Chez un individu infecté, le VHC circule sous la forme d’une population de variants viraux appelés quasi-espèce. Lors d’un évènement de transmission, certains variants viraux sont préférentiellement transmis et un effondrement de la diversité virale chez l’individu nouvellement infecté est souvent observé. Les propriétés électives de ces variants ainsi que leur rôle dans l’évolution clinique sont méconnus. L’objectif de cette étude est d’identifier si des déterminants moléculaires situés au niveau des glycoprotéines d’enveloppe du VHC sont associés à une plus grande capacité de transmission. Les propriétés fonctionnelles des variants transmis et non transmis seront étudiées, en particulier la sensibilité à la neutralisation autologue. Les échantillons étudiés proviennent de couples mère-enfant infectés chroniquement par le VHC issus de d’un essai clinique réalisé en Thaïlande. La composition des populations virales au sein de 3 paires a été étudiée à l’aide d’une technique d’amplification après dilution limite (SGA) suivie d’un séquençage profond (Illumina). Le variant majoritaire chez la mère était retrouvé majoritaire chez l’enfant pour les paires 1 et 3. Pour 2 paires (2 et 3), une moindre diversité génétique a été observée chez l’enfant par rapport à la mère témoignant d’un goulot d’étranglement génétique lors de la transmission. Après clonage des gènes E1E2, des tests d’infectivité sur cellules hépatocytaires ainsi que des tests de neutralisation par le sérum maternel sont réalisés avec le modèle de rétrovirus pseudotypés (VHCpp). Pour la 1ère paire, le variant majoritaire chez la mère (variant transmis à l’enfant) est infectieux et résistant au sérum autologue. Pour la deuxième paire, le variant minoritaire (transmis) est légèrement résistant à la neutralisation autologue. Un variant majoritaire non transmis apparait sensible à la neutralisation autologue. Des études complémentaires en système de virus réplicatifs issus de la culture cellulaire (VHCcc) sont en cours. Au final, les résultats de cette étude contribuent à comprendre les étapes précoces de l’infection par le VHC, afin de mieux appréhender de futures approches immunoprophylactiques ou vaccinales. / In infected individuals, HCV circulates as a complex mixture of genetically different, but closely related viral variants named quasispecies. In a transmission event, some viral variants are preferentially transmitted. The genetic and functional properties of these variants are still unknown. The aim of our work was to identify molecular determinants of E1E2 associates with a greater capacity of transmission. We also intend to study the functional properties of transmitted and no transmitted variants, as for example sensibility to autologous neutralization. Studied sera samples were obtained from three women and their child infected by the HCV, who were participating in HIV prevention clinical trial for the prevention of perinatal transmission of HIV in Thailand. Quasispecies were studied with single genome amplification (SGA) followed by deep sequencing (Illumina). A decrease in intra-host diversity (genetic bottleneck) was observed in the viral population of child near birth (week 6) compared with that observed in the mother (just before delivery). For 2 pairs, the major variant observed in the mother was the same as the major one identified in the child. Retroviral pseudotypes (HCVpp), bearing each transmitted and non-transmitted envelope glycoproteins were produced. For each one, the level of infectivity on HuH7 cells was measured as well as the neutralizing activity of the autologous sera. For the first pair, the major variant (transmitted) appears resistant to autologous neutralization. For the second pair, the transmitted minor variant appears slightly resistant to autologous neutralization. A non-transmitted major variant is sensitive to autologous neutralization. Complementary studies with HCV derived from cell culture (HCVcc) are in progress We hope that the results of this study may be helpful to better understand early steps of HCV infection, which is of great interest for the development of immunoprophylaxis and vaccine strategies.

