The HIV latent reservoir in resting memory CD4+ T cells prevents cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not in pediatric populations. We determined, ex vivo, the inducibility and size of the latent reservoir in perinatal compared with adult infections using the Tat/rev Induced Limiting Dilution Assay (TILDA), in which a single-round (12hr) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply-spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Despite similar rates of T cell activation with PMA/ionomycin, the latent reservoir in perinatal infection is slower to reactivate and of lower magnitude compared to adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and for 18 hours augmented proviral expression in perinatal but not adult infection. Baseline HLA–DR+ CD4+ T cells was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support differences in baseline immune activation and kinetics of latency reversal in perinatal compared with adult infections, with implications for latency reversal strategies towards reservoir clearance and remission.
Adit Dhummakupt, Jessica H. Rubens, Thuy Anderson, Laura Powell, Bareng A.S. Nonyane, Lilly V. Siems, Aleisha Collinson-Streng, Tricia L. Nilles, R. Brad Jones, Vicki Tepper, Allison Agwu, Deborah Persaud
In the RV144 HIV-1 phase III trial, vaccine efficacy directly correlated with the magnitude of the variable region 2–specific (V2-specific) IgG antibody response, and in the presence of low plasma IgA levels, with the magnitude of plasma antibody-dependent cellular cytotoxicity. Reenrollment of RV144 vaccinees in the RV305 trial offered the opportunity to define the function, maturation, and persistence of vaccine-induced V2-specific and other mAb responses after boosting. We show that the RV144 vaccine regimen induced persistent V2 and other HIV-1 envelope–specific memory B cell clonal lineages that could be identified throughout the approximately 11-year vaccination period. Subsequent boosts increased somatic hypermutation, a critical requirement for antibody affinity maturation. Characterization of 22 vaccine-induced V2-specific mAbs with epitope specificities distinct from previously characterized RV144 V2-specific mAbs CH58 and CH59 found increased in vitro antibody-mediated effector functions. Thus, when inducing non-neutralizing antibodies, one method by which to improve HIV-1 vaccine efficacy may be through late boosting to diversify the V2-specific response to increase the breadth of antibody-mediated anti–HIV-1 effector functions.
David Easterhoff, Justin Pollara, Kan Luo, Benjamin Janus, Neelakshi Gohain, LaTonya D. Williams, Matthew Zirui Tay, Anthony Monroe, Kristina Peachman, Misook Choe, Susie Min, Paolo Lusso, Peng Zhang, Eden P. Go, Heather Desaire, Mattia Bonsignori, Kwan-Ki Hwang, Charles Beck, Matina Kakalis, Robert J. O’Connell, Sandhya Vasan, Jerome H. Kim, Nelson L. Michael, Jean-Louis Excler, Merlin L. Robb, Supachai Rerks-Ngarm, Jaranit Kaewkungwal, Punnee Pitisuttithum, Sorachai Nitayaphan, Faruk Sinangil, James Tartaglia, Sanjay Phogat, Kevin Wiehe, Kevin O. Saunders, David C. Montefiori, Georgia D. Tomaras, M. Anthony Moody, James Arthos, Mangala Rao, M. Gordon Joyce, Gilad A. Ofek, Guido Ferrari, Barton F. Haynes
Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues yet its impact upon the heart is unknown. Here we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy and show that a novel mouse model of cardiac specific prelamin A accumulation exhibited a phenotype consistent with ‘inflammatory cardiomyopathy’ which we observed to be similar to HIV associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings: (1) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease suggesting protease inhibitors should be replaced with alternative therapies i.e. non-nucleoside reverse transcriptase inhibitors; and (3) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.
Daniel Brayson, Andrea Frustaci, Romina Verardo, Cristina Chimenti, Matteo Antonio Russo, Robert Hayward, Sadia Munir Ahmad, Gema Vizcay-Barrena, Andrea Protti, Peter S. Zammit, Cristobal G. dos Remedios, Elisabeth Ehler, Ajay M. Shah, Catherine M. Shanahan
We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population prior to initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) – control (non-failure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine /tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/OCTANE Trial 1 among women who had received prior single dose nevirapine. Ultrasensitive single genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual-class) and failure of three drug ART to suppress viremia. By contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.
