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Selective decrease in hepatitis C virus-specific immunity among African Americans and outcome of antiviral therapy: Virahep-C Study
 
 
  Hepatology August 2007
 
Hugo R. Rosen 1 *, Scott J. Weston 2, KyungAh Im 3, Huiying Yang 4, James R. Burton Jr. 1, Henry Erlich 5, Jared Klarquist 1, Steven H. Belle 3, Virahep-C Study Group 1Integrated Program in Immunology and Hepatitis C Research Center, Division of Gastroenterology & Hepatology, University of Colorado, Denver, CO 2University of California at San Francisco School of Dentistry, San Francisco, CA 3Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 4Genetics, Cedars-Sinai Medical Center, Los Angeles, CA 5Roche Molecular Systems, Pleasanton, CA
 
Funded by:
National Institute of Diabetes and Digestive and Kidney Diseases
National Center on Minority Health and Health Disparities (NCMHD)
Intramural Research Program of the National Cancer Institute (NCI)
Roche Laboratories, Inc.; Grant Number: U01 DK60329, U01 DK60340, U01 DK60324, U01 DK60344, U01 DK60327, U01 DK60335, U01 DK60352, U01 DK60342, U01 DK60345, U01 DK60309, U01 DK60346, U01 DK60349, U01 DK60341
National Center for Research Resources (NCRR) General Clinical Research Centers Program; Grant Number: M01 RR00645, M02 RR000079, M01 RR16500, M01 RR000042, M01 RR00046
 
Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, end-stage liver disease, and hepatocellular carcinoma throughout the world. Considerable evidence indicates that the risk of viral persistence, natural history, and response to antiviral therapy varies among racial groups, but limited data exist on potential mechanisms to account for these differences. Type 1 helper (Th1) responses to HCV proteins and cytomegalovirus (CMV) antigens were examined using a sensitive interferon (IFN)-y enzyme-linked immunospot (ELISPOT) assay in 187 Caucasian American (CA) and 187 African American (AA) patients with chronic genotype 1 infection. ELISPOT responses were examined relative to human leukocyte antigen (HLA) class II alleles and outcome of therapy with pegylated IFN and ribavirin. Th1 responses specific to hepatitis C core protein and combined HCV antigens were significantly lower in AAs compared to CAs, but CMV responses were comparable in the 2 races. The HCV difference in immunity remained after adjusting for gender, serum alanine aminotransferase, histologic severity, and viral level, and was not accounted for by the differential prevalence of human leukocyte antigen class II alleles. Pretreatment total HCV-specific CD4+ T cell response was associated with sustained virologic response (SVR) to pegylated IFN and ribavirin; 43% of patients who had more than 168 ELISPOTs/106 peripheral blood mononuclear cells (above background) experienced SVR compared to 28% of those who did not (P= 0.007). ELISPOT response was independently associated with SVR by multivariable analysis.
 
Conclusion: Compared to CAs, AAs have weaker HCV-specific immunity. Pretreatment HCV-specific immunity is associated with response to combination antiviral therapy.
 
Discussion

The precise mechanisms by which the host immune response determines the outcome of acute HCV infection, natural history/progression in chronic hepatitis, and response to antiviral therapy are not completely elucidated. CD4+ T cells play an essential role in antiviral immunity by providing help for priming and sustaining CD8+ CTLs.[2][26] In individuals who ultimately fail to control the virus, a wide range of CD4+ T cell activity has been reported.[27] In the current study, we found that AAs, who as a group have been previously reported to demonstrate lower rates of spontaneous clearance of acute HCV, milder degrees of liver injury, and lower rates of response to antiviral therapy,[6-15] show relatively abrogated HCV-specific CD4+ T cell responses, yet intact responses to CMV. Considering the importance of CD4+ Type 1 helper (Th1) response in promoting the generation of CTLs, it is conceivable that impairment in HCV-specific cell-mediated immunity might result in less hepatic immunopathology but also be associated with a greater likelihood of nonresponse to antiviral therapy.[28] We recognize that race is a construct that reflects social stratification of groups according to phenotypic and cultural characteristics;[29] nonetheless, our findings have important implications in understanding the basis for variable outcomes in HCV infection, including differential response to antiviral therapy.
 