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No. of bitstreams: 2 Tese - Angela Ferreira Lopes de Teive e Argolo - 2014.pdf: 1773502 bytes, checksum: 393b63ce506fdd2be70f95e14be99755 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Dengue is an endemic disease in Goias state with current circulation of the four serotypes. In the last five years has been occurred a significant increase in cases, hospitalizations and deaths caused by dengue in the region. In this thesis the first manuscript analyzed data from the virological surveillance of DENV in Goias during 1994-2013. Virus isolation in C6/36 (A.albopictus) cell culture followed by indirect immunofluorescence using monoclonal antibodies were performed in LACEN-GO. During the study period the four serotypes have been identified with introduction of DENV-1 in 1994, DENV-2(1998), DENV-3(2002) and DENV-4(2011). Among 21,684 virus isolation tests performed 4,839(22.3%) were positive. DENV-1 was isolated in 3,023(62.5%), DENV-3 in 1,051 (21.7%), DENV-4 in 413(8.5%) and DENV-2 in 352(7.3%) samples respectively. There were changes in the predominance of serotypes throughout the study period with dominance of DENV-1 between 1994-2002 and 2009-2011, DENV-3 in 2003-2008 and DENV-4 in 2013. The DENV-2 was never prevalent among serotypes isolated in LACEN-GO. The time series of the virological surveillance of dengue in Goias showed the introduction and the temporal sequence of circulation of DENV in the state. This study contributes to characterize the epidemiological scenario of dengue in Goias with possible implications for epidemiological surveillance of the disease in the region. The second manuscript evaluated the presence of dengue serotype-specific neutralizing antibodies (NAb) in patients involved in a clinical dengue cohort established during epidemic of 2012-2013 in Goiania, state of Goias. Among 452 participants of the cohort we analyzed the paired samples from 60 patients classified as severe dengue (n=40) or dengue fever (n=20). The eligibility criteria were confirmation of the infection by IgM, NS1Ag and /or RT-PCR; IgG positive in at least one of the samples. Monotypic or multitypic response were defined: PRNT50≥1/20 for only one serotype or ≥1/20 for two or more serotypes simultaneously. The infecting serotype was defined by ≥4 fold increase in the title of NAb in paired samples. Overall 73.3% of patients had NAb against DENV-4, 68.3% against DENV-1, 68.3% against DENV-2 or 61.6% against DENV-3. The patients' ages ranged from 3 to 81 years. Regardless of age group 85% of patients had multitypic response while 15% had monotypic response. Patients infected with DENV-4 showed the greatest difference in NAb titers in comparison to other serotypes indicating predominance of seroconversion for serotype 4. There was no correlation among preexistent NAb and disease severity. The characterization of dengue serotype-specific immune response is important to study the relationship of preexistence of neutralizing antibodies with clinical outcomes of disease also facing the perspective of introducing of antidengue vaccines to identify protective immunity. / A dengue é uma doença endêmica em Goiás com atual circulação dos quatro sorotipos virais com aumento expressivo no número de casos, hospitalizações e óbitos pela doença de forma acentuada nos últimos cinco anos. Na presente tese o primeiro manuscrito analisou os dados da vigilância virológica dos DENV em Goiás no período de 1994-2013. Os testes de isolamento viral em cultura celular C6/36(A.albopictus) seguido de imunofluorescência indireta utilizando anticorpos monoclonais foram realizados no LACEN-GO. No período do estudo, os quatro sorotipos virais foram identificados no estado com introdução do DENV-1 em 1994, DENV-2(1998), DENV-3(2002) e DENV-4(2011). Do total de 21.684 testes de isolamento viral 4.839(22,3%) foram positivos. DENV-1 foi isolado em 3.023(62,5%), DENV-3 em 1.051(21,7%), DENV-4 em 413(8,5%) e DENV-2 em 352(7,3%) amostras. A predominância dos sorotipos virais alternou ao longo do período com DENV-1 dominante entre os anos de 1994-2002 e 2009-2011, DENV-3 entre 2003-2008 e DENV-4 em 2013. O DENV-2 nunca foi predominante entre os sorotipos isolados no LACEN-GO. A série histórica da vigilância virológica da dengue em Goiás mostrou a introdução e a sequência temporal da circulação dos DENV no estado. Este estudo contribui para a caracterização do cenário epidemiológico da dengue em Goiás com possíveis implicações nas ações de vigilância epidemiológica da doença na região. O segundo manuscrito avaliou a presença de anticorpos neutralizantes (AcN) antidengue sorotipo específico em pacientes de uma coorte clínica de dengue estabelecida durante a epidemia de 2012-2013, em Goiânia-GO. Entre os 452 participantes da coorte, analisamos amostras pareadas de 60 pacientes classificados como dengue grave (n=40) ou dengue (n=20). Os critérios de elegibilidade foram a confirmação da infecção por IgM, NS1Ag e/ou RT-PCR; IgG positivo em pelo menos uma das amostras. Resposta monotípica ou multitípica foram definidas por PRNT50≥1/20 para apenas um sorotipo viral ou ≥1/20 para dois ou mais sorotipos virais simultaneamente. O sorotipo viral infectante foi definido por um aumento ≥4 vezes no título dos AcN em amostras pareadas. No total 73,3% dos pacientes apresentaram AcN contra DENV-4, 68,3% contra DENV-1, 68,3% contra DENV-2 ou 61,6% contra DENV-3. A idade dos pacientes variou de 3 a 81 anos. Independente da faixa etária 85% dos pacientes apresentaram resposta multitípica enquanto 15% tiveram resposta monotípica. Pacientes infectados por DENV-4 apresentaram a maior diferença de títulos de AcN em comparação com outros sorotipos virais indicando predominância de soroconversão para o sorotipo 4. Não houve correlação entre preexistência de AcN com a gravidade da doença. A caracterização de resposta imune sorotipo específica é importante para estudar a relação de anticorpos neutralizantes preexistentes com a gravidade clínica da doença, bem como frente à perspectiva de introdução de vacinas antidengue para identificar a imunidade protetora.

