The HIV-1 envelope glycoprotein, Env, facilitates the translocation of the viral capsid across the cellular membrane. Env is a trimer of hetero-dimers composed of a gp120 subunit and gp41 transmembrane protein. The gp120 subunit binds the primary receptor, CD4, leading to conformational changes of Env that then promote binding to the coreceptor, principally CCR5 or CXCR4. As the sole protein on the surface of the virion, Env is under continuous pressure from the host's antibody response. Two classes of antibodies target the highly conserved receptor-binding sites of gp120: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies.

Zehn Jahre nach dem Ausbruch des Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV, ist ein neues Betacoronavirus, das Middle East Respiratory Syndrome Coronavirus, MERS-CoV, auf der arabischen Halbinsel entdeckt worden. Seine anhaltende Ausbreitung stellt eine Bedrohung für die öffentliche Gesundheit dar. Das Spike (S) Protein der Coronaviren vermittelt den viralen Eintritt in Wirtszellen und bestimmt wesentlich den viralen Tropismus und die virale Pathogenese. Das Verständnis der Determinanten des MERS-CoV Spike (MERS-S)-vermittelnden Eintritts in Zellen könnte daher wichtige Einblicke in die MERS-CoV-Biologie liefern und war somit das erste Ziel dieser Arbeit. Um den Eintritt in die Zelle zu ermöglichen, muss das Coronavirus S-Protein durch Wirtszellproteasen aktiviert werden, welche potentielle Ziele für die therapeutischen Intervention darstellen. Daher sollten im zweiten Ziel dieser Arbeit Proteasen identifiziert werden, die MERS-S aktivieren. Das S-Protein ist das Hauptangriffsziel neutralisierender Antikörper und experimentelle Systeme zur S-Analyse können für die Diagnostik eingesetzt werden. Das letzte Ziel dieser Arbeit war es daher, die MERS-CoV Seroprävalenz in Saudi Arabien zu ermitteln. Es wurde ein lentivirales Vektorensystem etabliert, welches die Analyse des MERS-S-getriebenen Zelleintritts ermöglicht. Mit Hilfe dieses Systems konnte gezeigt werden, dass MERS-S den Eintritt in ein breites Spektrum humaner Zelllinien, wie Lungen-, Nieren- und Darmzellen vermittelt, was mit der klinischen Manifestation von MERS einhergeht. Der Wirtszelleintritt war unabhängig von bereits beschriebenen Coronavirus Eintrittsrezeptoren, wurde jedoch durch die endosomale Cysteinprotease Cathepsin L und die Transmembranserinprotease TMPRSS2 gefördert. Im Gegensatz dazu war die Aktivität von Proprotein Konvertasen für den S-Protein-vermittelnden Eintritt entbehrlich. Schließlich zeigten Neutralisationstests, dass Seren von Patienten aus der östlichen Provinz Saudi Arabiens, die zwischen 2010-2011 und 2012 entnommen wurden, keine MERS-S-neutralisierenden Antikörper enthielten. Dies deutet darauf hin, dass MERS-CoV-Infektionen vor dem Ausbruch 2012 nur selten vorkamen. Die gewonnen Ergebnisse tragen wesentlich zum Verständnis des MERS-CoV-Eintritts in Zellen bei und liefern wichtige Informationen zur MERS-CoV-Epidemiologie. Weiterhin könnte die Beobachtung, dass der Protease-Inhibitor Camostat, der für den Einsatz im Menschen zugelassen ist (in Japan), TMPRSS2 blockiert und damit den MERS-CoV Eintritt inhibiert, helfen, Behandlungsstrategien für MERS-Patienten zu etablieren.

