Infection & Immunity / Currently, there are 38 million people living with human immunodeficiency virus (HIV-1) worldwide and there were 680,000 HIV-related deaths in 2020 alone. The greatest cause of mortality in people living with HIV (PLHIV) is infection with opportunistic pathogens such as tuberculosis (TB), which accounts for one third of HIV-related deaths. PLHIV are 20 times more susceptible to TB and co-infection leads to significantly worsened outcomes in terms of both diseases. Humanized mouse (hu-mouse) models, which possess human immune cells for HIV to infect, have been useful for HIV research. Our aim is to create hu- mouse models of HIV, TB and co-infection to investigate disease progression, immune responses, therapeutics, prevention and vaccination. NOD-Rag1null-IL2rgnull (NRG) mice are highly immunocompromised mice that are traditionally used to generate hu-mouse models. We are also developing NRG mice that are transgenic for human HLA-DR4 and HLA-A2 (DRAG-A2) and similar mice have been reported to have improved immune responses. NRG and DRAG-A2 mice were humanized with hematopoietic stem cells obtained from human umbilical cord blood. DRAG-A2 mice had significantly higher engraftment success rates (defined as the percentage of mice with >10% hCD45+) as well as higher overall CD45+ leukocyte, CD4+ T cell, CD19+ B cell and CD14+ monocyte reconstitution in the blood compared to huNRGs. huNRG mice were permissive to infection with JR-CSF or NL4.3-Bal-Env HIV-1 intravaginally or systemically. huDRAG-A2 mice were also infected intravaginally with NL4.3-Bal-Env HIV-1. huDRAG-A2 mice, but not huNRGs, produced HIV-specific IgG, indicating improved immune responses. huNRG mice were infected intranasally with mCherry-Erdman, YFP-H37Rv or H37Rv Mtb. huDRAG-A2 mice were also infected with H37Rv. Human immune cell involvement and human-like granuloma formation was observed using flow cytometry and immunohistopathology. These findings show that the DRAG-A2 model may be optimal for investigating HIV, TB and co-infection, which continue to be serious global health concerns. / Thesis / Master of Science (MSc) / Human immunodeficiency virus (HIV) and tuberculosis (TB) are infectious diseases that affect millions of people worldwide every year. The greatest cause of death in people living with HIV is co-infection with TB and HIV-positive individuals are much more likely to get TB. Humanized mouse (hu-mouse) models possess human immune cells for HIV to infect and are useful for studying HIV. Our goal is to create hu-mouse models of HIV, TB and HIV/TB co-infection that will allow us to study how these diseases interact. We are currently developing a traditional hu-mouse model (known as NRG), as well as an improved next-generation model (known as DRAG-A2) with a more functional immune system. Both models have been successfully infected with HIV or TB. Only DRAG-A2 mice were able to make antibodies against HIV. The improved DRAG-A2 model will enable future studies on HIV, TB and co-infection, which continue to be understudied global problems.

