HIV eradication studies have focused on developing latency-reversing agents (LRAs). However, it is not understood how the rate of latent reservoir reduction is affected by different steps in the process of latency reversal. Furthermore, as current LRAs are host-directed, LRA treatment is likely to be intermittent to avoid host toxicities. Few careful studies of the serial effects of pulsatile LRA treatment have yet been done. This lack of clarity makes it difficult to evaluate the efficacy of candidate LRAs or predict long-term treatment outcomes. We constructed a mathematical model that describes the dynamics of latently infected cells under LRA treatment. Model analysis showed that, in addition to increasing the immune recognition and clearance of infected cells, the duration of HIV antigen expression (i.e., the period of vulnerability) plays an important role in determining the efficacy of LRAs, especially if effective clearance is achieved. Patients may benefit from pulsatile LRA exposures compared with continuous LRA exposures if the period of vulnerability is long and the clearance rate is high, both in the presence and absence of an LRA. Overall, the model framework serves as a useful tool to evaluate the efficacy and the rational design of LRAs and combination strategies.
Ruian Ke, Jessica M. Conway, David M. Margolis, Alan S. Perelson
The presence of a reservoir of latently infected cells in HIV-infected patients is a major barrier towards finding a cure. One active cure strategy is to find latency-reversing agents that induce viral reactivation, thus leading to immune cell recognition and elimination of latently infected cells, known as the shock-and-kill strategy. Therefore, the identification of molecules that reactivate latent HIV and increase immune activation has the potential to further these strategies into the clinic. Here, we characterized synthetic molecules composed of a TLR2 and a TLR7 agonist (dual TLR2/7 agonists) as latency-reversing agents and compared their activity with that of the TLR2 agonist Pam2CSK4 and the TLR7 agonist GS-9620. We found that these dual TLR2/7 agonists reactivate latency by 2 complementary mechanisms. The TLR2 component reactivates HIV by inducing NF-κB activation in memory CD4+ T cells, while the TLR7 component induces the secretion of TNF-α by monocytes and plasmacytoid dendritic cells, promoting viral reactivation in CD4+ T cells. Furthermore, the TLR2 component induces the secretion of IL-22, which promotes an antiviral state and blocks HIV infection in CD4+ T cells. Our study provides insight into the use of these agonists as a multipronged approach targeting eradication of latent HIV.
Amanda B. Macedo, Camille L. Novis, Caroline M. De Assis, Eric S. Sorensen, Paula Moszczynski, Szu-han Huang, Yanqin Ren, Adam M. Spivak, R. Brad Jones, Vicente Planelles, Alberto Bosque
A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection–induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection–induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system–humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver–derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.
Jasmine Samal, Samantha Kelly, Ali Na-Shatal, Abdallah Elhakiem, Antu Das, Ming Ding, Anwesha Sanyal, Phalguni Gupta, Kevin Melody, Brad Roland, Watfa Ahmed, Aala Zakir, Moses Bility
Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti–PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody–treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.
Geetha H. Mylvaganam, Lynette S. Chea, Gregory K. Tharp, Sakeenah Hicks, Vijayakumar Velu, Smita S. Iyer, Claire Deleage, Jacob D. Estes, Steven E. Bosinger, Gordon J. Freeman, Rafi Ahmed, Rama R. Amara
Clonal expansion of T cells harboring replication-competent virus has recently been demonstrated in patients on suppressive antiretroviral therapy (ART) regimens. However, there has not been direct evidence of this phenomenon in settings of natural control, including in posttreatment controllers who maintain control of viral replication after treatment when ART is discontinued. We present a case of an individual who has had undetectable viral loads for more than 15 years following the cessation of ART. Using near-full-genome sequence analysis, we demonstrate that 9 of 12 replication-competent isolates cultured from this subject were identical and that this identity was maintained 6 months later. A similar pattern of replication-competent virus clonality was seen in a treatment-naive HLA-B*57 elite controller. In both cases, we show that CD8+ T cells are capable of suppressing the replication of the clonally expanded viruses in vitro. Our data suggest that, while clonal expansion of replication-competent virus can present a barrier to viral eradication, these viral isolates remain susceptible to HIV-specific immune responses and can be controlled in patients with long-term suppression of viral replication.
Rebecca T. Veenhuis, Abena K. Kwaa, Caroline C. Garliss, Rachel Latanich, Maria Salgado, Christopher W. Pohlmeyer, Christopher L. Nobles, John Gregg, Eileen P. Scully, Justin R. Bailey, Frederic D. Bushman, Joel N. Blankson
Central nervous system (CNS) immune activation is an important driver of neuronal injury during several neurodegenerative and neuroinflammatory diseases. During HIV infection, CNS immune activation is associated with high rates of neurocognitive impairment, even during sustained long-term suppressive antiretroviral therapy (ART). However, the cellular subsets that drive immune activation and neuronal damage in the CNS during HIV infection and other neurological conditions remain unknown, in part because CNS cells are difficult to access in living humans. Using single-cell RNA sequencing (scRNA-seq) on cerebrospinal fluid (CSF) and blood from adults with and without HIV, we identified a rare (<5% of cells) subset of myeloid cells that are found only in CSF and that present a gene expression signature that overlaps significantly with neurodegenerative disease–associated microglia. This highlights the power of scRNA-seq of CSF to identify rare CNS immune cell subsets that may perpetuate neuronal injury during HIV infection and other conditions.
