BACKGROUND. Early antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAb) after acute/early ARTi is relevant, but poorly understood. METHODS. We characterize antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after infection) and early HIV (60-128 days after infection). RESULTS. Plasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 Envs representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In two of the three acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter. CONCLUSIONS. Results indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants. TRIAL REGISTRATION. NCT02656511 FUNDING. National Institutes of Health grants U01AI169767; R01AI162646; UM1AI164570; UM1AI164560; U19AI096109; K23GM112526; T32AI118684, P30-AI-045008, P30 AI027763, R24 AI067039. Gilead Sciences grant INUS2361354; Viiv healthcare grant A126326.
Gregory D. Whitehill, Jaimy Joy, Francesco E. Marino, Ryan J. Krause, Suvadip Mallick, Hunter M. Courtney, Kyewon Park, John W. Carey, Rebecca Hoh, Heather Hartig, Vivian Pae, Sannidhi Sarvadhavabhatla, Maria Sophia B. Donaire, Steven G. Deeks, Rebecca M. Lynch, Sulggi A. Lee, Katharine J. Bar
Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti–PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239–infected rhesus macaques (RMs). Adoptive transfer of anti–PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti–PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti–PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.
Karsten Eichholz, Yoshinori Fukazawa, Christopher W. Peterson, Francoise Haeseleer, Manuel Medina, Shelby Hoffmeister, Derick M. Duell, Benjamin D. Varco-Merth, Sandra Dross, Haesun Park, Caralyn S. Labriola, Michael K. Axthelm, Robert D. Murnane, Jeremy V. Smedley, Lei Jin, Jiaxin Gong, Blake J. Rust, Deborah H. Fuller, Hans-Peter Kiem, Louis J. Picker, Afam A. Okoye, Lawrence Corey
CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL757 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV highlights their responsiveness to neuroinflammation.
Sonny R. Elizaldi, Chase E. Hawes, Anil Verma, Yashavanth Shaan Lakshmanappa, Ashok R. Dinasarapu, Brent T. Schlegel, Dhivyaa Rajasundaram, Jie Li, Blythe P. Durbin-Johnson, Zhong-Min Ma, Pabitra B. Pal, Danielle Beckman, Sean Ott, Reben Raeman, Jeffrey Lifson, John H. Morrison, Smita S. Iyer
Background: Persistent controllers (PC) maintain antiretroviral-free HIV-1 control indefinitely over time while transient controllers (TC) eventually lose virological control. It is essential to characterize the quality of the HIV reservoir of these phenotypes to identify the factors that lead to HIV progression and to open new avenues in HIV cure strategies. Methods: The characterization of HIV-1 reservoir, from peripheral blood mononuclear cells, was performed using next-generation sequencing techniques, such as full-length individual and matched integration site proviral sequencing (FLIP-seq; MIP-seq). Results: PC and TC before losing virological control, presented significantly lower total, intact and defective proviruses compared to participants on antiretroviral therapy (ART). No differences were found in total and defective proviruses between PC and TC. However, intact provirus levels were lower in PC compared to TC, being the intact/defective HIV-DNA ratio significantly higher in TC. Clonally expanded intact proviruses were found only in PC and located in centromeric satellite DNA or zinc-finger genes, both associated with heterochromatin features. In contrast, sampled intact proviruses were located in permissive genic euchromatic positions in TC. Conclusions: These results suggest the need for, and can give guidance to the design of, future research to identify a distinct proviral landscape that may be associated with the persistent control of HIV-1 without ART. Funding: Instituto de Salud Carlos III (FI17/00186, FI19/00083, MV20/00057 PI18/01532, PI19/01127 and PI22/01796), Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (PI20/1276), Gilead Fellowships (GLD22/00147) and I+D+iFEDER Andalucía 2014-2020 (US-1380938).
Carmen Gasca-Capote, Xiaodong Lian, Ce Gao, Isabelle C. Roseto, María Reyes Jiménez-León, Gregory Gladkov, María Inés Camacho-Sojo, Alberto Pérez-Gómez, Isabel Gallego, Luis E. Lopez-Cortes, Sara Bachiller, Joana Vitalle, Mohammed Rafii-El-Idrissi Benhnia, Francisco J. Ostos, Antonio R. Collado-Romacho, Jesús Santos, Rosario Palacios, Cristina Gomez-Ayerbe, Leopoldo Muñoz-Medina, Andrés Ruiz-Sancho, Mario Frias, Antonio Rivero-Juarez, Cristina Roca-Oporto, Carmen Hidalgo-Tenorio, Anna Rull, Julian Olalla, Miguel A. Lopez-Ruz, Francesc Vidal, Consuelo Viladés, Andrea Mastrangelo, Matthias Cavassini, Nuria Espinosa, Matthieu Perreau, Joaquin Peraire, Antonio Rivero, Luis F. López-Cortes, Mathias Lichterfeld, Xu G. Yu, Ezequiel Ruiz-Mateos
Productively infected cells are generally thought to arise by HIV infection of activated CD4+ T cells, and these infected activated cells are also thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate by direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues restricted to resting CD4+ T cells and expression of pTEFb in these resting cells to enable productive infection, and we documented persistence of HIV producing resting T cells during ART. We thus provide evidence of a mechanism by which direct infection of resting T cell populations in lymphoid tissues to generate productively and latently infected cells could continually replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.