Lors de la primo-infection, la plupart des patients infectés par le VIH-1 développent des anticorps neutralisants autologues dirigés contre la glycoprotéine d’enveloppe virale. Ces anticorps exercent une pression de sélection conduisant à l’apparition de variants d’échappement. Nous avons montré que cette pression de sélection se répercute à l’échelle populationnelle, le VIH-1 en tant qu’espèce s’étant adapté au cours de l’épidémie à la réponse immunitaire de la population humaine en devenant de moins en moins sensible aux anticorps neutralisants. Cette adaptation du VIH‐1 a un impact sur les propriétés fonctionnelles de l’enveloppe. Nous avons ainsi observé une augmentation de l’infectivité associée à une augmentation de la cinétique d’entrée des virus qui circulent actuellement. Les virus contemporains montrent également une plus grande résistance à l’enfuvirtide, un inhibiteur de fusion, associée à une meilleure utilisation du co-récepteur CCR5 ainsi qu’une résistance accrue à l’inhibiteur du CD4 M48U1. L’ensemble de nos résultats est en faveur d’une adaptation progressive de l’espèce virale du VIH-1 à son hôte au cours de l’épidémie. / Most of HIV-1 infected patients develop autologous neutralizing antibodies against the viral envelope glycoprotein during primary infection. These antibodies exert a selective pressure that leads to the selection of escape variants. We showed that HIV-1 evolved at the population level towards an enhanced resistance to antibody neutralization over the course of the epidemic, subsequently to the selective pressure exerted by the individual autologous neutralizing antibodies responses. This antigenic drift has an impact on the functional properties of the viral envelope. We showed an increasing infectivity associated with an increasing entry kinetic of the most recently transmitted viruses. The contemporary viruses are also more resistant to the inhibitor of fusion enfuvirtide, related to a better use of the CCR5 co-receptor as well as a progressive increasing resistance to the CD4 inhibitor M48U1. Together our results are in favor of a progressive adaptation of HIV-1 species to humans over the course of the epidemic.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Background: Aberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive. Methods: With the goal to investigate the molecular basis of MET amplification (MET amp ) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring MET amp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo. Results: We find that cancer cells driven by MET amp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In MET amp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit MET amp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors. Conclusions: Our results demonstrate that MET amp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring MET amp , a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

Le VIH est la cause de la pandémie de SIDA depuis les années 1980. Avec plus d’un million de nouvelles infections chaque année, un vaccin prophylactique est indispensable pour bloquer de façon définitive la propagation du virus. Parmi les stratégies vaccinales, l’induction d’anticorps neutralisants à large spectre est une des plus prometteuses, car ceux-ci pourraient protéger contre l’infection par la grande diversité génétique des souches de VIH circulantes dans le monde. A ce jour, aucun immunogène n’a permis l’induction de tels anticorps, mais ils ont été isolés à partir de personnes infectées par le VIH. En effet, une faible fraction d’individus infectés développe des anticorps neutralisants à large spectre qui ciblent des régions vulnérables et conservées de la glycoprotéine d’enveloppe. La région du patch riche en mannose, centrée autour du glycane en position N332 de la gp120, est la plus fréquemment ciblée, et est à cet égard attractive d’un point de vue vaccinal.Afin de mieux comprendre comment se développent les anticorps ciblant le patch riche en mannose, nous avons étudié un donneur sélectionné de la cohorte du Protocole C de l’International AIDS Vaccine Initiative, et ayant une activité neutralisante sérique exceptionnelle. Nous avons isolé, à partir des cellules sanguines de cet individu, deux lignées d’anticorps ciblant la région N332, que nous avons caractérisées pour leur activité neutralisante et dont nous avons cartographié l’épitope. Nous avons également cartographié le paratope d’une lignée d’anticorps issue d’un autre donneur du Protocole C ciblant également la région N332. Nos résultats font apparaître la diversité de solutions adoptées pour atteindre une neutralisation à large spectre contre cette région. Les études de lignées, telles que nous l’avons entrepris, permettent d’appréhender comment la coévolution anticorps-virus conduit à la sélection d’anticorps neutralisants à large spectre. Le but ultime est d’utiliser les connaissances ainsi générées, pour mettre au point des immunogènes et des protocoles d’immunisations, visant à induire des lignées d’anticorps spécifiques et à conduire leur évolution vers la neutralisation à large spectre. / HIV has been the cause of the AIDS pandemic since the 1980s. With over a million new infections each