The effect of IFN-λ1 on the production of HBsAg and HBeAg by PEB8 was examined by ELISA (Fig. 12). Compared to control, IFN-λ1 did not have significant effect on the secretion of HBsAg and HBeAg both at 24 h and 48 h, but poly(I:C) significantly reduced the secretion of HBeAg at both times and HBsAg at 24 h, suggesting that antiviral mechanism by IFN-λ1 may be different from that by poly(I:C).
Fig. 11. The effect of IFN-λ1 on HBV transcription in PEB8. PEB8 was treated with MBP (1,000 ng/ml), MBP-IFN-λ1 (3,000 ng/ml) and poly(I:C) (0.5 ug/ml).
After 24 h or 48 h, total HBV RNAs were isolated from the cells and subjected to Northern blotting. Upper panel: three transcripts of HBV; lower panel: ribosomal RNA (rRNA). The figure is representative of two independent experiments.
(A) HBsAg
(B) HBeAg
Fig. 12. The effect of IFN-λ1 on the secretion of HBsAg and HBeAg by PEB8.
PEB8 was treated with MBP (1,000 ng/ml), MBP-IFN-λ1 (3,000 ng/ml) or poly(I:C) (0.5 ug/ml). After 24 h or 48 h, the production of HBsAg (A) and HBeAg (B) was analyzed by ELISA. Relative Ag production was calculated as (OD of each condition/OD of negative control) x100.
H. Generation of the polyclonal and the monoclonal Ab against IFN-λ1
Although it was reported that IFN-λ1 mRNA expression was induced or enhanced by IFN-α and other stimulators, the amount of IFN-λ1 was not quantitated at protein level (Siren et al., 2005; Osterlund et al., 2005). The reason may be because polyclonal or monoclonal anti-IFN-λ1 Ab was not commercially available at that time. In order to establish a quantitation system to measure the production of IFN-λ1, the polyclonal and the monoclonal anti-IFN-λ1 Abs were generated.
First, from three immunized mice 25 hybridoma cells producing Ab against IFN-λ1 were selected and three clones of these, designated as HL1, HL2 and HL3, were established through limiting dilution. The binding activity of these monoclonal Abs to IFN-λ1 was revealed by ELISA using MBP-IFN-λ1 as well as His-IFN-λ1 (Fig. 13A) and by Western blotting using His-IFN-λ1 (Fig. 13B). The isotype of each monoclonal Ab was determined; for HL1, G1 and for HL2 and HL3, G2b.
Next, the reactivity of polyclonal rabbit anti-IFN-λ1 Abs to native and denatured form of IFN-λ1 was shown in Westren blotting and ELISA (Fig. 13B and Fig. 14A).
(A)
(B)
Fig. 13. The reactivity of monoclonal Abs against IFN-λ1. (A) The reactivity of supernatants of monoclonal Abs to IFN-λ1 was examined by ELISA using recombinant MBP-IFN-λ1 (1 ug/well) and His-IFN-λ1 (1 ug/well). (B) The reactivity of supernatants of monoclonal Abs to denatured IFN-λ1 was examined by Western blotting using His-IFN-λ1 (500 ng/lane) and HRP conjugated anti-mouse Ig Ab. (-):
myeloma cell culture supernatant. (+): rabbit polyclonal anti-IFN-λ1 Ab.
0
I. Establishment of ELISA for IFN-λ1
Indirect ELISA for IFN-λ1 was established using polyclonal anti-IFN-λ1 Ab (Fig. 14A) and HL2 monoclonal anti-IFN-λ1 Ab (Fig. 14B). The detection limits were 40 ng/ml for both polyclonal and monoclonal anti-IFN-λ1 Abs. Sandwich ELISA using both polyclonal and monoclonal Abs was performed but the detection limit was not better than indirect ELISA (data not shown).
(A) Polyclonal anti-IFN-λ1 Ab
(B) HL2 monoclonal anti-IFN-λ1 Ab
Fig. 14. The sensitivity of ELISA for IFN-λ1 using polyclonal and monoclonal anti-IFN-λ1 Abs. Serially diluted IFN-λ1 was detected by ELISA (A) using
polyclonal rabbit anti-IFN-λ1 Ab (1:1,000) or (B) using HL1 monoclonal anti-IFN-λ1 Ab (10 ug/ml). IFN-λ1 expressed in eukaryotic cells was purchased from R & D systems Inc. Values represent the mean ± S.D. of
quadriplicates.
