scholarly journals Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary Cell Model of HIV Latency but not from Ex Vivo Reservoirs

2021 ◽  
Author(s):  
Yanqin Ren ◽  
Szu Han Huang ◽  
Amanda B. Macedo ◽  
Adam R. Ward ◽  
Winiffer D. Conce Alberto ◽  
...  
Keyword(s):  
T Cells ◽  
Antiretroviral Therapy ◽  
Cell Model ◽  
Ex Vivo ◽  
Family Members ◽  
Primary Cell ◽  
Bryostatin 1 ◽  
Selective Toxicity ◽  
Hiv Dna ◽  
Infected Cells

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T-cells, indicating increased tension between pro-apoptotic and pro-survival family members – and suggesting that inhibition of pro-survival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T-cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T-cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination PMA + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T-cells from ART-suppressed donors. Our results add to growing evidence that bonafide reservoir-harboring cells are resistant to multiple “kick and kill” modalities - relative to latency models. We also interpret our results as encouraging of further exploration of BCL-XL antagonists for cure, where combination approaches – including with immune effectors – may unlock the ability to eliminate ex vivo reservoirs. Importance Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in ‘reservoirs’ of infected cells that re-initiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a pro-survival factor – BCL-XL – that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model, but were insufficient to reduce ‘natural’ reservoirs in ex vivo CD4+ T-cells – adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

scholarly journals Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary Cell Model of HIV Latency but not from Ex Vivo Reservoirs

2020 ◽  
Author(s):  
Yanqin Ren ◽  
Szu Han Huang ◽  
Amanda B. Macedo ◽  
Adam R. Ward ◽  
Winiffer D. Conce Alberto ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Model ◽  
Ex Vivo ◽  
Family Members ◽  
Primary Cell ◽  
Bryostatin 1 ◽  
Viral Reservoirs ◽  
Hiv Persistence ◽  
Hiv Dna ◽  
Infected Cells

AbstractHIV persists, despite antiviral immune responses and effective antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T-cells, indicating increased tension between pro-apoptotic and pro-survival family members – as well as raising the possibility that the inhibition of pro-survival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to further study BCL2L1 (encoding the protein BCL-XL), due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T-cells with either a clinical isolate, a CCR5-tropic strain, or a CXCR4-tropic strain of HIV resulted in increased BCL-XL protein expression; and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to disproportionate cell death compared to uninfected CD4+ T-cells. In a primary cell model of latency, both BCL-XL antagonists drove significant reductions in total HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1, or in combination with the highly potent latency reversing agent combination PMA + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T-cells from ART-suppressed donors. Our results add to growing evidence that bonafide reservoir-harboring cells are resistant to multiple “kick and kill” modalities - relative to latency models - and uncover BCL-XL antagonists as a facile approach to probing mechanistic underpinnings. We also interpret our results as encouraging of further exploration of BCL-XL antagonists for cure, where combination approaches may unlock the ability to eliminate ex vivo reservoirs.

scholarly journals Th1/17 Polarization of CD4 T Cells Supports HIV-1 Persistence during Antiretroviral Therapy

2015 ◽  
Vol 89 (22) ◽  
pp. 11284-11293 ◽  
Author(s):  
Hong Sun ◽  
Dhohyung Kim ◽  
Xiaodong Li ◽  
Maja Kiselinova ◽  
Zhengyu Ouyang ◽  
...  
Keyword(s):  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Memory Cell ◽  
Viral Reservoir ◽  
Target Cells ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTThe ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection inex vivoassays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy.IMPORTANCECurrent antiretroviral therapy is very effective in suppressing active HIV-1 replication but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally attributed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.

scholarly journals Human Immunodeficiency Virus (HIV)–Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy

2019 ◽  
Vol 221 (5) ◽  
pp. 744-755 ◽  
Author(s):  
Jenny L Anderson ◽  
Gabriela Khoury ◽  
Rémi Fromentin ◽  
Ajantha Solomon ◽  
Nicolas Chomont ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
People Living With Hiv ◽  
Hiv Persistence ◽  
Hiv Dna ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Living With Hiv

