Andrew H. Talal

Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy.

BACKGROUND In patients infected with human immunodeficiency virus type 1 (HIV-1), combination antiretroviral therapy can result in sustained suppression of plasma levels of the virus. However, replication-competent virus can still be recovered from latently infected resting memory CD4 lymphocytes; this finding raises serious doubts about whether antiviral treatment can eradicate HIV-1. METHODS We looked for evidence of residual HIV-1 replication in eight patients who began treatment soon after infection and in whom plasma levels of HIV-1 RNA were undetectable after two to three years of antiretroviral therapy. We examined whether there had been changes over time in HIV-1 proviral sequences in peripheral-blood mononuclear cells, which would indicate residual viral replication. We also performed in situ hybridization studies on tissues from one patient to identify cells actively expressing HIV-1 RNA. We estimated the rate of decrease of latent, replication-competent HIV-1 in resting CD4 lymphocytes on the basis of the decrease in the numbers of proviral sequences identified during primary infection and direct sequential measurements of the size of the latent reservoir. RESULTS Six of the eight patients had no significant variations in proviral sequences during treatment. However, in two patients there was sequence evolution but no evidence of drug-resistant viral genotypes. In one patient, extensive in situ studies provided additional evidence of persistent viral replication in lymphoid tissues. Using two independent approaches, we estimated that the half-life of the latent, replication-competent virus in resting CD4 lymphocytes was approximately six months. CONCLUSIONS These findings suggest that combination antiretroviral regimens suppress HIV-1 replication in some but not all patients. Given the half-life of latently infected CD4 lymphocytes of about six months, it may require many years of effective antiretroviral treatment to eliminate this reservoir of HIV-1.

Naturally Occurring Dominant Resistance Mutations to Hepatitis C Virus Protease and Polymerase Inhibitors in Treatment-Naïve Patients

Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000‐fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment‐naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT‐C) in the population, we analyzed HCV genome sequences from 507 treatment‐naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication‐competent, drug‐resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN‐191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG‐021541; and to the NS4A antagonist ACH‐806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug‐resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. Conclusion: Naturally occurring dominant STAT‐C resistance mutations are common in treatment‐naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin. (HEPATOLOGY 2008;48:1769–1778.)

Hepatitis C virus infection in USA: an estimate of true prevalence.

The recent National Health and Nutrition Examination Survey (NHANES) sampled only the civilian, non‐institutionalized population of USA and may have underestimated the prevalence of hepatitis C virus (HCV) in this country. We searched the database MEDLINE, the Bureau of Justice Statistics, Center for Medicare and Medicaid and individual states Department of Corrections for all epidemiological studies regarding the prevalence of HCV in populations not sampled by the NHANES survey namely the incarcerated, homeless, nursing home residents, hospitalized and those on active military duty. Because of their relatively low frequency in the NHANES sample, we also expanded our search to include healthcare workers and long‐term dialysis patients. Although included in the NHANES sample, we also performed searches on drug users (injection and non‐injection) and veterans to confirm the findings of the NHANES study. Based on the prevalence of studies identified meeting our inclusion criteria, our most conservative estimates state that there at least 142 761 homeless persons, 372 754 incarcerated persons and 6805 persons on active military duty unaccounted for in the NHANES survey. While the NHANES estimates of drug users (both injection and non‐injection) appear to be reasonable, the survey seems to have underestimated the number of HCV‐positive veterans. Our most conservative estimates suggest that there are at least 5.2 million persons living with HCV in USA today, approximately 1.9 million of whom were unaccounted for in the NHANES survey.

The Effect of Commencing Combination Antiretroviral Therapy Soon after Human Immunodeficiency Virus Type 1 Infection on Viral Replication and Antiviral Immune Responses

Twelve subjects were treated with zidovudine, lamivudine, and ritonavir within 90 days of onset of symptoms of acute infection to determine whether human immunodeficiency virus type 1 (HIV-1) infection could be eradicated from an infected host. In adherent subjects, with or without modifications due to intolerance, viral replication was suppressed during the 24-month treatment period. Durable suppression reduced levels of HIV-1-specific antibodies and cytotoxic T lymphocyte responses in selected subjects. Proviral DNA in mononuclear cells uniformly persisted. The persistence of HIV-1 RNA expression in lymphoid tissues and peripheral blood mononuclear cells suggests that elimination of this residual pool of virus should be achieved before considering adjustments in antiretroviral therapeutic regimens. In addition, given the reduction in levels of virus-specific immune responses, it would seem prudent to consider enhancing these responses using vaccine strategies prior to the withdrawal of antiviral therapy.