IV. DISCUSSION
In this study, it was demonstrated that IFN-λ1 can inhibit HBV replication in PEB8 but not in WT10. Additionally, it was shown that HBV does not modulate the expression of CRF2-12, an IFN-λ1 receptor subunit. Also, ELISA for analyzing of production of IFN-λ1 was established.
Although antiviral activity of IFN-λ1 against several RNA viruses and cytomegalovirus has been reported (Robek et al., 2005), the effect of IFN-λ1 on HBV replication in human cells has not. A recent report showed that HBV replication in a murine hepatocyte cell line was inhibited by IFN-λ treatment (Robek et al., 2005). They treated murine hepatocyte cells with 1 to 10 ng/ml of murine IFN-λ2 expressed in E.coli (PEPROTCH,Rocky Hill, NJ, USA), analyzed HBV replication by Southern blotting at 24 h after the addition of IFN-λ2 and showed about 90%
reduction of HBV DNA replication intermediates. My results using human hepatoma cell lines revealed that 1,000 ng/ml of IFN-λ1 (equivalent of 3,000 ng/ml of MBP-IFN-λ1) reduced the level of viral DNA by 30% (corresponding to 100 fold reduction of viral progeny) in PEB8 cells but not in WT10 cells. Similar results were obtained in cells treated with 3,000 ng/ml of IFN-λ1 (data not shown). In this study recombinant MBP-λ1 was used because when I started these experiments IFN-λ1 was commercially unavailable. Then why did IFN-IFN-λ1 suppress HBV replication in PEB8, but not in WT10? Both cell lines originated from HepG2 cells. In both cell lines, comparable level of expression of IFN-λRs and IFN-λ-mediated antiviral gene
induction were observed. The known difference is that PEB8 was established by transfection of plasmid pcDNA containing a 1.05 HBV genome-length DNA of adwR9 subtype, while WT10 was made using retrovirus containing a 1.1 HBV genome length DNA of adr subtype (Fu L and Cheng YC, 2000). HBV adwR9 subtype belongs to genotype A, B and G, and HBV adr subtype belongs to genotype C. HBV genotype was reported to be an important predictor of response to treatment of IFN-α; better responses in A and B genotypes than those in C genotype was observed (Wai et al., 2002; Janssen et al., 2005; Thuy et al., 2005). Thus my results raise a question whether the antiviral activity of IFN-λ may vary in HBV genotypes.
Neither HBV transcription nor secretion of viral Ags was suppressed by the treatment of PEB8 with IFN-λ1. Similar to IFN-λl, type I IFN does not affect either viral transcription or production of secretory Ag. IFN-α-mediated suppression of HBV replication in HBV transgenic mouse occurs by reducing the intracellular content of HBV RNA-containing capsids without altering either HBV gene expression, translation, capsid maturation or virus secretion (Wieland et al., 2000).
IFN-β treatment of immortalized HBV transgenic hepatocytes results in rapid clearance of HBV RNA-containing capsids (Pasquetto et al., 2002). In an inducible HBV replication system, type I IFN suppresses HBV replication by preventing the formation of replication-competent core particles not by destabilization of pgRNA-containing capsids (Robek et al., 2005). To dissect the stage of HBV replication process affected by IFN-λl, further study is needed
IFN-λ l up-regulated the transcripts of MxA and 2’5’-OAS in HBV replicating
cell lines. The involvement of MxA in inhibitory activity of IFN-α on HBV replication has been reported (Gordien et al., 2001). In MxA transgenic mouse model, HBV replication was downregulated. By contraries in MxA deficient cells HBV replication was still inhibited by IFN-α (Rang A et al., 2002). Since MxA was induced by IFN-λl in WT10 which was nonresponsive to IFN-λl, it may be reasonable that MxA is not a major molecule for the suppression of HBV replication or at least MxA is not working in WT10 cellular context. Identification of antiviral proteins responsible for the activity of IFN-λl against HBV remains to be solved.
The expression level of an IFN-λ1 receptor subunit, CRF2-12 was not different in hepatoma cell lines regardless of HBV replication. It indicates that HBV does not modulate the expression of IFN-λR in order to resist to IFN-λ. As an escaping mechanism from the antiviral activity of IFN-α, downregulation of MxA promoter by HBV through direct interaction of it with precore/core proteins was reported (Fernandez et al., 2003). Also SOCS-1 expression in livers of patients with chronic HCV infection seems to be involved in resistance to IFN-α or IFN-λ (Brand et al., 2005; Imanaka et al., 2005). Based on the failure of IFN-λ in suppressing HBV replication in WT10 cells, it is conceivable that there may be an escaping mechanism against IFN-λ adopted by HBV.
