Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Therapeutically restricted sites present a formidable barrier in medicine. Herein, chemical strategies to overcome two restricted sites, HIV reservoirs and intracellular bacteria, will be discussed. First, cellular and anatomical HIV reservoirs, such as those in the brain, limit HIV eradication using currently known therapeutic regimes. HIV therapies are unable to localize in the brain, in part, due to high expression of efflux transporters, such as P-glycoprotein (P-gp), at the BBB, because many of these therapies are P-gp substrates. In an effort to overcome therapeutically restricted HIV sanctuaries, a dimerized combination HIV therapy was designed to act two-fold. First, the dimeracts as a P-gp inhibitor allowing therapeutic access to restricted sites. Second, the dimeractsas a prodrug, which once in the reducing environment of the cell, may release monomeric HIV therapies. The dual conjugate, Abacavir-S2-Darunavir, was shown to potently inhibit P-gp across two separate cell lines, was able to regenerate the component monomers in a reducing environment and contained modest anti-HIV activity.<div><br><div>Further, mammalian cells create sanctuary sites for bacteria to grow and proliferate, because many common antibiotic therapies are unable to cross the mammalian cell membrane. Therefore, these pathogens are able to proliferate without therapeutic constraint. Here, a chemical strategy was developed to deliver a dual antibiotic therapy inside mammalian cells in an effort to clear these intracellular pathogens. First, a new synthetic strategy was developed for facile synthesis of dual conjugates, composedof an aminoglycoside and a cell penetrating peptide (CPP) linked with a reversible disulfide tether, using kanamycin and the known CPP Arg8as a model system. Next, this synthetic methodology was expanded for use with theaminoglycoside tobramycin and theknown broad-spectrum antibiotic and cell penetrating peptide, P14LRR, once again linked via the reversible disulfide tether (TobP14). Two distinct isomers of TobP14 were synthesized, isolated, and fully characterized by 2D NMR. The TobP14 isomers were shown to be an effective antibiotic across various Gram positive and negative pathogens such as MRSA, S. epidermidis, P. aeruginosa, and A. baumannii. Further, the isomers effectively releasedthe monomeric therapies (tobramycin and P14-SH) in a reducing environment and werenontoxic to mammalian cells up to 16 μM. Finally, the dual conjugate isomers significantly reduce two different strains of intracellular A. baumanniiwithin macrophages.<br><div><br></div><div><br></div></div></div>

Malgré les améliorations significatives apportées par la thérapie antirétrovirale à la durée et à la qualité de vie des personnes vivant avec le VIH, elle ne permet pas de complètement éliminer le virus de l’organisme. La persistance du virus est due à l’existence de réservoirs viraux, des cellules infectées de manière latente par le VIH. Ces réservoirs nécessitent un traitement antirétroviral à vie, car le virus réapparait en cas d’interruption du traitement, signifiant que l’immunité des cellules T spécifiques du VIH n’est pas restaurée. Bien que cela soit théoriquement possible, seule une fraction de personne vivant avec le VIH, appelée Contrôleurs Élites, parvient à contrôler le virus en absence de traitement. Pour la majorité des individus, l’infection par le VIH entraîne une évasion virologique ainsi qu’un épuisement et une altération des réponses cellulaires spécifiques au VIH. À ce jour, les stratégies thérapeutiques visant à éliminer les réservoirs viraux ont échoué, en partie en raison de la présence de provirus principalement défectifs dans ces réservoirs. Dans cette thèse, nous avons identifié et caractérisé les provirus défectifs latents du VIH pouvant être transcrits et/ou traduits, ainsi que la relation entre ces réservoirs et les réponses immunitaires spécifique du virus. Dans un premier temps, nous avons montré que bien que défectifs et potentiellement incapable de donner lieu à la réplication virale, ces provirus peuvent être transcrits et traduits soit par réactivation à l’aide d’agents de réversion de la latence, soit de manière spontanée. Ces réservoirs donnent lieu à plusieurs populations de réservoirs, en fonction de la présence ou de l’absence certains gènes viraux. Nous avons déterminé que ces différentes populations sont régies par le profil génomique des cellules infectées. Les provirus identifiés étaient très rarement intacts, mais l’intégrité du génome était associée à la processivité de la transcription et de la traduction. Dans un second objectif, nous avons caractérisé les réponses T CD4+ et CD8+ spécifiques du VIH avant et après le début du traitement antirétroviral. Nous avons observé que les réponses T CD4+ spécifiques étaient comparables pendant l’infection chronique et après le traitement. En revanche, les réponses T CD8+ diminuaient considérablement après l’initiation de la thérapie antirétrovirale. Nous avons également constaté que la taille du réservoir traductionnellement actif pendant le traitement antirétroviral était négativement associée aux réponses T CD8+ spécifiques avant le début de la thérapie, tandis que le réservoir incapable de traduire les protéines du VIH subsistait. Ces observations mettent en évidence le rôle des cellules T CD8+ dans le contrôle de l’infection par le VIH, comme nous l’avons observé chez les Contrôleurs Élites. Nos travaux contribuent à une meilleure compréhension des réservoirs viraux du VIH, qui pourraient potentiellement être impliqués dans l’inflammation chronique et la dysfonction immunitaire associé à la pathogénèse du VIH. / Despite the significant improvement brought by antiretroviral therapy in the duration and quality of life for people living with HIV, it does not completely eliminate the virus from the body. The persistence of the virus is due to the existence of viral reservoirs, which are cells latently infected with HIV. These reservoirs require lifelong antiretroviral treatment because of the viral rebound reoccurring in case of treatment interruption. This suggests that HIV-specific T cell immunity is not restored. Although theoretically possible, only a fraction of people living with HIV, known as Elite Controllers, are able to control the virus in the absence of treatment. For the majority of individuals, HIV infection leads to virologic escape, as well as exhaustion and altered cellular responses to HIV. To date, therapeutic strategies aimed at eliminating viral reservoirs have failed, partly due to the presence of predominantly defective proviruses in these reservoirs. In this thesis, we have identified and characterized latent defective proviruses of HIV that can be transcribed and/or translated. We also have characterized the relationship between these reservoirs and the specific immune responses to the virus. Firstly, we have shown that although defective and potentially replication-incompetent, these proviruses can be transcribed and translated either through reactivation using latency reversal agents or spontaneously. These reservoirs give rise to several populations of reservoirs, depending on the presence or absence of certain viral genes. We have determined that these different populations are governed by the genomic profile of infected cells. The identified proviruses were rarely intact, and genome integrity was associated with the processivity of transcription and translation. Then, we characterized the specific CD4+ and CD8+ T cell responses to HIV before and after the initiation of antiretroviral treatment. We observed that specific CD4+ T cell responses were comparable during chronic infection and after treatment. However, CD8+ T cell responses decreased significantly after the initiation of antiretroviral therapy. We also found that the size of the translationally active reservoir during antiretroviral treatment was negatively associated with the specific CD8+ T cell responses prior to treatment initiation, while the translation-incompetent cells persisted. These observations highlight the role of CD8+ T cells in the control of HIV infection, as observed in Elite Controllers. Our work contributes to a better understanding of HIV viral reservoirs, which could potentially be involved in chronic inflammation and immune dysfunction associated with HIV pathogenesis.