HIV and TB are endemic in many regions around the world but disproportionately affect low-income countries. HIV/TB co-infections significantly worsen disease outcome with TB being the leading cause of death in people living with HIV (PLWH). Currently, the only vaccine for TB is the BCG vaccine which is not protective against adult pulmonary TB and not recommended for PLWH. Antiretroviral therapy (ART) can suppress viral loads and improve the life span in PLWH but is ultimately unable to eliminate the virus. Therefore, better therapeutic options are needed to improve outcomes for both HIV and TB infections. IL-17 is a proinflammatory cytokine characteristically produced by Th17 cells. Utilizing a humanized mouse model, we aimed to investigate the role of IL-17 and Th17 cells in HIV and TB. HIV infection preferentially depleted Th17 cells in the blood and tissues of humanized mice. In TB infection, significantly increased numbers of Th17 cells were observed at 2 weeks post infection (p.i) in the lungs of M.tb infected mice compared to control and 4 weeks p.i. IL-17 levels trended higher in the lungs at 4 weeks p.i compared to control and 2 weeks. IL-17 depletion in acute TB infection showed slight decrease in bacterial load in the lungs of the treated mice compared to the control. This could suggest a potential pathological role of IL-17 in TB infection where IL-17 promotes M.tb replication. When exogenous IL-17 was administered to the lungs prior to TB infection, significantly higher numbers of CD4+ and CD8+ T cells in the lungs of treated mice were seen compared to the control but this did not affect bacterial load. Our current experiment administers exogenous IL-17 to HIV infected mice to assess how it would affect disease progression. Further investigation is needed to explore how IL-17 affects HIV and TB disease pathogenesis. / Thesis / Master of Science (MSc) / HIV and TB are diseases that place a huge burden on healthcare systems globally. Co-infections with HIV and TB worsen a patient’s prognosis significantly. Currently, antiretroviral therapy is used to treat HIV which can control infection but cannot eliminate the virus. The current vaccine for TB, the BCG vaccine is not completely efficacious for adults with TB, highlighting the urgent need for better vaccines and treatments for HIV and TB. IL-17 is a signalling molecule that has many effects on the immune system. Using mouse models that mimic human immune responses, we aimed to assess how IL-17 is involved in HIV and TB infections and if it can be used to develop new therapeutics for HIV and TB. We found adding IL-17 to TB infected mice led to increased immune cell recruitment suggesting a beneficial role of IL-17. Further investigation is needed to fully assess IL-17’s role in infectious diseases.

Une nouvelle lignée homozygote de souris « humanisé » HLA transgéniques : HLA-A2.1+/+HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-β2m-/- (HLA-A2/DP4), a été obtenue en croisant entre les souris HLA transgéniques HLA-A2.1+/+β2m-/-(A2) et les souris HLA transgéniques HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-(DP4). Chez les souris HLA-A2/DP4, les réponses cellulaire T HLA-A2 restreint ou HLA-DP4 restreint contre l’antigène HBs du virus de l’Hépatite B suite à une immunisation avec le vaccin anti-AgHBs sont similaires à ceux observés chez les souris A2, chez les souris DP4, chez les humains infectés par le virus ou immunisés avec le même vaccin. Ces résultats montrent que les réponses cellulaires induites les souris HLA-A2/DP4 miment fidèlement les réponses homologues humaines. Ainsi, ces souris représentent un excellent modèle d’expérimentations précliniques animal pour évaluer ou comparer l’efficacité des réponses « humain » induites in vivo par des candidates vaccins. Le modèle facilitera également l'identification de nouvelles épitopes HLA-A2 et HLA-DP4 restreints, qui constituera de futurs réactives de suivi clinique des réponses contre l'infection chez les humains.En exploitant ces souris HLA-A2/DP4, nous avons identifié quatre nouveaux HLA-DP4-restricted épitopes issus de l'AgHBs et deux nouveaux HLA-A2 restreint épitopes dérivés de protéines M1. / A new homozygous humanized HLA transgenic mouse strain, HLA-A2.1+/+HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-β2m-/- (HLA-A2/DP4), was obtained by crossing the HLA transgenic HLA-A2.1+/+β2m-/-(A2) mice and HLA transgenic HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-(DP4) mice. In HLA-A2/DP4 mice, HLA-A2 restricted or HLA-DP4 restricted T cell responses against HBs antigen of hepatitis B virus after immunization with the HBsAg vaccine are similar to those induced in A2 mice, in DP4 mice, in HBV-infected or HBsAg-vaccinated humans. These results show that cellular responses induced in HLA-A2/DP4 mice faithfully mimic human responses counterparts. Thus, these mice represent an excellent animal model for preclinical experimentations to evaluate or compare the effectiveness of responses "human" induced in vivo by candidate vaccines. The model will also facilitate the identification of new epitopes HLA-A2 and HLA-DP4 restricted, which will be of future reactive for clinical monitoring response against infection in humans. By exploiting these HLA-A2/DP4 mice, we identified four new HLA-DP4-restricted epitopes from HBsAg and two new HLA-A2 restricted epitopes derived from protein M1.