The first study to suggest racial differences in HCV-specific immunity was performed by Sugimoto et al.,[30] showing that although AAs had a higher frequency of proliferative responses to HCV antigens, as a group, they did not produce IFN-y. In keeping with those results, we found an impaired HCV-specific Th1 response in AAs as compared to CAs. The study by Sugimoto et al.[30] was limited, however, by the fact it included only 52 patients with chronic infection. Moreover, AAs in the Sugimoto study also had a higher incidence of genotype 1 (90% versus 72% in CAs), and liver function parameters that were better preserved (significantly lower prevalence of cirrhosis), an important consideration given the reported inverse correlation between the peripheral CD4+ T cell frequency and extent of liver injury.[31] Our large sample size allowed us to adjust for the possible interactions of liver injury (i.e., HAI and ALT) with immune response by multivariable modeling. We found race to be independently associated with decreased HCV but not generalized immune responsiveness (i.e., intact CMV and PHA responses). In general, the magnitude of T cell responses and thus immunity are the sum effect of activation/expansion, death and stability, or memory.[32] Thus, the lower level of HCV-specific responsiveness in AAs could reflect differential antigen processing and presentation, defects in the priming or expansion of naive HCV-specific helper T cells, selective deletion of memory cells, preferential sequestration in the hepatic compartment, or more robust counterregulatory mechanisms.[33]
 
The immunogenetic background of HCV-infected individuals might in part account for the observed variation in viral-specific immunity. In this regard, binding of HCV peptides to HLA molecules is a critical step for the initiation of an antigen-specific immune response. We found significant differences in the frequencies of HLA class II alleles in the 2 racial groups (Supplementary Data). Of interest, the DRB1*11 allele, previously associated with protection from progressive HCV infection,[34] was found in AAs at a higher frequency than in CAs; we found no association between this allele and histologic severity (data not shown). Furthermore, inclusion of HLA class II alleles into the multivariable model did not modify the association between race and immunity, indicating that perhaps other immune modulating genes or factors may be of greater significance in explaining the basis for racial differences in HCV immunity.
 
Prior studies addressing the question of baseline HCV-specific immunity as a correlate of virologic outcome to therapy have led to conflicting results, likely in part related to the use of less sensitive assays,[35] small sample size,[36] inclusion of non-1 genotype infected patients,[37][38] and limited repertoire of HCV antigens analyzed.[39] The finding in our study that CD4+ T cell responses independently correlate with treatment outcome (irrespective or race) suggests that novel therapeutic approaches aimed at augmentation of T cell immunity (e.g., vaccination, adoptive transfer) might potentially lead to a higher rate of virological response.
 
Article Text
 
Hepatitis C virus (HCV) infection affects approximately 180 million people and is one of the leading causes of chronic hepatitis, end-stage liver disease, and hepatocellular carcinoma throughout the world.[1] Host immune responses appear to be crucial in the control of infection, and patients with self-limited courses of acute infection demonstrate vigorous proliferation and interferon (IFN)- production by HCV-specific CD4+ T cells, whereas weaker T cell responses have been associated with viral persistence.[2] In persistent infection, cellular immune responses may also offer varying degrees of protection against viral replication and tissue damage,[3] as suggested by the inverse correlation between levels of HCV-specific cytotoxic T lymphocytes (CTL) and viral levels in several studies.[4][5]
 
The prevalence, natural history, and response to antiviral therapy appear to differ significantly among racial groups.[6] The Fourth National Health and Nutrition Examination Survey has shown a 2-fold greater prevalence of HCV antibodies among African Americans (AAs) as compared to Caucasian Americans (CAs), including a remarkable 18% prevalence of anti-HCV positivity among AAs aged 45 to 49 years[7]; moreover, a greater proportion of seropositive AA than CA patients have HCV viremia, indicating a higher rate of persistence after acute HCV infection among AAs.[8] AAs also demonstrate a 2-fold higher incidence than CAs of primary hepatocellular carcinoma related to chronic HCV infection.[9] Interestingly, independent cohort studies have demonstrated that, compared to CAs, AAs have milder hepatic necroinflammatory activity and fibrosis progression[10][11] and are 8 times more likely to have normal serum ALT.[12] Indeed, one study estimated that the average time to development of cirrhosis was 22 years in CAs and 40 years in AAs.[12] It is well established that AAs have significantly diminished response rates to IFN-based antiviral therapy.[11][13-15] Differential host immunity to HCV may be implicated as a potential unifying mechanism to explain this set of observations.
 
