Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein–1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-κB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.
Mireille Centlivre, Peter Sommer, Marie Michel, Raphaël Ho Tsong Fang, Sandrine Gofflo, Jenny Valladeau, Nathalie Schmitt, Françoise Thierry, Bruno Hurtrel, Simon Wain-Hobson, Monica Sala
HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1–specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.
Thomas J. Scriba, Hua-Tang Zhang, Helen L. Brown, Annette Oxenius, Norbert Tamm, Sarah Fidler, Julie Fox, Jonathan N. Weber, Paul Klenerman, Cheryl L. Day, Michaela Lucas, Rodney E. Phillips
The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4+ T lymphocytes from HIV-1–infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4+ T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.
Feng-Xiang Wang, Yan Xu, Julie Sullivan, Emily Souder, Elias G. Argyris, Edward A. Acheampong, Jaime Fisher, Maria Sierra, Michael M. Thomson, Rafael Najera, Ian Frank, Joseph Kulkosky, Roger J. Pomerantz, Giuseppe Nunnari
The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell–tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.
Ilona Hauber, Dorian Bevec, Jochen Heukeshoven, Friedrich Krätzer, Florian Horn, Axel Choidas, Thomas Harrer, Joachim Hauber
Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone–induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor κB ligand–induced (RANKL-induced) activation of NF-κB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor–associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART.
Michael W.-H. Wang, Shi Wei, Roberta Faccio, Sunao Takeshita, Pablo Tebas, William G. Powderly, Steven L. Teitelbaum, F. Patrick Ross
Antiplatelet GPIIIa49–66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS.
Michael Nardi, Steven J. Feinmark, Liang Hu, Zongdong Li, Simon Karpatkin
HIV infection in humans and simian immunodeficiency virus (SIV) infection in macaques result in encephalitis in approximately one-quarter of infected individuals and is characterized by infiltration of the brain with infected and activated macrophages. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195) is a ligand specific for the peripheral benzodiazepine receptor abundant on macrophages and is expressed in low levels in the noninfected brain. We hypothesized that positron-emission tomography (PET) with the carbon-11–labeled, R-enantiomer form of PK11195 ([11C](R)-PK11195) could image brain macrophages and hence the development of encephalitis in vivo. [11C](R)-PK11195 binding was assessed in the brain using PET in 11 SIV infected macaques, six of which showed increased binding in vivo. Postmortem examination of the brain in these six macaques demonstrated encephalitis, while macaques that did not show an increase in [11C](R)-PK11195 binding did not develop SIV encephalitis. Brain tissue from SIV encephalitic macaques also showed increased [3H](R)-PK11195 binding compared with binding in nonencephalitic macaques. Increased PK11195 binding in vivo and in postmortem brain tissue correlated with abundance of macrophages but not astrocytes. Our results suggest that PET [11C](R)-PK11195 imaging can detect the presence of macrophages in SIV encephalitis in vivo and may be useful to predict the development of HIV encephalitis and in studies of the pathogenesis and treatment of HIV dementia.
Sriram Venneti, Brian J. Lopresti, Guoji Wang, Stephanie J. Bissel, Chester A. Mathis, Carolyn C. Meltzer, Fernando Boada, Saverio Capuano III, Geraldine J. Kress, Denise K. Davis, James Ruszkiewicz, Ian J. Reynolds, Michael Murphey-Corb, Anita M. Trichel, Stephen R. Wisniewski, Clayton A. Wiley
Transcutaneous immunization (TCI), the application of vaccines on the skin, induces robust systemic and mucosal antibodies in animal models and in humans. The means by which mucosal immune responses to vaccine antigens are elicited by TCI has not been well characterized. We examined the effect of TCI with an HIV peptide vaccine on the induction of mucosal and systemic CTL responses and protective immunity against mucosal challenge with live virus in mice. Robust HIV-specific CTL responses in the spleen and in the gut mucosa were detected after TCI. The responses were dependent upon the addition of an adjuvant and resulted in protection against mucosal challenge with recombinant vaccinia virus encoding HIV gp160. Although it is clear that adjuvant-activated DCs migrated mainly to draining lymph nodes, coculture with specific T cells and flow cytometry studies with DCs isolated from Peyer’s patches after TCI suggested that activated DCs carrying skin-derived antigen also migrated from the skin to immune-inductive sites in gut mucosa and presented antigen directly to resident lymphocytes. These results and previous clinical trial results support the observation that TCI is a safe and effective strategy for inducing strong mucosal antibody and CTL responses.
Igor M. Belyakov, Scott A. Hammond, Jeffrey D. Ahlers, Gregory M. Glenn, Jay A. Berzofsky
In vivo blockade of CD28 and CD40 T cell costimulation pathways during acute simian immunodeficiency virus (SIV) infection of rhesus macaques was performed to assess the relative contributions of CD4+ T cells, CD8+ T cells, and Ab responses in modulating SIV replication and disease progression. Transient administration of CTLA4-Ig and anti–CD40L mAb to SIV-infected rhesus macaques resulted in dramatic inhibition of the generation of both SIV-specific cellular and humoral immune responses. Acute levels of proliferating CD8+ T cells were associated with early control of SIV viremia but did not predict ensuing set point viremia or survival. The level of in vivo CD4+ T cell proliferation during acute SIV infection correlated with concomitant peak levels of SIV plasma viremia, whereas measures of in vivo CD4+ T cell proliferation that extended into chronic infection correlated with lower SIV viral load and increased survival. These results suggest that proliferating CD4+ T cells function both as sources of virus production and as antiviral effectors and that increased levels of CD4+ T cell proliferation during SIV infections reflect antigen-driven antiviral responses rather than a compensatory homeostatic response. These results highlight the interrelated actions of CD4+ and CD8+ T cell responses in vivo that modulate SIV replication and pathogenesis.
David A. Garber, Guido Silvestri, Ashley P. Barry, Andrew Fedanov, Natalia Kozyr, Harold McClure, David C. Montefiori, Christian P. Larsen, John D. Altman, Silvija I. Staprans, Mark B. Feinberg
Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor–dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor–induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor–induced atherosclerosis. Finally, ritonavir increased PPAR-γ and CD36 mRNA levels in a PKC- and PPAR-γ–dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.
James Dressman, Jeanie Kincer, Sergey V. Matveev, Ling Guo, Richard N. Greenberg, Theresa Guerin, David Meade, Xiang-An Li, Weifei Zhu, Annette Uittenbogaard, Melinda E. Wilson, Eric J. Smart