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Interferon (IFN) was discovered as an antiviral agent during studies on virus interference. Isaacs and Lindenmann reported in 1957 that influenza virus-infected chick cells produced a secreted factor that mediated the transfer of a virus-resistant state active against both homologous and heterologous viruses¹. IFNs belong to a multigene family of inducible cytokines. IFNs are commonly grouped into two types; Type I IFNs are induced by virus infection, whereas Type II IFNs are induced by mitogenic or antigenic stimuli. IFN signaling involves an IFN-mediated heterodimerization of the cell surface receptor subunits, IFNAR-1 and IFNAR-2 with IFN-a/ß and IFNGR-1 and IFNGR-2 with IFN-?.² IFN binding leads to activation of overlapping pairs of Jak/Stat transcription factors by tyrosine phosphorylation and subsequent translocation, along with IRF-9 (p48), to the nucleus. The complex of these three proteins, known as IFN-stimulated gene factor 3 (ISGF-3), trimeric complex binds to a cis-acting DNA element, designated ISRE (IFN-stimulated response element), found in IFN-a/ß-inducible genes which results in an activation of the transcription of IFN-a/ß–inducible genes like protein kinase PKR, 2’, 5’-oligoadenylate synthetase, RNase L, RNA-specific adenosine deaminase, protein Mx GTPase, major histocompatibility complex proteins and inducible nitric oxide synthase^3,4.

The 2’,5’-Adenine (2-5A) response leading to the degradation of RNA requires two enzymes, Oligoadenylate Synthetase (OAS) and RNase L. OAS catalyzes the synthesis of oligoadenylates of the general structure ppp(A2’p)nA, commonly abbreviated 2-5A^5,6. As their name implies, they possess a 2’,5’-phosphodiester bond linkage. RNase L, a latent endoribonuclease, becomes activated after binding to 2-5A oligonucleotides. The expression, regulation, and function of the OAS and the 2-5A-dependent RNase L has been characterized extensively in IFN treated and virus-infected cells^5-7 (Figure 1).

A number of studies, by different groups have established that the 2-5A system functions in the antiviral effects of IFN. For example, elevated levels of 2-5A and the appearance of specific rRNA cleavage products characteristic of RNase L activation are correlated with IFN-mediated inhibition of encephalomyocarditis virus (EMCV)^8,9, vaccinia virus¹⁰ and reovirus infections¹¹. Introduction of 2-5A into cells has been shown to reduce the cytopathic effects of several viruses, including vesicular stomatitis virus (VSV), EMCV, poliovirus, and Semliki Forest virus¹², whereas a 2-5A analog inhibitor of RNase L has reduced the anti-EMCV activity of IFN in intact cells¹³. Further, in cell lines in which 2-5A pathway activity is defective, EMCV replication is resistant to the inhibitory effects of IFN¹⁴. The cloning of cDNAs encoding 2-5A synthetase and RNase L has provided a means of definitively addressing the antiviral function of the 2-5A system in cells¹⁵. Constitutive expression of the 40-kDa form of 2-5A synthetase conferred resistance to EMCV and mengovirus^16,17. The therapy for chronic hepatitis C has evolved steadily since alpha interferon was first approved for use in this disease more than 10 years ago. At present, the optimal regimen appears to be a 24 or 48-week course of the combination of pegylated alpha interferon and ribavirin¹⁸.

Studies have demonstrated the presence of IFNa antibodies (IFNa Ab) in the chronic hepatitis C patients receiving IFN therapy^19-21. There is no information in this context from India. Nonetheless, the presence of IFNa Abs has been seen in non-responding chronic hepatitis C patients^22-25. These antibodies could be interrupting the antiviral effect of IFN therapy by neutralizing them and influencing the changes in expression levels of OAS1 gene. This study aims to correlate the presence of IFNa Abs and their effect on OAS1 expression in patients of chronic hepatitis C with response to therapy.