Foi investigada a existência de proteção cruzada entre bacterinas bivalentes formuladas com um de três representantes do Sorogrupo Sejroe: hardjo (bacterina A), wolffi (bacterina B) e guaricura (bacterina C), e uma estirpe do sorovar pomona, empregada por ser patogênica para hamsters (Mesocricetus auratus) e possibilitar a realização do teste de potência com desafio. Os ensaios foram efetuados em hamsters machos, comparando-se os níveis de anticorpos aglutinantes e neutralizantes, respectivamente obtidos nos testes de soroaglutinação microscópica (SAM) e inibição de leptospiras in vitro (ICL). Os animais receberam duas doses de vacinas via subcutânea; aos dez dias da segunda dose, foram inoculados com culturas não inativadas dos respectivos sorovares do Sorogrupo Sejroe. Aos 21 dias pós-infecção (d.p.i.), os animais foram sangrados, os soros (n=8) foram agrupados em pools e submetidos aos testes de SAM e ICL. O teste de potência com desafio para o sorovar pomona foi adaptado do protocolo preconizado pelo United States Department of Agriculture, mas as vacinas não foram diluídas e o esquema de imunização empregou duas aplicações de 1,0mL pela via subcutânea em intervalo de dez dias; o desafio foi efetuado aos dez dias da segunda aplicação; os óbitos por leptospirose foram registrados e, aos 21 d.p.i., os sobreviventes foram sacrificados e a condição de portadores renais foi investigada através de cultivos de tecido renal para isolamento de leptospiras. No teste de potência com o sorovar pomona, o número de doses infectantes empregado para desafio (100) situou-se dentro da faixa preconizada (10 a 100); respectivamente para as bacterinas A, B e C, as proporções de mortes por leptospirose entre os animais vacinados foram de 1/10, 0/10 e 3/10, e as de portadores renais de leptospiras entre os sobreviventes foram 2/9, 1/10 e 2/7. Os resultados do teste SAM revelaram que a bacterina A induziu reações para os sorovares hardjo e wolffi; a bacterina B, para hardjo, wolffi e guaricura, e a bacterina C, apenas para a guaricura, e do teste de ICL, que animais vacinados com as bacterinas B ou C apresentaram proteção para hardjo, wolffi e guaricura; entretanto, a bacterina A conferiu proteção apenas para wolffi. Apesar das variações no poder imunogênico segundo a estirpe de leptospira empregada para a produção das bacterinas, houve proteção cruzada entre os sorovares hardjo, wolffi e guaricura. / The existence of cross-protection among bivalent bacterins, produced with one of three leptospires belonging to Serogroup Sejroe: hardjo (bacterin A), wolffi (bacterin B) and guaricura (bacterin C), and a strain of serovar pomona (included because of its pathogenicity to hamsters and the possibility of performing potency assay with challenge), was investigated in male hamsters (Mesocricetus auratus) by comparison of agglutinating and neutralizing antibodies titers, respectively measured by microscopic agglutination (MAT) and in vitro growth inhibition (GIT) tests. All animals received two doses of bacterins by subcutaneous route; after ten days from the second dose, they were inoculated with non-inactivated cultures of respective serovars of Serogroup Sejroe. At 21 post-challenge day (p.c.d.), all animals were bled and their sera (n=8) were joined in pools and tested by MAT and GIT. The potency assay with challenge performed only with serovar pomona was modified from protocol of USDA, but vaccines were not diluted and the immunization schedule employed two 1.0 mL vaccine doses by subcutaneous route with 10?day interval; the challenge was performed after ten days from the second dose; the number of deaths due to leptospirosis was registered; at 21 p.c.d., the survivors were sacrificed and their renal carrier state was investigated by culture of renal tissue for leptospires isolation. In the potency assay with serovar pomona, the number of infectious doses employed for challenge (100 infective units) was in accordance with the recommended range (10-1,000 infective units); respectively to bacterins A, B and C, proportions of deaths due to leptospirosis among vaccinated animals were 1/10, 0/10 and 3/10, and proportions of leptospires renal carrier among survivors were 2/9, 1/10 and 2/7. Results of MAT showed that bacterin A induced reactions against serovars hardjo and wolffi; bacterin B, against hardjo, wolffi and guaricura, and bacterin C, against guaricura; results of GIT revealed that vaccinated animals with bacterins B or C presented protection against serovar hardjo, wolffi and guaricura; however, bacterin A induced protection only against wolffi. A cross?protection was observed among serovars hardjo, wolffi and guaricura, although variations exist in the immunogenic capacity according to the strain of leptospires used for the bacterins production.

