÕES: Nossos resultados enfatizam a necessidade de novos estudos e intervenções entre pessoas infectadas por HIV a fim de melhorar a adesão às recomendações de uso da vacina, reduzir a ativação imune excessiva associada à pior resposta vacinal, e determinar o intervalo de tempo ideal para administração de reforço vacinal nessa população / INTRODUCTION: The yellow fever vaccine is the main prevention strategy against the disease, and is rarely associated with severe adverse events for which HIV-infected persons present theoretical increased risk. Studies suggest that the immune response to the vaccine is reduced in this population, but predictors of the vaccine immunogenicity are not well known. In this study, we assessed yellow fever vaccine immunogenicity and reactogenicity among HIV-infected persons and controls by comparing yellow fever-specific neutralizing antibody titers, detection of viremia by the vaccine virus and adverse events following vaccination. We also investigated potential predictors of vaccine response. Furthermore, we assessed knowledge and perceptions about yellow fever, and adherence to yellow fever vaccine recommendations among HIV-infected individuals. METHODS: In Study 1, HIV-infected participants and controls with indication to receive yellow fever vaccine were enrolled in a prospective cohort study and followed for one year with serial assessments of adverse events, viremia by the vaccine virus and yellow fever-specific neutralizing antibody titers after vaccination. In study 2, HIV-infected individuals under antiretroviral therapy and controls with a history of a single dose of yellow fever vaccine in the past were enrolled in a cross sectional study to evaluate yellow fever-specific neutralizing antibody titers after vaccination. Finally, in Study 3, HIV-infected persons under clinical follow up were invited to complete a survey assessing knowledge and perceptions about yellow fever and adherence to yellow fever prevention recommendations. RESULTS: We found no increased risk for the occurrence of viremia by the vaccine virus or adverse events, and no significant difference in yellow fever-specific antibody titers among HIVinfected participants in the first three months after vaccination. However, the duration of antibody response was reduced in HIV-infected persons, and was inversely associated to CD4+/CD8+ ratio, a biomarker of immune activation and inflammation of increasing importance. In the survey responses, although participants demonstrated awareness about yellow fever and yellow fever prevention, the prevalence of discrepancy between vaccine recommendation and actual compliance was 19%. CONCLUSIONS: Our results reinforce the need for new studies and interventions among HIVinfected persons to improve adherence to yellow fever vaccine recommendations, reduce excessive immune activation associated with impaired vaccine response, and to determine the ideal interval for a booster vaccination in this population

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-12Bitstream added on 2014-06-13T19:03:33Z : No. of bitstreams: 1 dias_fc_dr_jabo.pdf: 950154 bytes, checksum: 170fddcca2fc4c7004161e93f093a97f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A ocorrência de anticorpos neutralizantes contra os genótipos do vírus da diarréia viral bovina (BVDV 1 e BVDV 2) foi verificada, pelo teste de virusneutralização (VN), em 260 amostras de soro sangüíneo de 26 rebanhos não vacinados contra o BVDV, provenientes dos Estados de Minas Gerais e São Paulo. Do total de amostras, 102 (39,2%) reagiram ao BVDV, das quais 81 (31,1 %) foram reagentes ao BVDV 1 e ao BVDV 2, sete (2,7%) reagiram apenas ao BVDV 1 e 14 (5,4%) reagiram apenas ao BVDV 2. Nos mesmos rebanhos, verificou-se também a presença sugestiva de animais persistentemente infectados (PI) por meio da pesquisa de anticorpos neutralizantes em cinco amostras de soro sangüíneo de bezerros sentinelas com idade entre 6 e 12 meses. A ocorrência de animais PI foi pesquisada em três dos 26 rebanhos, dos quais foram colhidas amostras pareadas de sangue de todos os animais do rebanho. Todas as amostras foram submetidas ao teste de VN contra o BVDV 1 e o BVDV 2, e naquelas não reagentes a pelo menos um dos genótipos, bem como nas amostras provenientes de bovinos com menos de seis meses de idade, foi realizada a pesquisa do vírus pela RT-PCR. Em um dos rebanhos foram detectados dois animais PI, cujas estirpes foram caracterizadas geneticamente como pertencentes ao subgenótipo eVOV 1 b. A infecção natural pelo BVDV foi monitorada nos três rebanhos selecionados para a pesquisa de animais PI, sendo que em dois deles constatou-se a eliminação do BVDV. No entanto, a infecção permaneceu no rebanho em que haviam sido detectados dois animais PI, pois foram diagnosticados posteriormente um animal transitoriamente infectado (TI) e novos animais reagentes ao vírus. Foram submetidos à análise estatística os resultados dos testes de VN contra o BVDV 1 e o BVDV 2, realizados em 1925 amostras de soro sangüíneo obtidas no período de execução... / The occurrence of