This prospective cross-sectional study includes 122 chronic hepatitis C patients diagnosed as per American Association for Study of Liver Disease (AASLD) practice guidelines¹⁸, attending the medical wards and outpatient departments of Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India. The healthy group comprises of 50 blood samples from voluntary donors and 10 liver tissue autopsies from victims of acciodental deaths (as per ethical grounds) who did not have any evidence of hepato-billiary disease. Liver biopsy was carried out in 64 patients of chronic hepatitis C who gave consent. The liver tissues were collected in RNA. Later, blood samples were collected from subjects using aseptic precautions for viral serology and biochemical investigations. The remaining serum/plasma and tissue were stored at -70OC for HCV genotyping, viral load, quantification of serum IFNa Abs levels and hepatic OAS1 expression analysis. Out of these 122 CHC patients, 54 patients received interferon-alpha, 3 million units (3-MU) thrice weekly for 24 weeks for genotype 2/3 or 48 weeks for genotype 1. Patients were considered as responders while they attain no detectable levels of HCV RNA during therapy and remained in same condition even after one year of follow-up. Otherwise, they were treated as non-responders¹⁸. The study protocol was approved by the institutional ethical committee of Maulana Azad MedicalCollege, New Delhi, and was performed in accordance with the Declaration of Helisinki. Informed consent was taken from all the patients.

Histological examination was performed, and the severity of liver disease was assessed according to Knodell score for histologic activity index (HAI) and Ishak’s fibrosis staging using sections from formalin-fixed and paraffin-embedded liver tissue samples by hematoxylin-eosin staining²⁶.

HCV RNA was extracted using the TRI® reagent (Sigma) from plasma and Geno-sen’s real time PCR kit was used to quantify the HCV RNA.  The genotyping was done by type specific primers in a multiplex PCR system as described by Kazuaki Chayama²⁷.

The serum concentrations of IFNa Ab in HCV infected and healthy individuals were measured using anti-IFNa quantitative ELISA kit (Abnova; Taiwan). The IFNa Ab levels were determined by comparing the optical density of the samples with a standard curve containing the increasing amounts of IFNa Ab, as per the manufacturer’s protocol.

Total mRNA from liver tissue biopsies was isolated using Qiagen RNesy Mini Kit and cDNA library was constructed. These cDNA were used for the expression analysis of 2', 5'-OAS1 by relative quantification method with SYBR green dye in reference to housekeeping gene ß-actin on the Real Time PCR machine (Corbett Research; RotorGene-3000) using 2–??Ct method as explained by Livak et.al28. Normal autopsy liver tissues were used as calibrators for normalization. 

Statistical significance was determined by Chi-square test with Yates correction (wherever needed) or by 2-sided Fisher exact test, Mann Whitney U test and student’s t- test wherever applicable, using EpiInfo and statistical package for social science, version 10 (SPSS, Chicago, IL). The p values =0.05 were considered significant.

Out of 122 chronic hepatitis C cases having an average age of 35.93 ± 12.33 years (range 20-51 years), 42 patients were female (34.43%) and 80 patients were male (65.57%). In 50 healthy control group, 19 were females (38%) and 31 were males (62%) having an average age of 34.14±11.02 years (range 19-55 years). In chronic hepatitis C group (CHC), 68 patients were treatment naïve and 54 patients were treated with standard IFN-a and weight guided ribavirin for 24 weeks.

The IFNa Abs were present in 74 (60.65%) out of 122 chronic hepatitis C patients and only 5 (10%) healthy individuals were carrying the antibodies out of 50 (p value < 0.01; OR =13.87; 95% CI). Antibodies among the HCV patients did not show any correlation with age and sex. Out of 68 treatment naïve patients 28 (41.17%) had IFNa Ab mean concentration of 6.7 ±8.74 ng/ml and 46 (85.18%) out of 54 treated patients had mean of 34.80±52.11 ng/ml (p < 0.01; Mann-Whitney U) (Figure 2A).

Treatment responders relatively carries less IFNa Ab with mean 3.79±4.78 ng/ml when compared with non-responders with mean 36.25±53.68 ng/ml (p <0.01; Mann-Whitney U) (Figure 2D). Forty-six treated cases with IFNa Abs, were analysed for the correlation of IFNa Ab levels in non-responders by ROC curve. Cases with IFNa Ab levels more than 7.45 ng/ml (cutoff) have shown AUC of 0.882±0.06 with 73.7% sensitivity and 75.0% specificity towards non-responsiveness. In treated group 24 patients with low IFNa Ab levels (<7.45ng/ml) had significantly lesser viral loads of mean 1.87±3.76 (x105 IU/ml) than remaining treated 30 patients who had higher IFNa Ab levels (=7.45ng/ml) with mean 3.47±5.83 (x105 IU/ml) (p =0.01; Mann-Whitney U) (Table 1).