Les infections chroniques de l’Immunodéficience Humaine et de l’Hépatite C (VIH et VHC) sont à l’origine de pandémies. Malgré des traitements avancés, leurs relations avec le système immunitaire ne sont pas résolues et restent nécessaires pour établir de nouvelles stratégies thérapeutiques. Les cellules Natural Killer (NK) sont des effecteurs majeurs antiviraux et sont importants pour l’immunité innée et adaptative. Ils contrôlent leur cytotoxicité et leur fonction immunorégulatrice via de multiples récepteurs activateurs et inhibiteurs qui sont enclenchés par interaction avec leurs ligands respectifs. Parmi tous les récepteurs, je me suis particulièrement intéressé aux Natural Cytotoxicity Receptors NKp30 et NKp44. Il est intéressant de noter que le laboratoire a précédemment identifié un épitope conservé de la gp41 du VIH-1 qui induit l’expression du ligand de NKp44 sur des LT CD4+ les rendant susceptibles à la lyse par des cellules NK-NKp44+. Après plusieurs études, le laboratoire a mis en place une stratégie vaccinale basée sur un peptide de l’épitope conservé de la gp41 qui induit chez la souris des Anticorps Neutralisants (AcNs W614A-3S) contre l’infection VIH-1. Alors que le VIH-2 est considéré comme un modèle unique d’une infection VIH contrôlée, les données sur les cellules NK y sont très limitées. Nous avons observé une dérégulation du récepteur NKp30 et une augmentation de ses ligands qui conduisent à des déficiences de leurs fonctions et représentent un nouveau mécanisme de persistance virale. Ensuite, nous avons observé pendant l’infection chronique par le VHC une forte proportion de cellules NK intra-hépatiques exprimant NKp44 qui corrèle avec la fibrose et la charge virale. De plus, nous avons identifié un épitope conservé de la protéine Core du VHC qui induit le ligand de NKp44 sur des lignées hépatiques. Ces données suggèrent que la déplétion des hépatocytes passe par un mécanisme similaire à celui observé pendant l’infection VIH-1. Enfin, une étude sur des patients VIH-1 contrôleurs nous a permis d’identifier la présence d’AcNs W614A-3S qui sont associées au contrôle viral et au maintien de LT CD4+ fonctionnelles. Ces données ont confirmé le potentiel de ces AcNs et, dont leur production par vaccination, ont été confirmée chez le lapin et le singe. Ainsi, ces études apportent de nouvelles données dans les relations entre les cellules NK et le VIH ou le VHC ainsi que de nouvelles stratégies thérapeutiques. Ces études ont notamment confirmé le pouvoir des AcNs W614A-3S dans un vaccin contre le VIH-1. / Human Immunodeficiency and Hepatitis C (HIV and HCV) chronic infections are at the origin of pandemics. Despite advance drug treatments, their relationship with the immune system is not resolved and is still required to establish new therapeutic strategies. Natural Killer (NK) cells are major antiviral effectors of the immune system and are important for innate and adaptive immune processes. They mediate cytotoxicity and immunoregulation via various activator and inhibitor receptors that are triggered upon interaction with their cognate ligands. Among all receptors, I particularly took an interest in Natural Cytotoxicity Receptors NKp30 and NKp44. Interestingly, the lab previously identified a conserved HIV-1 gp41 épitope that induce expression of NKp44 ligand on CD4+ T cells making them susceptible to lyses by NK-NKp44+ cells. After various studies, the lab established a vaccine strategy based on a peptide from the conserved gp41 épitope that induced in mice Neutralizing Antibodies (Nab W614A-3S) against HIV-1 infection. Whereas HIV-2 infection could be considered as a HIV control infection unique model, data on NK cells are very limited. We found a down-modulation of NKp30 receptor and an increased of its ligands that lead to functional impairments of NK cells and could represent a new viral persistence mechanism. Then, during the HCV chronic infection we found a high proportion of intrahepatic NK cells expressing NKp44 that correlates with fibrosis and viral load. Furthermore we identified a conserved épitope of HCV core protein that induced NKp44 ligand on hepatic cell lines. These data suggest that destruction of hepatocyte might occur by a similar mechanism observed during HIV-1 infection. Finally, a study on HIV-1 controllers patients allow us to identify the presence of Nab W614A-3S that correlates with viral control and the preservation of functional CD4+ T cells. These data confirm the potency of this Nab and their induction by vaccination has been also confirmed in rabbit and macaques. Thus, these studies highlight new data regarding relationship between NK cells and HIV or HCV that could represent new therapeutic approaches. These studies especially confirm the potency of Nab W614A-3S to implement a vaccine against HIV-1.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.

Passive immunization with neutralizing antibodies (nAbs) may prevent mother-to-child transmission (MTCT) of HIV-1 and/or impact HIV-1 exposed infant outcomes. This study compared plasma neutralizing activity against heterologous HIV-1 variants and the quasispecies present in the infected mothers among exposed uninfected infants (HEU) to infants that eventually acquired infection and between transmitting versus non-transmitting mothers. HEU (n = 42), compared to those that eventually acquired infection (n = 21), did not possess higher nAb responses against heterologous envelopes (p = 0.46) or their mothers’ variants (p = 0.45). Transmitting as compared to non-transmitting mothers, however, had significantly higher plasma neutralizing activity against heterologous envelopes (p = 0.03), although these two groups did not have significant differences in their ability to neutralize autologous strains (p = 0.39). Furthermore, infants born to mothers with greater neutralizing breadth and potency were significantly more likely to have a serious adverse event (p = 0.03). These results imply that pre-existing anti-HIV-1 neutralizing activity does not prevent breast milk transmission. Additionally, high maternal neutralizing breadth and potency may adversely influence both frequency of breast milk transmission and subsequent infant morbidity. In addition to neutralization, passively acquired maternal antibodies that mediate antibody dependent cellular cytotoxicity (ADCC) may impact both breast milk transmission and infant outcomes. To date, no study has rigorously compared ADCC activity against a broad panel of heterologous strains or circulating maternal viruses among HEU versus infants that eventually acquire infection and among transmitting versus non-transmitting mothers. We developed a high-throughput assay that measures the lysis of infected reporter target cells in the presence and absence of antibodies. This assay yields similar results as other methods that use decreases in reporter signal from target cells or lysis of primary cells estimated by flow cytometry. In contrast to other ADCC methods, we show that our assay allows assessment of ADCC breadth and potency in a relatively high throughput manner because it uses novel target cells that are susceptible to infection by diverse HIV-1 variants. Estimating ADCC breadth and potency and activity against maternal strains will have important insights about the impact of passively acquired maternal antibodies on MTCT.