A new homozygous humanized HLA transgenic mouse strain, HLA-A2.1+/+HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-β2m-/- (HLA-A2/DP4), was obtained by crossing the HLA transgenic HLA-A2.1+/+β2m-/-(A2) mice and HLA transgenic HLA-DP4+/+hCD4+/+mCD4-/-IAβ-/-(DP4) mice. In HLA-A2/DP4 mice, HLA-A2 restricted or HLA-DP4 restricted T cell responses against HBs antigen of hepatitis B virus after immunization with the HBsAg vaccine are similar to those induced in A2 mice, in DP4 mice, in HBV-infected or HBsAg-vaccinated humans. These results show that cellular responses induced in HLA-A2/DP4 mice faithfully mimic human responses counterparts. Thus, these mice represent an excellent animal model for preclinical experimentations to evaluate or compare the effectiveness of responses "human" induced in vivo by candidate vaccines. The model will also facilitate the identification of new epitopes HLA-A2 and HLA-DP4 restricted, which will be of future reactive for clinical monitoring response against infection in humans. By exploiting these HLA-A2/DP4 mice, we identified four new HLA-DP4-restricted epitopes from HBsAg and two new HLA-A2 restricted epitopes derived from protein M1.

Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

Parmi les nouvelles stratégies thérapeutiques, la thérapie génique contre le VIH connaît actuellement un regain d'intérêt. La description du premier cas de guérison avéré après greffe de cellules souches mutantes pour le corécepteur majeur du VIH (CCR5) a relancé l'intérêt de la communauté pour développer des stratégies de thérapies géniques ciblant CCR5. Durant ma thèse, j'ai développé une stratégie basée sur l'inhibition de l'entrée du virus dans les lymphocytes T CD4 humains. Nos gènes thérapeutiques comprennent un inhibiteur de fusion membranaire (peptide C46) et un inhibiteur d'expression de CCR5. L'expertise de l'équipe dans les modèles de souris humanisées m'a permis de valider l'intérêt pré-clinique de cette stratégie dans un modèle de transfert de lymphocytes T modifiés. J'ai pu montrer que les lymphocytes T modifiés possédaient un avantage sélectif massif in vivo comparé à des cellules non protégées, ainsi qu’une protection totale contre la délétion induite par le VIH. De plus, dans une autre étude, j'ai montré que la dynamique virale dans les modèles de souris humanisées par transfert de CSH est plus proche des patients. Cette étude nous montre qu'il n'est pas nécessaire d'invoquer la réponse immune, pour expliquer les profils de réplication virale observés dans la phase primaire de l'infection. Ce résultat suggère donc qu'une partie de la dynamique virale est dépendante du nombre de cellules à infecter et du nombre de virions disponibles. L'ensemble des résultats de ma thèse a permis (i) de faire progresser notre compréhension de la dynamique virale, et (ii) de valider la combinaison d'inhibiteurs d'entrée dans un vecteur lentiviral. / Among new therapeutic strategies to fight the infection, gene therapy targeting CCR5 will be an asset. Indeed, the first patient to be cured from the infection received hematopoietic progenitors deprived of CCR5. This finding has ignited a flurry of research on genetic means to decrease CCR5 from the cell surface.During my PhD, I developed a gene therapy strategy based on entry inhibition of HIV in CD4 T cells. Our therapeutic genes are a membrane fusion inhibitor (the C46 peptide) and a CCR5 expression inhibitor, based on a super-agonist ligand. The experience of the team in humanized mice models allowed the validation of the strategy in vivo in a model of gene-modified T cell transfer. I first showed that the combination of the transgenes was able to prevent HIV infection in vitro in a dose dependent manner. Most importantly, I then showed that this protection conferred modified T cells with a huge selective advantage in vivo. This advantage was associated with a complete protection of CD4 T cells from HIV-induced deletion. Moreover, in another study, I showed that the viral dynamics in mouse models humanized by transferring HSC is closer to patients. This study also teaches us that it is not necessary to invoke the immune response to HIV, to explain the profiles of viral replication observed early after infection. This result suggests that part of the viral dynamic is dependent on the target/virus ratio. Together, results collected during my PhD thesis provides new information on viral dynamic and establish the interest of combining two viral entry inhibitors for gene therapy of HIV infection.

