Philosophiae Doctor - PhD / Antimicrobial peptides (AMPs) play a key role in the innate immune response. They can be ubiquitously found in a wide range of eukaryotes including mammals, amphibians, insects, plants, and protozoa. In lower organisms, AMPs function merely as antibiotics by permeabilizing cell membranes and lysing invading microbes. Prediction of antimicrobial peptides is important because experimental methods used in characterizing AMPs are costly, time consuming and resource intensive and identification of AMPs in insects can serve as a template for the design of novel antibiotic. In order to fulfil this, firstly, data on antimicrobial peptides is extracted from UniProt, manually curated and stored into a centralized database called dragon antimicrobial peptide database (DAMPD). Secondly, based on the curated data, models to predict antimicrobial peptides are created using support vector machine with optimized hyperparameters. In particular, global optimization methods such as grid search, pattern search and derivative-free methods are utilised to optimize the SVM hyperparameters. These models are useful in characterizing unknown antimicrobial peptides. Finally, a webserver is created that will be used to predict antimicrobial peptides in haemotophagous insects such as Glossina morsitan and Anopheles gambiae. / South Africa

Dans le cas d'une infection chronique par le virus de l'hépatite B (HBV), les traitements actuels (IFN et analogues de nucléos(t)ides) ne permettent pas d'éradiquer l'infection du fait de la persistance de l'ADNccc et des phénomènes de résistance observés chez les patients. S'agissant des traitements par l'IFN, 70 % des patients porteurs chroniques sont non-répondeurs. En effet, le virus HBV aurait développé des stratégies immunosuppressives pour établir une infection persistante. La compréhension des mécanismes impliqués dans cette viro-immunosuppression devient ainsi un enjeu majeur dans la mise en place de nouvelles stratégies antivirales. Les objectifs de ma thèse ont consisté en l'étude des relations précoces entre HBV et les hépatocytes. Nous avons pu mettre en évidence que cette absence d'activation du système immunitaire était le résultat, non pas d'une « invisibilité » du virus, mais d'une inhibition active des réponses IFN de type I/III et proinflammatoires, précocement établie par le virus HBV pour établir une infection persistante. De façon intéressante, nous avons pu démontrer que la protéine HBc était capable d'inhiber spécifiquement l'activation des voies IFN via son interaction avec les promoteurs des gènes de l'immunité innée et l'installation de marques épigénétiques (H3K9me2/3) répressives sur ces gènes par recrutement d'histone méthyl-transférases. Ces résultats prônent l'utilisation de stratégies antivirales utilisant des anticapsides dégradant et/ou prévenant la localisation nucléaire de la protéine HBc, restaurant ainsi le potentiel immunitaire des hépatocytes, pouvant dès lors être exacerbé par des agonistes de PRRs / Current treatments against Hepatitis B virus (HBV) chronic infection (IFNs and nucleos(t)ide analogues) are inefficient due to the persistence of cccDNA and emergence of viral resistance observed in infected patients. So far, up to 70 % of these patients are non responders to IFN treatments and it seems that the virus itself is able to counteract actively the host innate immune responses to establish a persistent infection. Therefore, the understanding of molecular mechanisms involved in this immunosuppression is crucial to design new immunotherapeutic strategies. In this context, the aim of my thesis was to investigate the early interactions between HBV and the hepatocyte antiviral responses. We have determined that HBV is not only a weak inducer of the host immune response, but is also able to inhibit very early and actively type I/III IFNs and proinflammatory pathways to persist in the hepatocytes. Furthermore, we have identified HBc protein as the major determinant involved specifically in the inhibition of IFN responses by counteracting host innate immune gene activations leading to repressive epigenetic marks such as H3K9/K27me3, or the recruitment of histone methyl transferase enzymes to the host IFN gene promoters. These results highlight new immunotherapeutic strategies and proposed the use of anticapsids components to degrade or block the nuclear localization of HBc proteins in order to restore a potent immune response in the hepatocytes. These anticapsid treatments may be also combined to PRRs agonists in order to improve the host antiviral state and HBV replication control

