The Enhanced Liver Fibrosis Index Predicts Hepatic Fibrosis Superior to FIB4 and APRI in HIV/HCV Infected Patients.

BACKGROUND: Accurate noninvasive biomarkers of fibrotic progression are important for hepatitis C virus (HCV) management, but commonly used modalities may have decreased efficacy in human immunodeficiency virus (HIV)/HCV-coinfected persons. The enhanced liver fibrosis (ELF) index is a highly sensitive noninvasive marker of hepatic fibrosis that has had limited assessment in the HIV/HCV population. We compared ELF index performance to FIB4 and aspartate to platelet ratio index (APRI) at different stages of liver fibrosis as determined by liver histology, and validated the efficacy of the three noninvasive biomarkers in HIV/HCV-coinfected versus HCV-monoinfected. METHODS: The ELF index was determined in 147 HIV/HCV-coinfected and 98 HCV-monoinfected persons using commercial ELISA assays for the component elements of the index. Area under the receiver-operator curve was used to validate ELF and to compare its performance to liver histology as well as to other noninvasive biomarkers of liver fibrosis, FIB4, and APRI. RESULTS: The ELF index increased with histological stage of liver fibrosis and exhibited a linear relationship with Metavir score in all subjects. ELF performance was comparable between HIV/HCV and HCV with advanced liver fibrosis/cirrhosis. In the HIV/HCV cohort ELF cutoffs of 8.45 and 9.23 predicted mild and moderate fibrosis with 85% sensitivity, whereas the ELF cutoff of 9.8 had the highest specificity for advanced fibrosis and the cutoff of 10.4 was 99% specific for cirrhosis. ELF performance was superior to FIB4 and APRI in all subjects regardless of HIV status. CONCLUSIONS: ELF index demonstrated excellent characteristics toward accurate prediction of liver fibrosis and cirrhosis with superior performance to APRI and FIB4 in HIV/HCV coinfection. Applying this noninvasive biomarker index for diagnosis of liver fibrosis and progression in HIV/HCV is warranted.

Validation and Refinement of Noninvasive Methods to Assess Hepatic Fibrosis: Magnetic Resonance Elastography Versus Enhanced Liver Fibrosis Index.

BACKGROUND: Noninvasive fibrosis markers are routinely used in patients with liver disease. Magnetic resonance elastography (MRE) is recognized as a highly accurate methodology, but a reliable blood test for fibrosis would be useful. We examined performance characteristics of the Enhanced Liver Fibrosis (ELF) Index compared to MRE in a cohort including those with HCV, HIV, and HCV/HIV. METHODS: Subjects enrolled in the Miami Adult Studies on HIV (MASH) cohort underwent MRE and blood sampling. The ELF Index was scored and receiver-operator curves constructed to determine optimal cutoff levels relative to performance characteristics. Cytokine testing was performed to identify new markers to enhance noninvasive marker development. RESULTS: The ELF Index was determined in 459 subjects; more than half were male, non-white, and HIV-infected. MRE was obtained on a subset of 283 subjects and the group that had both studies served as the basis of the receiver-operator curve analysis. At an ELF Index of > 10.633, the area under the curve for cirrhosis (Metavir F4, MRE > 4.62 kPa) was 0.986 (95% CI 0.994-0.996; p < 0.001) with a specificity of 100%. For advanced fibrosis (Metavir F3/4), an ELF cutoff of 10 was associated with poor sensitivity but high specificity (98.9%, 95% CI 96.7-99.8%) with an AUC of 0.80 (95% CI 0.749-0.845). ELF Index performance characteristics exceeded FIB-4 performance. HCV and age were associated with increased fibrosis (p < 0.05) in a multivariable model. IP-10 was found to be a promising biomarker for improvement in noninvasive prediction algorithms. CONCLUSIONS: The ELF Index was a highly sensitive and specific marker of cirrhosis, even among HIV-infected individuals, when compared with MRE. IP-10 may be a biomarker that can enhance performance characteristics further, but additional validation is required.