Valerie F. Boltz, Wei Shao, Michael J. Bale, Elias K. Halvas, Brian Luke, James A. McIntyre, Robert T. Schooley, Shahin Lockman, Judith S. Currier, Fred Sawe, Evelyn Hogg, Michael D. Hughes, Mary F. Kearney, John M. Coffin, John W. Mellors
Broadly neutralizing antibodies (bNAbs) against HIV-1 are under evaluation for both prevention and therapy. HIV-1 sequence diversity observed in most HIV-infected individuals and archived variations in critical bNAb epitopes present a major challenge for the clinical application of bNAbs, as preexistent resistant viral strains can emerge, resulting in bNAb failure to control HIV. In order to identify viral resistance in patients prior to antibody therapy and to guide the selection of effective bNAb combination regimens, we developed what we believe to be a novel Bayesian machine-learning model that uses HIV-1 envelope protein sequences and foremost approximated glycan occupancy information as variables to quantitatively predict the half-maximal inhibitory concentrations (IC50) of 126 neutralizing antibodies against a variety of cross clade viruses. We then applied this model to peripheral blood mononuclear cell–derived proviral Env sequences from 25 HIV-1–infected individuals mapping the landscape of neutralization resistance within each individual’s reservoir and determined the predicted ideal bNAb combination to achieve 100% neutralization at IC50 values <1 μg/ml. Furthermore, predicted cellular viral reservoir neutralization signatures of individuals before an analytical antiretroviral treatment interruption were consistent with the measured neutralization susceptibilities of the respective plasma rebound viruses, validating our model as a potentially novel tool to facilitate the advancement of bNAbs into the clinic.
Wen-Han Yu, David Su, Julia Torabi, Christine M. Fennessey, Andrea Shiakolas, Rebecca Lynch, Tae-Wook Chun, Nicole Doria-Rose, Galit Alter, Michael S. Seaman, Brandon F. Keele, Douglas A. Lauffenburger, Boris Julg
Background. HIV-infected patients with poor virologic control and multi-drug resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705-matched progressor. Methods. Approximately 22 billion cells were collected from an elite controller by lymphaphersis and infused within 6 hours into a recipient with a pre-infusion CD4+ T cell count of 10 cells/μL (1%) and HIV plasma viral load of 114,993 copies/mL. Results. Donor cells were cleared from the recipient's peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8+ T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8+ T cells also expressed increased expression of these markers, especially in HIV-specific tetramer positive cells. Conclusions. These results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways.
Stephen A. Migueles, Cheryl Chairez, Siying Lin, Noah V. Gavil, Danielle M. Rosenthal, Milad Pooran, Ven Natarajan, Adam Rupert, Robin Dewar, Tauseef Rehman, Brad T. Sherman, Joseph Adelsberger, Susan Leitman, David Stroncek, Caryn G. Morse, Mark Connors, H. Clifford Lane, Joseph A. Kovacs
During chronic HIV infection, immune cells become increasingly dysfunctional and exhausted. Little is known about how immune functions are restored after initiation of antiretroviral therapy (ART). In this study, we assessed cellular and metabolic activity and evaluated the effect of individual antiretrovirals on cellular subsets ex vivo in ART-treated and treatment-naive chronically HIV-infected individuals. We observed that cellular respiration was significantly decreased in most immune cells in chronic HIV infection. The respiration was correlated to immune activation and the inhibitory receptor programmed cell death 1 on CD8+ T cells. ART restored the metabolic phenotype, but the respiratory impairment persisted in CD4+ T cells. This was particularly the case for individuals receiving integrase strand transfer inhibitors. CD4+ T cells from these individuals showed a significant reduction in ex vivo proliferative capacity compared with individuals treated with protease inhibitors or nonnucleoside reverse transcriptase inhibitors. We noticed a significant decrease in respiration of cells treated with dolutegravir (DLG) or elvitegravir (EVG) and a switch from polyfunctional to TNF-α–dominated “stress” immune response. There was no effect on glycolysis, consistent with impaired mitochondrial function. We detected increased levels of mitochondrial ROS and mitochondrial mass. These findings indicate that EVG and DLG use is associated with slow proliferation and impaired respiration with underlying mitochondrial dysfunction, resulting in overall decreased cellular function in CD4+ T cells.
