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
BACKGROUND Mosaic and consensus HIV-1 immunogens provide two distinct approaches to elicit greater breadth of coverage against globally circulating HIV-1 and have shown improved immunologic breadth in nonhuman primate models.METHODS This double-blind randomized trial enrolled 105 healthy HIV-uninfected adults who received 3 doses of either a trivalent global mosaic, a group M consensus (CON-S), or a natural clade B (Nat-B) gp160 env DNA vaccine followed by 2 doses of a heterologous modified vaccinia Ankara–vectored HIV-1 vaccine or placebo. We performed prespecified blinded immunogenicity analyses at day 70 and day 238 after the first immunization. T cell responses to vaccine antigens and 5 heterologous Env variants were fully mapped.RESULTS Env-specific CD4+ T cell responses were induced in 71% of the mosaic vaccine recipients versus 48% of the CON-S recipients and 48% of the natural Env recipients. The mean number of T cell epitopes recognized was 2.5 (95% CI, 1.2–4.2) for mosaic recipients, 1.6 (95% CI, 0.82–2.6) for CON-S recipients, and 1.1 (95% CI, 0.62–1.71) for Nat-B recipients. Mean breadth was significantly greater in the mosaic group than in the Nat-B group using overall (P = 0.014), prime-matched (P = 0.002), heterologous (P = 0.046), and boost-matched (P = 0.009) measures. Overall T cell breadth was largely due to Env-specific CD4+ T cell responses.CONCLUSION Priming with a mosaic antigen significantly increased the number of epitopes recognized by Env-specific T cells and enabled more, albeit still limited, cross-recognition of heterologous variants. Mosaic and consensus immunogens are promising approaches to address global diversity of HIV-1.TRIAL REGISTRATION ClinicalTrials.gov NCT02296541.FUNDING US NIH grants UM1 AI068614, UM1 AI068635, UM1 AI068618, UM1 AI069412, UL1 RR025758, P30 AI064518, UM1 AI100645, and UM1 AI144371, and Bill & Melinda Gates Foundation grant OPP52282.
Kristen W. Cohen, Andrew Fiore-Gartland, Stephen R. Walsh, Karina Yusim, Nicole Frahm, Marnie L. Elizaga, Janine Maenza, Hyman Scott, Kenneth H. Mayer, Paul A. Goepfert, Srilatha Edupuganti, Giuseppe Pantaleo, Julia Hutter, Daryl E. Morris, Stephen C. De Rosa, Daniel E. Geraghty, Merlin L. Robb, Nelson L. Michael, Will Fischer, Elena E. Giorgi, Harman Malhi, Michael N. Pensiero, Guido Ferrari, Georgia D. Tomaras, David C. Montefiori, Peter B. Gilbert, M. Juliana McElrath, Barton F. Haynes, Bette T. Korber, Lindsey R. Baden, the NIAID HVTN 106 Study Group
Brooke D. Kennedy, Jana Blazkova, Jesse S. Justement, Victoria Shi, M. Ali Rai, Maegan R. Manning, Lauren Praiss, Kathleen Gittens, Paul A. Wender, Sean Patro, Xiaolin Wu, Susan Moir, Tae-Wook Chun
BACKGROUND. Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown. METHODS. We carried an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5’-Leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets. RESULTS. Clones carrying proviruses with 5’-Leader defects can cause persistent NSV up to ~103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor site (MSD) and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced non-infectious virions containing viral RNA but lacking Envelope. CONCLUSION. These findings show that proviruses with 5’-Leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5’-Leader can help understanding failure to completely suppress viremia. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases, NIH, to the PAVE, BEAT-HIV and DARE Martin Delaney collaboratories.
Jennifer A. White, Fengting Wu, Saif Yasin, Milica Moskovljevic, Joseph Varriale, Filippo Dragoni, Angelica Camilo Contreras, Jiayi Duan, Mei Y. Zheng, Ndeh F. Tadzong, Heer B. Patel, Jeanelle Mae C. Quiambao, Kyle Rhodehouse, Hao Zhang, Jun Lai, Subul A. Beg, Michael Delannoy, Christin Kilcrease, Christopher J. Hoffmann, Sébastien Poulin, Frédéric Chano, Cecile Tremblay, Jerald Cherian, Patricia Barditch-Crovo, Natasha Chida, Richard D. Moore, Michael F. Summers, Robert F. Siliciano, Janet D. Siliciano, Francesco R. Simonetti
Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors which influence MMc detection and level across infancy and the effect of MMc on T cell responses to BCG vaccination in a cohort of HIV exposed, uninfected and HIV unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day one and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy with levels ranging from 0-1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal-infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc levels in HIV exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence MMc, which may subsequently impact infant T cell responses.
Christina Balle, Blair Armistead, Agano Kiravu, Xiaochang Song, Anna-Ursula Happel, Angela A. Hoffmann, Sami B. Kanaan, J. Lee Nelson, Clive M. Gray, Heather B. Jaspan, Whitney E. Harrington