year, a prophylactic vaccine is needed to stop the virus spread. Among vaccine strategies, the induction of broadly neutralizing antibodies is one of the most promising, as they could protect against infection by the huge genetic diversity of circulating HIV strains. To date, no immunogen has induced such antibodies, but they have been isolated from HIV infected people. Indeed, a small fraction of infected individuals eventually develops broadly neutralizing antibodies that target vulnerable and conserved sites of the envelope glycoprotein. The region of the high-mannose patch, centred around a glycan at position N332 of gp120, is the most frequently targeted, and is therefore attractive from a vaccination standpoint.In order to better understand how antibodies targeting the high-mannose patch develop, we studied a donor selected from the International AIDS Vaccine Initiative Protocol C cohort with exceptional serum neutralizing activity. We isolated two antibody lineages targeting the N332 region from this individual's blood cells, which we characterized for their neutralizing activity and mapped their epitope. We also mapped the paratope of an antibody lineage from another Protocol C donor, also targeting the N332 region. Our results show the great diversity of solutions to achieve broad neutralization against this region. Lineage studies, as we have undertaken, provide an understanding of how antibody-virus coevolution leads to the selection of broadly neutralizing antibodies. The ultimate goal is to use this knowledge to develop immunogens and immunization protocols, to induce specific antibody lineage and drive their evolution towards broad neutralization.

No description available.

La problématique de la neutralisation par les anticorps constitue un enjeu majeur dans la perspective de la conception d’un vaccin efficace contre le VIH et les connaissances récemment acquises conforte NT l’absolue nécessité de maintenir une recherche cognitive fondamentale sur le sujet. L’un des objectifs de ce travail de thèse a été de documenter les propriétés biologiques en terme de sensibilité à la neutralisation de variants présents chez certains patients asymptomatiques à long terme (ALT) et présentant des caractéristiques particulières. Nous avons pu identifié certains déterminants moléculaires associés à la sensibilité ou à la résistance à l’anticorps monoclonal 2G12 tels le site potentiel de glycosylation (PNGS) en position N302 et la longueur de la boucle V1V2 du gène env. Nous avons ensuite caractérisé la relation entre l’évolution du gène env et la sensibilité à la neutralisation dans un contexte d’évolution tardive chez un patient ALT. Ces travaux ont permis de mettre en évidence une poursuite de l’évolution du gène env plus de 10 ans après l’infection et ceci malgré la présence d’anticorps largement neutralisants et d’une réponse autologue croissante au cours du temps. Le contexte de la transmission mère enfant (TME) constitue un modèle de choix afin d’étudier le rôle des anticorps neutralisants. Afin d’identifier d’éventuels corrélats de protection, mon travail a consisté à étudier la réponse neutralisante dans une population de 114 couples mères-enfants. Nous avons pu confirmer que le spectre de neutralisation des sérums maternels n’était pas associé à une moindre TME du VIH-1, mais que les anticorps neutralisant certains isolats pourraient constituer des indicateurs d’intérêt associé à un moindre risque de transmission. L’ensemble de ces travaux souligne à nouveau la complexité et la pertinence à poursuivre les investigations relatives à l’identification d’éventuels corrélats de protection. / Basic research on neutralizing antibodies. still remains relevant in term of HIV vacccine development. One of the aim of this thesis was to document the neutralization sensitivity of particular HIV-1variants from long term non progressor (LTNP) patients. We first identified molecular signatures associated with sensitivity to 2G12, such as a potential N-linked glycosylation site (PNGS) at N302 and a longer V1V2 loop of gp120. We also studied the relationship between long-term evolution of the virus and neutralization sensitivity in a LTNP patient. We showed that HIV-1 may continue to evolve in presence of both broadly neutralizing antibodies and increasing autologous neutralizing activity more than 10 years post-infection. Mother-to-child transmission provides a natural model for studying the role of neutralizing antibodies. In previous studies, we showed that the presence or high titers of neutralizing antibodies against a CRF01_AE strain, MBA, was associated with a lower rate of HIV-1 intrapartum transmission in Thailand (Barin et al., 2006; Samleerat et al., 2009). In order to confirm this observation and to identify potential correlates of protection in the MTCT context, we examined the breadth and levels of neutralizing antibodies in 57 transmitting and 57 non-transmitting untreated HIV-1 infected mothers. Our study confirmed that the breadth of maternal neutralizing antibodies was not associated with protection of infants from infection, but that neutralizing antibodies to particular strains might be associated with a lower rate of MTCT of HIV-1.