Because of the underrepresentation of AAs and subject heterogeneity (e.g., different HCV genotypes) in previous clinical studies of HCV, the National Institute of Diabetes and Digestive and Kidney Diseases has sponsored a multicenter study of viral resistance to antiviral therapy to hepatitis C (Virahep-C). The clinical results of the Virahep-C study have been recently published and confirm lower rates of sustained virologic response (SVR) in AAs (28%) as compared to CAs (52%).[14] In the current analysis, we measured CD4+ T cell responses to recombinant HCV genotype 1-derived antigens in order to determine whether there are racial differences in HCV-specific immunity and whether pre-treatment immunity was associated with outcome of combination antiviral therapy.
 
Patients and Methods
 
Study Design

Virahep-C is a multicenter collaborative treatment study using combination therapy (pegylated IFN alfa-2a and ribavirin) for treating naive participants (205 CA and 196 AA) with HCV genotype 1 (see Supplementary Materials).
 
HCV Genotyping and Quantitation
We determined HCV genotype by a line-probe hybridization assay (VERSANT HCV Genotype Assay, Tarrytown, NY) and we quantified viral level from simultaneously collected serum specimens by the Roche Cobas Amplicor HCV monitor version 2.0.
 
Cell Preparation
We obtained blood samples during the second screening visit (prior to initiation of antiviral therapy). We isolated peripheral blood mononuclear cells (PBMCs) from whole blood using cellular preparation tubes (Becton-Dickinson, Franklin Lakes, NJ; anticoagulant sodium citrate). PBMCs were viably frozen in 80% fetal bovine serum (BioWhittaker, Walkersville, MD), 10% dimethyl sulfoxide, and 10% Roswell Park Memorial Institute (RPMI) 1640 Media (Life Technologies, Grand Island, NY) in liquid nitrogen. After thawing, we cultured PBMC in medium consisting of RPMI 1640 Media (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (BioWhittaker), 50 ug/mL gentamicin sulfate (BioWhittaker), 5 _ 10-5 M 2 ME (Sigma, St. Louis, MO), and 2 mM glutamine (Life Technologies).
 
HCV and Control Antigens
Purified recombinant HCV genotype 1-derived proteins were either kindly provided by Michael Houghton (E2, NS4, NS5; Chiron; Emeryville, CA) or purchased from Mikrogen (core, NS3; Munich, Germany). HCV proteins (10 ug/mL) included HCV core (aa 1-115) and NS3 (aa 1007-1534), both derived from genotype 1b, and were expressed in Eschericia coli, purified by ion exchange chromatography followed by preparative SDS gel electrophoresis, and suspended in Tris/glycine buffer. Bacterial lipopolysaccharide content was <30 pg endotoxin/g protein. HCV E2 (383-715), NS4 (1569-1931), NS5 (2054-2995) were expressed as COOH-terminal fusion proteins with human superoxide dismutase in yeast. We used yeast and superoxide dismutase (and for Mikrogen antigens, SDS buffer) as controls for nonspecific stimulation in enzyme-linked immunospot (ELISPOT) assays. We purchased gamma radiation-inactivated cytomegalovirus (CMV) antigen that had been cultured in MRC-5 monolayers from Microbix Biosystems (Encinitas, CA). PBMC with media but no antigen served as the control for CMV stimulation. We used phytohemagglutinin (PHA, 50 ug/mL Sigma; #L2769) and superantigen staphylococcal enterotoxin B (SEB, 1 ug/mL; Pharmingen, San Diego) as positive controls in each assay. We included only patients who had viable PBMC that produced IFN-y following stimulation with PHA or SEB in the analyses; we excluded results from 14 patients because they lacked responses to these positive controls, indicating that the cells were not viable. Another 11 subjects had unusable PBMC samples, and we did not run ELISPOTs on samples from 2 subjects, so we excluded 27 Virahep-C participants from this analysis. These 27 subjects were not significantly different from the 374 who were included in terms of age, gender, ALT, total histologic activity index (HAI), and fibrosis scores, but had slightly lower viral level (median log10 5.97 versus 6.50; P = 0.049).
 