Intrahepatic levels of CXCR3‐associated chemokines correlate with liver inflammation and fibrosis in chronic hepatitis C

Chemokines, chemotactic cytokines, may promote hepatic inflammation in chronic hepatitis C virus (HCV) infection through the recruitment of lymphocytes to the liver parenchyma. We evaluated the association between inflammation and fibrosis and CXCR3‐associated chemokines, interferon‐γ (IFN‐γ)–inducible protein 10 (IP‐10/CXCL10), monokine induced by IFN‐γ (Mig/CXCL9), and interferon‐inducible T cell α chemoattractant (I‐TAC/CXCL11), in HCV infection. Intrahepatic mRNA expression of these chemokines was analyzed in 106 chronic HCV‐infected patients by real‐time PCR. The intrahepatic localization of chemokine producer cells and CXCR3+ lymphocytes was determined in selected patients by immunohistochemistry. We found elevated intrahepatic mRNA expression of all three chemokines, most markedly CXCL10, in chronic HCV‐infected patients with higher necroinflammation and fibrosis. By multivariable multivariate analysis, intrahepatic CXCL10 mRNA expression levels were significantly associated with lobular necroinflammatory grade and HCV genotype 1. In the lobular region, CXCL10‐expressing and CXCL9‐expressing hepatocytes predominated in areas with necroinflammation. Strong CXCL11 expression was observed in almost all portal tracts, whereas CXCL9 expression varied considerably among portal tracts in the same individual. Most intrahepatic lymphocytes express the CXCR3 receptor, and the number of CXCR3+ lymphocytes was increased in patients with advanced necroinflammation. Conclusion: These findings suggest that the CXCR3‐associated chemokines, particularly CXCL10, may play an important role in the development of necroinflammation and fibrosis in the liver parenchyma in chronic HCV infection. (Hepatology 2008.)

The role of chemokines as inflammatory mediators in chronic hepatitis C virus infection.

Summary.  Hepatitis C virus (HCV) is a leading cause of chronic liver disease that can progress to cirrhosis and/or hepatocellular carcinoma. Intrahepatic inflammation and liver cell injury are defining features of chronic HCV infection. Chemokines, chemotactic cytokines that attract leucocytes to inflammatory sites, may be important in the development of intrahepatic inflammation. As T‐helper (Th)1 inflammatory cells, characterized by interferon (IFN)‐γ and interleukin (IL)‐2 secretion, predominate in the liver during chronic HCV infection, chemokines that attract these cells might be particularly important in disease progression. In this review, we focus on the role of Th1 chemokines, which are all members of the CXC or CC subfamilies. Among the CXC chemokines, the non‐ELR group comprised of IFN‐γ‐inducible protein 10 (IP‐10), monokine induced by IFN‐γ (Mig) and IFN‐inducible T‐cell‐α chemoattractant (I‐TAC), attract Th1 cells through the interaction with their receptor, CXCR3. Among the CC subfamily, Th1‐associated chemokines include regulated upon activation, normal T‐cell expressed and secreted (RANTES) and macrophage inflammatory proteins (MIP)1α and β. These chemokines attract cells through an interaction with their receptor, CCR5. While peripheral blood and intrahepatic levels of all of these chemokines are elevated in chronic hepatitis C patients, only select chemokines have been found to be correlated with hepatic inflammation. Among the six chemokines, IP‐10 has uniquely been shown to have prognostic utility as a marker of treatment outcome. In the future, chemokines might be used to monitor the natural course and progression of HCV‐associated liver disease, to identify patients with a high likelihood of achieving a therapeutic response, and they may even have potential as therapeutic targets.

