Ce travail a confirmé le rôle protecteur de certains anticorps neutralisants dans la TME du VIH-1, a permis de suggérer que certaines souches seraient de bons indicateurs d’anticorps neutralisants associés à la protection, et a confirmé le rôle de la région V2 de l’enveloppe virale en tant que cible des anticorps neutralisants. Les caractéristiques moléculaires des virus transmis dans le contexte de la TME confortent les données en faveur de la transmission à l’enfant d’une population virale restreinte génétiquement. Une gp 120 plus compacte et une moindre glycosylation ne sont pas des caractéristiques des virus transmis de la mère à l’enfant. Cependant, deux sites de N-glycosylation semblent être sélectionnés chez les virus transmis. L’identification de deux cas de TME liés à des variants issus de recombinaisons entre variants maternels a confirmé la présence d’un « hot spot » dans la région C2 du gène env, et a révélé pour la première fois un second « hot spot » dans la région C3. / A lower risk of MTCT was associated with higher NAb titers against the CRF01_AE strain, MBA, in Thailand. The results suggest that some primary isolates may be useful indicators for identifying protective antibodies, and confirm the role of the V2 region in neutralization. We found that only viruses of a restricted subset were transmitted to the infant. We did not find that shorter gp120 or fewer PNGS were characteristics of viruses transmitted from mother to infant. However, a limited number of PNGS, particularly at positions N301 and N384, may confer an advantage on the virus to be transmitted. Moreover, we identified two cases that suggest that recombination probably contributed to adaptation of HIV-1 to its environment to be successfully transmitted from mothers to their infants. In addition, our data allow both to confirm, in natural in vivo conditions, a hot spot for recombination in the C2 region of HIV-1 envelope gene, and to suggest another hot spot in the C3 region.

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.

Lors d’un rapport sexuel, le VIH-1, sous forme libre ou associé aux cellules, pénètre dans les tissus du tractus génital féminin. Les premières étapes de l’infection par le VIH-1 dans ces tissus sont très controversées. Afin d’analyser ces premiers évènements impliqués dans la transmission sexuelle, nous avons développé au cours de ma thèse, deux modèles d’infection de cellules isolées de tissu. Nous avons montré que les cellules dendritiques sont préférentiellement infectées et que les anticorps neutralisants inhibent ces premiers évènements de transmission du VIH-1. Dans le cadre de nouvelles stratégies vaccinales, l’inhibition de l’infection des cellules dendritiques sera à considérer afin de prévenir la transmission par voie sexuelle. / During intercourse, HIV free virus particles and cell-associated virus, penetrate into the female genital mucosa. The first events following HIV transmission in tissues are still controversial. In order to analyze these first events of transmission, two different models were developed during my thesis: a heterologous model of HIV transmission with cells generated from blood and a model with cells isolated from cervico-vaginal tissues. We found that dendritic cells are preferentially infected. Moreover, neutralizing antibodies efficiently inhibit these first events of HIV transmission. Future HIV prophylactic vaccine design should take into account the potential infection of dendritic cells and new strategies should be developed to prevent the infection of these particular cell populations.

Les cellules dendritiques plasmacytoïdes (pDC) sont infectées par le VIH-1 et la diminution de leur nombre dans la circulation sanguine est corrélée avec la virémie des patients. Au cours de mes travaux de thèse, nous avons montré que les anticorps neutralisants (AcN) spécifiques du VIH-1 inhibent l’infection des pDC par des isolats primaires de VIH-1. Contrairement aux mDC, le mécanisme d’inhibition de l’infection des pDC est indépendant du RFcγII présent à leur surface. En parallèle, nos résultats indiquent que les pDC produisent de l’interféron-α et d’autres cytokines et chimiokines en réponse au VIH-1, même lorsque l’infection des cellules est inhibée par les AcN. Enfin, nous avons observé l’inhibition du transfert en cis et en trans du VIH-1 des pDC aux lymphocytes T CD4 par les AcN.Dans un contexte d’induction d’AcN par vaccination, l’inhibition de la réplication du VIH-1 dans les pDC associé au maintien de la sécrétion de cytokines pro-inflammatoire par ces cellules pourrait favoriser l’élimination du virus et ralentir sa dissémination dans l’organisme. / Plasmacytoid dendritic cells (pDC) are able to replicate HIV-1, and the decrease of pDC number in blood is correlated with HIV-1 viremia in patients. During my thesis, we showed that HIV-1-specific neutralizing antibodies (NAb) inhibited the infection of pDC by HIV-1primary isolates. Unlike mDC, the mechanism of inhibition of pDC infection was independent of FcγRII expressed on these cells. In parallel, our results indicated that pDC produce interferon-α and other cytokines and chemokines in response to HIV-1, even when HIV-1 infection of these cells was inhibited by NAb. Finally, we showed that NAb were able to inhibit HIV-1 transfer in cis and trans from pDC to CD4 T cells.In the context of antibodies induction by vaccination, the inhibition of HIV-1 replication in pDC associated with the maintenance of pro-inflammatory cytokines released by these cells may help to eliminate the virus and impede its dissemination in the body.

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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.

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.

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.