Shyamasundaran Kottilil

Sofosbuvir and Ribavirin for Hepatitis C Genotype 1 in Patients With Unfavorable Treatment Characteristics: A Randomized Clinical Trial

IMPORTANCE The efficacy of directly acting antiviral agents in interferon-free regimens for the treatment of chronic hepatitis C infections needs to be evaluated in different populations. OBJECTIVE To determine the efficacy and safety of sofosbuvir with weight-based or low-dose ribavirin among a population with unfavorable treatment characteristics. DESIGN, SETTING, AND PATIENTS Single-center, randomized, 2-part, open-label phase 2 study involving 60 treatment-naive patients with hepatitis C virus (HCV) genotype 1 enrolled at the National Institutes of Health (October 2011-April 2012). INTERVENTIONS In the studys first part, 10 participants with early to moderate liver fibrosis were treated with 400 mg/d of sofosbuvir and weight-based ribavirin for 24 weeks. In the second part, 50 participants with all stages of liver fibrosis were randomized 1:1 to receive 400 mg of sofosbuvir with either weight-based or low-dose 600 mg/d of ribavirin for 24 weeks. MAIN OUTCOMES AND MEASURES The primary study end point was the proportion of participants with undetectable HCV viral load 24 weeks after treatment completion (sustained virologic response of 24 weeks [SVR24]). RESULTS In the first part of the study, 9 participants (90%; 95% CI, 55%-100%) achieved SVR24. In the second part, 7 participants (28%) in the weight-based group and 10 (40%) in the low-dose group relapsed after treatment completion leading to SVR24 rates of 68% (95% CI, 46%-85%) in the weight-based group and 48% (95% CI, 28%-69%; P = .20) in the low-dose group. Twenty individuals participated in a pharmacokinetic-viral kinetic substudy, which demonstrated a slower loss rate of infectious virus in relapsers than in participants who achieved SVR (clearance, 3.57/d vs 5.60/d; P = .009). The most frequent adverse events were headache, anemia, fatigue, and nausea. There were 7 grade 3 events including anemia, neutropenia, nausea, hypophosphatemia, and cholelithiasis or pancreatitis. No one discontinued treatment due to adverse events. CONCLUSION AND RELEVANCE In a population of patients with a high prevalence of unfavorable traditional predictors of treatment response, a 24-week regimen of sofosbuvir and weight-based or low-dose ribavirin resulted in SVR24 rates of 68% and 48%, respectively. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01441180.

Decreased Survival of B Cells of HIV-viremic Patients Mediated by Altered Expression of Receptors of the TNF Superfamily

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21low B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21low B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21low B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Both activated and resting CD4+ T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin α4β7 (α4β7), the gut mucosal homing receptor. We find that α4β7high CD4+ T cells are more susceptible to productive infection than are α4β7low-neg CD4+ T cells in part because this cellular subset is enriched with metabolically active CD4+ T cells. α4β7high CD4+ T cells are CCR5high and CXCR4low; on these cells, α4β7 appears in a complex with CD4. The specific affinity of gp120 for α4β7 provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.

Compromised B cell responses to influenza vaccination in HIV-infected individuals.

BACKGROUND Yearly influenza vaccination, although recommended for human immunodeficiency virus (HIV)-infected individuals, has not received thorough evaluation in the era of antiretroviral therapy. We assessed the impact of HIV disease on B cell responses to influenza vaccination. METHODS Sixty-four HIV-infected and 17 HIV-negative individuals received the 2003-2004 trivalent inactivated influenza vaccine. Frequencies of influenza-specific antibody-secreting cells (ASCs) were measured by enzyme-linked immunospot (ELISPOT) assay, and antibody responses were measured by hemagglutination-inhibition (HI) assay. Memory responses to influenza were measured by ELISPOT assay after polyclonal activation of B cells in vitro. RESULTS Prevaccination HI titers were significantly higher in HIV-negative than in HIV-infected individuals. Peak HI titers and influenza-specific ASC frequencies were directly correlated with CD4+ T cell counts in HIV-infected individuals. Influenza-specific memory B cell responses were significantly lower in HIV-infected than in HIV-negative individuals and were directly correlated with CD4+ T cell counts. CONCLUSIONS HIV infection is associated with a weak antibody response to influenza vaccination that is compounded by a poor memory B cell response. CD4+ T cell count is a critical determinant of responsiveness to influenza vaccination, and the contribution of plasma HIV RNA level is suggestive and warrants further investigation.

Endogenous intrahepatic IFNs and association with IFN-free HCV treatment outcome

BACKGROUND. Hepatitis C virus (HCV) infects approximately 170 million people worldwide and may lead to cirrhosis and hepatocellular carcinoma in chronically infected individuals. Treatment is rapidly evolving from IFN-α-based therapies to IFN-α-free regimens that consist of directly acting antiviral agents (DAAs), which demonstrate improved efficacy and tolerability in clinical trials. Virologic relapse after DAA therapy is a common cause of treatment failure; however, it is not clear why relapse occurs or whether certain individuals are more prone to recurrent viremia. METHODS. We conducted a clinical trial using the DAA sofosbuvir plus ribavirin (SOF/RBV) and performed detailed mRNA expression analysis in liver and peripheral blood from patients who achieved either a sustained virologic response (SVR) or relapsed. RESULTS. On-treatment viral clearance was accompanied by rapid downregulation of IFN-stimulated genes (ISGs) in liver and blood, regardless of treatment outcome. Analysis of paired pretreatment and end of treatment (EOT) liver biopsies from SVR patients showed that viral clearance was accompanied by decreased expression of type II and III IFNs, but unexpectedly increased expression of the type I IFN IFNA2. mRNA expression of ISGs was higher in EOT liver biopsies of patients who achieved SVR than in patients who later relapsed. CONCLUSION. These results suggest that restoration of type I intrahepatic IFN signaling by EOT may facilitate HCV eradication and prevention of relapse upon withdrawal of SOF/RBV. TRIAL REGISTRATION. ClinicalTrials.gov NCT01441180.

