The differential short- and long-term effects of HIV-1 latency-reversing agents on T cell function.

Despite the extraordinary success of HIV-1 antiretroviral therapy in prolonging life, infected individuals face lifelong therapy because of a reservoir of latently-infected cells that harbor replication competent virus. Recently, compounds have been identified that can reverse HIV-1 latency in vivo. These latency- reversing agents (LRAs) could make latently-infected cells vulnerable to clearance by immune cells, including cytolytic CD8+ T cells. We investigated the effects of two leading LRA classes on CD8+ T cell phenotype and function: the histone deacetylase inhibitors (HDACis) and protein kinase C modulators (PKCms). We observed that relative to HDACis, the PKCms induced much stronger T cell activation coupled with non-specific cytokine production and T cell proliferation. When examining antigen-specific CD8+ T cell function, all the LRAs except the HDACi Vorinostat reduced, but did not abolish, one or more measurements of CD8+ T cell function. Importantly, the extent and timing of these effects differed between LRAs. Panobinostat had detrimental effects within 10 hours of drug treatment, whereas the effects of the other LRAs were observed between 48 hours and 5 days. These observations suggest that scheduling of LRA and CD8+ T cell immunotherapy regimens may be critical for optimal clearance of the HIV-1 reservoir.

HIV eradication symposium: will the brain be left behind?

On 18 July 2014, the National Institute of Mental Health in collaboration with ViiV Health Care and Boehringer Ingelheim supported a symposium on HIV eradication and what it meant for the brain. The symposium was an affiliated event to the 20th International AIDS Conference. The meeting was held in Melbourne, Australia, and brought together investigators currently working on HIV eradication together with investigators who are working on the neurological complications of HIV. The purpose of the meeting was to bring the two fields of HIV eradication and HIV neurology together to foster dialogue and cross talk to move the eradication field forward in the context of issues relating to the brain as a potential reservoir of HIV. The outcomes of the symposium were that there was substantive but not definitive evidence for the brain as an HIV reservoir that will provide a challenge to HIV eradication. Secondly, the brain as a clinically significant reservoir for HIV is not necessarily present in all patients. Consequently, there is an urgent need for the development of biomarkers to identify and quantify the HIV reservoir in the brain. Lastly, when designing and developing eradication strategies, it is critical that approaches to target the brain reservoir be included.

Prospects for treatment of latent HIV.

Recent advances in antiretroviral therapy (ART) have drastically improved the quality of life for people with HIV infection. However, owing to the persistence of latent HIV in the presence of therapy, patients must remain on therapy indefinitely. Currently, the solution to the HIV pandemic rests on the prevention of new infections and many decades of ART for the steadily expanding number of people infected worldwide. ART is costly, requires ongoing medical care, and can have side effects, thereby preventing its universal availability. Therefore, to escape the ironic burdens of therapy, efforts have begun to develop treatments for latent HIV infection. Current approaches propose either complete eradication of infection or induction of a state of stringent control over viral replication without ART. This review will discuss these strategies in detail and their potential for clinical development.

Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy.

Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Neurocognitive effects of treatment interruption in stable HIV-positive patients in an observational cohort.

OBJECTIVE: Prior studies have shown improved neurocognition with initiation of antiretroviral treatment (ART) in HIV. We hypothesized that stopping ART would be associated with poorer neurocognitive function. METHODS: Neurocognitive function was assessed as part of ACTG 5170, a multicenter, prospective observational study of HIV-infected subjects who elected to discontinue ART. Eligible subjects had CD4 count >350 cells/mm(3), had HIV RNA viral load <55,000 cp/mL, and were on ART (>or=2 drugs) for >or=6 months. Subjects stopped ART at study entry and were followed for 96 weeks with a neurocognitive examination. RESULTS: A total of 167 subjects enrolled with a median nadir CD4 of 436 cells/mm(3) and 4.5 median years on ART. Significant improvements in mean neuropsychological scores of 0.22, 0.39, 0.53, and 0.74 were found at weeks 24, 48, 72, and 96 (all p < 0.001). In the 46 subjects who restarted ART prior to week 96, no significant changes in neurocognitive function were observed. CONCLUSION: Subjects with preserved immune function found that neurocognition improved significantly following antiretroviral treatment (ART) discontinuation. The balance between the neurocognitive cost of untreated HIV viremia and the possible toxicities of ART require consideration. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that discontinuing ART is associated with an improvement in 2 neuropsychological tests (Trail-Making Test A & B and the Wechsler Adult Intelligence Scale-Revised Digit Symbol subtest) for up to 96 weeks. Resuming ART was not associated with a decline in these scores for up to 45 weeks.

