- Email: [email protected]
Long-term response to interferon alpha is unrelated to“interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-1b infection
Journal of Hepatology 1999;30: 1023-1027 Printed in Denmark All rights reserved Munksgaard . Copenhagen
Copyright 0 European Association for the Study of the Liver I999
Journal of Hepatology ISSN 01684278
Long-term response to interferon alpha is unrelated to “interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-lb infection Giovanni Squadrito ‘, Maria Elena Orlando’, Irene Cacciola’, Maria Grazia Rumi2, Marco Artini3, Antonio Picciotto4, Oreste Loiacono5, Rocco Siciliano6, Massimo Levrero3 and Giovanni Raimondo’ ‘Dipartimento Medicina Interna, Messina University; 2Dipartimento Medicina Interna. Milan University: ‘Laboratorio Espressione Genica Fondazione A. Cesalpino Universitd La Sapienza, Rome University; 4Cattedra Gastroenterologia, Genoa University; ‘Istituto Clinica Medica, Palermo University; and 61stituto Medicina Interna, Catania University, Italy
Background/Aims: Contradictory data have been reported about the predictive value of the variability in interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV) genotype-lb on response to interferon-alpha (IFN-a) therapy. The aim of this study was to examine this issue in a series of patients with long-term response to IFN treatment. Methods: We retrospectively analyzed 24 patients with chronic HCV genotype-lb infection treated with IFN-a (total dose median 677, range 216-1350 MU) selected in 6 Italian Liver Units. These patients were defined as true long-term responders (LTR) since they showed persisting biochemical and virological responses to IFN treatment (mean follow-up 38 months). HCV genomes from pretreatment serum samples were amplified and directly sequenced. The ISDR amino-acid sequences obtained were aligned and compared with the published sequence of HCV-J. Results: Amino-acid substitutions were found in 23 of
the 24 patients, and 22 of them showed an H to R amino-acid change at codon 2218. Fourteen patients showed only one mutation (at codon 2218), two had 2, five had 3, one had 4 and one had 5 mutations. When we compared the ISDR sequences from the 24 LTR with those of non-responders (NR), we found no significant correlation between the number of mutations and the response to therapy. Conclusions: Our results demonstrate that the persisting efficacy of IFN treatment in patients with chronic HCV is not related to the number of ISDR amino acid susbstitutions of the infecting viruses. Further studies are needed to verify whether other NSSA sequences outside the ISDR might be involved in the mechanisms of IFN resistance.
H
have shown the association between genotype lb and a low rate of long-term response to IFN-a (2,3). Insight regarding the resistance of genotype 1b infection to therapy has been reported from Japan by Enomoto et al., suggesting that mutations in a 40-amino-acid stretch, named the “interferon sensitivity determining region” (ISDR), in the nonstructural viral protein 5A (NSSA) modulates sensitivity to IFN-a (4,5). According to these authors, high variability in the ISDR before therapy could thus predict the response to IFN-ol in most cases. However, data reported by subsequent papers have been contradictory, since some studies confirmed the above-mentioned results by Enomoto et
C virus (HCV) is a major cause of progressive chronic hepatitis worldwide and the management of therapy for HCV infection is therefore a fundamental medical issue. The only approved drug for chronic hepatitis C is interferon alpha (IFN-a), although long-term response to this therapy is achieved in around 25% of treated patients (1). Several studies EPATITIS
Received 10 November:
revised I7 December:
accepted 17 December
1998
Giovanni Raimondo, Dipartimento Medicina Interna, Policlinico Universitario, 98 100 Messina, Italy. Tel: 39 0 90 2212393. Fax 39 0 90 2935162. E-mail [email protected] Correspondence:
Key words: Chronic hepatitis C; Direct sequencing; Non-structural 5A protein; Polymerasae chain reaction; Viral genomic heterogeneity.
1023
G. Squadrito et al. taneously at a dose of 3 to 6 MU of IFN for 6 to 12 months (total dose median 677, range 2161350 MU). All patients had normal alanine aminotransferase (ALT) activity during and after treament and they were constantly HCV-RNA negative for a mean follow up of 38 months (range 1%84). Thirteen of them underwent a needle liver biopsy 12 to 60 months after stopping therapy; seven showed histological recovery and six marked improvement (Table 1). The study was performed according to the principles of the Declaration of Helsinki, and informed consent was obtained in every case. HCV genotyping was performed by restriction fragment length polymorphism (RFLP) analysis. RNA extraction, reverse transcription of NSSA region and polymerase chain reaction (PCR) were performed as previously reported (11). PCR products were analyzed by gel electrophoresis using 2% agarose stained with ethidium bromide. Precautions were taken to avoid carryover. and one negative control (extraction buffer) for each processed sample was inserted at the extraction step and run to the end of the test. The amplified DNA products were sequenced using the Sequitherm Cycle Sequencing Kit (Epicentre Technologies, Madison, WI, USA). Sense and antisense oligonucleotide primers used for sequencing analyses were located. respectively, at genomic position 689996919 (5’-GGC CAG GGG GTC TCC CCC CTC-3’) and at position 71517131 (3’-GGA ACG GAT ACT TCC CTC TCA-5’) of the published nucleotide sequence of HCV-J (16).