Abstract Background Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. Methods Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. Results Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. Conclusions HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

scholarly journals The combination of venetoclax and ixazomib selectively and efficiently kills HIV infected cell lines, but has unacceptable toxicity in primary cell models

2021 ◽  
Author(s):  
Alecia Alto ◽  
Sekar Natesampillai ◽  
Aswath P. Chandrasekar ◽  
Ashton Krogman ◽  
Anisha Misra ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Model ◽  
Ex Vivo ◽  
The Body ◽  
Primary Cells ◽  
Reversal Agent ◽  
Latently Infected ◽  
Infected Cells ◽  
Unacceptable Toxicity

The anti-apoptotic protein BCL2 inhibits death of HIV-infected cells. Previously, we have shown that the BCL2 inhibitor venetoclax selectively kills acutely HIV-infected cells and reduces HIV DNA in latently infected CD4 T cells ex vivo after reactivation with αCD3/αCD28. However, there is a need to identify a combination therapy with venetoclax and a clinically relevant latency reversal agent. Ixazomib is an oral proteasome inhibitor which we have shown reactivates latent HIV and predisposes reactivated cells to cell death. Here, we determined that the combination of venetoclax and ixazomib kills more latently HIV-infected cells and leads to greater reduction in HIV replication compared to either treatment alone in vitro in a T cell model. However, combination treatment of ex vivo CD4 T cells from ART-suppressed, HIV-positive participants resulted in unanticipated and unacceptable non-specific toxicity in primary cells. Therefore, while we show proof of concept that multiple agents can enhance selective killing of HIV infected cells, the combination of venetoclax and ixazomib has unacceptable toxicity in primary cells, and so further investigation is needed to identify a clinically relevant latency reversal agent to combine with venetoclax as a novel strategy to reduce the size of the HIV reservoir. IMPORTANCE: A cure for HIV would require eliminating cells that contain the virus in a latent form from the body. Current antiretroviral medications are unable to rid the body of latently infected cells. Here we show that a combination of investigational agents – ixazomib plus venetoclax- which reactivate latent virus, and predispose infected cells to apoptosis, may reduce latent virus in a T cell model, but at the expense of non-specific toxicity in primary cells.

scholarly journals Analysis of HIV Reservoirs in Cellular Conjugates from Peripheral Blood

2021 ◽  
Author(s):  
Liliana Pérez ◽  
Daniel Crespo-Vélez ◽  
Max Lee ◽  
Saami Zakaria ◽  
April Poole ◽  
...  
Keyword(s):  
T Cells ◽  
Dna Sequences ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Single Cells ◽  
Cell Types ◽  
Hiv Dna ◽  
Infected Cells ◽  
Hiv Controllers

AbstractDefining distinctive attributes of HIV-infected cells will inform development of HIV cure-directed therapies. Prior ex vivo studies of blood and tissue have suggested that some HIV-infected CD4 T cells are found in conjugates with other cell types. Here, we analyzed levels and sequences of HIV nucleic acids in sorted cellular conjugates from PBMC. Compared to single CD4 T cells, conjugates containing CD4 T cells showed no enrichment for HIV DNA or RNA. However, in several HIV controllers, HIV DNA sequences from sorted conjugates were enriched for sequences closely related to plasma viruses. In ART-treated people, although subgenomic HIV DNA sequences in sorted conjugates and single cells were genetically intermingled, intact proviruses were more frequent in whole blood cells than in magnetically-purified CD4 T cells. We conclude that some HIV-infected cells have attributes that predict preferential loss during sample processing, and that may also reflect vulnerability to therapeutic targeting in vivo.

scholarly journals Long-Term Spontaneous Control of HIV-1 Is Related to Low Frequency of Infected Cells and Inefficient Viral Reactivation

