Comparison of HIV-1 specific T cell immunity in the female genital tract and blood of HIV-infected women : impact of in vitro T cell expansion on HIV-specific T cell specificity, maturational status and functional complexity

Includes bibliographical references (leaves 161-184).This study shows that HIV-specific cervical T cells can be isolated by cytobrushing and in vitro polyclonal expansion is a useful approach to increase the number of T cells available from mucosal sites. Dynal beads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the best yields of cervical T cells while anti-CD3 in the presence of IL-2 best conserved the ex vivo T cell profile. Expanded T cell lines, irrespective of expansion method used, generally maintain their cytokine response profile to HIV anti- gens. This study shows that HIV Gag-specific blood and cervical T cells were largely mono-functional with polyfunctional T cells being detected in women with high blood CD4 count and low plasma viral load. This study confirms that HIV-specific Gag T cell responses detected in the polyclonal expanded female genital tract T cells are associated with those measured in blood during HIV infection

HIV Reverse Transcriptase Fidelity And Inhibition Are Modulated By Divalent Cations In A Concentration-Dependent Manner In Vitro

Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions

Identification des réservoirs viraux chez le macaque rhésus suite à un traitement antirétroviral précoce

Un des problèmes majeurs au cours de l'infection par le virus de l'immunodéficience humaine (VIH) est la persistance virale. En effet, bien qu'un traitement antirétroviral (ART) supprime la réplication virale, un rebond viral survient lors de l'arrêt de la ART, indiquant une dissémination virale précoce et l'absence d'une éradication complète du virus. Par conséquent, l'identification de la nature des cellules infectées et des tissus qui contribuent au rebond viral est cruciale afin de guérir de l'infection par le VIH. Dans le but d'identifier les cibles précoces du virus, celles qui contribuent à la persistance virale sous ART et au rebond viral à l'arrêt du traitement, nous avons utilisé un modèle d'infection expérimental du macaque rhésus (RM) par le SIVmac251. L'utilisation de ce modèle animal nous a permis d'analyser des organes difficilement accessibles chez l'homme. Ainsi, nous avons analysé ces réservoirs durant la primo-infection, sous ART précoce (initiée à 4 jours postinfection) et après l'interruption du traitement (ATi). À partir de ces animaux, différents tissus ont été étudiés comme la rate, les ganglions mésentériques, les ganglions axillaires/inguinaux, et l'intestin. Nous avons trié les sous-populations de cellules cibles du virus à savoir les cellules CD4 et les monocytes/macrophages par cytométrie en flux. Nous avons quantifié la charge virale plasmatique, ainsi que l'ADN viral, les transcrits d'ADN R-U5, et l'ARN viral associés aux cellules par qRT-PCR. Mes travaux ont permis de démontrer qu'en l'absence de ART, les sous-populations de cellules lymphocytaires CD4 et monocytaires contiennent des transcrits R-U5, de l'ADN et de l'ARN viral. De plus, ces cellules infectées sont aptes à produire du virus infectieux après stimulation. Nos résultats ont également démontré que les cellules Tfh et TEM sont les principales cibles du SIV. Par ailleurs, les cellules Tfh produisent davantage de virus après activation que les autres cellules. Au niveau tissulaire mes travaux ont montré que dans la rate la réplication virale s'effectue très tôt après infection comparativement à d'autres tissus comme les ganglions périphériques ou mésentériques. Nous avons détecté de l'ADN et de l'ARN viral dans ce compartiment dès le jour 7. Nous avons montré que la ART précoce, administrée au jour 4 post-infection, réduit considérablement l'inflammation, empêche efficacement la dissémination virale dans les monocytes, en revanche le virus persiste dans les lymphocytes T CD4 au niveau des ganglions mésentériques et de la rate, en particulier dans les cellules Tfh et TEM. L'interruption de la ART est associée à un rebond viral en moins de deux semaines, conduisant à la dissémination virale provenant fort probablement des cellules Tfh et TEM des tissus lymphoïdes viscéraux et ciblant à la fois les monocytes et les sous-populations de lymphocytes T. Ainsi, mes travaux ont permis de faire progresser la compréhension sur la dissémination virale précoce pour laquelle les tissus lymphoïdes viscéraux sont cruciaux dans le maintien des sanctuaires au sein desquels les cellules Tfh et TEM sont les principales cellules réservoirs. Afin de guérir de l'infection par le VIH, ces sanctuaires viraux devraient être ciblés par les nouvelles stratégies thérapeutiques.One of the major problems during HIV infection is viral persistence. Although early antiretroviral therapy (ART) suppresses viral replication, ART discontinuation results in viral rebound, indicating early viral seeding and absence of eradication. Therefore, identification of the infected cells and tissues that contribute to viral rebound are crucial for HIV cure. In order to identify the early targets of the virus, those that contribute to viral persistence on ART and to viral rebound upon discontinuation of treatment, we used an experimental model of SIVmac251 infected rhesus macaques (RMs). This allowed us to analyze organs that are difficult to access in humans. We analyzed these reservoirs during the primary infection, under early ART (day 4 post infection) and after ART interruption (ATi). From these animals, different tissues were investigated such as spleen, mesenteric LNs, axillary/inguinal LNs, and intestine (colon, ileum, and jejunum parts). We sorted the target cell subsets of the virus including CD4 cells and monocytes by flow cytometry. We quantified plasma viral load, as well as cell-associated viral DNA, R-U5 transcripts, and viral RNA by RT-PCR. My results show that, in the absence of ART, CD4 T and monocyte cell subsets harbor viral R-U5 transcripts, DNA and RNA. Furthermore, these infected cells are able to produce infectious virus after stimulation. Our results also demonstrated that TEM and Tfh cells are the main SIV target cells. Tfh cells produce more viruses than other cells after activation. Compared to mesenteric LNs and peripheral LNs, viral replication occurs rapidly in the spleen. Viral DNA and RNA are detected in this organ since day 7. We provided evidence that early ART, administered at day 4 post-infection, drastically reduced inflammation, efficiently prevents viral dissemination in monocytes, but the virus persisted in CD4 T cells in the mesenteric LNs and the spleen, particularly in Tfh and TEM cells. ART interruption led to viral rebound in less than 2 weeks, leading to viral dissemination most likely from Tfh and TEM cells in visceral lymphoid tissues and targeting both monocytes and T cell subsets. Thus, my work contributed to the understanding of early viral dissemination for which visceral lymphoid tissues are crucial in maintaining sanctuaries in which TEM and Tfh cells are the main viral reservoirs. In order to cure HIV infection, these viral sanctuaries should be considered when evaluating new treatment strategies