neutralizing antibodies against to bovine viral diarrhoea virus genotypes (BVDV 1 and BVDV 2) has been confirmed by virusneutralization test (VN) in 260 samples of blood serum undertaken in the states of Minas Gerais and São Paulo, Brazil. Samples were retrieved from 26 cattle herds which were not BVDV vaccinated. One hundred and two samples (39.2%) were reagents to BVDV, or rather, 81 (31.1%) were reagents to BVDV 1 and BVDV 2, seven (2.7%) were reagents to BVDV 1 only and 14 (5.4%) were reagents to BVDV 2 only. In the same herds, the significant presence of persistently infected (PI) animais was also verified by a research involving neutralizing antibodies in five samples of blood serum in 6 to 12¬month-old calves. The occurrence of PI animais was researched in three out of 26 herds by paired blood samples of total animais in the herdo Ali samples were analyzed by VN test against to BVDV 1 and BVDV 2, and virus research was undertaken by RT-PCR in samples which were not reagent to at least one of the genotypes and in samples of less than 6-month-old cattle. Two PI animais, genetically characterized as belonging to BVDV 1 b subgenotype, were detected in one of the herds. BVDV natural infection was monitored in three herds selected for PI animais research. BVDV was eliminated in two herds. However, infection remained in the herd in which two PI animais were diagnosed, because a transiently infected animal (TI) and other virus-reagent animais were detected later on. Statistical analysis evaluated VN test results against BVDV 1 and BVDV 2 with 1925 samples of blood serum obtained during the research period. Although no significant ditterences were found between BVDV 1 reagent bovines and BVDV 2 reagent ones (p>0.05), a significant ditterence was detected among titles of antibodies from samples which were reagent to both genotypes (p<0.0001).

La néphropathie à BK virus (BKV) est l'une des complications les plus fréquentes de la transplantation rénale. La prise en charge consiste en la réduction préemptive de l'immunosuppression basée sur le suivi de la charge virale, mais cette stratégie n’est pas complètement efficace et augmente le risque de rejet. Dans un premier volet, nous avons évalué la mesure de la charge virale par PCR quantitative en temps réel, permettant de mettre en évidence des facteurs de variabilité comme le polymorphisme du BKV et de valider la technique utilisée pour le suivi de notre cohorte. L’intérêt des anticorps neutralisants (AcNs) anti-BKV en tant que marqueur prédictif de la réplication BKV a ensuite été évalué dans une cohorte de 168 transplantés rénaux. Nous avons montré i) que le virus responsable de l’infection provenait du donneur ; ii) que les AcNs jouent un rôle dans la prévention de la réactivation et le contrôle de la réplication virale et iii) qu’un seuil d’AcNs de 4 log10 permettait de stratifier le risque de réplication BKV. Ce travail ouvre la voie à un suivi personnalisé en fonction du risque de réplication BKV et à de nouvelles approches immunothérapeutiques. / BK virus (BKV)-associated nephropathy is one of the major causes of graft dysfunction and loss in kidney transplant recipients. Since no BKV-specific antiviral therapies are available, management relies on preemptive immunosuppression reduction based on viral load monitoring. However, this strategy does not fully eliminate the risk of nephropathy and can increase the risk of graft rejection. In this work, we evaluated viral load measurement by quantitative real-time PCR in an interlaboratory comparison. Variability factors such as BKV polymorphism or pre-PCR steps have been highlighted and the method used for monitoring our cohort has been validated. The role of anti-BKV neutralizing antibodies (NAbs) as a predictive marker of BKV replication has been investigated in a cohort of 168 kidney transplant recipients. We showed that i) viral infection is caused by the donor strain; ii) NAbs play an essential role in viral replication prevention and control and iii) a NAbs cutoff of 4 log10 allows to stratify BKV replication risk. This work paves the way for personalized monitoring according to BKV replication risk and for new preventive or therapeutic strategies.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The feline calicivirus (FCV) and the felid herpesvirus type 1 (FeHV-1) are the main agents of the respiratory tract diseases of felines. Both viruses are distributed worldwide, however its distribution and prevalence in Brazil are not well known. In the present thesis we describe the isolation and one serosurvey of FCV and FeHV-1 in some counties of the Rio Grande do Sul State, Brazil; and the molecular characterization of the capsid protein gene of FCV isolates is also described. In Chapter 1, we describe the epidemiologic survey of FCV and FeHV-1 through investigation of the conjunctival, nasal, oral and oropharyngeal swabs from 302 domestic cats. The viral isolation was performed in Crandell-Reese feline kidney cells and the isolates were submitted to PCR and RT-PCR for confirmation of the presence of the FeHV-1 and