Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.
Julia A.M. Sung, Joy Pickeral, Liqin Liu, Sherry A. Stanfield-Oakley, Chia-Ying Kao Lam, Carolina Garrido, Justin Pollara, Celia LaBranche, Mattia Bonsignori, M. Anthony Moody, Yinhua Yang, Robert Parks, Nancie Archin, Brigitte Allard, Jennifer Kirchherr, JoAnn D. Kuruc, Cynthia L. Gay, Myron S. Cohen, Christina Ochsenbauer, Kelly Soderberg, Hua-Xin Liao, David Montefiori, Paul Moore, Syd Johnson, Scott Koenig, Barton F. Haynes, Jeffrey L. Nordstrom, David M. Margolis, Guido Ferrari
Group 3 innate lymphoid cells (ILC3s) have demonstrated roles in promoting antibacterial immunity, maintaining epithelial barrier function, and supporting tissue repair. ILC3 alterations are associated with chronic inflammation and inflammatory disease; however, the characteristics and relevant regulatory mechanisms of this cell population in HIV-1 infection are poorly understood due in part to a lack of a robust model. Here, we determined that functional human ILC3s develop in lymphoid organs of humanized mice and that persistent HIV-1 infection in this model depletes ILC3s, as observed in chronic HIV-1–infected patients. In HIV-1–infected mice, effective antiretroviral therapy reversed the loss of ILC3s. HIV-1–dependent reduction of ILC3s required plasmacytoid dendritic cells (pDCs), IFN-I, and the CD95/FasL pathway, as targeted depletion or blockade of these prevented HIV-1–induced ILC3 depletion in vivo and in vitro, respectively. Finally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. We conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis.
Zheng Zhang, Liang Cheng, Juanjuan Zhao, Guangming Li, Liguo Zhang, Weiwei Chen, Weiming Nie, Natalia J. Reszka-Blanco, Fu-Sheng Wang, Lishan Su
Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1–transmitting mothers and 165 propensity score–matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1–infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3–specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.
Sallie R. Permar, Youyi Fong, Nathan Vandergrift, Genevieve G. Fouda, Peter Gilbert, Robert Parks, Frederick H. Jaeger, Justin Pollara, Amanda Martelli, Brooke E. Liebl, Krissey Lloyd, Nicole L. Yates, R. Glenn Overman, Xiaoying Shen, Kaylan Whitaker, Haiyan Chen, Jamie Pritchett, Erika Solomon, Emma Friberg, Dawn J. Marshall, John F. Whitesides, Thaddeus C. Gurley, Tarra Von Holle, David R. Martinez, Fangping Cai, Amit Kumar, Shi-Mao Xia, Xiaozhi Lu, Raul Louzao, Samantha Wilkes, Saheli Datta, Marcella Sarzotti-Kelsoe, Hua-Xin Liao, Guido Ferrari, S. Munir Alam, David C. Montefiori, Thomas N. Denny, M. Anthony Moody, Georgia D. Tomaras, Feng Gao, Barton F. Haynes
Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100FY, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100FY fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100FY, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies.
Jordan R. Willis, Gopal Sapparapu, Sasha Murrell, Jean-Philippe Julien, Vidisha Singh, Hannah G. King, Yan Xia, Jennifer A. Pickens, Celia C. LaBranche, James C. Slaughter, David C. Montefiori, Ian A. Wilson, Jens Meiler, James E. Crowe Jr.
Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.