Stephen W. Wietgrefe, Jodi Anderson, Lijie Duan, Peter J. Southern, Paul Zuck, Guoxin Wu, Bonnie J. Howell, Cavan Reilly, Eugène Kroon, Suthat Chottanapund, Supranee Buranapraditkun, Carlo Sacdalan, Nicha Tulmethakaan, Donn J. Colby, Nitiya Chomchey, Peeriya Prueksakaew, Suteeraporn Pinyakorn, Rapee Trichavaroj, Julie L. Mitchell, Lydie Trautmann, Denise C. Hsu, Sandhya Vasan, Sopark Manasnayakorn, Mark de Souza, Sodsai Tovanabutra, Alexandra Schuetz, Merlin L. Robb, Nittaya Phanuphak, Jintanat Ananworanich, Timothy W. Schacker, Ashley T. Haase
BACKGROUND. HIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (EC) and people on long-term therapy suggests that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing. METHODS. In this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells. RESULTS. The proviral landscape was dominated by two large clones with replication-competent proviruses integrated into Zinc Finger genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-folds less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene. CONCLUSION. We provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.
Filippo Dragoni, Abena Kwaa, Caroline C.G. Traut, Rebecca T. Veenhuis, Bezawit A. Woldemeskel, Angelica Camilo-Contreras, Hayley E. Raymond, Arbor G. Dykema, Eileen P. Scully, Amanda M. Rosecrans, Kellie N. Smith, Frederic D. Bushman, Francesco R. Simonetti, Joel N. Blankson
HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first seven years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After seven years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected infected cell proliferation. Most (79.8%) inducible, replication-competent viruses had env sequences identical to other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.
Natalie F. McMyn, Joseph Varriale, Emily J. Fray, Carolin Zitzmann, Hannah J MacLeod, Jun Lai, Anushka Singhal, Milica Moskovljevic, Mauro A. Garcia, Brianna M. Lopez, Vivek Hariharan, Kyle Rhodehouse, Kenneth Lynn, Pablo Tebas, Karam Mounzer, Luis J. Montaner, Erika Benko, Colin Kovacs, Rebecca Hoh, Francesco R. Simonetti, Gregory M. Laird, Steven G. Deeks, Ruy M. Ribeiro, Alan S. Perelson, Robert Siliciano, Janet M. Siliciano
Long-acting antiretroviral agents for pre-exposure prophylaxis (PrEP) represent a promising new alternative to daily oral regimens for HIV prevention. Lenacapavir (LEN) is a first-in-class long-acting capsid inhibitor approved for the treatment of HIV-1 infection. Here, we assessed the efficacy of LEN for PrEP using a single high-dose simian-human immunodeficiency virus (SHIV) rectal challenge macaque model. In vitro, LEN showed potent antiviral activity against SHIV, similar to HIV-1. In macaques, a single subcutaneous administration of LEN demonstrated dose proportional increases in and durability of drug plasma levels. A high-dose SHIV inoculum for the PrEP efficacy evaluation was identified via virus titration in untreated macaques. LEN-treated macaques were challenged with high-dose SHIV 7 weeks post drug administration and the majority remained protected from infection as confirmed by plasma PCR, cell-associated proviral DNA, and serology testing. Complete protection and superiority to the untreated group was observed among animals whose LEN plasma exposure exceeded its model-adjusted clinical efficacy target at the time of challenge. All infected animals had subprotective LEN concentrations and showed no emergent resistance. These data demonstrate effective SHIV prophylaxis in a stringent macaque model at clinically relevant LEN exposures and support the clinical evaluation of LEN for HIV PrEP in humans.
Elena Bekerman, Stephen R. Yant, Laurie A. VanderVeen, Derek Hansen, Bing Lu, William Rowe, Kelly Wei Wang, Christian Callebaut
Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.
Yuyang Tang, Antoine Chaillon, Sara Gianella, Lilly M. Wong, Dajiang Li, Theresa L. Simermeyer, Magali Porrachia, Caroline Ignacio, Brendon Woodworth, Daniel Zhong, Jiayi Du, Eduardo de la Parra Polina, Jennifer Kirchherr, Brigitte Allard, Matthew L. Clohosey, Matt Moeser, Amy L. Sondgeroth, Gregory D. Whitehill, Vidisha Singh, Amir Dashti, Davey M. Smith, Joseph J. Eron, Katherine J. Bar, Ann Chahroudi, Sarah B. Joseph, Nancie M. Archin, David M. Margolis, Guochun Jiang
HSV-2 coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well-defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2 infected and bystander 2D10 cells. Bulk and single-cell RNA sequencing studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms including upregulation of MALAT1 to release epigenetic silencing.
Carl A. Pierce, Lip Nam Loh, Holly R. Steach, Natalia Cheshenko, Paula Preston-Hurlburt, Fengrui Zhang, Stephanie Stransky, Leah Kravets, Simone Sidoli, William M. Philbrick, Michel N. Nassar, Smita Krishnaswamy, Kevan C. Herold, Betsy C. Herold