FCV, respectively. Fifty five (18.2%) of the 302 cats analyzed were positive for the viral isolation; when tested by PCR, 29 cats (52.7%) were shedding the FCV, 21 (38.2%) shedding FeHV-1, and 5 cats (9.1%) shedding both viruses. In addition, isolates of FCV and FeHV-1 were standardized to be used as control in the PCR and virus neutralization (VN) assays (serological technical used in chapter 3 of the thesis). The SV65/90 (FCV) and the SV534/00 (FeHV-1) isolates were defined as standard viruses for the assays. In Chapter 2, the molecular characterization of 13 FCV isolates is described; ten isolates came from the survey performed in chapter 1, two were deposited in the virology laboratory of the UFSM and one was obtained from a cat with gingivitis-stomatitis syndrome. The ORF2 (open reading frame) region that codifies the major protein of the viral capsid was sequenced, which includes an immunodominant region of the virus. The ORF 2 was analyzed at the nucleotide and amino acid levels and these data was used for phylogenetic studies of the 13 Brazilian isolates of FCV. These isolates were compared to ten reference strains of FCV and to the San Miguel sea lion virus type 1 (SMSV-1) as an outgroup in phylogenetic study. The primers were designed using the complete sequence of FCV-F9. The comparison of the 13 Brazilian isolates with the F9 strain revealed a large molecular diversity among them, concentrated mainly along the linear epitopes of the region. The Chapter 3 describes a serosurvey against FCV and FeHV-1 in serum samples from domestic feline. From the 630 samples tested by the VN assay, 53.6% (338/630) were positive to one or both viruses with a prevalence of 39.2% for neutralizing antibodies against FCV and 30.6% for FeHV-1. The results from the present study demonstrated that the FCV and FeHV-1 are circulating among the feline population examined, and also, the presence of carriers of the viruses among these cats. Besides, the molecular characterization of the FCV evidenced the great genetic variability of the Brazilian isolates when compared to vaccine and reference strains. / O calicivírus felino (feline calicivirus FCV) e o herpesvírus felino tipo 1 (felid herpesvirus type 1 FeHV-1) são os principais agentes das doenças do trato respiratório dos felinos. Ambos os vírus são mundialmente distribuídos, mas no Brasil sua distribuição e prevalência são ainda pouco conhecidas. Na presente tese descrevemos o isolamento e sorologia do FCV e do FeHV-1 em alguns municípios do estado do Rio Grande do Sul (RS), Brasil; e a caracterização molecular do gene da proteína do capsídeo de isolados de FCV. No Capítulo 1, descreve-se o isolamento do FCV e do FeHV-1 por meio da coleta de suabes (conjuntival, nasal, oral e orofaringeano) de 302 gatos domésticos. O material coletado foi inoculado em cultivo celular de origem felina (CRFK) e submetidos a PCR e RT-PCR para confirmação em FeHV-1 e FCV, respectivamente. Dos 302 gatos analisados, 55 (18,2%) foram positivos no isolamento; e quando testados por PCR, 29 dos 55 gatos (52,7%) excretavam o FCV, 21 (38,2%) excretavam o FeHV-1 e 5 gatos (9,1%) apresentavam ambos os vírus. Além dessa descrição, realizou-se a padronização de um isolado de FCV e de FeHV-1, para serem utilizados como controle nas técnicas de PCR e na vírus neutralização [VN] (técnica sorológica utilizada no capítulo 3 da tese). O SV65/90 (FCV) e o SV534/00 (FeHV-1) foram os isolados definidos como vírus-padrão. No Capítulo 2, incluímos o estudo sobre a análise molecular de 13 isolados de FCV; dez isolados foram obtidos a partir do material descrito no capítulo 1, dois estavam armazenados no laboratório de virologia da UFSM e um foi obtido de um gato acometido pela síndrome gengivo-estomatite. A região analisada foi o gene da proteína do capsídeo, codificado pela ORF2 (open reading frame), na qual se localizam as regiões imunodominantes do FCV. A ORF2 foi analisada em nível de nucleotídeos e aminoácidos; e uma análise filogenética dos 13 isolados brasileiros de FCV, incluindo dez cepas de referência e o vírus dos leões marinhos de San Miguel tipo 1 (San Miguel sea lion virus type 1 SMSV-1) como um outgroup, foi também realizada. Os primers foram desenhados com base na sequência completa do genoma da cepa de referência a FCV-F9. Quando os 13 isolados brasileiros de FCV foram comparados a cepa F9, detectou-se grande diversidade molecular nas regiões variáveis do gene e nos epitopos lineares mapeados. O Capítulo 3 contem um estudo sorológico contra FCV e FeHV-1 em amostras de soro de felinos domésticos. Das 630 amostras testadas pela VN, 53,6% (338/630) foram positivas para um ou ambos os vírus; sendo a prevalência de anticorpos neutralizantes contra o FCV de 39,2% e do FeHV-1 de 30,6%. Os resultados aqui apresentados demonstram a circulação do FCV e do FeHV-1 na população de gatos domésticos analisados, assim como, a presença de gatos portadores para ambos os vírus. Além disso, a caracterização molecular do FCV demonstrou a grande variabilidade genética dos isolados brasileiros em comparação às cepas vacinais e às de referência.

Made available in DSpace on 2015-03-26T13:46:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 157447 bytes, checksum: 535cd3ca6a89ff6d3d230a356a1b6922 (MD5) Previous issue date: 2004-02-19 / Observations of clinical cases suspected of distemper, attended at the Veterinary Hospital at the Federal University of Viçosa, indicate that the dogs vaccinated in stores which commercialize agropecuary products showed a greater frequency of the disease compared to the ones vaccinated at veterinary clinics. Having as the objective of this study the evaluation of the answer to the vaccines commercialized in different stores and following or not the vaccination protocol indicated by the literature, the antibody titers against the distemper in dogs serum in the urban area of the city of Viçosa-MG was determined. To carry out this experiment, a blood serum sample was obtained from each of the 150 selected dogs. These animals were healthy and were between six months to six years old. The animals belong to several breeds and both sex. The dogs were selected following pre-established characteristics to form five groups of 30 animals each: A) dogs vaccinated in veterinary clinics following the literature patterns; B) dogs vaccinated in veterinary clinics not following the indicated protocol; C) dogs vaccinated in agropecuary product stores following the indicated protocol; D) dogs vaccinated in agropecuary product stores not following the indicated protocol; E) dogs not vaccinated. The antibody titers were measured in each animal serum through the serum neutralizing test. According to the results of this study, it was concluded that the vaccination against distemper is important, considering the difference between the titers of vaccinated and non-vaccinated dogs. There is no difference in relation to vaccinating in veterinary clinics or agropecuary product stores, if the vaccination is carried out following the vaccination protocol indicated by the literature. The quality of the vaccines commercialized in veterinary clinics and agropecuary products stores should be evaluated due to the great percentage of negative titer and titers below the ones considered protective in all the vaccinated dogs groups. / Observações de casos clínicos suspeitos de cinomose, atendidos no Hospital Veterinário da Universidade Federal de Viçosa, indicam que os cães vacinados em lojas que comercializam produtos agropecuários apresentam maior freqüência da doença, quando comparados aos cães vacinados em clínicas veterinárias. Com o objetivo de avaliar a resposta às vacinas comercializadas em diferentes estabelecimentos e seguindo ou não o protocolo de vacinação indicado pela literatura, foi determinado o título de anticorpos contra a cinomose no soro de cães da área urbana do município de Viçosa-MG. Para tanto, uma amostra de soro sanguíneo foi obtida de cada um dos 150 cães selecionados. Estes animais estavam sadios e tinham entre seis meses e seis anos de idade. Os animais pertenciam a diversas raças e eram de ambos os sexos. Os cães foram selecionados segundo características pré-estabelecidas para compor cinco grupos de 30 animais: A) cães vacinados em clínicas veterinárias, seguindo o protocolo indicado na literatura; B) cães vacinados em clínicas veterinárias, não seguindo o protocolo indicado; C) cães vacinados em lojas de produtos agropecuários, seguindo o protocolo indicado; D) cães vacinados em lojas de produtos agropecuários, não seguindo o protocolo indicado; E) cães não vacinados. O título de anticorpos foi mensurado no soro de cada animal, por meio do teste de soroneutralização. De acordo com os resultados deste estudo, pôde-se concluir que a vacinação contra cinomose é importante, considerando a diferença encontrada entre o título de anticorpos dos cães vacinados e não vacinados. Não há diferença em vacinar os cães contra cinomose em clínicas veterinárias ou em lojas de produtos agropecuários, desde que a vacinação seja realizada seguindo o protocolo indicado pela literatura. A qualidade das vacinas comercializadas em clínicas veterinárias e lojas de produtos agropecuários devem ser avaliadas, diante da grande porcentagem de títulos negativos e de títulos abaixo do considerado protetor em todos os grupos de cães vacinados.