Gregory M. Laird, C. Korin Bullen, Daniel I.S. Rosenbloom, Alyssa R. Martin, Alison L. Hill, Christine M. Durand, Janet D. Siliciano, Robert F. Siliciano
Pierre-Alexandre Bart, Yunda Huang, Shelly T. Karuna, Samuel Chappuis, Julien Gaillard, Nidhi Kochar, Xiaoying Shen, Mary A. Allen, Song Ding, John Hural, Hua-Xin Liao, Barton F. Haynes, Barney S. Graham, Peter B. Gilbert, M. Juliana McElrath, David C. Montefiori, Georgia D. Tomaras, Giuseppe Pantaleo, Nicole Frahm
Heme oxygenase-1 (HO-1) is an inducible, detoxifying enzyme that is critical for limiting oxidative stress, inflammation, and cellular injury within the CNS and other tissues. Here, we demonstrate a deficiency of HO-1 expression in the brains of HIV-infected individuals. This HO-1 deficiency correlated with cognitive dysfunction, HIV replication in the CNS, and neuroimmune activation. In vitro analysis of HO-1 expression in HIV-infected macrophages, a primary CNS HIV reservoir along with microglia, demonstrated a decrease in HO-1 as HIV replication increased. HO-1 deficiency correlated with increased culture supernatant glutamate and neurotoxicity, suggesting a link among HIV infection, macrophage HO-1 deficiency, and neurodegeneration. HO-1 siRNA knockdown and HO enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with nonselective pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. Furthermore, IFN-γ, which is increased in CNS HIV infection, reduced HO-1 expression in cultured human astrocytes and macrophages. These findings indicate that HO-1 is a protective host factor against HIV-mediated neurodegeneration and suggest that HO-1 deficiency contributes to this degeneration. Furthermore, these results suggest that HO-1 induction in the CNS of HIV-infected patients on antiretroviral therapy could potentially protect against neurodegeneration and associated cognitive dysfunction.
Alexander J. Gill, Colleen E. Kovacsics, Stephanie A. Cross, Patricia J. Vance, Lorraine L. Kolson, Kelly L. Jordan-Sciutto, Benjamin B. Gelman, Dennis L. Kolson
The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one
Shuying S. Li, Peter B. Gilbert, Georgia D. Tomaras, Gustavo Kijak, Guido Ferrari, Rasmi Thomas, Chul-Woo Pyo, Susan Zolla-Pazner, David Montefiori, Hua-Xin Liao, Gary Nabel, Abraham Pinter, David T. Evans, Raphael Gottardo, James Y. Dai, Holly Janes, Daryl Morris, Youyi Fong, Paul T. Edlefsen, Fusheng Li, Nicole Frahm, Michael D. Alpert, Heather Prentice, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Jaranit Kaewkungwal, Sorachai Nitayaphan, Merlin L. Robb, Robert J. O’Connell, Barton F. Haynes, Nelson L. Michael, Jerome H. Kim, M. Juliana McElrath, Daniel E. Geraghty
Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.
Lela Kardava, Susan Moir, Naisha Shah, Wei Wang, Richard Wilson, Clarisa M. Buckner, Brian H. Santich, Leo J.Y. Kim, Emily E. Spurlin, Amy K. Nelson, Adam K. Wheatley, Christopher J. Harvey, Adrian B. McDermott, Kai W. Wucherpfennig, Tae-Wook Chun, John S. Tsang, Yuxing Li, Anthony S. Fauci
Interaction of the chemokine CXCL12 with its receptor CXCR4 promotes neuronal function and survival during embryonic development and throughout adulthood. Previous studies indicated that μ-opioid agonists specifically elevate neuronal levels of the protein ferritin heavy chain (FHC), which negatively regulates CXCR4 signaling and affects the neuroprotective function of the CXCL12/CXCR4 axis. Here, we determined that CXCL12/CXCR4 activity increased dendritic spine density, and also examined FHC expression and CXCR4 status in opiate abusers and patients with HIV-associated neurocognitive disorders (HAND), which is typically exacerbated by illicit drug use. Drug abusers and HIV patients with HAND had increased levels of FHC, which correlated with reduced CXCR4 activation, within cortical neurons. We confirmed these findings in a nonhuman primate model of SIV infection with morphine administration. Transfection of a CXCR4-expressing human cell line with an iron-deficient FHC mutant confirmed that increased FHC expression deregulated CXCR4 signaling and that this function of FHC was independent of iron binding. Furthermore, examination of morphine-treated rodents and isolated neurons expressing FHC shRNA revealed that FHC contributed to morphine-induced dendritic spine loss. Together, these data implicate FHC-dependent deregulation of CXCL12/CXCR4 as a contributing factor to cognitive dysfunction in neuroAIDS.
Jonathan Pitcher, Anna Abt, Jaclyn Myers, Rachel Han, Melissa Snyder, Alessandro Graziano, Lindsay Festa, Michele Kutzler, Fernando Garcia, Wen-Jun Gao, Tracy Fischer-Smith, Jay Rappaport, Olimpia Meucci