Murine monoclonal Abs and rabbit polyclonal Ab able to react with native form as well as denatured form of IFN-λ1 was produced. Indirect ELISA using these Abs has the detection limit of 40 ng/ml. Although the sensitivity of ELISA need to be improved, analysis of IFN-λ1 production at protein level is now possible.
For many years, administration of IFN-α has been the mainstay of therapy for chronic HBV. In about 30 percent of patients who tolerated this regimen, successful responses have been reported. However, the side effects of therapy with IFN-α (fever, myalgias, thrombocytopenia, and depression) make it untolerable for many patients.
Moreover, in some patients autoimmunity occurs during administration of IFN-α.
This phenomenon may reflect the immunomodulatory activity of IFN-α, such as upregulation of MHC class I antigens, inhibition of apoptosis of cytotoxic T lymphocytes and direction of helper T cell differentiation to TH1 (Ganem and Prince, 2004). Although IFN-λ receptor is broadly expressed on non-hematopoietic tissues, it does not seem to be present on leukocytes. This may provide a possible therapeutic advantage for IFN-λs as a therapeutic agent with less adverse effects (Brand et al., 2005).
This study suggests that IFN-λl may be useful to repress HBV replication in selected chronic hepatitis B patients. I raised the possibility that HBV genotype may be a determinant for the responsiveness of IFN-λl treatment. Further studies are required to test this possibility and to identify other determinants leading to suppression of HBV replication by IFN-λl.
V. CONCLUSION
IFN-λ family has antiviral activity against some viruses, but its effect against HBV in human hepatocytes, natural host cells of HBV, has not been known. I examined whether IFN-λ1 has the suppressive effect on HBV replication in human hepatoma cells. It was demonstrated that HBV replication was inhibited by IFN-λ1 in PEB8, one of two HBV-replicating human hepatoma cell lines examined. Although it is not explainable why HBV replication was not inhibited in the other cell line, in both cell lines the transcription of the antiviral proteins, MxA and 2’5’-OAS was induced by IFN-λ1. In PEB8, treatment of IFN-λ1 did not suppress transcription of HBV nor did it reduce the secretion of HBsAg and HBeAg, indicating that the posttranscriptional stage of HBV replication may be affected by IFN-λ1.
The expression of the unique receptor subunit for IFN-λ1 was analyzed in HBV-replicating human hepatoma cell lines. In both cell lines, the amount of transcripts and protein of CRF2-12 was similar to that of their parent cell line, suggesting that HBV may not modulate the expression of IFN-λ1 receptor.
Finally, I produced the polyclonal and the monoclonal Abs reactive to native form and denatured form of IFN-λ1. Using these Abs the ELISA system with sensitivity of 40 ng/ml was established, which enables further study to analyze the production of IFN-λ1 by HBV infection.
My results indicated that the suppressive effect of IFN-λ1 on HBV replication may be affected by factors of cellular origin and/or viral origin. IFN-λ1 can be useful
in the treatment of HBV infection.
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-국문요약국문요약국문요약- 국문요약
B형 간염 바이러스의 감염과 관련된 인터페론 람다 시스템의 역할과 조절
아주대학교 대학원 의학과 홍 승 호
(지도교수 : 박 선)
연구배경 및 목적: B형 간염 바이러스 (HBV)에 의해 진행되는 만성
감염은 간경변과 간암을 유발할 수 있으므로 효과적인 치료법이 요구된다.
현재 사용되고 있는 인터페론 알파 (IFN-α)와 라미부딘 (lamivudine) 등은 치료효율과 부작용 등에서 개선이 요구된다. 인터페론 람다 (IFN-λ)는
CRF2-12와 IL-10R2로 이루어진 고유의 수용체에 결합하며 몇몇
바이러스의 증식을 억제하는 것으로 최근 보고되어 HBV 치료에 이용될 가능성을 보인다. 본 연구에서는 IFN-λ가 HBV의 증식을 억제하는가를 알아보고, CRF2-12 발현이 HBV 증식에 의해 조절되는 지에 대해 알아보고자 하였다. 또한 HBV에 의해 IFN-λ의 생산이 조절되는가를 알아보기 위해 IFN-λ에 대한 ELISA를 확립하고자 하였다.
연구 방법: 두 종류의 IFN-λ1을 세균에서 발현시켰다: 항바이러스
연구 방법: 두 종류의 IFN-λ1을 세균에서 발현시켰다: 항바이러스