Orientador: Samir Issa Samara / Banca: Claudia Del Fava / Banca: Amauri Alcindo Alfieri / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Aramis Augusto Pinto / Resumo: A ocorrência de anticorpos neutralizantes contra os genótipos do vírus da diarréia viral bovina (BVDV 1 e BVDV 2) foi verificada, pelo teste de virusneutralização (VN), em 260 amostras de soro sangüíneo de 26 rebanhos não vacinados contra o BVDV, provenientes dos Estados de Minas Gerais e São Paulo. Do total de amostras, 102 (39,2%) reagiram ao BVDV, das quais 81 (31,1 %) foram reagentes ao BVDV 1 e ao BVDV 2, sete (2,7%) reagiram apenas ao BVDV 1 e 14 (5,4%) reagiram apenas ao BVDV 2. Nos mesmos rebanhos, verificou-se também a presença sugestiva de animais persistentemente infectados (PI) por meio da pesquisa de anticorpos neutralizantes em cinco amostras de soro sangüíneo de bezerros sentinelas com idade entre 6 e 12 meses. A ocorrência de animais PI foi pesquisada em três dos 26 rebanhos, dos quais foram colhidas amostras pareadas de sangue de todos os animais do rebanho. Todas as amostras foram submetidas ao teste de VN contra o BVDV 1 e o BVDV 2, e naquelas não reagentes a pelo menos um dos genótipos, bem como nas amostras provenientes de bovinos com menos de seis meses de idade, foi realizada a pesquisa do vírus pela RT-PCR. Em um dos rebanhos foram detectados dois animais PI, cujas estirpes foram caracterizadas geneticamente como pertencentes ao subgenótipo eVOV 1 b. A infecção natural pelo BVDV foi monitorada nos três rebanhos selecionados para a pesquisa de animais PI, sendo que em dois deles constatou-se a eliminação do BVDV. No entanto, a infecção permaneceu no rebanho em que haviam sido detectados dois animais PI, pois foram diagnosticados posteriormente um animal transitoriamente infectado (TI) e novos animais reagentes ao vírus. Foram submetidos à análise estatística os resultados dos testes de VN contra o BVDV 1 e o BVDV 2, realizados em 1925 amostras de soro sangüíneo obtidas no período de execução... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The occurrence of neutralizing antibodies against to bovine viral diarrhoea virus genotypes (BVDV 1 and BVDV 2) has been confirmed by virusneutralization test (VN) in 260 samples of blood serum undertaken in the states of Minas Gerais and São Paulo, Brazil. Samples were retrieved from 26 cattle herds which were not BVDV vaccinated. One hundred and two samples (39.2%) were reagents to BVDV, or rather, 81 (31.1%) were reagents to BVDV 1 and BVDV 2, seven (2.7%) were reagents to BVDV 1 only and 14 (5.4%) were reagents to BVDV 2 only. In the same herds, the significant presence of persistently infected (PI) animais was also verified by a research involving neutralizing antibodies in five samples of blood serum in 6 to 12¬month-old calves. The occurrence of PI animais was researched in three out of 26 herds by paired blood samples of total animais in the herdo Ali samples were analyzed by VN test against to BVDV 1 and BVDV 2, and virus research was undertaken by RT-PCR in samples which were not reagent to at least one of the genotypes and in samples of less than 6-month-old cattle. Two PI animais, genetically characterized as belonging to BVDV 1 b subgenotype, were detected in one of the herds. BVDV natural infection was monitored in three herds selected for PI animais research. BVDV was eliminated in two herds. However, infection remained in the herd in which two PI animais were diagnosed, because a transiently infected animal (TI) and other virus-reagent animais were detected later on. Statistical analysis evaluated VN test results against BVDV 1 and BVDV 2 with 1925 samples of blood serum obtained during the research period. Although no significant ditterences were found between BVDV 1 reagent bovines and BVDV 2 reagent ones (p>0.05), a significant ditterence was detected among titles of antibodies from samples which were reagent to both genotypes (p<0.0001). / Doutor