Loss of memory B cells occurs from the onset of HIV-1 infection and persists into the chronic stages of infection. Lack of survival of these cells, even in subjects being treated, could primarily be the consequence of an altered local microenvironment induced by HIV infection. In this study we showed that memory B cell survival was significantly decreased in aviremic successfully treated (ST) subjects compared with subjects who control viral load as a result of natural immunity (elite controller [EC]) or with uninfected control (HIV–) subjects. The lower survival levels observed in memory B cells from ST subjects were the result of disrupted IL-2 signaling that led to increased transcriptional activity of Foxo3a and increased expression of its proapoptotic target TRAIL. Notably, memory B cell survival in ST subjects was significantly enhanced by the addition of exogenous IL-2 in a Foxo3a-dependent manner. We further showed that Foxo3a silencing by siRNA resulted in decreased expression of TRAIL and apoptosis levels in memory B cells from ST subjects. Our results thus establish a direct role for Foxo3a/TRAIL signaling in the persistence of memory B cells and provide a mechanism for the reduced survival of memory B cells during HIV infection. This knowledge could be exploited for the development of therapeutic and preventative HIV vaccines.
Julien van Grevenynghe, Rafael A. Cubas, Alessandra Noto, Sandrina DaFonseca, Zhong He, Yoav Peretz, Abdelali Filali-Mouhim, Franck P. Dupuy, Francesco A. Procopio, Nicolas Chomont, Robert S. Balderas, Elias A. Said, Mohamed-Rachid Boulassel, Cecile L. Tremblay, Jean-Pierre Routy, Rafick-Pierre Sékaly, Elias K. Haddad
Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor–mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor–like–4 (FCRL4) and sialic acid–binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell–associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections.
Lela Kardava, Susan Moir, Wei Wang, Jason Ho, Clarisa M. Buckner, Jacqueline G. Posada, Marie A. O’Shea, Gregg Roby, Jenny Chen, Hae Won Sohn, Tae-Wook Chun, Susan K. Pierce, Anthony S. Fauci
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Lee Adam Wheeler, Radiana Trifonova, Vladimir Vrbanac, Emre Basar, Shannon McKernan, Zhan Xu, Edward Seung, Maud Deruaz, Tim Dudek, Jon Ivar Einarsson, Linda Yang, Todd M. Allen, Andrew D. Luster, Andrew M. Tager, Derek M. Dykxhoorn, Judy Lieberman
High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4+ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1–induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4+ and CD8+ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4+ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.
Margalida Rotger, Judith Dalmau, Andri Rauch, Paul McLaren, Steve Bosinger, Raquel Martinez, Netanya G. Sandler, Annelys Roque, Julia Liebner, Manuel Battegay, Enos Bernasconi, Patrick Descombes, Itziar Erkizia, Jacques Fellay, Bernard Hirschel, Jose M. Miró, Eduard Palou, Matthias Hoffmann, Marta Massanella, Julià Blanco, Matthew Woods, Huldrych F. Günthard, Paul de Bakker, Daniel C. Douek, Guido Silvestri, Javier Martinez-Picado, Amalio Telenti
Elite controllers represent a unique group of HIV-1–infected persons with undetectable HIV-1 replication in the absence of antiretroviral therapy. However, the mechanisms contributing to effective viral immune defense in these patients remain unclear. Here, we show that compared with HIV-1 progressors and HIV-1–negative persons, CD4+ T cells from elite controllers are less susceptible to HIV-1 infection. This partial resistance to HIV-1 infection involved less effective reverse transcription and mRNA transcription from proviral DNA and was associated with strong and selective upregulation of the cyclin-dependent kinase inhibitor p21 (also known as cip-1 and waf-1). Experimental blockade of p21 in CD4+ T cells from elite controllers resulted in a marked increase of viral reverse transcripts and mRNA production and led to higher enzymatic activities of cyclin-dependent kinase 9 (CDK9), which serves as a transcriptional coactivator of HIV-1 gene expression. This suggests that p21 acts as a barrier against HIV-1 infection in CD4+ T cells from elite controllers by inhibiting a cyclin-dependent kinase required for effective HIV-1 replication. These data demonstrate a mechanism of host resistance to HIV-1 in elite controllers and may open novel perspectives for clinical strategies to prevent or treat HIV-1 infection.