A prospective evaluation of the effect of atazanavir on the QTc interval and QTc dispersion in HIV-positive patients.

BACKGROUND: Atazanavir (ATV), an HIV protease inhibitor (PI) that may be preferred for the treatment of HIV-infected patients with cardiovascular comorbidities because of its favourable effects on plasma lipids, has been associated with cardiac rhythm disturbances. OBJECTIVE: To quantify the effect of ATV on corrected QT (QTc) and QTc dispersion (QTd), markers of the potential for cardiac dysrhythmia, in patients switching from other PIs to ATV. METHODS: In this prospective, single-centre, open-label study, 12-lead electrocardiograms were performed for subjects at baseline, 2 h after the first dose of ATV, and 1 month after initiation of ATV. RESULTS: Twenty-one patients (19 received ritonavir-boosted ATV) completed the study. There was a trend towards an increase in the QTc at 2 h after the first dose [mean+/-standard deviation 3.19+/-8.0 ms; 95% confidence interval (CI) -0.47 to 6.85 ms; P=0.084]. There was no difference between QTc values at baseline and at 1 month (-1.5+/-8.75 ms; 95% CI -5.50 to 2.46; P=0.43). There was a nonsignificant decrease in the QTd between baseline and 2 h (-5.1+/-15.19 ms; 95% CI -13.22 to 2.96; P=0.197) and between baseline and 1 month (-0.61+/-15.04 ms; 95% CI -8.1 to 6.87; P=0.865). A significant increase in the PR interval (7.4+/-10.7 ms; 95% CI 2.5 to 12.25 ms; P=0.005) was observed at 1 month. CONCLUSIONS: The use of ATV did not result in increases in the QTc interval or QTd. However, PR interval monitoring may be warranted in patients with underlying heart block or those treated with atrioventricular nodal blocking agents.

A pilot study of the use of mycophenolate mofetil as a component of therapy for multidrug-resistant HIV-1 infection.

Mycophenolic acid (MPA) increases the activity of both abacavir (ABC) and didanosine (ddI) in vitro against wild-type and multinucleoside-resistant HIV. We treated 7 patients with diagnosed AIDS who did not respond to eight or more antiretroviral therapies in an open label pilot study with mycophenolate mofetil (MMF), ABC, ddI, amprenavir (APV), and ritonavir (RTV), with or without efavirenz (EFV). Therapy was well tolerated despite the patients' advanced disease states. No significant decline in lymphocyte or other blood counts was observed. Median HIV RNA was 5.26 log10 copies/ml at entry, 4.53 log10 copies/ml at 4 weeks, and 5.13 log10 copies/ml at 16 weeks. Median CD4+ count was 34 cells/microl at entry and 39 cells/microl at 16 weeks of therapy. CD4+ counts increased further in five study subjects on extended therapy to 25 weeks (median 27 cells/microl at entry, 66 cells/microl at close), despite loss of virologic suppression in 4 of 5 cases. MPA can induce apoptosis in lymphocytes in vitro. However despite viral rebound, cell surface markers of apoptosis and activation declined in total CD3+ cells and CD3+/CD4+ cells twofold to fourfold in 4 of 5 adherent study subjects at 16 weeks, reaching levels comparable with those found in seronegative donors. Although low-dose MMF appears safe in late-stage HIV disease, this study did not demonstrate virologic efficacy. Higher doses of MMF may be more effective. With careful monitoring of toxicities and pharmacokinetics, MMF deserves further testing in HIV therapy.

The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1.

Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity of CXCR4, a coreceptor for HIV-1 entry.

Transcription factors USF1 and USF2 up-regulate gene expression (i.e. , HIV-1 long terminal repeats) via interaction with an E box on their target promoters, which is also a binding site for c-Myc. The c-Myc oncoprotein is important in control of cellular proliferation and differentiation, while Yin-Yang 1 (YY1) has been shown to control the expression of a number of cellular and viral genes. These two proteins physically interact with each other and mutually inhibit their respective biological functions. In this study, we show that USF/c-Myc up-regulates, while YY1 down-regulates the promoter activity of CXCR4, a coreceptor for T cell-tropic HIV-1 entry. We have identified an E box around -260 and a YY1 binding site around -300 relative to the transcription start site. Mutation of the E box abolished USF/c-Myc-mediated up-regulation of CXCR4 promoter activity, and mutation of the YY1 binding site was associated with unresponsiveness to YY1-mediated inhibition. These data suggest that USF/c-Myc and YY1 may play an important role in the HIV-1-replicative cycle, by modulating both the viral fusion/entry process and viral expression.

In Vitro Techniques for Studies of HIV-1 Promoter Activity.

A rather unique feature of the human immunodeficiency virus type-1 (HIV-1) is the structural complexity of the regulatory sequences located in the long-terminal repeat (LTR) promoter region and the number of cellular and viral transcription factors known to interact with these sequences and modulate HIV gene expression see ref. 1). The HIV-1 LTR can be schematically divided into four functional regions: (1) the negative regulatory element (NRE) encompassing nuceotides -350 to -190 with respect to the transcription start site; (2) the enhancer (-140 to-81), containing two binding sites for the transcription factor NFκB; (3) the basal promoter (located between -80 and +1), including a typical TATAA box and three binding sites for the transcription factor Sp1; and (4) the trans-activation response (TAR) element, a bulged stem-and-loop structure present in the nascent RNA (+1 to +59) transcript that provides a binding site for Tat activation of HIV-1 transcription. In addition, a novel regulatory DNA element, named IST (Initiator of Short Transcripts), has been shown to be present in the HIV-1 LTR (position (-)5 to (+)26), encompassing the binding site for transcription factors YY1 and late SV40 transcription factor (LSF, or CP-2, or LBP-1) (see refs. 2 and 3). IST directs the RNA polymerase II to synthesize short (59-61 nt), correctly initiated, nonpolyadenylated transcripts that prematurely terminate at the TAR stem-loop structure. The function of these transcripts remains unclear (see ref. 4).

Activation of CD8+ T lymphocytes through the T cell receptor turns on CD4 gene expression: implications for HIV pathogenesis.

T cell activation through the T cell receptor is necessary to achieve a specific and effective immune response. We report here that stimulation of CD8+ T cells through the T cell receptor complex leads to de novo expression of the CD4 antigen on the cell surface that results in susceptibility of CD8+ T cells to HIV infection. In addition, activation of peripheral blood mononuclear cells from HIV-infected individuals results in the appearance of double-positive CD4+/CD8+ T cells, which become infected by endogenous HIV. HIV DNA sequences could be detected in uncultured and sorted mature CD3+CD8+ T cells from HIV+ individuals. These results suggest a new mechanism by which HIV could attack the immune system and may help to explain the CD8+ T cell defects in AIDS patients.

A 74-year-old man with persistent fevers.

An elderly man with a history of extensive world travel presents with a chronic illness and fevers. The febrile illness has been present for eight years, and no diagnosis has been made despite extensive evaluation and testing. The differential diagnosis of this unusual case of fever of unknown origin is discussed.

Repression of human immunodeficiency virus type 1 through the novel cooperation of human factors YY1 and LSF.

