Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Les traitements actuels dirigés contre le VIH ne sont que partiellement efficaces en raison de l'apparition de mutations qui confèrent au virus une grande capacité de résistance aux antirétroviraux existants. Un moyen d'améliorer la lutte contre le virus consiste par conséquent à trouver de nouvelles stratégies d'inhibition. Le complexe de reverse transcription est une des principales cibles pour le développement de traitement anti-SIDA, il catalyse une étape obligatoire du cycle de réplication du virus. Cependant, l'ensemble des inhibiteurs de la transcriptase inverse sont limités par l'apparition rapide de souches résistances. Dans ce contexte, mes travaux de thèse ont permis de développer des inhibiteurs ciblant spécifiquement la reverse transcriptase (RT) du VIH-1, basée sur des fragments d'anticorps dérivés des anticorps chaînes lourdes de dromadaire appelés VHHs ou encore Nanobodies. Associé à une stratégie de vectorisation non invasive basée sur l'utilisation de peptides vecteurs pénétrants, les Nanobodies ont été délivré efficacement dans les cellules et par conséquent ils présentent tous une forte activité antivirale de l'ordre du nanomolaire. L'étude du mécanisme d'action du Nanobody leader NbRT20 montre qu'il agit en tant qu'inhibiteur conformationnel. Il interagit avec la forme intermédiaire inactive de la RT et empêche la mobilité du sous-domaine thumb requis pour le positionnement correct de la matrice/amorce sur la RT et inhibant l'incorporation des nucléotides dans la chaîne d'ADN naissante déstabilisant l'enzyme dans une conformation inactive, non processive. Pris ensemble, ces résultats montrent que la plate-forme Nanobody peut être très efficace pour générer des intracorps extrêmement puissants et sélectifs pour neutraliser la RT et la réplication virale.Mots clés : HIV-1, RT, Nanobodies, peptide vecteur pénétrant / The rapid emergence of drug-resistant viruses against all approved HIV clinical drugs together with inaccessible latent virus reservoirs and side effects of currently used compounds have limited the potency of existing anti-HIV-1 therapeutics. Therefore, there is a critical need for new safer drugs, active against resistant viral strains. Reverse transcriptase (RT) plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1) and remains a primary target of anti-HIV-1 drugs. To develop specific HIV inhibitors, we have elaborated a new strategy based on short antibody fragments derived from the unique Heavy-chain antibodies present in Camelidae called Nanobodies that targets RT-activation. The immunization of dromedaries with RT has lead to the isolation of a panel of Nanobodies that tightly bind the two subunits of RT and inhibit its DNA-dependent DNA polymerase activity at nanomolar range. From that screen we have elaborated an intrabody (cell penetrating anti-RT Nanobody) NbRT20 that constitutes a potential interesting anti-HIV compound.We demonstrated that NbRT20 inhibits RT polymerase activity and exhibiting a potent antiviral activity with a subnanomolar IC50. NbRT20 binds the thumb subdomain and restricts its flexibility and mobility resulting in an inactive/non processive dimeric conformation of the enzyme. From a mechanistic point of view, we have showed that NbRT20 is a conformational inhibitor. it prevents proper binding of primer/template and of dNTP and destabilizes the enzyme in an inactive/non processive dimeric conformation.Taken together, these results demonstrated that, the Nanobody platform may be highly effective at generating extremely potent and selective intrabody to neutralize RT and HIV proliferation.Key words: HIV-1, RT, Nanobodies, cell penetrating peptide

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Les récepteurs à la vasopressine V1bR et à la sérotonine 5HT3R jouent des rôles physiologiques importants dans la détection des signaux extracellulaires, les mécanismes de transmission nerveuse et diverses pathologies dont le cancer, le diabète et des maladies des SNC et SNP. Mes études avaient pour but de générer ou trouver des modulateurs peptidiques de ces deux récepteurs. Pour le V1bR, j’ai développé plusieurs anticorps de type VHH et les ai caractérisés aux plans biochimique et fonctionnel. L’un de ces VHHs agit comme un agoniste allostérique complet et spécifique du V1bR humain (hV1bR). In vitro ce VHH est capable d’activer les voies de signalisation de l’inositol phosphate et des MAP kinases et d’induire l'internalisation du hV1bR. Dans des îlots pancréatiques surexprimant le hV1bR, il induit une augmentation du Ca2+ intracellulaire et une sécrétion d'insuline. Pour le 5HT3R, j’ai criblé par SPR 31 venins de serpents sur des récepteurs recombinants immobilisés et mis en évidence une interaction à partir d’un de ces venins. Suite à purification par chromatographie liquide et identification par spectrométrie de masse, j’ai identifié une toxine préalablement caractérisée comme une enzyme à activité Ca2+-dépendante. Cette toxine interagit avec les 5HT3R A et AB indépendamment du Ca2+ et avec des valeurs de Kd ≤ 10 nM. L’analyse fonctionnelle par électrophysiologie suggère qu’elle agit comme un PAM de l’activité canal du 5HT3R. Des images de ME en coloration négative montrent la toxine fixée sur le domaine extracellulaire du 5HT3R, à distance du site pour la 5HT. Le VHH et la toxine pourraient être utilisés comme outils pharmacologiques et/ou agents thérapeutiques. / The vasopressin V1bR and serotonin 5HT3R receptors play important physiological roles in the detection of extracellular signals, in the mechanisms for neuronal transmission, and in various pathologies including cancer, diabetes, and CNS and PNS diseases. My studies were aimed at generating or finding peptidic modulators of these two receptors. For the V1bR, I developed several antibodies of the VHH type and characterized them biochemically and functionally. One of these VHHs acts as a complete allosteric agonist specific for the human V1bR (hV1bR). In vitro this VHH is able to activate the signaling pathways of inositol phosphate and MAP kinases and to induce the internalization of hV1bR. In pancreatic islets overexpressing hV1bR, it induces an increase in intracellular Ca2+ and a secretion of insulin. For the 5HT3R, using SPR I screened 31 snake venoms on immobilized recombinant receptors and for one of these venoms, evidenced an interaction. Following purification by liquid chromatography and identification by mass spectrometry, I identified a toxin previously characterized as an enzyme with Ca2+-dependent activity. This toxin interacts with the 5HT3R A and AB independently of Ca2+ and with Kd values ≤ 10 nM. Functional analysis by electrophysiology suggests that it acts as a PAM of the 5HT3R channel activity. Images recorded by negative staining EM show that the toxin binds to the 5HT3R extracellular domain, at a distance from the 5HT binding site. Both this VHH and this toxin could be used as pharmacological tools and / or therapeutic agents.