Les modèles animaux actuellement disponibles pour l’étude des leucémies humaines ne sont pas adaptés pour le développement optimal de nouvelles thérapies ciblées. Au cours de ce projet, qui s’inscrit dans une double perspective fondamentale et industrielle, nous avons cherché à générer un modèle versatile de leucémogénèse humaine chez la souris humanisée BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD). Les animaux sont greffés avec des progéniteurs hématopoïétiques transduits par des vecteurs lentiviraux surexprimant les oncogènes MYC et BCL2 et placés sous le contrôle d’un promoteur ubiquitaire (EF1α ou SFFV). Le suivi longitudinal des animaux sur une période de 5 mois montre que seule la construction SFFV/Myc-T2A-Bcl2 entraîne la transformation des progéniteurs hématopoïétiques. Entre 12 à 14 semaines post-greffe, >90% des animaux développent des lympho-proliférations de type pro-B (CD19+CD10+CD9+CD20-cytIgM-) infiltrant principalement la rate et la moelle osseuse, et circulant en abondance dans le sang. Le caractère transmissible des tumeurs est validé par des greffes secondaires de tumeurs spléniques. Les cultures in vitro de progéniteurs hématopoïétiques suggèrent que l’émergence des blastes est liée à la réactivation d’un programme B latent dans les précurseurs T, dont le développement est bloqué. Nous avons développé en parallèle un modèle de tumeur autologue. L’ensemble de ces résultats valide le modèle de leucémogénèse humaine développé chez la souris humanisée BRGS et ouvre des perspectives pour la caractérisation fonctionnelle des mécanismes de leucémogénèse, et la validation préclinique de nouvelles stratégies anti-tumorales. / Existing animal models for the study of human leukemia are not accurate for the proper development of innovative, targeted therapies. The aim of this project, which contains both a fundamental and an industrial perspective, therefore was to develop a new, versatile model of human leukemogenesis in the BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD) humanized mouse. Animals are grafted with hematopoietic progenitors transduced with lentivirals vectors to allow overexpression of MYC and BCL2 proteins under the control of an ubiquitous promotor (EF1α or SFFV). Longitudinal monitoring of the animals over five months shows that only the SFFV/Myc-T2A-Bcl2 construction induces the transformation of humans hematopoietics progenitors. Between 12 and 14 weeks post-transplantation, more than 90% of the animals develop pro-B lympho-proliferations (CD19+CD10+CD9+CD20-cytIgM-), with tumor cells being mainly found in the spleen, the bone marrow and in blood. Tumor transferability is achievable through secondary transplantation in immunodeficient mouse recipients. In vitro culture of bone marrow T cell progenitors suggest that the blasts arise from these cells after reactivation of a latent B cell program with blockade of their T cell development. In parallel, we have also developed an autologous tumor model. Altogether, these results validate the human leukemogenesis model constructed here in humanized BRGS mice and provide attractive prospects regarding the functional characterization of leukemogenesis and a preclinical validation of new anti-tumor strategies.