IFN-y ELISPOT Assay
We performed ELISPOT assays as detailed, with minor modifications.[16] We coated a total of 96-well nitrocellulose-backed plates (MAHA S4510; Millipore, Bedford, MA) as recommended by the manufacturer with 10 ug/mL capture mouse anti-IFN-y (1-D1K; Mabtech AB, Nacka, Sweden) overnight at room temperature. We then washed plates 3 times with PBS/0.05% Tween 20 (Sigma), and blocked them with RPMI/10% human serum for 1 hour at room temperature. We added thawed whole PBMC at 2.5 _ 105 cells per well in triplicate with RPMI + 10% human serum and low dose interleukin (IL)-2 [0.50 ng/mL (10 U/mL); Chiron Corporation, Emeryville, CA] for the HCV and CMV antigens, and when permitting, in 6 wells for negative controls. For PHA and SEB control wells, we used 1.25 _ 105 PBMC (no IL-2 added for PHA or SEB). We incubated the plate for 44 hours at 37C. We quantitated spots using a Zeiss Axioplan 2 microscope with 3200 K incident illumination equipped with a Epiplan Neofluar 5_/0.15 objective, Sony DXC 950 CCD camera, Mrzhuser scanning stage, MCP4 control unit, Pentium PC computer, and KS ELISPOT software (Carl Zeiss Vision; Hallbergmoos, Germany; http://www.zeiss.de).
 
HLA Class II Typing
We established an Epstein-Barr virus-transformed cell line for each subject who consented, for genetic testing, and we extracted genomic DNA from these cell lines. We performed class II molecular high-resolution genotyping for DRB1, DQA1, DQB1, DPA1, and DPB1 loci by amplifying genomic DNA with biotinylated primers and hybridization to an immobilized probe array of (sequence specific oligonucleotides). We scanned the probe reactivity patterns and interpreted them as human leukocyte antigen (HLA) genotypes using StripScan software (Roche Molecular System). We constructed 3 marker haplotypes (DRB1-DQA1-DQB1) by using the PHASE v2.0.2 Program[17] (available from http://www.stat.washington.edu/stephens/phase.html).
 
Statistical Analysis
We generated summary statistics to describe the study sample at baseline. We used descriptive statistics, including measures of central tendency (e.g., means, medians) and estimates of spread (e.g., SD) for continuous variables. We used Pearson's chi-squared test (uncorrected), or Fisher's Exact Test, to test for racial differences in categorical variables, whereas we used the Wilcoxon rank sum test for continuous variables. To account for the multiple comparisons when examining HLA allele frequencies between the racial groups, we used Holm's step-down method[18] to obtain adjusted P-values. We masked the laboratories performing the ELISPOT assays and HLA typing to all demographic and clinical patient information; we sent the results to the Data Coordinating Center, where all analyses were performed.
 
We calculated the quantitative response measurements by taking differences in the mean ELISPOT counts between active and control wells for Core, E2, NS3, NS4, and NS5 antigens and CMV. We calculated both numbers of spots and areas of spots. We calculated Spearman's correlation coefficient to assess the strength of association between number of spots and area. We defined qualitative response as having at least 2 (of 3) active wells (containing antigen) with number of spots both: (1) greater than the average of the controls plus 10 spots; and (2) greater than the average of the controls plus twice the SD of the number of spots across the control wells.
 
