La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire. / Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.

Helper T cells (Th) are a vital component of the immune system responsible for directing other immune cells to eliminate pathogens and cancer. Specifically, Th cells facilitate B cell and cytotoxic T cell (Tc) activation and recruitment and enhance their function against cancer and infectious diseases. Th cells are a valuable resource for improving Tc responses in cancer treatment and have become a focus of immunotherapeutic research. While it is increasingly clear that helper T cells serve an important role, the details about which entities produce an effective Th cell response remain unclear. CD5 is a T cell co-receptor that negatively regulates T cell activation and helps fine-tune the TCR repertoire by altering TCR signaling during the selection process in the thymus. This work discusses the role of the co-receptor CD5 in influencing Th cell metabolism, as well as the study of two T cells called LLO118 and LLO56 that have different CD5 expression levels, and their functional response to altered peptides. Antibodies have revolutionized the world of cancer research and accelerated the development of therapies that trigger the immune system to target disease. In recent years, many antibody-based immunotherapies have emerged as effective candidates for combating cancer due to their refined specificity and ability to target a variety of epitopes. However, many therapies, such as those that target CD19 on B cell cancers, are also present on healthy cells, destroying both cancerous and healthy cells alike. Thymidine kinase 1 (TK1) is an enzyme involved in the DNA salvage pathway that converts thymidine into the nucleotide thymine. Recently, TK1 has been shown to be overexpressed on the surface of many cancers such as acute lymphoblastic leukemia. Importantly, TK1 is not expressed on the surface of healthy cells, making it an ideal cancer-specific antigen that can be targeted for cancer treatment. This work discusses our efforts to characterize TK1-specific single-chain antibodies from a yeast display library. According to the World Health Organization, genomics is defined as the study of all genes and their related functions. In contrast to genetics, genomics analyzes the entire DNA makeup of an organism rather than a single gene. In the past 20 years, the cost of genomic sequencing has decreased dramatically, making it affordable and accessible. A key area that must be addressed with genomic testing involves education about their promise, challenges, potential consequences, and ethical considerations. Genomic testing provides a powerful opportunity to educate everyone on scientific and ethical issues to increase understanding on the subject. This work discusses the influence of personal genomics in society and focuses on the importance, benefits, and consequences of genomics education in the classroom, clinic, and the public.

En l'absence d'un vaccin efficace et avec des thérapies antirétrovirales incapables d'éradiquer le virus, le VIH-1 reste un problème de santé publique mondial. Des immunothérapies à base d'anticorps sont à l'étude pour éliminer les réservoirs cellulaires, qui représentent un obstacle incontournable à la guérison du VIH-1. Les glycoprotéines d'enveloppe du VIH-1 (Env) représentent le seul antigène du virus exposé à la surface des cellules infectées et constituent donc la principale cible des anticorps. L’Env non-liée adopte sa conformation « fermée », reconnue préférentiellement par les anticorps neutralisants. L'interaction avec CD4 fait passer Env dans sa conformation « ouverte », exposant des épitopes conservés reconnus par les anticorps non-neutralisants (nnAbs) présents dans le sérum d’individus infectés par le VIH-1 (sérums VIH+). Les nnAbs peuvent éliminer les cellules infectées par la cytotoxicité cellulaire dépendante des anticorps (ADCC). Cependant, les protéines accessoires Nef et Vpu diminuent l’expression de surface de CD4 et BST-2 afin d’évader à la reconnaissance et l'élimination des cellules infectées par les nnAbs. Dans cette thèse, nous caractérisons en détail la contribution d’Env, Nef et Vpu pour échapper aux réponses humorales et explorons de nouvelles stratégies pour sensibiliser les cellules infectées à l’ADCC. Pour quantifier plus adéquatement la réponse ADCC, nous avons identifié des biais dans les tests largement utilisés, notamment pour évaluer les corrélats de protection vaccinale. Il s'agit de l'incapacité à faire la distinction entre l'élimination des cellules infectées et des cellules non-infectées, et l'utilisation de constructions virales comportant un gène rapporteur empêchant l’expression de Nef. En utilisant un nouveau marquage intracellulaire, nous avons confirmé l’effet protecteur de Nef et Vpu contre l’ADCC. Ensuite, nous avons