2016 ◽  
Vol 90 (13) ◽  
pp. 6148-6158 ◽  
Author(s):  
Nicolas Noel ◽  
Ruth Peña ◽  
Annie David ◽  
Veronique Avettand-Fenoel ◽  
Itziar Erkizia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Ex Vivo ◽  
Low Frequency ◽  
T Cell Response ◽  
Viral Reactivation ◽  
Viral Control ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTHIV establishes reservoirs of infected cells that persist despite effective antiretroviral therapy (ART). In most patients, the virus begins to replicate soon after treatment interruption. However, a low frequency of infected cells at the time of treatment interruption has been associated with delayed viral rebound. Likewise, individuals who control the infection spontaneously, so-called HIV-1 controllers (HICs), carry particularly low levels of infected cells. It is unclear, however, whether and how this small number of infected cells contributes to durable viral control. Here we compared 38 HICs with 12 patients on effective combined antiretroviral therapy (cART) and found that the low frequency of infected cells in the former subjects was associated both with less efficient viral reactivation in resting CD4+T cells and with less efficient virion productionex vivo. We also found that a potent HIV-specific CD8+T cell response was present only in those HICs whose CD4+T cells produced virusex vivo. Long-term spontaneous control of HIV infection in HICs thus appears to be sustained on the basis of the inefficient reactivation of viruses from a limited number of infected cells and the capacity of HICs to activate a potent HIV-specific CD8+T cell response to counteract efficient viral reactivation events.IMPORTANCEThere is a strong scientific interest in developing strategies to eradicate the HIV-1 reservoir. Very rare HIV-1-infected patients are able to spontaneously control viremia for long periods of time (HIV-1 controllers [HICs]) and are put forward as a model of HIV-1 remission. Here, we show that the low viral reservoirs found in HICs are a critical part of the mechanisms underlying viral control and result in a lower probability of HIV-1 reactivation events, resulting in limited HIV-1 release and spread. We found that those HICs in whom viral reactivation and spread from CD4+T cellsin vitrowere the most difficult were those with diminished CD8+T cell responses. These results suggest that, in some settings, low HIV-1 reservoirs decisively contribute to at least the temporary control of infection without antiretroviral therapy. We believe that this work provides information of relevance in the context of the search for HIV-1 remission.

Bryostatin-1 decreases HIV-1 infection and viral production in human primary macrophages

2021 ◽  
Author(s):  
Laurent Hany ◽  
Marc-Olivier Turmel ◽  
Corinne Barat ◽  
Michel Ouellet ◽  
Michel J. Tremblay
Keyword(s):  
T Cells ◽  
Myeloid Cells ◽  
People Living With Hiv ◽  
Bryostatin 1 ◽  
Viral Production ◽  
Human Macrophages ◽  
Reversal Agents ◽  
Infected Cells ◽  
Hiv 1 ◽  
Latency Reversal Agents

While combination antiretroviral therapy maintains undetectable viremia in People Living With HIV (PLWH), a life-long treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long lived HIV-1 latently infected cells reversing to a viral productive status. Reversing latency and elimination of these cells by the so-called shock and kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4 + T cells. However, LRAs impact on de novo infection or HIV-1 production in productively infected macrophages remain elusive. Nontoxic doses of bryostatin-1, JQ1 and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effect rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance neither HIV-1 production nor gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4 + T cells are responding differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. Importance HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing a gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo . Yet, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging anti-replicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists which is specific to myeloid cells. This study provides new insights on HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1 targeted cells.

scholarly journals Adaptive and Innate Immune Cells in Fetal Human Cytomegalovirus-Infected Brains

2020 ◽  
Vol 8 (2) ◽  
pp. 176 ◽  
Author(s):  
Yann Sellier ◽  
Florence Marliot ◽  
Bettina Bessières ◽  
Julien Stirnemann ◽  
Ferechte Encha-Razavi ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Immune Cells ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Fetal Brain ◽  
Brain Lesions ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Infected Cells

