People living with HIV (PLWH) who are Immune Non-Responders (INR) persons are at greater risk of comorbidity and mortality than are Immune Responders (IR) who restore their CD4 T cells count (IR) after anti-retroviral therapy (ART). INR have low CD4-T cell counts (<350 c/ul), heightened systemic inflammation, and increased CD4-T cell cycling (Ki67+). Here we report the findings that memory CD4-T cells and plasma samples of INR from several cohorts are enriched in gut-derived bacterial solutes (GDBS) p-cresol-sulfate (PCS) and indoxyl sulfate (IS) that both negatively correlated with CD4-T cell counts. In vitro PCS or IS blocked CD4-T cell proliferation, induced apoptosis, and diminished the expression of mitochondrial proteins. Electron microscopy imaging (EMI) revealed perturbations of mitochondria networks similar to those found in INR following incubation of healthy memory CD4-T cells with PCS. Using the bacterial 16S rDNA, INR stool samples were found enriched with proteolytic bacterial genera that metabolize tyrosine and phenylalanine amino acids to produce PCS. We propose that toxic solutes from the gut bacterial flora may impair CD4-T cell recovery during ART and may contribute to CD4-T cell lymphopenia characteristic of INR.
Brian Ferrari, Amanda Cabral Da Silva, Ken H. Liu, Evgeniya V. Saidakova, Larisa B. Korolevskaya, Konstantin V. Shmagel, Carey Shive, Gabriela Pacheco Sanchez, Mauricio Retuerto, Ashish Arunkumar Sharma, Khader Ghneim, Laura Noel-Romas, Benigno Rodriguez, Mahmoud A. Ghannoum, Peter P. Hunt, Steven G. Deeks, Adam D. Burgener, Dean P. Jones, Mirela A. Dobre, Vincent C. Marconi, Rafick-Pierre Sekaly, Souheil-Antoine Younes
Proliferation of latently-infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to target this latent cell expansion is to inhibit the mechanistic target of rapamycin (mTOR), a regulatory kinase involved with cell growth, metabolism and proliferation. Here we determined the effects of chronic mTOR inhibition with rapamycin +/- T cell activation in SIV-infected rhesus macaques (RM) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequencies of proliferating CD4+ memory T cells (TM) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RM relative to controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RM on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin + CD3LALA-treated and control-treated RM rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that while rapamycin can decrease the proliferation of CD4+ TM, chronic mTOR inhibition alone or in combination with T cell activation, was not sufficient to disrupt the stability of the SIV reservoir.
Benjamin D. Varco-Merth, William Brantley, Alejandra Marenco, Derick D. Duell, Devin N. Fachko, Brian Richardson, Kathleen Busman-Sahay, Danica Shao, Walter Flores, Kathleen Engelman, Yoshinori Fukazawa, Scott W. Wong, Rebecca L. Skalsky, Jeremy Smedley, Michael K Axthelm, Jeffrey D. Lifson, Jacob D. Estes, Paul T. Edlefsen, Louis Picker, Cheryl M.A. Cameron, Timothy J. Henrich, Afam A. Okoye
Interleukin (IL)-10 is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper cell (TFH) differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph node (LN) were induced by infection and not normalized with ART. During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including TFH, and predicted the frequency of CD4+ TFH and their cell-associated SIV-DNA content during ART, respectively. In ART-treated RMs, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B-cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and by extension LN memory CD4+ T-cells, including TFH and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T-cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.
Justin Harper, Susan P. Ribeiro, Chi Ngai Chan, Malika Aid, Claire Deleage, Luca Micci, Maria Pino, Barbara Cervasi, Gopalan Raghunathan, Eric Rimmer, Gulesi Ayanoglu, Guoxin Wu, Neeta Shenvi, Richard J.O. Barnard, Gregory Q. Del Prete, Kathleen Busman-Sahay, Guido Silvestri, Deanna A. Kulpa, Steven E. Bosinger, Kirk Easley, Bonnie J. Howell, Dan Gorman, Daria J. Hazuda, Jacob D. Estes, Rafick-Pierre Sekaly, Mirko Paiardini
Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T-cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naïve and memory CD4+ T-cell subsets isolated from 24 participants. We found that the proportion of genetically-intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T-cells when compared with naïve, central, and transitional memory CD4+ T-cells. Interestingly, we found that escape mutations remained stable over time within effector memory T-cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically-intact HIV-1. These findings posit effector memory T-cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.