INTRODUCTION: SARS-CoV-2 is a novel virus from the coronavirus family that emerged in the Hubei province of China in December 2019 and rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared a global pandemic. Infection with SARS-CoV-2 causes coronavirus disease 19 (COVID19) which can be fatal. There is an obvious and pressing need for research surrounding SARS-CoV-2 that will aid in eradication of this pandemic. OBJECTIVE: The goal of this study was to absolve the dire need for small animal models of human disease that demonstrate hallmark pathological features of infection. Due to ethical and financial obstacles, the use of animals that closely resemble human immunity, such as non-human primates, is often not a viable option. For this reason, there is a push to develop small animal models that can mimic human disease responses, particularly those in viral infections that have a narrow species tropism. To achieve this in the context of the novel coronavirus, SARS-CoV-2, we studied various mouse models and their capacity to become infected with and mount an immune response to SARS-CoV-2. Our goal was to identify a model that sufficiently mimics severe COVID19 seen in humans as well as provide molecular insight into pathways that prevent the development of severe disease. METHODS: NRG-L and HIS-NRGF-L mice were subcutaneously implanted with human fetal lung xenografts and infected with SARS-CoV-2. Tissues were stained with H&E for histopathological scoring. NRG-L and HIS-NRGF-L tissues were fluorescently labeled using 2 different multiplex immunohistochemistry panels. Slides were digitized by a Vectra Polaris™ fluorescent whole slide scanner and digital analysis was completed using HALO™. Statistical analysis was conducted using GraphPad Prism™ 9.0.1. RESULTS: Infected NRG-L mice present extensive histopathological manifestations when compared to uninfected controls. Cumulative histology scores at both 2 and 7DPI were increased when compared to uninoculated fLX. Neutrophil influx, intra-airspace necrosis, capillary fibrin thrombi, and presence of syncytial cells were the most significant independent observations that contributed to the increased cumulative score. Together these findings indicate that fLX inoculated with SARS-CoV-2 faithfully recapitulate several features of diffuse alveolar damage (DAD) described in severe COVID-19. HIS-NRGF-L mice displayed decreased influx of neutrophils, intra-airway necrosis, and syncytial cells when compared to NRG-L fLX. Hemorrhage was decreased at both 2 and 7 DPI for HIS-NRGF-L fLX compared to NRG-L fLX. Cumulative histology scores were decreased in HIS-NRGF-L fLX at 7 DPI when compared to NRG-L fLX. Taken together these findings support the hypothesis that human myeloid and lymphoid infiltrates suppress or prevent the disparate host response observed in NRGL-L fLX that manifested in pronounced diffuse alveolar damage. CONCLUSION: Using digital image analysis of multiplex immunohistochemistry paired with semi-quantitative histopathological scoring, this study characterized important hallmark lesions observed in severe COVID19 as seen in small animal models. NRG-L and HIS-NRGF-L mice that are subcutaneously implanted with human fetal lung xenografts are susceptible to infection with SARS-CoV-2 and can produce severe and moderate disease phenotypes respectively. Co-engraftment with human fetal lung tissue and human immune system components in HIS-NRGF-L mice suppresses the divergent host response that is observed in NRG-L mice. For this reason, NRG-L mice engrafted with fLX are an ideal small animal model of severe COVID19, whereas HIS-NRGF-L mice severe as a valuable and informative model for deciphering molecular mechanisms driving severe COVID-19 that will serve as targets for therapeutic development.

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Die Sepsis ist ein gefürchtetes Krankheitsbild, das in hochentwickelten Industrienationen mit einer hohen Mortalität verknüpft ist und damit zu den häufigsten Todesursachen gehört. Die Pathomechanismen dieses komplexen und heterogenen Krankheitsbildes zu entdecken, gehört momentan zu den Hauptinteressengebieten der Sepsisforschung. Da die Interpretation klinischer Studien aufgrund der Heterogenität des Patientenguts schwierig ist, kommt der Entwicklung adäquater Tiermodelle eine entscheidende Bedeutung zu. Die hierbei gängigen Tiermodelle in Mäusen weisen jedoch Unzulänglichkeiten auf, die die Übertragung der in Tierexperimenten gewonnen Daten auf den klinischen Kontext nur teilweise ermöglichen. Eine Brücke kann hierbei das Tiermodell der humanisierten Maus schlagen, in der, durch Transplantation mit humanen hämatopoetischen Stammzellen, ein humanes Immunsystem reift. Die vorliegende Arbeit beschäftigt sich mit der Fragestellung, inwieweit die humanen Immunzellen in der humanisierten Maus in der Lage sind, auf LPS als Stimmulus zu reagieren. Darüberhinaus wird die Nutzung der Endotoxämie in der humanisierten Maus als alternatives Sepsismodell im Bezug zum klinischen Kontext untersucht. Hierbei ergab sich eine mögliche Nutzung des Endotoxämiemodells in der humanisierten Maus zur genaueren Erforschung des Zytokinmilieus, sowie neuer Surrogatmarker wie Pentraxin 3. Bezüglich der Reaktion einzelner immunologischer Subpopulationen und deren Bedeutung für die Klinik scheint eine Untersuchung an Modellen, die eine B- und T-Zell-Reifung nachvollziehen können und in der murine Residualzellen möglichst gering vorhanden sind, als sinnvoll.