Shelli F. Farhadian, Sameet S. Mehta, Chrysoula Zografou, Kevin Robertson, Richard W. Price, Jenna Pappalardo, Jennifer Chiarella, David A. Hafler, Serena S. Spudich
The genomic integration of HIV into cells results in long-term persistence of virally infected cell populations. This integration event acts as a heritable mark that can be tracked to monitor infected cells that persist over time. Previous reports have documented clonal expansion in people and have linked them to proto-oncogenes; however, their significance or contribution to the latent reservoir has remained unclear. Here, we demonstrate that a directed pattern of clonal expansion occurs in vivo, specifically in gene pathways important for viral replication and persistence. These biological processes include cellular division, transcriptional regulation, RNA processing, and posttranslational modification pathways. This indicates preferential expansion when integration events occur within genes or biological pathways beneficial for HIV replication and persistence. Additionally, these expansions occur quickly during unsuppressed viral replication in vivo, reinforcing the importance of early intervention for individuals to limit reservoir seeding of clonally expanded HIV-infected cells.
Kevin G. Haworth, Lauren E. Schefter, Zachary K. Norgaard, Christina Ironside, Jennifer E. Adair, Hans-Peter Kiem
The peripheral blood represents only a small fraction of the total number of lymphocytes in the body. To develop a more thorough understanding of T cell dynamics, including the effects of SIV/SHIV/HIV infection on immune cell depletion and immune reconstitution following combination antiretroviral therapy (cART), one needs to utilize approaches that allow direct visualization of lymphoid tissues. In the present study, noninvasive in vivo imaging of the CD4+ T cell pool has revealed that the timing of the CD4+ T cell pool reconstitution following initiation of ART in SIV-infected nonhuman primates (NHPs) appears seemingly stochastic among clusters of lymph nodes within the same host. At 4 weeks following initiation or interruption of cART, the changes observed in peripheral blood (PB) are primarily related to changes in the whole-body CD4 pool rather than changes in lymphocyte trafficking. Lymph node CD4 pools in long-term antiretroviral-treated and plasma viral load–suppressed hosts appear suboptimally reconstituted compared with healthy controls, while splenic CD4 pools appear similar between the 2 groups.
Michele Di Mascio, Sharat Srinivasula, Insook Kim, Gorka Duralde, Alexis St. Claire, Paula DeGrange, Marisa St. Claire, Keith A. Reimann, Erin E. Gabriel, Jorge Carrasquillo, Richard C. Reba, Chang Paik, Henry C. Lane
Tenofovir gel and dapivirine ring provided variable HIV protection in clinical trials, reflecting poor adherence and possibly biological factors. We hypothesized that vaginal microbiota modulates pharmacokinetics and tested the effects of pH, individual bacteria, and vaginal swabs from women on pharmacokinetics and antiviral activity. Tenofovir, but not dapivirine, uptake by human cells was reduced as pH increased. Lactobacillus crispatus actively transported tenofovir leading to a loss in drug bioavailability and culture supernatants from Gardnerella vaginalis, but not Atopobium vaginae, blocked tenofovir endocytosis. The inhibition of endocytosis mapped to adenine. Adenine increased from 65.5 μM in broth to 246 μM in Gardnerella, but decreased to 9.5 μM in Atopobium supernatants. This translated into a decrease in anti-HIV activity when Gardnerella supernatants or adenine were added to cultures. Dapivirine was also impacted by microbiota, as drug bound irreversibly to bacteria, resulting in decreased antiviral activity. When drugs were incubated with vaginal swabs, 30.7% ± 5.7% of dapivirine and 63.9% ± 8.8% of tenofovir were recovered in supernatants after centrifugation of the bacterial cell pellet. In contrast, no impact of microbiota on the pharmacokinetics of the prodrugs, tenofovir disoproxil fumarate or tenofovir alafenamide, was observed. Together, these results demonstrate that microbiota may impact pharmacokinetics and contribute to inconsistent efficacy.
Ekaterina Taneva, Shada Sinclair, Pedro M.M. Mesquita, Brian Weinrick, Scott A. Cameron, Natalia Cheshenko, Kerry Reagle, Bruce Frank, Sujatha Srinivasan, David Fredricks, Marla J. Keller, Betsy C. Herold
Current strategies aimed to cure HIV infection are based on combined efforts to reactivate the virus from latency and improve immune effector cell function to clear infected cells. These strategies are primarily focused on CD8+ T cells and approaches are challenging due to insufficient HIV antigen production from infected cells and poor HIV-specific CD8+ T cells. γδ T cells represent a unique subset of effector T cells that can traffic to tissues, and selectively target cancer or virally infected cells without requiring MHC presentation. We analyzed whether γδ T cells represent a complementary/alternative immunotherapeutic approach towards HIV cure strategies. γδ T cells from HIV-infected virologically suppressed donors were expanded with bisphosphonate pamidronate (PAM) and cells were used in autologous cellular systems ex vivo. These cells (a) are potent cytotoxic effectors able to efficiently inhibit HIV replication ex vivo, (b) degranulate in the presence of autologous infected CD4+ T cells, and (c) specifically clear latently infected cells after latency reversal with vorinostat. This is the first proof of concept to our knowledge showing that γδ T cells target and clear autologous HIV reservoirs upon latency reversal. Our results open potentially new insights into the immunotherapeutic use of γδ T cells for current interventions in HIV eradication strategies.
Carolina Garrido, Matthew L. Clohosey, Chloe P. Whitworth, Michael Hudgens, David M. Margolis, Natalia Soriano-Sarabia
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