A subpopulation of stably infected CD4+ cells capable of producing virus upon stimulation has been identified in human immunodeficiency virus (HIV)-positive individuals (T.-W. Chun, D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, and R. F. Siliciano, Nat. Med. 1:1284-1290, 1995). Few host factors that directly limit HIV-1 transcription and could support this state of nonproductive HIV-1 infection have been described. YY1, a widely distributed human transcription factor, is known to inhibit HIV-1 long terminal repeat (LTR) transcription and virus production. LSF (also known as LBP-1, UBP, and CP-2) has been shown to repress LTR transcription in vitro, but transient expression of LSF has no effect on LTR activity in vivo. We report that both YY1 and LSF participate in the formation of a complex that recognizes the initiation region of the HIV-1 LTR. Further, we have found that these factors cooperate in the repression of LTR expression and viral replication. This cooperative function may account for the divergent effects of LSF previously observed in vitro and in vivo. Thus, the cooperation of two general cellular transcription factors may allow for the selective downregulation of HIV transcription. Through this mechanism of gene regulation, YY1 and LSF could contribute to the establishment and maintenance of a population of cells stably but nonproductively infected with HIV-1.

Human transcription factor YY1 represses human immunodeficiency virus type 1 transcription and virion production.

The transcriptional activity of human immunodeficiency virus type 1 (HIV-1) is affected by many cellular factors. Homologies near the HIV-1 initiator region to the DNA-binding sequences of YY1, a multifunctional transcription factor known to regulate diverse viral and cellular promoters, suggested that YY1 might regulate HIV-1. Antibody to YY1 blocked the formation of complexes by HeLa cell nuclear extract and a DNA oligonucleotide encoding the HIV-1 initiator region. HIV-1 long terminal repeat (LTR) expression, as measured the expression of a transfected LTR-CAT reporter gene, was repressed more than 12-fold by the cotransfection of a YY1 expression vector. HIV-1 production by both COS-1 and CEM cells after transfection of an infectious molecular HIV-1 clone was repressed 7- to 20-fold by cotransfection of a YY1 expression vector. HIV-1 production was also decreased threefold in a CD4-positive lymphocyte cell line chronically infected with HIV-1 (8E5) after transfection of YY1. In situ hybridization studies confirmed that YY1 reduced HIV-1 RNA expression. YY1 may play an important role in the regulation of HIV-1 LTR expression in vivo and virus production by infected cells.

Induction and enhancement of immune responses to herpes simplex virus type 2 in humans by use of a recombinant glycoprotein D vaccine.

A vaccine for a chronic or recurrent viral infection should induce immune responses that protect against primary disease or that augment preexisting defenses sufficiently to diminish the likelihood of disease recurrence or progression. Such a vaccine was sought for genital herpes, a sexually transmitted infection of epidemic proportion. Vaccine containing recombinant herpes simplex virus type 2 glycoprotein D expressed in CHO cells was given repeatedly and safely to 24 human volunteers. In previously uninfected subjects, the vaccine induced primary antigen-specific and neutralizing antibody responses nearing or exceeding those seen at entry in subjects with genital herpes. Primary cellular immune responses were also evoked. Vaccination of previously seropositive subjects boosted antibody titers to levels that remained, for > or = 1 year, severalfold above those attained in recurrent genital herpes. Either the quantity or mode of presentation of antigen permitted this vaccine to exhibit previously unachieved immunogenicity, which may prove adequate for antiviral immunoprophylaxis or treatment of genital herpes.

A cutaneous lesion in a patient with AIDS: an unusual presentation of infection due to Mycobacterium avium complex.

A patient with AIDS developed a purplish, necrotic skin lesion followed by fevers, constitutional symptoms, and watery diarrhea. Stains of samples from the skin lesion and of stool and bone marrow revealed acid-fast bacilli, and Mycobacterium avium was isolated from cultures of these specimens and blood. With the initiation of multiagent oral antimycobacterial therapy, the patient's symptoms abated and the cutaneous lesion reepithelialized. We believe this lesion to be a manifestation of disseminated infection due to Mycobacterium avium complex (MAC). As the population of patients with AIDS who have CD4 cell counts of < 100/mm3 increases, new and unusual manifestations of disseminated MAC infection can be expected. New oral agents with increased activity against MAC may make early recognition and treatment of MAC infections more rewarding.