Les cellules dendritiques classiques (cDC) jouent un rôle crucial dans l’efficacité des réponses immunitaires précoces conduisant au contrôle ou à la persistance virale. A cet égard, il a été montré que l’infection par le VIH induit des dysfonctions des cDC caractérisées par une inhibition de leur capacité à stimuler les cellules T et associées à la progression de la maladie. Parmi les mécanismes moléculaires impliqués dans ces dysfonctions, des études in vitro ont mis en évidence le rôle du récepteur inhibiteur LILRB2. Néanmoins, la dynamique d’expression de LILRB2 ainsi que son rôle dès les premiers stades de l'infection restent à démontrer. Chez des patients en primo-infection VIH-1, nous observons une augmentation de l’expression de LILRB2 et de ses ligands HLA-I à la surface des cDC. Par ailleurs, la cinétique d’expression de LILRB2 et CMH-I au cours de l’infection de macaques cynomolgus par le SIVmac251 montre une augmentation transitoire de l'expression de LILRB2 et du CMH-I sur les cDC du sang et des ganglions lymphatiques dès les premiers jours de l’infection. Parmi les mécanismes qui pourraient être impliqués dans la régulation de l’expression de LILRB2, nos résultats indiquent que la réplication du VIH-1, l'activation de voies TLR7/8 ainsi que la présence d’IL-10 et d’IFN-I induisent une forte expression de LILRB2. Enfin, cette expression exacerbée de LILRB2 sur les cDC semble être spécifique à l'infection par le VIH/SIV. En effet, l’infection de macaques cynomolgus par le virus chikungunya, qui est caractérisée par une réponse immunitaire antivirale robuste aboutissant à un contrôle de la virémie, est associée à une expression diminuée de LILRB2 sur les cDC dès les premiers jours de l’infection. L’ensemble de nos données suggèrent un rôle majeur de l’axe inhibiteur LILRB2/MHC-I dans les mécanismes de dérégulations des cDC qui pourrait participerait à l’inefficacité des réponses immunitaires adaptatives et à la persistance du VIH/SIV. / Conventional dendritic cells (cDCs) play a crucial role in setting up early immune responses leading to viral control or persistence. In this regard, it has been shown that HIV-1 infection induces cDC dysfunctions characterized by inhibitions in their ability to stimulate T-cells and associated with disease progression. In vitro studies have shown the implication of LILRB2 inhibitory receptor in cDC dysfunctions. However, the dynamic of LILRB2 expression and its role in the early stages of infection are yet to be characterized. In primary HIV-1 infected patients, we observe an increased expression of LILRB2 and its ligands, HLA-I, on the surface of cDCs. Kinetics of LILRB2 and MHC-I expressions during SIV infection of Cynomolgus macaques shows a transient increase in LILRB2 and MHC-I expressions on blood and lymph node cDCs during the first days of infection. We also show that HIV replication, activation of TLR7/8 pathways, and presence of IL-10 and IFN-I drive upregulated expression of LILRB2. Finally, this strong induced LILIRB2 expression seems specific to HIV/SIV infections. Indeed, chikungunya virus infection of cynomolgus macaques, which characterized by a robust antiviral immune response leading to viral control, is associated with decreased expression of LILRB2 on cDCs in the first days of infection. Taken together, our data suggest a major role of the LILRB2/HLA-I inhibitory axis, mediating cDC dysfunctions and thus contributing to inefficient adaptive immune responses and viral persistence.

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Enveloped RNA viruses like human immunodeficiency virus type-1 (HIV-1) and SARS-CoV-2 enter host cells through fusion with the plasma membrane, a process facilitated by specific viral envelope proteins that recognize and bind to receptors expressed on the host cell surface. These receptors can diverge based on the type of cell and virus. For HIV-1, the primary receptors on myeloid cells are CD4 and CCR5 or CXCR4. For SARS-CoV-2, although the primary receptor is ACE2, other myeloid-cell specific sialic acid binding lectins can also facilitate entry. Following cellular invasion, different viral RNA species can be detected by distinct host nucleic acid sensors, resulting in type I interferons and pro-inflammatory cytokine induction. While these innate immune responses are essential for controlling viral infections, overactivation can lead to chronic inflammation, tissue damage, and disease pathogenesis. Herein, I examine the contribution of HIV-1 and SARS-CoV-2 de-novo RNA expression and the molecular mechanisms that contribute to innate immune activation in myeloid cells. Despite advancements in combination antiretroviral therapy (ART) in suppressing systemic viral replication in individuals infected with HIV, residual viral RNA expression in tissue reservoirs remains a significant hindrance to curative efforts. I hypothesized that persistent expression of viral RNAs in myeloid cells triggers dysregulated innate immune activation, and inflammasomes activation. This study centers on the long-lived tissue-resident innate immune cells - macrophages and microglia, which, owing to their self-renewing nature, operate as reservoirs of viral RNA production, and are thought to lead to chronic