Background: The understanding of the pathogenesis of cytomegalovirus (CMV)-induced fetal brain lesions is limited. We aimed to quantify adaptive and innate immune cells and CMV-infected cells in fetal brains with various degrees of brain damage. Methods: In total, 26 archived embedded fetal brains were studied, of which 21 were CMV-infected and classified in severely affected (n = 13) and moderately affected (n = 8), and 5 were uninfected controls. The respective magnitude of infected cells, immune cells (CD8+, B cells, plasma cells, NK cells, and macrophages), and expression of immune checkpoint receptors (PD-1/PD-L1 and LAG-3) were measured by immunochemistry and quantified by quantitative imaging analysis. Results: Quantities of CD8+, plasma cells, NK cells, macrophages, and HCMV+ cells and expression of PD-1/PD-L1 and LAG-3 were significantly higher in severely affected than in moderately affected brains (all p values < 0.05). A strong link between higher number of stained cells for HCMV/CD8 and PD-1 and severity of brain lesions was found by component analysis. Conclusions: The higher expression of CD8, PD-1, and LAG-3 in severely affected brains could reflect immune exhaustion of cerebral T cells. These exhausted T cells could be ineffective in controlling viral multiplication itself, leading to more severe brain lesions. The study of the functionality of brain leucocytes ex vivo is needed to confirm this hypothesis.

scholarly journals Interleukin-7 promotes HIV persistence during antiretroviral therapy

Blood ◽  
2013 ◽  
Vol 121 (21) ◽  
pp. 4321-4329 ◽  
Author(s):  
Claire Vandergeeten ◽  
Rémi Fromentin ◽  
Sandrina DaFonseca ◽  
Mariam B. Lawani ◽  
Irini Sereti ◽  
...  
Keyword(s):  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
Viral Latency ◽  
Absolute Number ◽  
Hiv Persistence ◽  
Hiv Dna ◽  
Interleukin 7 ◽  
Key Points ◽  
Latently Infected

Key Points IL-7 does not disrupt viral latency in highly pure resting latently infected CD4+ T cells from HIV-infected subjects receiving ART. IL-7 therapy leads to a 70% increase in the absolute number of circulating CD4+ T cells harboring integrated HIV DNA 4 weeks posttherapy.

scholarly journals Continuous Prophylactic Antiretrovirals/Antiretroviral Therapy Since Birth Reduces Seeding and Persistence of the Viral Reservoir in Children Vertically Infected With Human Immunodeficiency Virus

2020 ◽  
Author(s):  
Marta Massanella ◽  
Thanyawee Puthanakit ◽  
Louise Leyre ◽  
Thidarat Jupimai ◽  
Panadda Sawangsinth ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Cross Sectional Study ◽  
Viral Reservoir ◽  
Cross Sectional ◽  
Hiv Reservoir ◽  
Hiv Dna ◽  
Art Initiation ◽  
Immunodeficiency Virus ◽  
Infected Cells

Abstract Background Early antiretroviral therapy (ART) restricts the size of the human immunodeficiency virus (HIV) reservoir in infants. However, whether antiretroviral (ARV) prophylaxis given to exposed vertically infected children exerts similar effects remains unknown. Methods We measured total and integrated HIV DNA, as well as the frequency of CD4 T cells producing multiply spliced RNA (msRNA) after stimulation (inducible reservoir) in vertically infected Thai infants. Eighty-five infants were followed longitudinally for up to 3 years. We compared the size of the reservoir in children who received continuous ARV prophylaxis since birth vs those who never received or discontinued prophylaxis before initiating ART. We used samples from a cross-sectional cohort of 37 Thai children who had initiated ART within 6 months of life to validate our findings. Results Before ART, levels of HIV DNA and the frequencies of cells producing msRNA were significantly lower in infants who received continuous ARV prophylaxis since birth compared to those in whom ARV prophylaxis was discontinued or never initiated (P &lt; .020 and P &lt; .001, respectively). Upon ART initiation, total and integrated HIV DNA levels decayed significantly in both groups (P &lt; .01 in all cases). Interestingly, the initial differences in the frequencies of infected cells persisted during 3 years on ART. The beneficial effect of prophylaxis on the size of the HIV reservoir was confirmed in the cross-sectional study. Importantly, no differences were observed between children who discontinued prophylactic ARVs before starting ART and those who delayed ART initiation without receiving prior prophylaxis. Conclusions Neonatal ARV prophylaxis with direct transition to ART durably limits the size of the HIV reservoir.

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