We used multiple linear regression analysis to examine the association between the number of spots and independent variables hypothesized to be related to response, including gender, baseline viral level (categorized as less than or at least 8 _ 105 IU/mL), and total inflammation HAI based on Knodell scores (subscores and sum of periportal, lobular, and portal inflammation scores).[19][20] Due to the presence of outliers, we performed robust regression with Tukey's biweight function,[21] and confirmed the results. We also examined the relationship between race and CD4+ T cell response accounting for differences in HLA class II allele or haplotype prevalence using multiple linear regression.
 
We assessed the adjusted association of race with the probability of SVR using stepwise modified Poisson regression modeling.[22-24] Selected factors with P < 0.25 in univariable analyses were eligible for entry in the regression model for predicting SVR. We added the number of spots for each individual antigen, and across all antigens, to the result of the stepwise model resulting in 6 models; 1 for each antigen (Core, E2, NS3, NS4, and NS5) and for all antigens combined. We employed classification and regression tree (CART) analysis to find a cutoff ELISPOT value for all antigens combined that maximally discriminated SVR from non-SVR. For these latter analyses, we set all negative responses (i.e., greater response in control wells than active wells) to 0.
 
A proportion of the sample had more spots in the control wells than active wells in at least 1 of the antigens. In other words, a response in the negative control wells occurred when the average number of spots across replicates in the control wells was greater than the average number of spots across replicates in the active wells. Though we performed analyses both including and excluding data from people with a response in the negative control wells, the results were qualitatively the same so we reported only results that include all observations.
 
For all analyses, we set statistical significance at a= 0.05. We carried out all statistical analyses in STATA 7.0 (Stata Corporation, College Station, TX), SAS 8.02 (Statistical Analysis Software; SAS Institute, Cary, NC), and CART (http://www.salford-systems.com).
 
Results
 
AAs Demonstrate Decreased HCV-specific But Intact CMV-specific CD4+ Th1 Responses

Of the 401 patients with chronic HCV infection enrolled in the Virahep-C study, 374 had viable PBMC stored at baseline prior to initiation of antiviral therapy. The demographic and clinical characteristics of the study population that could be evaluated with immune assays are shown in Table 1 H(and are not significantly different from the total enrolled population). AAs were significantly more likely to have genotype 1b infection than CAs, whereas CAs were more likely than AAs to have mixed genotype infection. The percentages of patients in each racial group with cirrhosis (Ishak fibrosis score 5 or 6)[19] and severity of HAI[20] were similar. There were no significant differences in age, gender, or risk factors for HCV acquisition between CAs and AAs at enrollment, but AAs had significantly lower serum ALT, in accord with previous reports.[11][12]
 
In order to test the hypothesis that AAs have relatively diminished HCV-specific responses, we used sensitive IFN-y ELISPOT assays to quantify CD4+ T cell responses to individual recombinant HCV antigens and to CMV. As described in Patients and Methods, we used recombinant proteins derived from genotype 1a (E2, NS4, and NS5) and 1b (core and NS3). The peripheral CD4+ T cell response directed against HCV core was lower in AAs than in CAs (P = 0.022) (Fig. 1). Moreover, the total HCV-specific immune response, derived from the sum of responses to HCV core, E2, NS3, NS4, and NS5, was significantly lower in AAs than in CAs (P = 0.007). For total HCV responses, the interquartile range (IQR) (25%, 75%), for CAs was 132 to 546 and for AAs was 68 to 437. In contrast, the response to CMV antigens (including early and late antigens, nuclear antigen, cytoplasmic antigen, and structural and nonstructural proteins) did not differ significantly between the racial groups. Comparison of non-HCV core responses (E2 + NS3 + NS4 + NS5) between races nears but does not reach statistical significance (P = 0.057; nonparametric Wilcoxon test).
 
The large Spearman correlation coefficients (rs) between ELISPOT number and area for each antigen (core, rs = 0.951; E2, rs = 0.909; NS3, rs = 0.919; NS4, rs = 0.947; NS5, rs = 0.897; CMV, rs = 0.961; all P < 0.0001) validate the internal consistency of our assay results. Accordingly, the median ELISPOT area for total HCV response was 2.56 _ 105 um2 and 4.11 _ 105 um2 for AAs and CAs, respectively (P = 0.011). There was not a significant difference in immune response in patients according to infecting HCV genotype (data not shown). The breadth of the HCV-specific response, i.e., proportion of patients with qualitative responses to recombinant proteins, was not statistically different according to race: 2 or more antigens (Ag) (29.8%), 3 or more Ag (11%), 4 or more Ag (3.7%), all 5 HCV Ag (0.6%).
 