al. (6-S) while others failed to find a correlation between ISDR variability and response to IFN-ar (9-13). A limitation of all the studies performed so far is the short follow-up after stopping therapy (usually 6 months) in patients considered long-term responders (LTR). The aim of our study was to examine the relationship between ISDR variability and response to IFN-a in a series of patients from different Italian centers who showed complete biochemical and virological responses for a mean time of 38 months.
Materials and Methods Twenty-four anti-HCV and serum HCV RNA positive patients infected with genotype lb, who had been treated with IFN-a, were retrospectively studied (Table 1). All patients had histologicallyproven mild or moderate chronic hepatitis or, in one case, cirrhosis. None of them were hepatitis B viral antigen (HBsAg) positive, HIV infected or alcohol abusers. The routes of infection were unknown in all patients. Recombinant IFN-a 2a or 2b was administered subcu-
TABLE
1
Clinical
and histological
characteristics
Cases
of study group
Sex
Age
Histology Pre-treatment
Post-treatment
M M M M M M
50 50 65 57 32 60
7
F
50
8
M
31
9 10 11
F F M
48 59 63
12 13 14 15 16
F M M M F
59 44 45 45 30
17 18
F F
42 49
19
M
50
20
M
52
21 22 23
M M M
65 65 38
24
F
65
CAH” CAH” CPH” CAH” CPH” Cirrhosis score 334b CAH score 322b CAH score 121b CAH” CAH” CAH” score 423b CAH” CAH” CAH” CAH” CAH score 332b CAH” CAH score 323b CAH score 322b CAH score 322b CAH” CAH” CAH score 331b CAH” hepatitis.
nd: not done. NL: normal
CAH: chronic active hepatitis. CPH: chronic a According to De Groote et al. (14). b According to Desmet et al. (15).
1024
persistent
Follow-up (months)
Total IFN dose (MU)
nd NL” nd CPH” NL” Cirrhosis score 224b NLb
30 30 24 25 18 58
432 1350 486 729 729 540
26
468
NLb
28
468
nd nd nd
48 51 25
432 432 432
nd NL” NL” nd NLb
48 36 65 24 51
288 288 216 216 504
nd CAH” score 112b CAH” score 10lb CAH score 222b nd nd CAH score lllb nd
24 64
432 468
28
468
46
504
36 28 61
432 480 468
35
216
liver.
ISDR
2209 HCVJ
2240
PSLKATCTTHHDSPDADLIEANLLWFtQEMGGNITFWESEN
CASES
1 2 3 4 5 6
___----__R-_________---------___________
7
--------_R__________------___--_________
8
---______~______________________________
9
____---_-R------_________------_-_______
10
-----____R__________--__________________
11
-________~______________________________
12
_------__R-_________-___________________
.13
_________R---------___---------_________
14
_______--R__________-___________________
15
_________R------____-------_____________
16
__-------R__T_______-__________________-
17
_________&----_____------_Q____________
18
_---g--__R_____-----_____----D-_-_________
19
----E____R__________-____L______________
20
_______--R______----______-----______C_D
21
-----____R__---S____-----L-_____________~-
22 23 24
--_______~_---_______-_---__________c_____D____P__R__S___-_________----_______
_-------_~_----S____p-*-r+--___________-
Fig. 1. Amino acid sequence in Nonstructural Protein 5A (NS5A2209_2248) in 24patients infected with HCVgenotype lb. The NS’.5AZZ09_2248 amino acid sequence of HCV-J is shown in standard single-letter codes at the top. The sequences of the 24 patients are shown consecutively; dashes indicate amino acid residues identical to those in HCV-J.
The resulting amino acid sequences of ISDR in the NSSA of HCV (AA 2209-2248) were aligned and compared with that of HCV-J. Statistical signiticances were evaluated by Student’s t-test.