Gabriel Duette, Bonnie Hiener, Hannah Morgan, Fernando G. Mazur, Vennila Mathivanan, Bethany A. Horsburgh, Katie Fisher, Orion Tong, Eunok Lee, Haelee Ahn, Ansari Shaik, Rémi Fromentin, Rebecca Hoh, Charline Bacchus-Souffan, Najla Nasr, Anthony Cunningham, Peter W. Hunt, Nicolas Chomont, Stuart G. Turville, Steven G. Deeks, Anthony D. Kelleher, Timothy E. Schlub, Sarah Palmer
Studies using the nonhuman primate model of M. tuberculosis /Simian Immunodeficiency Virus co-infection have revealed protective CD4+ T cell-independent immune responses that suppress LTBI reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV co-infection. Here, we administered cART at 2 weeks post-SIV co-infection to study if restoration of CD4+ T cell immunity occurred more broadly, and if this prevented reactivation of LTBI compared to cART initiated at 4 weeks post-SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication and reduced immune activation in the periphery and lung vasculature thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed post SIV co-infection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The novelty of these findings mainly relates to the development of a robust animal model of human Mtb/HIV co-infection that allows the testing of underlying mechanisms.
Riti Sharan, Shashank R. Ganatra, Allison N. Bucsan, Journey Cole, Dhiraj K. Singh, Xavier Alvarez, Maya Gough, Cynthia Alvarez, Alyssa Blakley, Justin Ferdin, Rajesh Thippeshappa, Bindu Singh, Ruby Escobedo, Vinay Shivanna, Edward J. Dick, Jr., Shannan Hall-Ursone, Shabaana A. Khader, Smriti Mehra, Jyothi Rengarajan, Deepak Kaushal
Early initiation of antiretroviral therapy (ART) in acute HIV infection (AHI) is effective in limiting seeding of the HIV viral reservoir, but little is known about how the resultant decreased antigen load affects long-term antibody development after ART. We report here that Env-specific plasma antibody levels and antibody-dependent cellular cytotoxicity (ADCC) increased during the first 24 weeks of ART and correlated with antibody levels persisting after 48 weeks of ART. Participants treated in AHI stage 1 had lower Env-specific antibodies levels and ADCC activity on ART than those treated later. Importantly, participants who initiated ART after peak viremia in AHI developed elevated cross-clade ADCC responses detectable one year after ART initiation even though clinically undetectable viremia was reached by 24 weeks. These data suggest that there is more germinal center activity in the later stages of AHI and that antibody development continues in the absence of detectable viremia during the first year of suppressive ART. Development of therapeutic interventions that can enhance earlier development of germinal centers in AHI and antibodies after ART initiation could provide important protection against the viral reservoir that is seeded in early treated individuals.
Julie L. Mitchell, Justin Pollara, Kenneth Dietze, R. Whitney Edwards, Junsuke Nohara, Kombo F. N'guessan, Michelle Zemil, Supranee Buranapraditkun, Hiroshi Takata, Yifan Li, Roshell Muir, Eugene Kroon, Suteeraporn Pinyakorn, Shalini Jha, Sopark Manasnayakorn, Suthat Chottanapund, Pattarawat Thantiworasit, Peeriya Prueksakaew, Nisakorn Ratnaratorn, Bessara Nuntapinit, Lawrence Fox, Sodsai Tovanabutra, Dominic Paquin-Proulx, Lindsay Wieczorek, Victoria R. Polonis, Frank Maldarelli, Elias K. Haddad, Praphan Phanuphak, Carlo P. Sacdalan, Morgane Rolland, Nittaya Phanuphak, Jintanat Ananworanich, Sandhya Vasan, Guido Ferrari, Lydie Trautmann
To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics which recapitulate antigen-specific T-cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently-tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent anti-viral activities. Intravenous injection of HIV or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens, respectively. Notably, these expanded HIV or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections, respectively, in the humanized mice providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.