Multiple linear regression analysis adjusting for gender, serum ALT, Knodell inflammatory score, and baseline viral level confirmed the independent association between race and HCV-specific immunity (Table 2). Therefore, assessment of IFN-y production of PBMC, which depends on T cells that have been exposed to antigen and are therefore clonally expanded in vivo, independently shows racial differences in the vigor of HCV-specific but not CMV-specific responses.
 
Racial Distribution of HLA Class II Alleles
Presentation of hepatitis C viral epitopes to CD4+ T cells depends on the HLA class II molecule(s) involved, which translate into structurally diverse peptide binding grooves.[25] We determined the frequency distribution of HLA class II alleles in the 2 racial groups, and whether HCV ELISPOT responses differed for specific alleles and haplotypes (see Supplementary Materials).
 
To evaluate whether the observed CD4+ ELISPOT response difference between racial groups could be explained by the race-related HLA class II allele distributions, we examined CD4+ ELISPOT response by the allele carrier status within each race (Supplementary Table 1). Although the distributions of response were significantly different by the allele carrier status for some allele types (such as DPA1*0103, DQA1*03, DQA1*04, and DRB1*11) within each race, none of these differences were consistent in both race groups and none remained significant after adjusting for multiple testing. In the adjusted multivariable regression models, we found no significant independent association between alleles, haplotypes, and the CD4+ ELISPOT responses, further underscoring that the polygenic nature of the interaction between HCV and host (including the racial effect on immunity) is not fully explained by HLA genes.
 
Pretreatment HCV-specific ELISPOT Associated with SVR to Antiviral Therapy We employed CART analyses to find a cutoff ELISPOT that maximally discriminated SVR from non-SVR. CART analyses failed to reveal an association with individual HCV antigens and SVR. We found that a difference of 168 HCV-specific IFN-y ELISPOTS per 106 PBMC (sum of HCV responses above negative control) discriminated between responders and nonresponders. Combining CAs and AAs, 43% of patients who exceeded this threshold experienced sustained virological response (SVR, HCV RNA negativity 24 weeks following end of therapy) compared to 28% of those who did not (P = 0.007) (Fig. 2A). Among CA patients, 53% who exceeded this threshold experienced SVR, as compared to 38% of CA who did not (P = 0.102). Among AA patients, 31% who exceeded this threshold experienced SVR, as compared to 22% who did not (P = 0.239) (Fig. 2B). Baseline ELISPOT response was significantly associated with SVR (relative risk = 1.37; 95% CI, 1.01-1.85; P = 0.042) after adjusting for potential confounding variables (race, gender, baseline viral level, the interaction between race and baseline viral level, histologic severity as assessed by activity index, and proportion of maximal pegylated IFN dose received in the first 24 weeks) (Table 3). Moreover, because dose reduction was associated with outcome, we performed a subset analysis in patients receiving full dose. We masked the laboratory personnel performing the ELISPOTs with regard to all demographic, clinical, and SVR data. SVR varied by baseline HCV-specific ELISPOT level (Fig. 2C). There were no significant differences in HCV-specific CD4+ T cell responses by SVR within each race among the fully-dosed patients but there were racial differences within the SVR group. In other words, CAs who experienced SVR had significantly higher ELISPOTs than AAs who experienced SVR (median and IQR, 376 and 195 to 668 as compared to 262 and 143 to 405, respectively; P = 0.010) or AAs who did not experience SVR (P = 0.023). There was a trend toward a statistical association in the non-SVR patients; the median and IQR for CAs and AAs were 340 (167 to 600) and 219 (110 to 544), P = 0.064 (Supplementary Table 2). Hence, the differences across race and SVR response groups among fully-dosed participants shown in Fig. 2C appear to be mostly due to the racial difference in CD4+ T cell response.
 
 
 
 
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