HIV-1 primarily utilizes the CCR5 receptor as a co-receptor, but over time, viruses can evolve to use the CXCR4 protein. Changes in the viral envelope V3 loop mediate this switch. The emergence of CXCR4-utilizing viruses has been presumed to occur as a consequence of decreased humoral immunity. We show that exclusively CXCR4-using (X4) viruses contain a 2 to 3 amino acid insertion in the V3 loop. Structural modeling revealed that this insertion caused a protrusion in the V3 loop, which impacts CCR5 receptor interaction. These genotypic and structural motifs affected neutralization susceptibility because X4, as compared to co-circulating CCR5-utilizing (R5) viruses, were less neutralization sensitive to autologous contemporaneous and heterologous plasma. Individuals with co-circulating X4 and R5, as compared to those with only R5, viruses had similar neutralization breadth and potency indicating that the emergence of X4 viruses is not associated with decreased humoral immunity. These results suggest that X4 viruses are neutralization escape variants and arise due to humoral selective pressure. This work has implications for future antibody-based therapeutics. Along with providing a framework for developing an HIV-1 vaccine, broadly neutralizing antibodies (bnAbs) are also being investigated as a potential therapeutic. BnAbs target a limited number of conserved HIV-1 envelope structures, including glycans in and around the V1/V2 and V3 domains. Along with the V3 loop, changes in V1/V2 are also known to impact co-receptor usage. We show that viruses that exclusively use the CXCR4 co-receptor, as compared to variants that only utilize CCR5, were less neutralization sensitive to V1/V2 and V3 directed bnAbs. In contrast, R5 and X4 viruses did not demonstrate neutralization differences to bnAbs that target non-V1/V2 and V3 envelope regions, such as the CD4 binding site and the membrane proximal external region. Structural modeling revealed that the predicted orientation of the V1/V2 loop among diverse HIV-1 variants predicts susceptibility to V3 loop directed bnAbs. In aggregate, our results suggest that viruses with different co-receptor usage have differing bnAb susceptibility. Furthermore, structural modeling may be used as a tool to predict neutralization susceptibility to bnAbs against regions associated with co-receptor usage. / 2020-06-12T00:00:00Z

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La transmission mère-enfant (TME) est un modèle naturel permettant d’explorer le rôle des anticorps neutralisants dans la protection vis-à-vis de l’infection par le VIH-1 ainsi que les caractéristiques des virus transmis aux enfants malgré la présence d’anticorps maternels. Au cours de mes travaux, nous avons confirmé l’importance de la région V2 de la glycoprotéine de surface (gp120) du VIH-1 dans la résistance du virus à la neutralisation. L’analyse des propriétés de variants transmis à l’enfant et de variants maternels a mis en évidence une grande hétérogénéité de leurs propriétés biologiques sans pour autant identifier de propriétés spécifiques conférant un avantage sélectif aux virus transmis. Cependant, nous avons montré que les virus transmis étaient plus sensibles à la neutralisation par deux anticorps monoclonaux humains largement neutralisants récemment identifiés, PG9 et PG16, une observation qui pourrait avoir des applications intéressantes en termes de prévention de la TME ou de développement vaccinal. Enfin, nous avons identifié deux résidus très conservés de la gp120 impliqués dans la sensibilité à la neutralisation par PG9 et/ou PG16. / Mother-to-child transmission (MTCT) provides a natural model for studying the role of neutralizing antibodies in preventing HIV-1 infection, and the characteristics of the virus transmitted to the infants despite the presence of maternal antibodies. During my thesis works, we confirmed that the V2 domain of the surface envelope glycoprotein (gp120) of HIV-1 plays a major role in resistance to neutralization. The analysis of properties of variants transmitted to infant and maternal variants showed a wide spectrum of their biological properties, but we did not identify any specific property conferring a selective advantage for transmission of the virus to the infant. Nevertheless, we showed that the transmitted variants were more sensitive to neutralization by the two recently described broadly neutralizing monoclonal antibodies PG9 and PG16. This observation should have interesting applications in terms of prevention of MTCT or vaccine development. Finally, we identified two gp120 cross-clade conserved residues involved in neutralization sensitivity to PG9 and/or PG16.