Mengyan Li, Scott J. Garforth, Kaitlyn E. O’Connor, Hang Su, Danica M. Lee, Alev Celikgil, Rodolfo J. Chaparro, Ronald D. Seidel, R. Brad Jones, Ravit Arav-Boger, Steven C. Almo, Harris Goldstein
Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (Mtb) followed by rapid progression to disease. Alveolar macrophages (AM) are the first cells of the innate immune system that engage Mtb, but how HIV and antiretroviral therapy (ART) impact on the anti-mycobacterial response of AM is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AM to Mtb, we obtained AM by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as pre-exposure prophylaxis (PrEP) to prevent HIV infection. Following in-vitro challenge with Mtb, AM from each group displayed overlapping but distinct profiles of significantly up- and down-regulated genes in response to Mtb. Comparatively, AM isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AM from HC subjects challenged with Mtb responded with pronounced chromatin accessibility changes while AM obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AM.
Wilian Correa-Macedo, Vinicius M. Fava, Marianna Orlova, Pauline Cassart, Ron Olivenstein, Joaquín Sanz, Yong Zhong Xu, Anne Dumaine, Renata H.M. Sindeaux, Vania Yotova, Alain Pacis, Josée Girouard, Barbara Kalsdorf, Christoph Lange, Jean-Pierre Routy, Luis B. Barreiro, Erwin Schurr
Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I–II predominated before, and stages V–VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.
Joana Dias, Giulia Fabozzi, Kylie March, Mangaiarkarasi Asokan, Cassandra G. Almasri, Jonathan Fintzi, Wanwisa Promsote, Yoshiaki Nishimura, John-Paul Todd, Jeffrey D. Lifson, Malcolm A. Martin, Lucio Gama, Constantinos Petrovas, Amarendra Pegu, John R. Mascola, Richard A. Koup
BACKGROUND. Chimeric antigen receptor (CAR)-modified T cells have emerged as a novel approach to treat malignant tumors. This strategy has also been proposed for the treatment of HIV-1 infection. We have developed a broadly neutralizing antibody (bNAb)-derived CAR-T cell therapy which can exerted specific cytotoxic activity against HIV-1-infected cells. METHODS. We conducted an open-label trial of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of bNAb-derived CAR-T cell therapy in HIV-1-infected individuals who were undergoing analytical interruption of antiretroviral therapy (ART). RESULTS. A total of 14 participants completed only a single administration of bNAb-derived CAR-T cells. CAR-T administration was safe and well tolerated. Six participants discontinued ART, and viremia rebound occurred in all of them, with a 5.3-week median time. Notably, the cell-associated viral RNA and intact proviruses decreased significantly after CAR-T treatment. Analyses of HIV-1 variants before or after CAR-T administration suggested that CAR-T cells exerted pressure on rebound viruses, resulting in a selection of viruses with less diversity and mutations against CAR-T-mediated cytotoxicity. CONCLUSIONS. No safety concerns were identified with adoptive transfer of bNAb-derived CAR-T cells. They reduced viral reservoir. All the rebounds were due to preexisting or emergence of viral escape mutations. TRIAL REGISTRATION. ClinicalTrials.gov number, NCT03240328. FUNDING. Ministry of Science and Technology of China, National Natural Science Foundation of China, and Department of Science and Technology of Guangdong Province.
Bingfeng Liu, Wanying Zhang, Baijin Xia, Shuliang Jing, Yingying Du, Fan Zou, Rong Li, Lijuan Lu, Shaozhen Chen, Yonghong Li, Qifei Hu, Yingtong Lin, Yiwen Zhang, Zhangping He, Xu Zhang, Xiejie Chen, Tao Peng, Xiaoping Tang, Weiping Cai, Ting Pan, Linghua Li, Hui Zhang