Improvement in Hepatic Fibrosis Biomarkers Associated With Chemokine Receptor Inactivation Through Mutation or Therapeutic Blockade.

BACKGROUND: The C-C chemokine receptor Type 5 (CCR5) is a key receptor for human immunodeficiency virus type 1 (HIV-1) entry into T-cells and a variant allele, CCR5 delta-32, is associated with decreased viral replication and disease progression. Active HIV-1 replication is highly associated with accelerated rates of hepatic fibrosis. We postulated that CCR5 plays a role in the development of hepatic fibrosis and evaluated the longitudinal effect of natural or drug-induced CCR5 mutation and blockade on biomarkers of liver fibrosis in HIV-1 patients. METHODS: To accomplish this goal, we examined 2 distinct cohorts. First, we evaluated fibrosis markers in the Multicenter Hemophilia Cohort Studies (MHCS), which included subjects with HIV and hepatitis C virus (HCV) coinfection with the CCR5 delta-32 allele. We also evaluated an HIV-1 infected cohort that was treated with a dual CCR5/CCR2 antagonist, cenicriviroc. The enhanced liver fibrosis (ELF) index was validated against liver histology obtained from HCV/HIV and HCV patients and demonstrated strong correlation with fibrosis stage. RESULTS: In both the MHCS patients and patients treated with cenicriviroc, CCR5 mutation or blockade was associated with a significant decrease in the ELF index. Among the patients with the delta-32 allele, the ELF index rate significantly decreased in sequential samples as compared to CCR5 wild-type patients (P = .043). This was not observed in control subjects treated with efavirenz nor with a lower dose of 100 mg cenicriviroc. CONCLUSION: These findings suggest that hepatic fibrosis in HIV-1 infected patients can be modulated by the mutation of CCR5 and/or use of CCR5/CCR2 blockade agents. CLINICAL TRIALS REGISTRATION: NCT01338883.

Ultra-Deep Genomic Sequencing of HCV NS5A Resistance-Associated Substitutions in HCV/HIV Coinfected Patients.

BACKGROUND AND AIMS: The prevalence of naturally occurring HCV-NS5A resistance-associated substitutions (RAS) to DAA drugs might affect the response to treatment in HCV/HIV coinfected subjects. There are limited data on the frequency of HCV-NS5A naturally occurring drug-RAS at baseline in HCV/HIV coinfected patients when ultra-deep sequencing methodologies are applied. METHODS: HCV-NS5A-RAS were evaluated among 25 subjects in each group. Patients were matched by age, gender, and hepatic fibrosis stage category to control for selection bias. RESULTS: Within subtype 1a, RAS were observed in 28% of HCV monoinfected and 48% of HCV/HIV coinfected subjects. More patients in the HCV/HIV coinfected group had clinically relevant mutations to DAA directed at NS5A. CONCLUSION: While the clinical significance of this observation may be limited in highly drug adherent populations, some HCV/HIV coinfected persons may be at greater risk of viral resistance if suboptimal dosing occurs.

Association between IL28B rs12979860 single nucleotide polymorphism and the frequency of colonic Treg in chronically HCV-infected patients.

The IL28B gene is associated with spontaneous or treatment-induced HCV viral clearance. However, the mechanism by which the IL28B single nucleotide polymorphism (SNP) affects the extra-hepatic HCV immune responses and its relationship to HCV pathogenesis have not been thoroughly investigated. To examine the mechanism by which IL28B affects HCV clearance. Forty Egyptian patients with chronic HCV infection receiving an Interferon/ribavirin treatment regimen were enrolled into this study. There were two groups: non-responders (NR; n = 20) and sustained virologic responders (SVR; n = 20). The initial plasma HCV viral loads prior to treatment and IL28B genotypes were determined by quantitative RT-PCR and sequencing, respectively. Liver biopsies were examined to determine the inflammatory score and the stage of fibrosis. Colonic regulatory T cell (Treg) frequency was estimated by immunohistochemistry. No significant association between IL28B genotypes and response to therapy was identified, despite an odds ratio of 3.4 to have the TT genotype in NR compared to SVR (95 % confidence interval 0.3-35.3, p = 0.3). Patients with the TT-IL28Brs12979860 genotype (unfavorable genotype) have significantly higher frequencies of colonic Treg compared to the CT (p = 0.04) and CC (p = 0.03) genotypes. The frequency of colonic Treg cells in HCV-infected patients had a strong association with the IL-28B genotype and may have a significant impact on HCV clearance.

HIV-Induced Epigenetic Alterations in Host Cells.

Human immunodeficiency virus (HIV), a member of the Retroviridae family, is a positive-sense, enveloped RNA virus. HIV, the causative agent of acquired immunodeficiency syndrome (AIDS) has two major types, HIV-1 and HIV-2 In HIV-infected cells the single stranded viral RNA genome is reverse transcribed and the double-stranded viral DNA integrates into the cellular DNA, forming a provirus. The proviral HIV genome is controlled by the host epigenetic regulatory machinery. Cellular epigenetic regulators control HIV latency and reactivation by affecting the chromatin state in the vicinity of the viral promoter located to the 5' long terminal repeat (LTR) sequence. In turn, distinct HIV proteins affect the epigenotype and gene expression pattern of the host cells. HIV-1 infection of CD4(+) T cells in vitro upregulated DNMT activity and induced hypermethylation of distinct cellular promoters. In contrast, in the colon mucosa and peripheral blood mononuclear cells from HIV-infected patients demethylation of the FOXP3 promoter was observed, possibly due to the downregulation of DNA methyltransferase 1. For a curative therapy of HIV infected individuals and AIDS patients, a combination of antiretroviral drugs with epigenetic modifying compounds have been suggested for the reactivation of latent HIV-1 genomes. These epigenetic drugs include histone deacetylase inhibitors (HDACI), histone methyltransferase inhibitors (HMTI), histone demethylase inhibitors, and DNA methyltransferase inhibitors (DNMTI).

Humoral and T-cell-mediated immunity to SARS-CoV-2 vaccination in patients with liver disease and transplant recipients.

BACKGROUND: SARS-CoV-2 vaccination induces a varied immune response among persons with chronic liver disease (CLD) and solid organ transplant recipients (SOTRs). We aimed to evaluate the humoral and T-cell-mediated immune responses to SARS-CoV-2 vaccination in these groups. METHODS: Blood samples were collected following the completion of a standard SARS-CoV-2 vaccination (2 doses of either BNT162b2 or mRNA-12732), and a subset of patients had a blood sample collected after a single mRNA booster vaccine. Three separate methods were utilized to determine immune responses, including an anti-spike protein antibody titer, neutralizing antibody capacity, and T-cell-mediated immunity. RESULTS: The cohort included 24 patients with chronic liver disease, 27 SOTRs, and 9 controls. Patients with chronic liver disease had similar immune responses to the wild-type SARS-CoV-2 compared with controls following a standard vaccine regimen and single booster vaccine. SOTRs had significantly lower anti-S1 protein antibodies (p < 0.001), neutralizing capacity (p < 0.001), and T-cell-mediated immunity response (p = 0.021) to the wild-type SARS-CoV-2 compared with controls following a standard vaccine regimen. Following a single booster vaccine, immune responses across groups were not significantly different but numerically lower in SOTRs. The neutralization capacity of the B.1.1.529 Omicron variant was not significantly different between groups after a standard vaccine regimen (p = 0.87) and was significantly lower in the SOTR group when compared with controls after a single booster vaccine (p = 0.048). CONCLUSION: The immunogenicity of the SARS-CoV-2 vaccine is complex and multifactorial. Ongoing and longitudinal evaluation of SARS-CoV-2 humoral and cellular responses is valuable and necessary to allow frequent re-evaluation of these patient populations.

Hepatitis E Infection in a Longitudinal Cohort of Hepatitis C Virus and HCV/HIV Coinfected Persons.

Hepatitis E virus (HEV) is thought to be common in the United States with increased prevalence in those with concomitant hepatitis C virus (HCV) or HCV/HIV coinfection. Little is known regarding true prevalence, incidence, and antibody seroreversion in these populations. We sought to define these rates among HCV and HCV/HIV coinfected persons in the Washington, DC area. Two longitudinal cohorts of HCV and HCV/HIV coinfected subjects from the Washington, DC area were evaluated. Multiple HEV test modalities were deployed including immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody testing, evaluation of antibody avidity, HEV RNA testing, and HEV enzyme-linked immune absorbent spot (ELISPOT) analysis. A total of 379 individuals were evaluated including 196 who were HCV monoinfected and 183 HCV/HIV coinfected. Anti-HEV IgG was detected and confirmed in 18.7% of the cohort at baseline. None demonstrated anti-HEV IgM positive or HEV RNA positive results. Proportions of HEV antibody prevalence did not significantly differ between groups. Longitudinal follow-up samples were available for 226 individuals with a mean follow-up time of 24 months. Seroreversion was noted in 1.8%. One HCV/HIV infected person seroconverted to HEV IgG positivity in the followed cohort. About 40% of the positive population demonstrated high avidity suggestive of more remote exposure. Interferon gamma ELISPOT was performed in 70 subjects and false negative and false positive HEV enzyme-linked immunosorbent assay antibodies were identified. In HIV-infected persons in the United States HEV exposure and seroconversion is frequent enough that HEV should be considered in the differential diagnosis of acute hepatitis. Seroreversion may lead to underestimation of true infection risk.

Soluble CD163 Identifies Those at Risk for Increased Hepatic Inflammation & Fibrosis.

BACKGROUND: Liver disease remains a significant cause of morbidity and mortality in HIV-infected persons. Soluble CD163 is a marker of Kupffer cell activation that is highly associated with development of hepatic fibrosis. The relative contributions of HIV-associated systemic immune activation vs other etiologies of injury are poorly characterized. METHODS: We utilized subjects in the Miami Adult Studies on HIV (MASH) cohort to evaluate 464 participants including 361 people with HIV (PWH) and 103 hepatitis C virus (HCV)/HIV-uninfected controls. Subjects underwent testing for hepatic fibrosis using both magnetic resonance elastography and the Enhanced Liver Fibrosis Index. Steatosis was evaluated by magnetic resonance imaging-derived proton density fat fraction. Immune activation markers and cytokines were quantitated using Luminex methodologies. RESULTS: Participants with HIV with or without HCV coinfection had higher levels of sCD163 than uninfected controls (P < .05). Soluble sCD163 was highly associated with elevated alanine aminotransferase, a key marker of inflammation/injury and with hepatic fibrosis. Hepatic steatosis was also associated with a cytokine pattern suggestive of Kupffer cell activation but was not associated with an increase in sCD14 or sCD27. CONCLUSIONS: Injury and resultant hepatic fibrosis occur by distinct though overlapping mechanistic pathways. In PWH, sCD163 is highly associated with both injury and fibrosis, suggesting that persistent systemic immune activation is a major contributor to long-term outcomes, adding to damage caused by alcohol, steatosis, and other hepatotoxic drug effects.

HBV and HIV/HBV Infected Patients Have Distinct Immune Exhaustion and Apoptotic Serum Biomarker Profiles.

BACKGROUND: Hepatitis B virus (HBV) infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Due to their shared routes of transmission, approximately 10% of HIV-infected patients worldwide are chronically coinfected with HBV. Additionally, liver disease has become a major cause of morbidity and mortality in HBV/HIV coinfected patients due to prolonged survival with the success of antiretroviral therapy. The relationship between immune exhaustion markers (PD-1/PD-L1) and apoptotic markers such as Fas/FasL, TGFß1, TNF-α, and Th1/Th2 cytokines are not clearly delineated in HBV/HIV coinfection. METHODS: Levels of soluble Fas/FasL, TGFß1, TNF-α, and sPD-1/sPD-L1 as well as Th1 and Th2 cytokines were evaluated in the sera of HBV-monoinfected (n = 30) and HBV/HIV-coinfected (n = 15) patients and compared to levels in healthy controls (n = 20). RESULTS: HBV-monoinfected patients had significantly lower levels of the anti-inflammatory cytokine IL-4 (P < 0.05) and higher levels of apoptotic markers sFas, sFasL, and TGFß-1 (P < 0.001) compared to healthy controls. Coinfection with HIV was associated with higher levels of sFas, TNF-α, and sPD-L1 (P < 0.005), and higher levels of the pro-inflammatory cytokines IL-6, IL-8, and IL-12p70 (P < 0.05) compared to healthy controls. Patients with HBV infection had a unique biomarker clustering profile comprised of IFN-γ, IL12p70, IL-10, IL-6, and TNF-α that was distinct from the profile of the healthy controls, and the unique HIV/HBV profile comprised GM-CSF, IL-4, IL-2, IFN-γ, IL12p70, IL-7, IL-10, and IL-1ß. In HBV monoinfection a significant correlation between sFasL and PD1(r = 0.46, P = < 0.05) and between sFas and PDL1 (r = 0.48, P = <0.01) was observed. CONCLUSION: HBV-infected and HBV/HIV-coinfected patients have unique apoptosis and inflammatory biomarker profiles that distinguish them from each other and healthy controls. The utilization of those unique biomarker profiles for monitoring disease progression or identifying individuals who may benefit from novel immunotherapies such as anti-PD-L1 or anti-PD-1 checkpoint inhibitors appears promising and warrants further investigation.

Characterization of HCV NS3 Protease Variants in HCV/HIV-Coinfected Patients by Ultra-Deep Sequence Analysis: Relationship with Hepatic Fibrosis.

BACKGROUND: Treatment of HCV/HIV coinfection is now largely based on utilization of direct acting agents. Pretreatment viral resistant-associated variants (RAVs) and host liver condition may affect the sustained virological response. In this study, we explored relative prevalence of protease resistance-associated mutations, the evolution of those RAVs after 12 weeks of pegylated interferon alfa exposure, and the role hepatic fibrosis might have on RAV display. METHODS: Thirty nonresponder HCV/HIV-coinfected subjects were evaluated before and after 12 weeks of PegIFN treatment. Ultra-deep sequence analysis of NS3 RAVs was performed. Hepatic fibrosis was determined by sensitive computer-assisted histomorphometry determination. RESULTS: At baseline, protease inhibitor RAVs were present in 73.3% of patients and expanded to 83.3% of patients after 12 weeks of PegIFN exposure. Q80K showed the highest prevalence before and after treatment at 46.7% and 56.7%, respectively. The presence of Q80K is positively correlated with percent collagen content of the liver tissue. CONCLUSIONS: Key RAVs for HCV protease inhibitors are present in a major portion of the HCV/HIV-coinfected population before therapy. Some variants get selected after exposure. Correlation of Q80K with collagen content of the liver suggests that compartmentalization within the liver may contribute to persistence of mutations less fit than wildtype.

Evaluating the Role of Cellular Immune Responses in the Emergence of HCV NS3 Resistance Mutations During Protease Inhibitor Therapy.

The efficacy of protease inhibitor drugs in hepatitis C virus (HCV) treatment is limited by the selection and expansion of drug-resistant mutations. HCV replication is error-prone and genetic variability within the dominant epitopes ensures its persistence. The aims of this study are to evaluate the role of cellular immune response in the emergence of HCV protease resistance mutations and its effects on treatment outcome. Ten chronically HCV-infected subjects were treated with boceprevir (BOC)-based triple therapy. HCV-RNA was tested for BOC resistance-associated viral variants. HCV protease resistance mutations were investigated pretreatment and 24 weeks post-treatment. Synthetic peptides representing the wild-type and the potential nonstructural (NS)3 variants were used to evaluate T cell responses and human leukocyte antigen binding. Sustained viral response was achieved in 70% of patients, two patients were treatment nonresponders (NRs) and one was classified as a relapse. Pretreatment, the proportion of drug-resistant variants within individuals was higher in sustained viral responders (SVRs) than in NR patients. However, resistance-associated variants increased in NRs after BOC combined triple therapy. In contrast to NR patients, significant stronger cell-mediated immune responses were observed at the baseline among those who achieved sustained viral response for all T cell epitopes tested. Despite the increase in cell-mediated immune responses at week 24 in NRs, they failed to control the virus replication, leading to development of overt drug-resistant variants. Our data suggest that strong NS3-specific T cell immune responses at the baseline may predict a positive outcome of directly acting antiviral-based therapy, and the presence of pre-existent resistance mutations does not play a significant role in the outcome of anti-HCV combined therapy.

Epigenetic modification of FOXP3 in patients with chronic HIV infection.

OBJECTIVES: HIV-1 modulates host cell epigenetic machinery to control its own replication and induce immune suppression. HIV-1 infection leads to activation of T regulatory cell (T(reg)), but the mechanism underlying this immune modulation is unclear. T(reg) plays a prominent role in gut-mucosal immune tolerance by restraining excessive effector T-cell responses, a mechanism that is known to be disturbed in chronic HIV-1 infection. DNA methylation plays a major role in T(reg) lineage commitment and immune homeostasis, which may be regulated by HIV. To investigate the mechanisms of aberrant methylation of the T(reg) marker FOXP3 in HIV-1 infection, we evaluated the expression pattern of methylation-related enzymes and its correlation to FOXP3 methylation. METHODS: FOXP3 promoter methylation in the colon mucosa and peripheral blood from HIV-infected patients and control subjects was measured using Pyrosequencing. Gene expression pattern of DNA methylation enzymes in the colon mucosa was investigated by Microarray and quantitative reverse transcriptase-polymerase chain reaction analysis in the same subjects. RESULTS: FOXP3 promoter was significantly (P ≤ 0.0001) demethylated in HIV-infected patients compared with control subjects in both tissues. Expression of DNA methyltransferase 1 (DNAMT1), DNA methyltransferase 1-associated protein 1(DMAP1), methyltransferase-like 7B (METTL7B), and methyltransferase-like 10 (METTL10) were significantly down regulated in HIV-infected patients compared with controls and had a significant positive correlation to FOXP3 promoter methylation. CONCLUSIONS: We present evidence suggesting that altered methylation pattern of FOXP3 and accordingly higher T(reg) frequency in gut mucosa of HIV-infected patients may be because of aberrant methylation processing in HIV.

Immune activation in HIV/HCV-infected patients is associated with low-level expression of liver expressed antimicrobial peptide-2 (LEAP-2).

BACKGROUND: Immune activation is one of the main features of HIV/Hepatitis C virus (HCV) infections and has been linked to the disturbance of the gut-associated lymphoid tissue (GALT). In chronic HIV infection, loss of GALT integrity results in translocation of microbial products and chronic immune activation. We explored the relationship between bacterial translocation and specific colonic proteins, including liver expressed antimicrobial peptide (LEAP 2) which may play a role in modulating the bacterial translocation process. METHODS: A total of 40 subjects (10 HIV/HCV, 10 HIV, 10 HCV-infected patients and 10 controls) were enrolled and underwent serum and colonic tissue sampling. The levels of immune activation were evaluated by measuring plasma sCD27, and the levels of selected proinflammatory, Th2 and regulatory cytokines in both the plasma and supernatant of CD3-stimulated intraepithelial lymphocytes. We also evaluated LEAP-2 expression in the colon biopsies using Affymetrix Human Gene 1.0 ST (HuGene) and fluorescent immunohistochemistry. RESULTS: Increased levels of sCD27 were observed in HIV/HCV coinfected (p=0.03) and HIV monoinfected (p=0.04) patients compared with controls consistent with the presence of immune activation. The chip array identified LEAP-2 expression as a key marker associated with immune activation. LEAP-2 expression in HIV, HCV and HIV/HCV-infected patients was significantly lower compared with controls, and was significantly negatively correlated (p=0.03, r=-0.44) with sCD27. CONCLUSIONS: Our data suggests that HCV and HIV infections are associated with decreased expression of LEAP-2 in colonic tissue. This may represent a key mechanism for enhanced microbial translocation and immune activation in HIV/HCV-infected patients.