Marek Korencak, Morgan Byrne, Enrico Richter, Bruce T. Schultz, Patrick Juszczak, Julie A. Ake, Anuradha Ganesan, Jason F. Okulicz, Merlin L. Robb, Buena de los Reyes, Sandra Winning, Joachim Fandrey, Timothy H. Burgess, Stefan Esser, Nelson L. Michael, Brian K. Agan, Hendrik Streeck
In HIV-infected individuals on long-term antiretroviral therapy (ART), more than 40% of the infected cells are in clones. Although most HIV proviruses present in individuals on long-term ART are defective, including those in clonally expanded cells, there is increasing evidence that clones carrying replication-competent proviruses are common in patients on long-term ART and form part of the HIV reservoir that makes it impossible to cure HIV infection with current ART alone. Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca. 1 × 105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III–V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6,500 independent integration sites from peripheral blood mononuclear cells before ART was initiated and after 0.5–18 years of suppressive ART. Some infected clones could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years.
John M. Coffin, David W. Wells, Jennifer M. Zerbato, Joann D. Kuruc, Shuang Guo, Brian T. Luke, Joseph J. Eron, Michael Bale, Jonathan Spindler, Francesco R. Simonetti, Shawn Hill, Mary F. Kearney, Frank Maldarelli, Xiaolin Wu, John W. Mellors, Stephen H. Hughes
Reduction/elimination of HIV-1 reservoirs that persist despite combination antiretroviral therapy (cART) will likely require induction of viral expression by residual infected cells and enhanced clearance of these cells. TLR7 agonists have potential to mediate these activities. We evaluated immunologic and virologic effects of repeated doses of the TLR7 agonist GS-9620 in SIV-infected rhesus macaques receiving cART, which was initiated at 13 days after infection and was continued for 75 weeks prior to GS-9620 administration. During cART, GS-9620 induced transient upregulation of IFN-stimulated genes in blood and tissues, increases in plasma cytokines, and changes in immune cell population activation and phenotypes but did not result in measurable increases in plasma viremia or viral RNA–to–viral DNA ratio in PBMCs or tissues nor decreases in viral DNA in PBMC or tissues. SIV-specific CD8+ T cell responses, negligible prior to GS-9620 treatment, were not measurably boosted by treatment; a second course of GS-9620 administration overlapping with later cART discontinuation was associated with increased CD8+ T cell responses during viral recrudescence. These results confirm and extend evidence for GS-9620–mediated enhancement of antiviral immune responses in SIV-infected macaques but suggest that GS-9620–mediated viral induction may depend critically on the timing of initiation and duration of cART and resulting characteristics of viral reservoirs.
Gregory Q. Del Prete, W. Gregory Alvord, Yuan Li, Claire Deleage, Mukta Nag, Kelli Oswald, James A. Thomas, Cathi Pyle, William J. Bosche, Vicky Coalter, Adam Wiles, Rodney Wiles, Brian Berkemeier, Michael Hull, Elizabeth Chipriano, Lorna Silipino, Randy Fast, Jacob Kiser, Rebecca Kiser, Tyler Malys, Joshua Kramer, Matthew W. Breed, Charles M. Trubey, Jacob D. Estes, Tiffany L. Barnes, Joseph Hesselgesser, Romas Geleziunas, Jeffrey D. Lifson
BACKGROUND. The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here, we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS. EXID’s clinical and immunological characteristics were compared to immunological responders (IRs), immunological nonresponders (INRs), healthy controls (HCs), and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS. EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/μl, compared with CD4+ increases of 193 cells/μl and 427 cells/μl in INR and IR, respectively. EXID had reduced naive CD4+ T cells, but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR, but the IL-7 axis was profoundly perturbed compared with HC, IR, INR, and ICL. Genes involved in T cell and monocyte/macrophage function, autophagy, and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC, while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS. EXID is a distinct immunological outcome compared with previously described INR. Anti–CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID.
Andrea Lisco, Chun-Shu Wong, Silvia Lucena Lage, Itzchak Levy, Jason Brophy, Jeffrey Lennox, Maura Manion, Megan V. Anderson, Yolanda Mejia, Christopher Grivas, Harry Mystakelis, Peter D. Burbelo, Ainhoa Perez-Diez, Adam Rupert, Craig A. Martens, Sarah L. Anzick, Caryn Morse, Shanna Chan, Claire Deleage, Irini Sereti
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