Results We found that all the cases had different ISDR nucleotide sequences and none of them were identical to the reference HCV J NSSA, although most of the mutations were silent (data not shown). The number of amino acid changes in the ISDR ranged from 0 to 5. Twenty-three of the 24 patients showed amino acid substitutions and all but one had the arginine for histidine substitution at codon 2218 which is common in genotype lb (Fig. 1). Among these 23 patients, 14 had
variability and cure
of chronic hepatitis C
only the 2218 mutation, two had 2 mutations, five had 3 mutations, one had 4 and one patient had 5 mutations. When we compared the ISDR sequences from the 24 long-term responders with that of our previous published series of 41 non-responders, we found no significant correlation between the number of mutations and the response to therapy (mean: 1.75? 1.22 vs 1.45 1.99 respectively, p=O.4 NS) (11). It should be pointed out that there are consensus data in the literature concerning the rate of ISDR mutations in NR (5-13). We further divided our patients into three categories according to the criteria used by Enomoto and colleagues: a “mutant” form, defined by HCV genomes with at least 4 ISDR mutations was shown in two patients; with the same criteria, the “intermediate” type (1 to 3 mutations) was found in 21 subjects, whereas one patient was infected with the “wild” type (no amino acid changes). It is worthy of note that both wild-type sequence and viral strains with a single mutation at codon 2218 have also been reported as being associated with IFN resistance in Japanese studies.
Discussion HCV lb is the most prevalent genotype in Japan and in European countries such as Italy. Several studies have indicated the lack of correlation between genotype lb and response to treatment. Promising data for a better understanding of the mechanisms of interferon resistance in patients infected with genotype lb have come from studies by Enomoto et al. (4,5). These authors showed that the finding of high variability before starting therapy (from 4 to 11 amino-acid substitutions) in a region (ISDR) of the amino-terminal part of the HCV NSSA gene could predict the response to IFN-ol in 100% of cases (5). However, several subsequent reports from eastern as well as western countries have provided contradictory results (613). In vitro studies performed by Gale et al. provided interesting insight regarding the resistance to therapy of HCV-lb (17,18). In fact, they have shown that the ISDR of HCV genotype lb interferes with the catalytic domain of the PKR kinase, a cellular protein with antiviral properties induced by interferon, confirming that this region of HCV may play a critical role in interfering with the therapeutic activity of IFN-(x. These authors hypothesized that the presence of multiple amino acid changes at the ISDR level inhibits the binding of the virus with the PKR. However, Pawlotsky et al., using SSCP analysis and sequencing of cloning products from the NSSA region, were unable to find an ISDR sequence intrinsically resistant or sensitive to IFN-cx (13).
1025
G. Squadrito et al.
In light of the consensus data in the literature concerning the rate of ISDR mutations in NR, we concentrated our attention on the 40-amino-acid stretch of ISDR in 24 patients selected on the basis of the very long sustained response to IFN therapy (5513). These patients were followed up for a period of 38 months (range 18-85 months) and 13 of them had a histological recovery or marked improvement. It is worthy of note that this is the Grst report to date on HCV variability in a considerable number of patients infected by lb genotype with such a sustained response to therapy, considering also that in a recent report non-lb genotype was recognized as one of the main predictors for a 4-year virological response to IFN-(x (3). In our study we found that the mutation rate of ISDR amino-acid sequence cannot be taken before therapy as a predictive factor of response to IFN-a in patients with chronic hepatitis C-genotype 1b, since the mutant strain of ISDR (from 4 to 11 amino-acid substitutions) was found in only two of 24 LTR, while 15 cases showed no or a single 2218 mutation. In a previous report in which we investigated ISDR variability in a series of Italian and French patients, we failed to show a significant statistical correlation between the number of amino acid substitutions in the NSSA and response to therapy (11). Two major criticisms were made with respect to this study: firstly, a relatively small number of LTR’s (seven patients) was included; secondly, differences in the total IFN-a doses were thought to be the cause of the discrepancies among studies from the two different geographical areas. The present study extends and largely confirms our previous report since we investigated a new and highly informative series of 24 HCV-lb LTR to interferon who had been treated with a dose of IFN-a similar to that given in the Japanese studies (5,7). Our data suggest that contradictory results obtained so far may be, at least in part, related to the duration of follow up in patients infected with genotype lb who had an apparent benefit from IFN-a. With this view it would be interesting to revise the previous data regarding ISDR variability in patients with chronic hepatitis C due to genotype lb. As mentioned above, in vitro studies have shown that NSSA protein may repress the IFN-induced protein kinase PKR (17,18). However, our results demonstrate that in viva the response to IFN-a is not related to the number of ISDR amino acid substitutions. At present there is no explanation for these discrepancies. However, we cannot exclude that NSSA sequences outside the ISDR might be involved in the mechanisms of IFN resistance. In this context it should be noted that recent reports indicate that binding of NSSA to PKR includes IO26
the region adjacent to the C-terminal extremity of the ISDR, and its variability might be involved in resistance to interferon during HCV infection (18,19).