The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection

BACKGROUND & AIMS GS-9620 is an oral agonist of toll-like receptor 7, a pattern-recognition receptor whose activation results in innate and adaptive immune stimulation. We evaluated the safety, pharmacokinetics, and pharmacodynamics of GS-9620 in patients with chronic hepatitis B. METHODS In two double-blind, phase 1b trials of identical design, 49 treatment-naïve and 51 virologically suppressed patients were randomized 5:1 to receive GS-9620 (at doses of 0.3mg, 1mg, 2mg, 4mg) or placebo as a single dose or as two doses seven days apart. Pharmacodynamic assessment included evaluation of peripheral mRNA expression of interferon-stimulated gene 15 (ISG15), serum interferon gamma-induced protein 10 and serum interferon (IFN)-alpha. RESULTS Overall, 74% of patients were male and 75% were HBeAg negative at baseline. No subject discontinued treatment due to adverse events. Fifty-eight percent experienced ⩾1 adverse event, all of which were mild to moderate in severity. The most common adverse event was headache. No clinically significant changes in HBsAg or HBV DNA levels were observed. Overall, a transient dose-dependent induction of peripheral ISG15 gene expression was observed peaking within 48 hours of dosing followed by return to baseline levels within seven days. Higher GS-9620 dose, HBeAg positive status, and low HBsAg level at baseline were independently associated with greater probability of ISG15 response. Most patients (88%) did not show detectable levels of serum IFN-alpha at any time point. CONCLUSIONS Oral GS-9620 was safe, well tolerated, and associated with induction of peripheral ISG15 production in the absence of significant systemic IFN-alpha levels or related symptoms.

Deleterious Effect of HIV-1 Plasma Viremia on B Cell Costimulatory Function

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4+ T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4+ T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4+ T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4+ T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4+ T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4+ T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4+ T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4+ T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.

Perturbations in B cell responsiveness to CD4+ T cell help in HIV-infected individuals

HIV infection induces a wide array of B cell dysfunctions. We have characterized the effect of plasma viremia on the responsiveness of B cells to CD4+ T cell help in HIV-infected patients. In HIV-negative donors, B cell proliferation correlated with CD154 expression on activated CD4+ T cells and with the availability of IL-2, whereas in HIV-infected viremic patients, reduced B cell proliferation was observed despite normal CD154 expression on activated CD4+ T cells. Reduced triggering of B cells by activated CD4+ T cells was clearly observed in HIV-infected viremic patients compared with aviremic patients with comparable CD4+ T cell counts, and a dramatic improvement in B cell function was observed in patients whose plasma viremia was controlled by effective antiretroviral therapy. The degree of B cell dysfunction in viremic patients correlated strongly with the inability of B cells to express CD25 in response to activated CD4+ T cells, resulting in an inability to mount a normal proliferative response to IL-2. Similar defects in responsiveness to IL-2 were observed in the B cells of HIV-infected viremic patients in the context of B cell receptor stimulation. These data provide new insight into the mechanisms associated with ineffective humoral responses in HIV disease.

Expression of Chemokine and Inhibitory Receptors on Natural Killer Cells: Effect of Immune Activation and HIV Viremia

We examined the cell-surface expression of chemokine and natural killer (NK) cell inhibitory receptors (iNKRs) on NK cells from individuals with human immunodeficiency virus (HIV) infection, chronic hepatitis C infection, and Wegeners granulomatosis (WG), an inflammatory, granulomatous vasculitis. The expression of CCR5 on NK cells was up-regulated in individuals with HIV viremia and in individuals with active WG, indicating that expression of this receptor is modulated by states of immune activation associated with viral infection and inflammatory or immune-mediated diseases. In contrast, iNKRs were shown to be up-regulated only on NK cells of individuals with HIV viremia, and they returned to a normal level when viremia was controlled with effective antiviral therapy. In individuals with HIV-1 viremia, there was a direct correlation between the level of expression of p58.1, p58.2, and CD94 receptors and plasma HIV viremia, suggesting that ongoing active HIV replication has an effect on the expression of such receptors on NK cells. These results suggest that immune activation leads to abnormal cell-surface expression of chemokine receptors on NK cells, whereas HIV-specific processes account for the up-regulation of iNKRs on NK cells; this may explain the NK cell-functional defects seen in HIV infection.

Single Cell Analysis of Lymph Node Tissue from HIV-1 Infected Patients Reveals that the Majority of CD4+ T-cells Contain One HIV-1 DNA Molecule

Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4+ T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4+ T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.