Normal Functional Capacity in Circulating Myeloid and Plasmacytoid Dendritic Cells in Patients with Chronic Hepatitis C

Initial reports analyzing dendritic cell (DC) function in patients with hepatitis C virus (HCV) infection have been controversial. Here, we enumerate and characterize the function of circulating myeloid and plasmacytoid DCs. The results show lower percentages of myeloid DCs (0.62 vs. 0.83; P = .05) and plasmacytoid DCs (0.11 vs. 0.34; P = .004) in patients with chronic HCV infection than in healthy, non-HCV-infected individuals. Despite the lower numbers of circulating myeloid DCs present, no phenotypic or functional defects were identified. The lower percentage of plasmacytoid DCs resulted in decreased absolute interferon (IFN)-alpha production; however, when analyzed on a per-cell basis, plasmacytoid DCs from HCV-infected patients generated levels of IFN-alpha equivalent to those generated by DCs from healthy, non-HCV-infected individuals. Contrary to data from previous models (which attributed HCV pathogenesis to defects in the DC compartment), our data reveal functional DC subsets in patients with chronic HCV infection. These results are encouraging for DC-based HCV immunotherapy trials.

Determinants of Hepatitis C Virus Treatment Completion and Efficacy in Drug Users Assessed by Meta-analysis

BACKGROUND Hepatitis C virus (HCV)-infected drug users (DUs) have largely been excluded from HCV care. We conducted a systematic review and meta-analysis of the literature on treatment completion and sustained virologic response (SVR) rates in DUs. We assessed the effects of different treatment approaches and services to promote HCV care among DUs as well as demographic and viral characteristics. METHODS Studies of at least 10 DUs treated with pegylated interferon/ribavirin that reported SVR were analyzed. Heterogeneity was assessed (Cochran test) and investigated (meta-regression), and pooled rates were estimated (random effects). RESULTS Thirty-six studies comprising 2866 patients were retrieved. The treatment completion rate among DUs was 83.4% (95% confidence interval [CI], 77.1%-88.9%). Among studies that included addiction-treated and untreated patients during HCV therapy, the higher the proportion of addiction-treated patients, the higher the HCV treatment completion rate (P < .0001). After adjusting for human immunodeficiency virus (HIV)/HCV coinfection, sex, and treatment of addiction, support services during antiviral therapy increased treatment completion (P < .0001). The pooled SVR rate was 55.5% (95% CI, 50.6%-60.3%). Genotype 1/4 (P = .0012) and the proportion of HIV-coinfected DUs (P = .0173) influenced the SVR rate. After adjusting for HCV genotype 1/4 and HIV/HCV coinfection, the SVR rate was positively correlated with involvement of a multidisciplinary team (P < .0001). CONCLUSIONS Treatment of addiction during HCV therapy results in higher treatment completion. Our pooled SVR rate is similar to that obtained in registration trials in the general population. Treatment of addiction during HCV therapy will likely be important for HCV-infected DUs undergoing treatment with more complex regimens including direct-acting antivirals.

Peripheral CXCR3-Associated Chemokines as Biomarkers of Fibrosis in Chronic Hepatitis C Virus Infection

BACKGROUND CXCR3-associated chemokines CXCL9-CXCL11 promote histologic progression in chronic hepatitis C virus (HCV) infection, as indicated by elevated intrahepatic levels of messenger RNA in patients with advanced inflammation and fibrosis. We evaluated the potential of peripheral chemokine levels to discriminate among patients with chronic HCV infection who had different stages of fibrosis. METHODS Peripheral levels of CXCR3-associated chemokines were measured by enzyme-linked immunosorbent assay of plasma samples obtained from 93 patients with chronic HCV infection. Of the subjects, 79 (85%) were white, and 68 (73%) were infected with HCV genotype 1. RESULTS Expression of all 3 chemokines, when analyzed as a group, was significantly associated with intrahepatic inflammation and fibrosis. Plasma levels of CXCL10 were significantly elevated in patients with advanced fibrosis, whereas CXCL9 levels were significantly elevated in patients with advanced inflammation. By proportional odds multivariate modeling, we observed an association between fibrosis and CXCL10 (P< .002) as well as between fibrosis and inflammation (P<.001). Of the individual parameters, the CXCL10 level was most useful in identifying patients with more-severe (stage 3-4) fibrosis. Discriminatory ability was improved by the combination of CXCL10 and CXCL9. CONCLUSIONS The strong association between CXCR3-associated chemokines and fibrosis suggests that they may have promise as noninvasive markers of hepatic fibrosis in a predominantly white HCV genotype 1-infected population.

Genome-Wide Association Study Identifies Variants Associated with Progression of Liver Fibrosis from HCV Infection

BACKGROUND & AIMS Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection. METHODS We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance. RESULTS In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages. CONCLUSIONS Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.