Neutralisierende Antikörper sind entscheidend in der Eindämmung der Virusinfektion, indem sie den Eintritt in die Wirtszelle hemmen bzw. die Aktivierung der Komplementkaskade initiieren. Distinkte wirtseigene Oberflächenrezeptoren für die Fc-Domäne von IgG (FcyR) sind für die Kommunikation von humoraler und zellulärer Immunantwort verantwortlich. Auch Mitglieder der Herpesviren kodieren für Fc-bindende Proteine, die Kandidaten für immunevasive Funktionen darstellen könnten. Der Nachweis der HCMV-kodierten Fc-bindenden Proteine gp34 und gp68 als Bestandteil der Virushülle lies auf eine immunevasive Funktion hinsichtlich neutralisierendem IgG und Komplement-vermittelter Virolyse schließen, wie für den HSV-1-kodierten FcyR gE beschrieben. Weder für gp34 noch für gp68 konnte in vitro ein hemmender Effekt auf Neutralisation und Virolyse beobachtet werden. In unserem Labor wurde jedoch gezeigt, dass gp34 und gp68 selektiv die IgG-abhängige Aktivierung zellulärer FcyR inhibieren. Die glykosylierungsunabhängige Ligandenbindung von gp34 und gp68 wies auf unterschiedliche Interaktionsmechanismen zwischen den zellulären und den viralen FcyR hin. Mithilfe eines mutierten Fc-Fragments konnte für gp68 eindeutig eine mit HSV-1 gE überlappende Bindestelle an IgG identifiziert werden. Die für die Ligandenbindung erforderlichen Aminosäuren 71-292 von gp68 binden Fc in einer 2:1 Stöchiometrie, wobei die N-, nicht aber die O-Glykosylierung des vFcyRs essentiell sind. Darüber hinaus formt gp34 auf infizierten Zellen und auf der Virushülle kovalente Homooligomere. gp34-Cysteinpunktmutanten auf Basis der für die Bindung notwendigen Aminosäuren 24-140 lassen vermuten, dass die Oligomerisierung Voraussetzung für die Fc-Bindung ist. Im Gegensatz zu gp68 scheint der Mechanismus der Fc-Bindung von gp34 einzigartig unter den bekannten Fcy-Rezeptoren zu sein. Diese Ergebnisse lassen vermuten, dass trotz redundanter Expression der HCMV-FcyR der Bindungs- und Wirkungsmechanismus selektiv ist. / Neutralizing IgGs play a key role in diminishing virus infectivity by inhibiting the entry into host cells. Additionally, IgG-bound particles may be inactivated by virolysis through the activation of complement. Surface receptors specific for the Fc domain of IgG represent host proteins, connecting humoral and cellular immune responses. Also members of the herpes virus family code for proteins with Fc binding properties, implying functions that could intervene with antibody-dependent effector mechanisms. The presence of gp34 and gp68 on the virion membrane raised the question whether they are able to inhibit neutralising IgG and complement-mediated virolysis. The HSV-1-encoded FcyR gE was described to affect neutralisation and virolysis. Despite extensive analysis, there were no implications found that gp34 or gp68 interfere with neutralising IgG or virolysis in vitro. However, our lab could demonstrate that gp34 and gp68 selectively inhibit the IgG-dependent activation of the different host FcyRs. In contrast to the cFcyRs Fc recognition by gp34 and gp68 occurs independently of N-linked glycosylation of IgG, which points to a different binding mechanism among host and viral FcyRs. By taking advantage of a mutated Fc fragment, overlapping binding regions of the HSV-1 gE and gp68 were identified. For gp68 the amino acids 71-292 including the N-glycans are strictly required for Fc binding in a 2:1 stoichiometry. Interestingly, gp34 forms covalently linked homo-oligomers in infected cells and on the virion. Based on the minimal binding domain comprising the amino acids 24-140 of gp34, targeted cysteine exchange mutants revealed that oligomer formation by gp34 is absolutely required for Fc binding. In contrast to gp68, the Fc binding characteristics of gp34 appears to be unique among the known FcyRs. These findings allow us to postulate that even if the HCMV-encoded FcyRs are redundantly expressed the mechanistic details and binding properties are selective.

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.