Acknowledgement This work was supported Schering Plough.
in part by a grant from
References I. Hoofnagle JH. Therapy of acute and chronic viral hepatitis 1994. Adv Intern Med 1994; 39: 241-75. 2. Martinot-Peignoux M, Marcellin P Pouteau M, Castelnau C, Boyer N, Poliquin M, et al. Pretreatment serum HCV RNA levels and HCV genotype are the main and independent prognostic factors of sustained response to alpha interferon therapy in chronic hepatitis C. Hepatology 199s; 22: 1050-6. 3. Manesis EK. Papaioannou C, Gioustozi A. Kaliri G, Koskinas J, Hadziyannis S. Biochemical and virological outcome of patients with chronic hepatitis C treated with interferon alfa-2b for 6 or 12 months: a 4-year follow-up of 211 patients. Hepatology 1997; 26: 7349. 4. Enomoto N. Sakuma I, Asahina Y. Kurosaki M, Murakami T, Yamamoto C. et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b sensitivity to interferon is conferred by amino acid substitutions in the NSSA region. J Clin Inves 1995: 96: 22430. 5. Enomoto N, Sakuma I, Asahina Y, Kurosaki M. Murakami T, Yamamoto C. et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b interferon. N Engl J Med 1996; 334: 77-81. 6. Kurosaky M, Enomoto N, Murakami T, Sakuma 1, Asahina Y, Yamamoto C. et al. Analysis of genotypes and amino acid residues 2209 to 224X of the NSSA region of hepatitis C virus in relation to response to interferon-/i’therapy. Hepatology 1997; 25: 750-3. 7. Chayama K, Tsubota A, Kobayashi M. Okamoto K, Hashimoto M, Miyano Y, et al. Pretreatment virus load and multiple amino acid substitutions in the interferon sensitivjity-determining region predict the outcome of interferon treatment in patients with chronic genotype lb hepatitis C virus infection. Hepatology 1997: 25: 7459. 8. S&z J-C, Lopez-Labrador F-X, Ampurdants S, Dopazo J. Form X. Sanchez-Tapias J-M, et al. The prognostic relevance of the nonstructural 5A gene interferon sensitivity determing region is different in infections with genotype lb and 3a isolates of hepatitis C virus, J Infect Dis 1997: 177: 839-47. 9. Khorsi H, Castelain S. Wyseur A. Izopet J, Canva A, Rombout D, et al. Mutations of hepatitis C virus lb NSSA 2209-2248 amino acid sequence do not predict the response to recombinant interferon alfa therapy in French patients. J Hepatol 1997; 27: 72.-7 10. Zeuzem S, Lee J-H, Roth WK. Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa. Hepatology 1997: 25: 74011. 11. Squadrito G. Leone F, Sartori M, Nalpas B, Berthelot P Raimondo G, et al. Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology 1997; 113: 567--l?: 12. Hofgartner W. Polyak SJ. Sullivan D, Carithers RL Jr.. Gretch DR. Mutations in the NSSA gene of hepatitis C virus in north american patients infected with HCV genotype la or lb. J Med Viral 1997; 53: 1IX. 13. Pawlotsky J-M, Germanidis G. Neumann AU, Pellerin M, Frainais P-O, Dhumeaux D. Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations. J Virol 1998: 72: 27955805. 14. De Groote J. Desmet VJ. Gedigk P Korb G. Popper H, Poulsen
ISDR H, et al. A classification of chronic hepatitis. Lancet 1968; ii: 6268. 15. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 1994; 19: 1513-20. 16. Kato N, Hijikata M, Oostuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Nat1 Acad Sci USA 1990; 87: 95248. 17. Gale MJ, Korth MJ, Tang SL, Hopkins DA, Dever TE, Polyak SJ, et al. Evidence that hepatitis C virus resistence to interferon
variability and cure of chronic hepatitis C
is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997; 230: 217-27. 18. Gale MJ, Blakely CM, Kwieciszewski B, Tang SL, Dosset M, Tang N, et al. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 1998; 18: 243143. 19. Duverlie G, Khorsi H, Castelain S, Jaillon 0, Izopet J, Lunel F, et al. Sequence analysis of the NSSA protein of European hepatitis C virus lb isolates and relation to interferon sensitivity. J Gen Virol 1998; 79: 1373-81.