immune activation even in the absence of productive replication. Our previous studies suggest that de novo expression of unspliced intron-containing HIV-1 RNA (herein referred to as icRNA) triggers activation of pro-inflammatory cytokines in myeloid cells. Here, I demonstrate that cytosolic expression of HIV-1 icRNA, but not multiply-spliced viral RNAs induces inflammasome activation, LDH release and IL-1β secretion in productively infected monocyte-derived macrophages (MDM) and induced pluripotent stem cell (iPSC)-derived microglia. Interestingly, knockdown of RLRs, RIG-I and MDA5 or endosomal TLRs failed to abrogate HIV-1 icRNA-induced IL-1β secretion. Rather, knockdown of NLRP1, but not NLRP3, inflammasome resulted in a significant reduction in IL-1β secretion, underscoring NLRP1's pivotal role in the HIV-1 icRNA-induced IL-1β secretion. Furthermore, Rev-Crm1-dependent nucleocytoplasmic export of HIV-1 icRNA was required for NLRP1-mediated Caspase-1 activation, IL-1β secretion, LDH release and cell death. Similarly, SARS-CoV-2, while not establishing productive infection in macrophages, can activate these cells, contributing to a hyper-inflammatory response marked by the heightened expression of pro-inflammatory cytokines, which is understood to be a principal driver of COVID-19 pathology. SARS-CoV-2 established an abortive infection in macrophages. CD169, a macrophage-specific sialic-acid binding lectin, mediated ACE2-independent SARS-CoV-2 entry in human macrophages and establishment of restricted infection. Interestingly, CD169-mediated SARS-CoV-2 entry in macrophages led to the expression of viral genomic and subgenomic RNAs, with negligible viral protein expression and no release of infectious virus particles, implying a post-entry restriction to SARS-CoV-2 replication in macrophages that was curbed by exogenous ACE2 expression. Despite restricted viral RNA expression, cytoplasmic RLRs, RIG-I and MDA5, sensed abortive viral transcripts, and induced pro-inflammatory responses in a MAVS dependent manner. This dissertation reveals striking parallels between the role of viral RNAs in driving pro-inflammatory responses in HIV-1 and SARS-CoV-2 infections. These findings collectively underscore the central role of cytoplasmic sensing of viral RNAs and their contribution to chronic inflammation in virus-infected myeloid cells. Elucidating these molecular mechanisms further may pave the way for novel therapeutic interventions to mitigate the persistent innate immune activation and immunopathology detected in HIV-1 and SARS-CoV-2 infected individuals.

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Foot-and-mouth disease virus (FMDV) is a highly infectious virus affecting cloven-hoofed animals. The most prominent of its clinical signs is the development of vesicular lesions on the feet and in or around the mouth, which are a consequence of extensive FMDV-induced epithelial cell death. Currently, there is no certain biological knowledge on why extensive epithelial cell death occurs in some FMDV-infected tissues, but not in others. Using the epithelial tissues of tongue and dorsal soft palate as examples of a tissue where lesions occur and one that does not visibly exhibit FMDV-induced cell death, this work aims to identify the potential drivers of epithelial cell death and survival. A partial differential equation (PDE) model informed by experimental data on epithelial structure, is used to test epithelium thickness and cell layer structure as potential determinants. A second PDE model investigates FMDV-interferon (IFN) dynamics and their impact on the levels of cell death and survival, while an experimental study is undertaken to provide data for model validation. The work carried out casts light on the important role of a variety of factors including FMDV replication, IFN production and release, and IFN antiviral action.

TRBP (TAR RNA Binding Protein), est un facteur activateur de la réplication du Virus de l'Immunodéficience Humaine de type 1 (VIH-1). Cette protéine cellulaire qui interagit avec les ARN double brins est connue pour son rôle crucial dans la voie des miRNA. Isolée pour sa capacité à interagir avec la séquence leader TAR présente à l'extrémité 5' de tous les ARN du VIH-1, TRBP favorise la réplication du VIH-1 au niveau post-transcriptionnel, en partie via l'inhibition de la PKR (Protéine Kinase ARN dépendante).Dans le but de mieux comprendre les mécanismes moléculaires par lesquels TRBP facilite la réplication du VIH-1, le complexe protéique associé à TRBP a été purifié par immunoprécipitation par double affinité et identifié par spectrométrie de masse. En plus des facteurs déjà connus, un nouveau partenaire à activité ARN 2'-O-méthyltransférase (2'-OMTase) potentielle a été copurifié : la protéine FTSJ3. Chez les eucaryotes supérieurs, deux 2'-OMTases permettent la méthylation des ARNm cellulaires au niveau de la position ribose 2'-O- du premier (coiffe 1) et du deuxième nucléotide (coiffe 2). Cette coiffe 1/2 est une signature moléculaire permettant de discriminer les ARNm endogènes et exogènes. Dans la cellule, MDA5, un senseur cytoplasmique, reconnait les ARN exogènes non coiffés et déclenche la production d'interférons (IFNs) de type I pour établir un état antiviral. Pour échapper à la réponse immune innée, certains virus ont développé des mécanismes leur permettant de mimer une coiffe 1/2.Le VIH ne code pas pour une activité 2'-OMTase. Cependant FTSJ3, de par son interaction avec TRBP, se retrouve à proximité de l'extrémité 5' de l'ARN viral. Cette 2'-OMTase méthyle l'ARN TAR in vitro, qui, transfecté dans les cellules monocytaires humaines U937 n'induit plus la production d'IFNs de type I. A l'inverse, le virus VIH-1 produit en l'absence de FTSJ3 déclenche une induction de l'expression des IFNs de type I dépendante de MDA5 dans les cellules U937. L'expression de ce virus est atténuée suite à un défaut d'import nucléaire. Ainsi, ces travaux montrent que la protéine FTSJ3, recrutée au niveau de l'extrémité 5' de l'ARN du VIH-1 par TRBP, facilite la réplication du VIH-1 en assurant la synthèse d'une coiffe 1/2 qui permet au VIH-1 d'échapper à la reconnaissance par le senseur MDA5 et à l'induction des IFNs de type I. Cette étude met en évidence un nouveau mécanisme permettant au VIH-1 d'échapper à la détection par le système d'immunité innée cellulaire. / TRBP (TAR RNA Binding Protein) is a cellular RNA binding protein that facilitates the replication of Human Immunodeficiency Virus type 1 (HIV-1). Isolated for its ability to bind HIV-1 TAR sequence present at the 5' end of all HIV-1 RNA, TRBP promotes HIV-1 replication at a post-transcriptional level by counteracting the antiviral activity of the protein kinase R (PKR).To gain more insight on how TRBP enhances HIV-1 replication, TRBP associated factors were purified using tandem immunoaffinity purification and identified by mass spectrometry. In addition to already known associated factors, a new protein with a putative RNA 2'-O-methyltransferase activity (2'OMTases) was copurified: FTSJ3. In higher eukaryotes, cellular mRNA are methylated on 2'-O ribose position on the first (Cap 1) and second nucleotide (Cap 2). This capping provides a molecular signature for the discrimination of endogenous versus exogenous mRNA. In the cell, MDA5, a cytoplasmic sensor, recognizes exogenous uncapped RNA and activate type I interferons (IFNs) production to establish an antiviral state. To evade innate immune response, some viruses have evolved mechanisms to mimics cap 1/2.HIV-1 does not encode a 2'O-MTase activity. However, owing to its interaction with TRBP, FTSJ3 is recruited at the 5' end of the viral genome and methylates TAR RNA in vitro. When capped by FTSJ3, TAR does not induce type I IFNs anymore when transfected in monocytic cell line U937. Conversely, HIV-1 viruses produced in FTSJ3 knock-down cells triggers type I IFNs expression through MDA5 sensing. This virus is attenuated, expressed in low amounts because of a block at the level of HIV-1 nuclear import. This study shows that FTSJ3 is recruited to HIV-1 5' end TAR sequence by TRBP and facilitates HIV-1 replication. HIV-1 RNA capping allows HIV-1 escape from MDA5 sensing and type I IFN induction. This study highlights a new way of HIV-1 escape from innate immune system.

Toll-like receptors (TLRs) are a major group of pattern recognition receptors expressed on the surface of immune cells that recognize molecular patterns associated with all classes of pathogenic microorganisms. TLR4 recognizes the lipopolysaccharide component of Gram-negative bacterial cell walls and is the only TLR known to induce signaling through both the MyD88 and TRIF pathways. Basigin, a ubiquitous cell adhesion molecule, is a member of the immunoglobulin superfamily that has the ability to influence cell signaling mediated by the MyD88 and TRIF pathways, the same signaling pathways induced by the TLR4 receptor protein. Analysis of the Basigin protein sequence indicates the presence of a hydrophilic glutamate residue within the hydrophobic transmembrane domain, but no consensus binding sites for MyD88 or TRIF. The purpose of this study was to determine if Basigin uses TLR4 for signal transduction. It is hypothesized that Basigin interacts with TLR4 and that the glutamate residue plays a role in the interaction. Enzyme-linked immunosorbent binding assays were performed using endogenous TLR4 and recombinant Basigin proteins. These analyses demonstrated that binding of Basigin to TLR4 was significantly greater than that of the control protein and that the glutamate residue in the Basigin transmembrane domain does play a role in the interaction between Basigin and TLR4 as well as many hydrophobic residues in the Basigin transmembrane domain. The data suggest that Basigin interacts with TLR4 to influence signaling cascades using MyD88 and TRIF.