A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

No Evidence for Decay of the Latent Reservoir in HIV‐1–Infected Patients Receiving Intensive Enfuvirtide‐Containing Antiretroviral Therapy

Human immunodeficiency virus type 1 (HIV-1) persists in a latent reservoir of infected resting memory CD4 cells in patients receiving antiretroviral therapy. We assessed whether multitarget therapy with enfuvirtide, 2 reverse-transcriptase inhibitors, and a ritonavir-boosted protease inhibitor leads to decay of this reservoir. Nineteen treatment-naive patients initiated this regimen; 9 experienced virologic suppression and continued enfuvirtide-containing therapy for at least 48 weeks. In enfuvirtide-treated patients with virological suppression, there was no decay of the latent reservoir (95% confidence interval for half-life, 11 months to infinity). The stability of the latent reservoir despite intensive therapy suggests that new strategies are needed to eradicate HIV-1 from this reservoir

Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research

HLA-B*57 Elite Suppressor and Chronic Progressor HIV-1 Isolates Replicate Vigorously and Cause CD4+ T Cell Depletion in Humanized BLT Mice

Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design

The Risk of Virologic Failure Decreases with Duration of HIV Suppression, at Greater than 50% Adherence to Antiretroviral Therapy

Background: We hypothesized that the percent adherence to antiretroviral therapy necessary to maintain HIV suppression would decrease with longer duration of viral suppression. Methodology: Eligible participants were identified from the REACH cohort of marginally housed HIV infected adults in San Francisco. Adherence to antiretroviral therapy was measured through pill counts obtained at unannounced visits by research staff to each participant's usual place of residence. Marginal structural models and targeted maximum likelihood estimation methodologies were used to determine the effect of adherence to antiretroviral therapy on the probability of virologic failure during early and late viral suppression. Principal Findings: A total of 221 subjects were studied (median age 44.1 years; median CD4+ T cell nadir 206 cells/mm3). Most subjects were taking the following types of antiretroviral regimens: non-nucleoside reverse transcriptase inhibitor based (37%), ritonavir boosted protease inhibitor based (28%), or unboosted protease inhibitor based (25%). Comparing the probability of failure just after achieving suppression vs. after 12 consecutive months of suppression, there was a statistically significant decrease in the probability of virologic failure for each range of adherence proportions we considered, as long as adherence was greater than 50%. The estimated risk difference, comparing the probability of virologic failure after 1 month vs. after 12 months of continuous viral suppression was 0.47 (95% CI 0.23–0.63) at 50–74% adherence, 0.29 (CI 0.03–0.50) at 75–89% adherence, and 0.36 (CI 0.23–0.48) at 90–100% adherence. Conclusions: The risk of virologic failure for adherence greater than 50% declines with longer duration of continuous suppression. While high adherence is required to maximize the probability of durable viral suppression, the range of adherence capable of sustaining viral suppression is wider after prolonged periods of viral suppression

The type 2C phosphatase Wip1: An oncogenic regulator of tumor suppressor and DNA damage response pathways

The Wild-type p53-induced phosphatase 1, Wip1 (or PPM1D), is unusual in that it is a serine/threonine phosphatase with oncogenic activity. A member of the type 2C phosphatases (PP2Cδ), Wip1 has been shown to be amplified and overexpressed in multiple human cancer types, including breast and ovarian carcinomas. In rodent primary fibroblast transformation assays, Wip1 cooperates with known oncogenes to induce transformed foci. The recent identification of target proteins that are dephosphorylated by Wip1 has provided mechanistic insights into its oncogenic functions. Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphorylating proteins that are substrates of both ATM and ATR, important DNA damage sensor kinases. Wip1 also suppresses the activity of multiple tumor suppressors, including p53, ATM, p16INK4a and ARF. We present evidence that the suppression of p53, p38 MAP kinase, and ATM/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers

An integrated modelling approach for R5-X4 mutation and HAART therapy assessment

We have modelled the within-patient evolutionary process during HIV infection using different methodologies. New viral strains arise during the course of HIV infection. These multiple strains of the virus are able to use different coreceptors, in particular the CCR5 and the CXCR4 (R5 and X4 phenotypes, respectively)influence the progression of the disease to the AIDS phase. We present a model of HIV early infection and CTLs response which describes the dynamics of R5 quasispecies, specifying the R5 to X4 switch and effects of immune response. We illustrate dynamics of HIV multiple strains in the presence of multidrug HAART therapy. The HAART combined with X4 strain blocker drugs might help to reduce infectivity and lead to slower progression of disease. On the methodology side, our model represents a paradigm of integrating formal methods and mathematical models as a general framework to study HIV multiple strains during disease progression, and will inch towards providing help in selecting among vaccines and drug therapies. The results presented here are one of the rare cases of methodological cross comparison (stochastic and deterministic) and a novel implementation of model checking in therapy validation

Anchors aweigh: the sources, variety, and challenges of mission drift

The growing number of studies which reference the concept of mission drift imply that such drift is an undesirable strategic outcome related to inconsistent organizational action, yet beyond such references little is known about how mission drift occurs, how it impacts organizations, and how organizations should respond. Existing management theory more broadly offers initial albeit equivocal insight for understanding mission drift. On the one hand, prior studies have argued that inconsistent or divergent action can lead to weakened stakeholder commitment and reputational damage. On the other hand, scholars have suggested that because environments are complex and dynamic, such action is necessary for ensuring organizational adaptation and thus survival. In this study, we offer a theory of mission drift that unpacks its origin, clarifies its variety, and specifies how organizations might respond to external perceptions of mission drift. The resulting conceptual model addresses the aforementioned theoretical tension and offers novel insight into the relationship between organizational actions and identity

Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 μM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART