Objectives: Chronic hepatitis C virus infection compromises hemodialysis patients and increases liver-related mortality. Interferon treatment is associated with improved sustained virological response rates and increased risk of graft loss after kidney transplant. This may be related to the development of antihuman leukocyte antigen antibodies, which may be a surrogate marker of potent immune response. We evaluated panel reactive antibody 1 and 2 levels for prediction of sustained viral response in patients with kidney transplant.

Materials and Methods: In this retrospective cohort study, we reviewed data from hepatitis C virus-infected hemodialysis patients who received interferon treatment before kidney transplant. Panel reactive antibody > 20% was considered positive. Sustained viral response rates for interferon treatment were obtained and compared with panel reactive antibody 1 and 2 values.

Results: There were 40 patients (16 female and 24 male patients; mean age, 41.5 y; range, 18-65 y). Sustained viral response rate was 18/40 (45%). Panel reactive antibody 1 was negative in 31 patients and positive in 9 patients. Sustained viral response ratio was not correlated with panel reactive antibody 1 positivity. Panel reactive antibody 2 was negative in 31 patients (sustained viral response: present, 11 patients; absent, 20 patients) and positive in 9 patients (sustained viral response: present, 7 patients; absent, 2 patients). Sustained viral response ratio was significantly correlated with panel reactive antibody 2 positivity.

Conclusions: We showed a correlation between panel reactive antibody 2 positivity and sustained viral response rates that may be a predictive tool for hepatitis C virus treatment response. In patients with other complications that compromise hepatitis C virus treatment, panel reactive antibody 2 may be a surrogate marker for sustained viral response prediction. The induction of cellular immunity may cause clearance of hepatitis C virus infection and formation of high panel reactive antibody 2 levels.

Key words : End-stage renal disease, Human leukocyte antigens, Interferon, Sustained viral response

Introduction

Chronic liver disease secondary to hepatitis C virus (HCV) is an important cause of morbidity and mortality in patients who receive hemodialysis treatment and kidney transplant.¹ The prevalence of HCV infection ranges from 5% to 15% in kidney transplant recipients.² The natural course of HCV in hemodialysis patients has not been fully elucidated. Monotherapy with nonpegylated interferon (IFN) (3 to 5 MU, 3 times weekly) or pegylated (PEG) IFN preparations (PEG IFN-α2a or PEG IFN-α2b) once weekly have shown efficacy defined by sustained viral response (SVR) rate from 30% to 60%.^3-6 Although IFN-based treatment regimens have been linked to low levels of tolerance, higher adverse effects, and marginal results,⁷ use of IFN in these patients is required because there are no evidence-based alternative treatment options.⁸ However, in kidney transplant recipients, numerous studies have reported an increased risk of acute rejection after IFN treatment for chronic active hepatitis C.^9,10

Human leukocyte antigens (HLAs) conduct the development of a xenophobic immune response against the transplanted organ.¹¹ Panel reactive antibody (PRA) is produced against HLA and induced by transfusions, pregnancies, infections, autoimmune diseases, and prior transplants for HLA alloimmunization.^12,13 Presence of PRA is a major risk factor for increased incidence of hyperacute or acute graft rejection and graft dysfunction.^12,14 The class, avidity, and affinity of PRAs to their HLA counterparts show variability according to the degree of immune response of the host, which may be affected by concurrent IFN treatment.

In contrast, HLA class I and II molecules play a central role in regulating host immune responses against microbial infections because they present foreign antigens to CD8+ (class I) and CD4+ (class II) T lymphocytes.^15-17 Several cytokines such as IFN up-regulate HLA class I and II gene expression.¹⁶ The IFN exhibits a wide spectrum of biological activities in target cells including antiviral, immuno­modulatory, antiangiogenic, and growth inhibitory effects.¹⁸ The IFN may cause increased cell surface expression of HLA antigens and induction of cytokine gene expression with subsequent stimulation of antibody production.^18,19 However, recent studies investigating the effect of HCV in HLA class I response have shown that IFN-stimulated HLA class I expression is reduced by HCV infection.²⁰ This gives rise to inhibition of antiviral response mediated by CD8+ lymphocytes. Therefore, the relation between IFN-stimulated HLA expressions in HCV infection is complex and may be affected by multiple factors. The levels of PRAs are increased by IFN treatment,²¹ and this increases risk of graft loss after kidney transplant. This may be related to the development of anti-HLA antibodies.

There are well known predictive factors related to patient and viral characteristics for HCV treatment including HCV genotypes 2 or 3, lower basal HCV viral load, rapid viral response (HCV RNA levels are undetectable at treatment week 4), IL-28B rs12979860 C/C genotype, IFNL3 genotypes, a lower degree of liver inflammation, and fibrosis. We hypothesized that the development of anti-HLA antibodies may be used as a surrogate marker of the development of a potent immune response against HCV. We evaluated the possibility of using PRA levels (PRA 1 for HLA class I and PRA 2 for class II) to predict SVR in patients after kidney transplant.

Materials and Methods

Study design and data collection
In this retrospective cohort study, we reviewed the data from IFN-treated HCV-infected patients who were evaluated for kidney transplant. We collected data of the patients who were followed in the nephrology and gastroenterology clinics between January 2000 and March 2013. The demographic data, serologic profile (including anti-HCV, HCV RNA, PRA 1, and PRA 2 levels), characteristics of IFN treatment (dose and duration), liver biopsies, and changes during follow-up were recorded and analyzed.

Definitions
To investigate a possible correlation, we compared PRA 1 and PRA 2 levels and presence of SVR in patients treated with conventional IFN or PEG IFN. Any patient receiving treatment for ≥ 12 weeks was accepted as having an adequate minimum duration of treatment for inclusion. Exclusion criteria included a history of immunosuppressive drug treatment, patients who underwent transplant (because of modification of PRA values by immunosuppressive treatment), treatment duration < 12 weeks, and patients with highly variable PRA levels.

Measurement of exposure
Qualitative detection of antibodies against HCV was performed using a microparticle enzyme immuno­assay (AxSYM HCV, Abbott, Abbott Park, IL, USA) and chemiluminescent microparticle immunoassay (The Architect System, Abbott). The HCV RNA was detected with a real-time polymerase chain reaction (PCR) from 2003 to 2005 (LightCycler, Roche, Basel, Switzerland) and another real-time PCR within a linear interval (serum HCV RNA between 10 and 20 IU/mL) from 2005 to 2013 (CobasTaqman 48 HCV, Roche). Serum samples were tested for immuno­globulin G anti-HLA class I- and II-specific anti­bodies (PRA 1 and PRA 2) with a commercial enzyme-linked immunosorbent assay kit according to the instructions from the manufacturer (LATM20x5, One Lambda, Canoga Park, CA, USA). A PRA value > 20% was considered positive. The SVR rates were calculated and compared with PRA 1 and PRA 2 values.

Statistical analyses
Statistical analyses were performed using software (SPSS version 11.0, SPSS Inc., Chicago, IL, USA). The percentages of anti-HLA antibodies, demographic characteristics, and HCV treatment results were compared with chi-square test) and Fisher exact test, and numeric variables were compared with Mann-Whitney U test. The level of significance was defined by P ≤ .05.

Results

After evaluating 112 patients, a total of 40 patients (16 female and 24 male patients) were included after application of exclusion criteria. The mean age was 41.5 years (range, 18-65 y). The average duration of dialysis therapy was 11.2 years (range, 4-21 y), and the average duration of IFN treatment was 37.5 months (range, 12-52 mo). The SVR rate was 18 of 40 patients (45%). The duration of renal failure and duration of IFN treatment were comparable between PRA 1 and PRA 2 groups (Table 1).

There were 31 patients with negative PRA 1 (13 with SVR and 18 with no SVR) and 9 patients with positive PRA 1 (5 with SVR and 4 with no SVR). The SVR ratio was not correlated with PRA 1 positivity (Fisher exact test, P > .05). There were 31 patients with negative PRA 2 (11 with SVR and 20 with no SVR) and 9 patients with positive PRA 2 (7 with SVR and 2 with no SVR). The SVR ratio was significantly correlated with PRA 2 positivity (Fisher exact test, P ≤ .05) (Table 1).

Discussion

The clinical importance of HLAs in transplant has been known for 40 years. The antibodies against HLA, when they have been formed before transplant, are important in allograft survival and they form a significant barrier in kidney transplant. Pretransplant positive PRAs are related to increased incidence of hyperacute/acute rejection, chronic rejection, and early/latent graft loss.^22,23 Kidney transplant recipients with high PRA have worse outcomes than those with lower PRA.

Genetic factors of the host determine the clinical outcome of viral infections and CD4+ T-cell-mediated immune response. The HLA class I and II genes also modify cellular and humoral immune responses. Class I and class II genes are expressed on the surfaces of activated T lymphocytes, B lymphocytes, Langerhans cells, and dendritic cells. These molecules function during presentation of internalized antigenic proteins to the CD4+ T-helper cells.

Previous evidence indicated that positive anti-HCV serology is inversely related to graft and overall survival.^2,24,25 This finding is partially explained by elevated PRA percentages (secondary to increased antigenic stimuli by HCV) which eventually results in graft rejection.

The IFN-α may activate an inflammatory response by a bystander mechanism, which may develop into a cellular or humoral response, depending on the genetic susceptibility of the individual. Although IFNs exert strong immuno­modulatory effects in several ways, the mechanisms by which they trigger anti-HLA antibody production are incompletely understood. The up-regulation of both HLA and cytokine genes and induction of long-lived antibody production and immunologic memory are possible explanations for this phenomenon.¹⁸

In a previous case report, IFN-α treatment resulted in increased percentage PRA levels and development of new specific PRAs, indicating that IFN may mediate changes in immune system responses.²⁶ The IFN-α-based therapy induces expression of various genes, which include genes involved in antigen presentation and T-cell activation.^27,28 The IFN-stimulated genes related to T-cell activation also are induced during treatment of HCV with IFN-α.²⁹

In the present study, we hypothesized that PRA change can be used as a surrogate marker of SVR prediction. Our preliminary findings might support such a novel use of PRAs. In contrast, IFN treatment itself might have resulted in an increase in PRA levels. Various secondary antibodies and auto­immune conditions have been related to chronic HCV infection.³⁰ Therefore, development of high PRAs may be explained by development of HCV-related autoimmunity. However, in a previous study in end-stage renal disease patients, PRA levels did not differ between anti-HCV-positive (PRA 1 [ABC], 28.9%; PRA 2 [DR], 21.8%) or negative subgroups (PRA 1 [ABC], 19.4%; PRA 2 [DR], 20.9%) (P > .05), indicating that there was no correlation between HCV-related autoimmunity and PRA levels.³¹

In our study, we observed that persistent PRA elevation in patients receiving IFN treatment had higher SVR rates. We believe that future studies can be performed to identify this specific risk by identifying anti-HLA antibodies and comparing them before and after IFN treatment. In addition, prospective studies are required to delineate the natural history of increased PRA levels induced after IFN treatment.

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