Joseph Kulkosky

IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

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.

Intensification and Stimulation Therapy for Human Immunodeficiency Virus Type 1 Reservoirs in Infected Persons Receiving Virally Suppressive Highly Active Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.

In a Subset of Subjects on Highly Active Antiretroviral Therapy, Human Immunodeficiency Virus Type 1 RNA in Plasma Decays from 50 to <5 Copies per Milliliter, with a Half-Life of 6 Months

ABSTRACT Three of five virally suppressed human immunodeficiency virus type I (HIV-1)-infected patients treated with highly active antiretroviral therapy and followed intensively with a supersensitive reverse transcriptase PCR assay with a lower limit of quantitation of 5 copies/ml showed statistically significant viral load decays below 50 copies/ml, with half-lives of 5 to 8 months and a mean of 6 months. This range of half-lives is consistent with the estimated half-life of the latent HIV-1 reservoir in the peripheral blood. Those patients without decay of viral load in plasma may have significant cryptic HIV-1 residual replication.

Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons.

Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extra-cellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.

HAART-persistent HIV-1 latent reservoirs: their origin, mechanisms of stability and potential strategies for eradication.

HIV-1 infection persists despite long-term administration of highly active antiretroviral therapy (HAART). The mechanism of this persistence appears to result primarily from viral infection of CD4+ T-lymphocytes that have the ability to duplicate and revert into a quiescent state. These infected resting cells are long-lived and evade immune surveillance or clearance. The inability to eradicate this class of cells, bearing the viral DNA, suggests life-long persistence of virus in HIV-1-infected individuals, even if HAART were administered for decades. This review discusses the origins and mechanisms accounting for stability of these latent HIV-1 cellular reservoirs. It further provides an overview of recent clinical trials aimed at their eradication. There have been a limited number of immune activation (IAT) trials directed at HAART-persistent, viral reservoir eradication. These trials have not resulted in purging of these highly stable viral reservoirs though results from such efforts suggest partial effects. The properties of novel compounds that might be included into IAT eradication protocols are continuing to be evaluated and their potential for inclusion into future IAT trials will be discussed.

Protein Kinase C: One Pathway towards the Eradication of Latent HIV-1 Reservoirs

An effective means to eradicate latent reservoirs in HIV-1-infected individuals remains elusive. Attempts to purge these reservoirs were undertaken over a decade ago without success. The subsequent lapse in further clinical attempts since may have been justified as our knowledge of the mechanisms which underpin the latent state still evolves. Although additional novel molecular antagonists of HIV-1 latency have subsequently been reported, these candidate agents have not been tested in human trials for reservoir ablation. This review provides an overview of the protein kinase C (PKC) pathway which can be modulated by small molecular agents to induce the expression of latent HIV-1 from within infected reservoir cells. Some of these agents have been tested against select cancers with seemingly tolerable side effects. As such, modulation of the PKC pathway may yet be a viable mechanism toward HIV-1 reservoir eradication.

High-Frequency Epigenetic Repression and Silencing of Retroviruses Can Be Antagonized by Histone Deacetylase Inhibitors and Transcriptional Activators, but Uniform Reactivation in Cell Clones Is Restricted by Additional Mechanisms

ABSTRACT Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(−)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(−) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(−) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(−) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(−) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

Pathogenesis of HIV-1 Infection Within Bone Marrow Cells

Mononuclear phagocytic cells and CD4+ T lymphocytes represent the major targets for infection by HIV-1 in vivo. The most severe pathogenic features associated with HIV-1 infection can be attributed to malfunction or premature death of these cells that are of hematopoietic origin. Patients with acquired immunodeficiency syndrome (AIDS), suffer from many hematologic disorders, particularly those persons with long-term infection of HIV-1. These disorders include anemia, lymphocytopenia, thrombocytopenia and neutropenia. The mechanisms that lead to the induction of these disorders are multi-factorial. However, sufficient evidence has accumulated which suggests that HIV-1 infection of cells within the microenvironment of the bone marrow can lead to the induction of hematopoietic deficits. Most studies indicate that marrow-derived hematopoeitic stem cells cannot be infected by HIV-1 until they undergo modest differentiation in order to express the appropriate receptors to enable virus entry and subsequent replication. Some cells within the mixed environment of the marrow stroma appear to support HIV-1 replication however. These cells include marrow microvascular endothelial cells, sometimes referred to as blanket cells, stromal fibroblasts, as well as mononuclear phagocytes. Our recent experiments suggest that the HIV-1 accessory protein, Vpr, plays some role in the activation of marrow-derived mononuclear phagocytes which appears to result in premature phagocytosis of non-adherent marrow cells present in the in vitro cultures. This phenomenon could account, in part, for the induction of cytopenias that are typical of individuals infected by HIV-1.

Genotypic alteration of HAART-persistent HIV-1 reservoirs in vivo

Three HIV-1-infected individuals, on virally-suppressive highly active anti-retroviral therapy (HAART), were treated in vivo with anti-retroviral inhibitor intensification and cell stimulatory therapies in attempting to eradicate latent viral reservoirs. Afterwards, the patients ceased all anti-retroviral drugs. Sequences of the V3 region of HIV-1 envelope protein (ENV) from patient peripheral blood mononuclear cell (PBMC) proviral DNA, patient blood plasma viral RNA and virion-associated RNA from viruses amplified by patient cell co-culture, were obtained before, during, and certain times after the clinical regimen. As anticipated, the V3 loop sequencing results indicate diversity in viral strain complexity among the individual patients. However, the detection of unique V3 ENV signature sequences or V3 signatures of low frequency, relative to those observed prior to therapy, indicate that the expression of specific viruses, or viruses of low abundance, can be induced through stimulation in vivo. Furthermore, this stimulation or general immune activation therapy (IAT) approach, consisting of administration of the anti-T-cell receptor antibody, OKT3, and IL-2 in vivo, appeared to have subsequently altered the genotype of the persistent viral reservoir in peripheral blood cells for two of the three patients.

In Vivo Effects of Antiviral Protein Kinase C Modulators on Zebrafish Development and Survival

Clinical interventions using protein kinase C (PKC) modulators have been proposed for eradication of HIV-1-infected cellular reservoirs which persist in patients despite prolonged antiretroviral therapy. The effects of some of these agents have not been assessed in a developing vertebrate model. This study examines the developmental and toxicological effects of these compounds on zebrafish embryos and larvae. Treatment of zebrafish through the first week of development with various PKC pathway modulators did not elicit gross physical defects or elevated incidences of death at lower doses. Higher concentrations resulted in rapid death for both later-stage embryos and larvae. Each compound had a threshold dose for lethality. The defined nonlethal doses may be useful toward assessing the effects of modulating PKC activity on zebrafish development. They may further provide some guidance for the potential dosing of PKC modulators in clinical trials toward the goal of HIV-1 reservoir eradication.