Huabiao Chen, Chun Li, Jinghe Huang, Thai Cung, Katherine Seiss, Jill Beamon, Mary F. Carrington, Lindsay C. Porter, Patrick S. Burke, Yue Yang, Bethany J. Ryan, Ruiwu Liu, Robert H. Weiss, Florencia Pereyra, William D. Cress, Abraham L. Brass, Eric S. Rosenberg, Bruce D. Walker, Xu G. Yu, Mathias Lichterfeld
The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.
Hung-Chih Yang, Sifei Xing, Liang Shan, Karen O’Connell, Jason Dinoso, Anding Shen, Yan Zhou, Cynthia K. Shrum, Yefei Han, Jun O. Liu, Hao Zhang, Joseph B. Margolick, Robert F. Siliciano
New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2–/–γc–/– mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.
Martha R. Neagu, Patrick Ziegler, Thomas Pertel, Caterina Strambio-De-Castillia, Christian Grütter, Gladys Martinetti, Luca Mazzucchelli, Markus Grütter, Markus G. Manz, Jeremy Luban
HIV infection results in CD4+ T cell deficiency, but efficient combination antiretroviral therapy (c-ART) restores T cells and decreases morbidity and mortality. However, immune restoration by c-ART remains variable, and prolonged T cell deficiency remains in a substantial proportion of patients. In a prospective open-label phase I/IIa trial, we evaluated the safety and efficacy of administration of the T cell regulator IL-7. The trial included 13 c-ART–treated HIV-infected patients whose CD4+ cell counts were between 100 and 400 cells/μl and plasma HIV RNA levels were less than 50 copies/ml. Patients received a total of 8 subcutaneous injections of 2 different doses of recombinant human IL-7 (rhIL-7; 3 or 10 μg/kg) 3 times per week over a 16-day period. rhIL-7 was well tolerated and induced a sustained increase of naive and central memory CD4+ and CD8+ T cells. In the highest dose group, 4 patients experienced transient increases in viral replication. However, functional assays showed that the expanded T cells responded to HIV antigen by producing IFN-γ and/or IL-2. In conclusion, in lymphopenic HIV-infected patients, rhIL-7 therapy induced substantial functional and quantitative changes in T cells for 48 weeks. Therefore, patients may benefit from intermittent therapy with IL-7 in combination with c-ART.
Yves Levy, Christine Lacabaratz, Laurence Weiss, Jean-Paul Viard, Cecile Goujard, Jean-Daniel Lelièvre, François Boué, Jean-Michel Molina, Christine Rouzioux, Véronique Avettand-Fénoêl, Thérèse Croughs, Stéphanie Beq, Rodolphe Thiébaut, Geneviève Chêne, Michel Morre, Jean-François Delfraissy
Genital coinfections increase an individual’s risk of becoming infected with HIV-1 by sexual contact. Several mechanisms have been proposed to explain this, such as the presence of ulceration and bleeding caused by the coinfecting pathogen. Here we demonstrate that Langerhans cells (LCs) are involved in the increased susceptibility to HIV-1 in the presence of genital coinfections. Although LCs are a target for HIV-1 infection in genital tissues, we found that immature LCs did not efficiently mediate HIV-1 transmission in an ex vivo human skin explant model. However, the inflammatory stimuli TNF-α and Pam3CysSerLys4 (Pam3CSK4), the ligand for the TLR1/TLR2 heterodimer, strongly increased HIV-1 transmission by LCs through distinct mechanisms. TNF-α enhanced transmission by increasing HIV-1 replication in LCs, whereas Pam3CSK4 acted by increasing LC capture of HIV-1 and subsequent trans-infection of T cells. Genital infections such as Candida albicans and Neisseria gonorrhea not only triggered TLRs but also induced TNF-α production in vaginal and skin explants. Thus, during coinfection, LCs could be directly activated by pathogenic structures and indirectly activated by inflammatory factors, thereby increasing the risk of acquiring HIV-1. Our data demonstrate a decisive role for LCs in HIV-1 transmission during genital coinfections and suggest antiinflammatory therapies as potential strategies to prevent HIV-1 transmission.
Marein A.W.P. de Jong, Lot de Witte, Menno J. Oudhoff, Sonja I. Gringhuis, Philippe Gallay, Teunis B.H. Geijtenbeek
Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Δ), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8–labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Δ cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Δ C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.
Guowu Hu, Moshe Hacham, Scott R. Waterman, John Panepinto, Soowan Shin, Xiaoguang Liu, Jack Gibbons, Tibor Valyi-Nagy, Keisuke Obara, H. Ari Jaffe, Yoshinori Ohsumi, Peter R. Williamson