The relative expression levels of OAS1 mRNA did not show significance when compared between 30 treated patients (16.03±31.55 fold) and 34 treatment naïve (13.94±25.29 fold).When the expression levels of OAS1 were compared with IFNa Ab levels, 43 patients carrying low IFNa Ab levels (<7.45ng/ml) had 19.23±32.46 fold rise, whereas 21 patients with high IFNa Ab levels (=7.45ng/ml) had 6.09±12.94 fold rise (p < 0.01; Mann-Whitney U) (Figure 2B). Treatment enhanced the expression of OAS1 (6.7±13.93-fold rise) in the patients with higher antibody levels when compared with treatment naïve (2.33±0.58 fold rise; p =0.02). This rise was higher in treated patients with lower antibody levels (30.00±44.37 fold rise; p =0.03) (Table 1). Treatment responders who had resolved the infection possesses better OAS1 expression (42.16±53.51-fold rise) compared to the non-responders (10.21±20.16-fold rise) (Figure 2D).

When OAS1 expression levels were analysed by ROC curves, assuming low IFNa Ab levels facilitateexpression, it showed AUC of 0.741±0.106 with 75% sensitivity and 89% specificity when OAS1 expression is 4.5-fold rise or more. Out of 34 treatment-naïve patients, 16 had enhanced and 18 had lower expression levels whereas out of 30 treated, 11 had enhanced and 19 had lower expression levels.

Patients with enhanced OAS1 expression (=4.5-fold rise) had significantly low HCV viral loads in 11 treated patients (1.34±3.39 x 105 IU/ml) when compared with 16 treatment naïve patients (8.98±10.32 x 105 IU/ml) (p < 0.01; Mann-Whitney U) (Figure 2C). Whereas patients with lower OAS1 expression (<4.5 fold rise) had failed to show significant drop in HCV viral loads, when compared between 19 treated patients (3.69±6.15 x 105 IU/ml) and 18 treatment naïve patients (9.45±16.26 x 105 IU/ml) (p = 0.19; Mann-Whitney U).

The genotype distribution among CHC is 11(9.02%) GT1, 3(2.46%) GT2 and 108(88.52%) GT3. Treatment is received by 43 GT3 and all 11 GT1 patients out of which non-responders were 33 and 10 respectively. When IFNa Ab levels were correlated in different genotypes, only GT3 patients had shown significant antibody levels among 10 responders (3.57±4.98 ng/ml) and 33 non-responders (39.79±60.03 ng/ml) (p < 0.01; Mann-Whitney U) (Table 2).

In non-responders irrespective of HCV genotype OAS1 expressions were low (5.94±14.17 fold rise) in 16 patients with higher IFNa Ab levels compared with 17.12±28.51 fold rise in 8 patients with low IFNa Ab levels (p = 0.02; Mann-Whitney U).

The HAI scores were observed in different genotypes, viral loads, OAS1 gene expression levels and IFNa Ab presence among chronic hepatitis C infected patients. No significant correlation was found within genotypes, viral loads and HAI score. However, it was observed that, patients with HAI score 5 and above had OAS1 expression enhanced when IFNa Ab levels were low than high. As the inflammation increases, there is a decrease in trend of OAS1 gene expression (Figure 3).

Humans have naturally occurring IFNa Abs in their serum and it is a tempting theory that human cytokines and lymphokines, are at least, partly regulated by these antibodies. Ross et al. found antibodies in systemic disorders like SLE and cancer, but not before treatment²¹. Another study by Hansen et al had proposed binding of IgG antibodies to IFN but this binding is non-specific. Also, they did not find these antibodies in normal subjects²⁹. 

However, in the present study conflicting results were obtained depicting the presence of IFNa Ab in 5 (10%) healthy subjects, 28 (41.17%) treatment naïve and 46 (85.18%) treated patients. In the treatment naïve group, the IFN secreted for viral clearance might have influenced the production of IFNa Abs as a counter-regulatory mechanism.

