A new concept quickly gaining ground in the field of cancer research is that the inflammatory process plays a key role in cancer development and metastasis; however, the molecular mechanisms of such an involvement in cancer progression remain largely unspecified. YKL-40, also known as human cartilage glycoprotein 39, is a secreted heparin-binding protein with ties to both cancers and inflammatory disease. In these diseases, YKL-40 has been suggested to play a role in regulating tissue and extracellular matrix remodeling. It has been found that in certain cancers, including breast, colorectal and brain, that high YKL-40 serum levels correlate with poor outcome, and consequently it may serve as a biomarker. Our recent study has shown that tumor-derived YKL-40 acts as an angiogenic factor due to its ability to up-regulate vessel formation and metastasis during tumor development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. The goal of this project was to better understand the importance of tumor-derived YKL-40 in angiogenesis through both functional and structural studies. By establishing a monoclonal YKL-40 antibody for blocking YKL-40, the function of tumor-derived YKL-40 in inducing endothelial cell angiogenesis and tumor cell survival was uncovered, confirming YKL-40's importance in tumor signaling as well as offering evidence in the benefit of its neutralization. Additionally, a postulated heparin-binding domain on YKL-40 was mutated in hopes of revealing the relevance of this binding ability on YKL-40's function and whether this could serve as a target in inhibiting YKL-40 signaling.

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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.

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.

INTRODUÇÃO: A Febre Amarela (FA) é uma doença viral aguda endêmica em grande parte do Brasil. A principal medida de prevenção é a vacinação. O presente estudo avaliou a prevalência e os títulos de anticorpos neutralizantes em pessoas com 60 anos ou mais que haviam recebido anteriormente a vacina de FA 17DD, em comparação a adultos saudáveis com 18 a 59 anos. Além disso, foram avaliadas a correlação entre os títulos de anticorpos e o tempo decorrido desde a vacinação, nos participantes que receberam apenas uma dose da vacina, e a prevalência de anticorpos nos vacinados há menos e há mais de dez anos. MÉTODOS: Os participantes foram recrutados entre pessoas que procuraram o Centro de Referência para Imunobiológicos Especiais (CRIE) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) para receber diferentes vacinas e que referiam ter recebido a vacina de FA anteriormente. Os seguintes dados foram coletados: idade, etnia, sexo, número de doses da vacina de FA recebidas, data da última vacinação de FA. Foi realizada contagem de linfócitos TCD4+ usando citometria de fluxo. Os anticorpos neutralizantes contra FA foram dosados pelo teste de neutralização por redução de 50% das placas de lise (PRNT50). RESULTADOS: Foram incluídos 94 indivíduos, 46 com idade de 60 anos ou mais (Grupo 1) e 48 com 18 a 59 anos (Grupo 2). Não houve diferença significativa entre os dois grupos na distribuição de gênero, etnia, número de doses de vacina de FA recebidas anteriormente, tempo desde a última dose e contagem de linfócitos TCD4+. Não houve diferença na prevalência de anticorpos neutralizantes anti-FA entre os dois grupos (87% e 93,8% nos Grupos 1 e 2, respectivamente, p=0,263). O título médio geométrico (GMT) dos anticorpos neutralizantes foi maior no grupo mais jovem (3,77 log10mUI/mL) comparado ao grupo mais velho (3,64 log10mUI/mL) e essa diferença foi estatisticamente significante (p=0,022). Não foi encontrada correlação entre os títulos de anticorpos neutralizantes e o tempo decorrido desde a vacinação entre os participantes que receberam apenas uma dose de vacina, tendo sido analisados os dois grupos conjuntamente. Também não foi encontrada diferença estatisticamente significativa na prevalência de anticorpos neutralizantes entre os participantes que receberam apenas uma dose da vacina de FA há mais de 10 anos ou há menos de 10 anos. CONCLUSÕES: São necessários outros estudos de persistência de anticorpos na população idosa devido à possibilidade de resposta vacinal alterada pela imunosenescência / INTRODUCTION. Yellow Fever (YF) is an acute viral disease endemic in large parts of Brazil. The main preventive measure is vaccination. This study aimed to assess the prevalence and titers of neutralizing antibodies in persons aged 60 years and older who had previously received YF 17DD vaccine, in comparison to healthy adults aged 18 to 59 years. The study also evaluated the correlation between the antibodies titers and the time elapsed since vaccination, in participants who had received a single dose of the vaccine, and the prevalence of antibodies in participants vaccinated within ten years and more than ten years before enrollment. METHODS. Participants were recruited among persons who came to the Reference Center for Special Immunobiologicals (Centro de Referência para Imunobiológcos Especiais, CRIE) of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) to receive any vaccine and who had previously received the YF vaccine. The following data were collected: age, ethnicity, gender, number of YF vaccine doses taken and date of last YF vaccination. CD4 T cells counts were performed using flow cytometry. YF neutralizing antibodies were measured using test of neutralization by 50% reduction of lysis plaques (PRNT50). RESULTS. Ninety-four subjects were enrolled: 46 persons aged 60 years and older (Group 1) and 48 persons aged 18 to 59 years (Group 2). There was no significant difference between the groups regarding gender, ethnicity, number of YF vaccine doses previously received, time since the last dose and CD4+T cells count. There was no difference in the prevalence of YF neutralizing antibodies between the groups (87% and 93.8% in Groups 1 and 2, respectively, p=0.263). The log-transformed Geometric Mean Titer (GMT) of YF neutralizing antibodies was higher in the younger group (3.77 log10mUI/mL) in comparison to the older group (3.64 log10mUI/mL), and this difference was statistically significant (p=0.022). There was no correlation between YF neutralizing antibodies titers and time elapsed since vaccination among the participants who had previously received a single dose of YF vaccine, with the two groups analyzed together. There was no significant difference in the prevalence of neutralizing antibodies among participants who received a single dose of YF vaccine within ten years or more than 10 years before enrollment. CONCLUSIONS. Further studies on antibodies persistence in the elderly are necessary, considering the possibility of compromised immune response to vaccines due to immunosenescence