étudié les déterminants d’Env et Vpu modulant la susceptibilité des cellules infectées à l’ADCC médiée par les nnAbs. Certaines caractéristiques structurelles d'Env modulent ses transitions conformationnelles, incluant le domaine d'association du trimère, le site de clivage de la furine et la cavité Phe43. L’altération de ces composantes augmente la sensibilité des cellules infectées à l'ADCC par les sérums VIH+. Outre l’inhibition de CD4 et BST-2, Vpu cible également NTB-A et PVR, des ligands de récepteurs activateurs des cellules NK. Cependant, la polyfonctionnalité de Vpu est compromise par l’augmentation de BST-2 par les interférons de type I (IFN-I), sensibilisant ainsi les cellules infectées aux réponses NK. En utilisant un modèle de souris humanisée, nous validons l'importance de Vpu pour échapper à la pression immunitaire des nnAbs in vivo. Enfin, nous avons exploré de nouvelles stratégies pour sensibiliser les cellules infectées à l'ADCC en modulant la conformation d’Env avec des mimétiques moléculaires de CD4 (CD4mc). Nous avons identifié des résidus bordant la cavité Phe43 modulant la sensibilité au CD4mc. L’accumulation d’Env induite par les IFN-I augmente la capacité du CD4mc à sensibiliser les cellules infectées à l'ADCC par les sérums VIH+. Globalement, cette thèse dévoile une caractérisation approfondie des déterminants viraux et cellulaires modulant la susceptibilité des cellules infectées par le VIH-1 aux réponses humorales. Une meilleure compréhension de ces mécanismes est nécessaire pour développer des nouvelles stratégies capables d’éradiquer les réservoirs viraux. / In the absence of an effective vaccine and with antiretroviral therapies unable to eradicate the virus, HIV-1 remains a global public health problem. Antibody-based immunotherapies are currently being investigated to eliminate cellular reservoirs, which represent a major obstacle towards an HIV-1 cure. HIV-1 envelope glycoproteins (Env) represent the only virus-specific antigen exposed at the surface of infected cells and therefore is the main target for antibodies. In its unliganded form, Env samples a ‘closed’ conformation, preferentially recognized by neutralizing antibodies. Interaction with CD4 drives Env into its ‘open’ conformation, exposing conserved epitopes recognized by non-neutralizing antibodies (nnAbs) present in sera from HIV-1 infected individuals (HIV+ sera). NnAbs can eliminate infected cells by antibody-dependent cellular cytotoxicity (ADCC). However, HIV-1 encodes for the accessory proteins Nef and Vpu which decrease cell surface levels of CD4 and BST-2, thus avoiding recognition and elimination of infected cells by nnAbs. In this thesis, we characterize in detail the contribution of Env, Nef, and Vpu to evade humoral responses and explore new strategies for sensitizing infected cells to ADCC. In an effort to develop a more adequate quantification of ADCC responses, we identified major biases in widely used assays, including the ones used to assess correlates of vaccine protection. These include the inability to distinguish between the elimination of infected and uninfected cells and the use of viral constructs coding for a reporter gene that prevents Nef expression. Using a novel intracellular staining, we confirmed the protective effect of Nef and Vpu against ADCC responses. Next, we studied the different Env and Vpu determinants modulating the susceptibility of infected cells to nnAbs-mediated ADCC responses. Certain Env structural features modulate its conformational transitions, including the trimer association domain, the furin cleavage site and the Phe43 cavity. Alterations of these components increase the susceptibility of HIV-1-infected cells to ADCC mediated by HIV+ sera. In addition to inhibiting CD4 and BST-2, Vpu also targets NTB-A and PVR, which act as ligands for NK cell activating receptors. However, we found that the polyfunctionality of Vpu can be compromised by the upregulation of BST-2 by type I interferons (IFN-I), thereby sensitizing infected cells to NK cell responses. Using a humanized mouse model, we validate the importance of Vpu to escape the immune pressure of nnAbs in vivo. Finally, we explored new strategies to bypass the protective effect of Vpu and Nef and sensitize HIV-1-infected cells to ADCC by modulating Env conformation using small CD4-mimetic compounds (CD4mc). We identified a network of residue lining the Phe43 cavity that modulates Env sensitivity to CD4mc. The enhanced surface expression of Env by type I IFNs boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by HIV+ sera. Overall, this thesis sheds light on a thorough characterization of viral and cellular determinants modulating the susceptibility of HIV-1-infected cells to humoral responses. A better understanding of these mechanisms is needed to develop new strategies able to eradicate viral reservoirs.