IFNa Abs in serum was observed in a significant number of chronic hepatitis C patients when compared with normal healthy persons. We observed that the IFNa Ab concentrations were higher in the treated group than the treatment naïve group. This data gives us an inference that IFNa Abs were present in patients of chronic hepatitis C, and the IFN therapy further enhances their concentrations. Ikeda et al postulated that though IFNa Abs were present in acute hepatitis, they do not block IFN activity³⁰. Querashiet al in 2007 observed that IFNa Abs was formed in a small percentage of cases receiving interferon therapy¹⁹. However, in this study, a higher concentration of IFNa Abs levels was observed in treated HCV patients. The current study suggests that some chronic hepatitis C patients, who have not received interferon therapy, harbor IFNa Abs even before treatment and concentrations were increased in treated patients (Figure 2A).

Kobayashi et al (1996) studied the affect of IFN antibody on IFN therapy in 58 chronic hepatitis C patients³¹. They found that the response rate was not significantly different between patients with the antibody and those without (29% and 41%, respectively). Our study suggests that some cases of chronic hepatitis C patients who have not received IFN therapy mayharbouranti-IFN antibodies even before treatment and concentrations were increased in treated patients. These patients may be needed to be screened for the presence of anti-IFN antibodies before initiating therapy.

The OASs are interferon-induced proteins characterised by their capacity to catalyze the synthesis of 2-prime, 5-prime oligomers of adenosine (2-5As). 2-5As bind to RNase L and activate it, to degrade viral and cellular RNA, leading to inhibition of cellular protein synthesis and impairment of viral replication^11,32 and ³³. In the current study, it was observed that in treatment naïve patients when IFNa Ab concentrations were low there was significantly higher OAS1 expressions and vice versa. Similarly, in treated patients, the antibody presence and OAS1 expressionsshow an inverse relationship with each other. Thus, it appears that OAS1 expression is inhibited by IFNa Abs (Table 1 & Figure 2B). When the HCV viral load was correlated with OAS1 expression, it was found that, as enhancement in the expression of OAS1 takes place there is a simultaneous decrease in the viral load, to reduce the load in 30 treated patients (p value < 0.05) (Figure 2C). Similar observations were seen in 34 treatment naïve patients though the inference was statistically not established (p value 0.05).

In fifteen non-responder patients, significantly high IFNa Ab concentrations and low OAS1 gene expression level was observed, supporting the contention that IFNa Abs interfere in viral clearance. Thus, this could be a matter of concern particularly for non-responders (Figure 2D). Our results were in concurrence with previous reports^22-25 which indicate an increase in IFNa Ab levels after treatment and show its effect on viral clearance.

It was observed that when HAI score is 0-4 signifying minimal inflammation of liver in chronic hepatitis C patients, expression of OAS 1 gene was maximal. As the HAI score or inflammation increased, expression of OAS1 decreased. This signifies that, in the initial stage of hepatitis C, as the virus makes in-roads into hepatocytes, the expression of OAS1 increases to combat HCV viral replication and reduces inflammation. So far, the correlation between HAI and OAS1 expression has not been studied. In the current study, it was found that among IFNa Ab carriers, the levels of OAS1 expression decreased with the progression of liver disease  (Figure 3). This might be due to disruption of the 2-5A system, which controls cell growth, differentiation, and apoptosis³³.

The neutralizing IFN-a-Abs are enhanced with administration of recombinant interferon and reduced OAS1 expression, eventually leading to failure/reduction in the effectiveness of the IFN and ribavirin treatment (Figure 1). Our study proposes a strategy of constant monitoring of IFN-a-Ab during therapy along with HCV RNA levels. The cut-off level of <7.4ng/ml for IFN-a-Ab was elucidated as the level that reduced the expression of OAS-1 gene which in turn affected viral clearance during therapy. This study infers the need for incorporating IFN-a-Ab as a parameter that needs to be considered in modulating the dosage of the current interferon treatment regimen. 

We are grateful to Dr Bhudev Chandra Das, Ambedkar Center for Biomedical Research, Delhi University, for his constructive criticism and helpful comments in the preparation of the manuscript.