INTRODUÇÃO: A Febre Amarela (FA) é uma doença viral aguda endêmica em grande parte do Brasil. A principal medida de prevenção é a vacinação. O presente estudo avaliou a prevalência e os títulos de anticorpos neutralizantes em pessoas com 60 anos ou mais que haviam recebido anteriormente a vacina de FA 17DD, em comparação a adultos saudáveis com 18 a 59 anos. Além disso, foram avaliadas a correlação entre os títulos de anticorpos e o tempo decorrido desde a vacinação, nos participantes que receberam apenas uma dose da vacina, e a prevalência de anticorpos nos vacinados há menos e há mais de dez anos. MÉTODOS: Os participantes foram recrutados entre pessoas que procuraram o Centro de Referência para Imunobiológicos Especiais (CRIE) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) para receber diferentes vacinas e que referiam ter recebido a vacina de FA anteriormente. Os seguintes dados foram coletados: idade, etnia, sexo, número de doses da vacina de FA recebidas, data da última vacinação de FA. Foi realizada contagem de linfócitos TCD4+ usando citometria de fluxo. Os anticorpos neutralizantes contra FA foram dosados pelo teste de neutralização por redução de 50% das placas de lise (PRNT50). RESULTADOS: Foram incluídos 94 indivíduos, 46 com idade de 60 anos ou mais (Grupo 1) e 48 com 18 a 59 anos (Grupo 2). Não houve diferença significativa entre os dois grupos na distribuição de gênero, etnia, número de doses de vacina de FA recebidas anteriormente, tempo desde a última dose e contagem de linfócitos TCD4+. Não houve diferença na prevalência de anticorpos neutralizantes anti-FA entre os dois grupos (87% e 93,8% nos Grupos 1 e 2, respectivamente, p=0,263). O título médio geométrico (GMT) dos anticorpos neutralizantes foi maior no grupo mais jovem (3,77 log10mUI/mL) comparado ao grupo mais velho (3,64 log10mUI/mL) e essa diferença foi estatisticamente significante (p=0,022). Não foi encontrada correlação entre os títulos de anticorpos neutralizantes e o tempo decorrido desde a vacinação entre os participantes que receberam apenas uma dose de vacina, tendo sido analisados os dois grupos conjuntamente. Também não foi encontrada diferença estatisticamente significativa na prevalência de anticorpos neutralizantes entre os participantes que receberam apenas uma dose da vacina de FA há mais de 10 anos ou há menos de 10 anos. CONCLUSÕES: São necessários outros estudos de persistência de anticorpos na população idosa devido à possibilidade de resposta vacinal alterada pela imunosenescência / INTRODUCTION. Yellow Fever (YF) is an acute viral disease endemic in large parts of Brazil. The main preventive measure is vaccination. This study aimed to assess the prevalence and titers of neutralizing antibodies in persons aged 60 years and older who had previously received YF 17DD vaccine, in comparison to healthy adults aged 18 to 59 years. The study also evaluated the correlation between the antibodies titers and the time elapsed since vaccination, in participants who had received a single dose of the vaccine, and the prevalence of antibodies in participants vaccinated within ten years and more than ten years before enrollment. METHODS. Participants were recruited among persons who came to the Reference Center for Special Immunobiologicals (Centro de Referência para Imunobiológcos Especiais, CRIE) of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) to receive any vaccine and who had previously received the YF vaccine. The following data were collected: age, ethnicity, gender, number of YF vaccine doses taken and date of last YF vaccination. CD4 T cells counts were performed using flow cytometry. YF neutralizing antibodies were measured using test of neutralization by 50% reduction of lysis plaques (PRNT50). RESULTS. Ninety-four subjects were enrolled: 46 persons aged 60 years and older (Group 1) and 48 persons aged 18 to 59 years (Group 2). There was no significant difference between the groups regarding gender, ethnicity, number of YF vaccine doses previously received, time since the last dose and CD4+T cells count. There was no difference in the prevalence of YF neutralizing antibodies between the groups (87% and 93.8% in Groups 1 and 2, respectively, p=0.263). The log-transformed Geometric Mean Titer (GMT) of YF neutralizing antibodies was higher in the younger group (3.77 log10mUI/mL) in comparison to the older group (3.64 log10mUI/mL), and this difference was statistically significant (p=0.022). There was no correlation between YF neutralizing antibodies titers and time elapsed since vaccination among the participants who had previously received a single dose of YF vaccine, with the two groups analyzed together. There was no significant difference in the prevalence of neutralizing antibodies among participants who received a single dose of YF vaccine within ten years or more than 10 years before enrollment. CONCLUSIONS. Further studies on antibodies persistence in the elderly are necessary, considering the possibility of compromised immune response to vaccines due to immunosenescence

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.

<p>One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. </p><p>We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, &#945;-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.</p><p>Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.</p> / Dissertation

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.