1027
Copyright 0 European Association for the Study of the Liver I999
Journal of Hepatology ISSN 01684278
Long-term response to interferon alpha is unrelated to “interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-lb infection Giovanni Squadrito ‘, Maria Elena Orlando’, Irene Cacciola’, Maria Grazia Rumi2, Marco Artini3, Antonio Picciotto4, Oreste Loiacono5, Rocco Siciliano6, Massimo Levrero3 and Giovanni Raimondo’ ‘Dipartimento Medicina Interna, Messina University; 2Dipartimento Medicina Interna. Milan University: ‘Laboratorio Espressione Genica Fondazione A. Cesalpino Universitd La Sapienza, Rome University; 4Cattedra Gastroenterologia, Genoa University; ‘Istituto Clinica Medica, Palermo University; and 61stituto Medicina Interna, Catania University, Italy
Background/Aims: Contradictory data have been reported about the predictive value of the variability in interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV) genotype-lb on response to interferon-alpha (IFN-a) therapy. The aim of this study was to examine this issue in a series of patients with long-term response to IFN treatment. Methods: We retrospectively analyzed 24 patients with chronic HCV genotype-lb infection treated with IFN-a (total dose median 677, range 216-1350 MU) selected in 6 Italian Liver Units. These patients were defined as true long-term responders (LTR) since they showed persisting biochemical and virological responses to IFN treatment (mean follow-up 38 months). HCV genomes from pretreatment serum samples were amplified and directly sequenced. The ISDR amino-acid sequences obtained were aligned and compared with the published sequence of HCV-J. Results: Amino-acid substitutions were found in 23 of
the 24 patients, and 22 of them showed an H to R amino-acid change at codon 2218. Fourteen patients showed only one mutation (at codon 2218), two had 2, five had 3, one had 4 and one had 5 mutations. When we compared the ISDR sequences from the 24 LTR with those of non-responders (NR), we found no significant correlation between the number of mutations and the response to therapy. Conclusions: Our results demonstrate that the persisting efficacy of IFN treatment in patients with chronic HCV is not related to the number of ISDR amino acid susbstitutions of the infecting viruses. Further studies are needed to verify whether other NSSA sequences outside the ISDR might be involved in the mechanisms of IFN resistance.
H
have shown the association between genotype lb and a low rate of long-term response to IFN-a (2,3). Insight regarding the resistance of genotype 1b infection to therapy has been reported from Japan by Enomoto et al., suggesting that mutations in a 40-amino-acid stretch, named the “interferon sensitivity determining region” (ISDR), in the nonstructural viral protein 5A (NSSA) modulates sensitivity to IFN-a (4,5). According to these authors, high variability in the ISDR before therapy could thus predict the response to IFN-ol in most cases. However, data reported by subsequent papers have been contradictory, since some studies confirmed the above-mentioned results by Enomoto et
C virus (HCV) is a major cause of progressive chronic hepatitis worldwide and the management of therapy for HCV infection is therefore a fundamental medical issue. The only approved drug for chronic hepatitis C is interferon alpha (IFN-a), although long-term response to this therapy is achieved in around 25% of treated patients (1). Several studies EPATITIS
Received 10 November:
revised I7 December:
accepted 17 December
1998
Giovanni Raimondo, Dipartimento Medicina Interna, Policlinico Universitario, 98 100 Messina, Italy. Tel: 39 0 90 2212393. Fax 39 0 90 2935162. E-mail [email protected] Correspondence:
Key words: Chronic hepatitis C; Direct sequencing; Non-structural 5A protein; Polymerasae chain reaction; Viral genomic heterogeneity.
1023
G. Squadrito et al. taneously at a dose of 3 to 6 MU of IFN for 6 to 12 months (total dose median 677, range 2161350 MU). All patients had normal alanine aminotransferase (ALT) activity during and after treament and they were constantly HCV-RNA negative for a mean follow up of 38 months (range 1%84). Thirteen of them underwent a needle liver biopsy 12 to 60 months after stopping therapy; seven showed histological recovery and six marked improvement (Table 1). The study was performed according to the principles of the Declaration of Helsinki, and informed consent was obtained in every case. HCV genotyping was performed by restriction fragment length polymorphism (RFLP) analysis. RNA extraction, reverse transcription of NSSA region and polymerase chain reaction (PCR) were performed as previously reported (11). PCR products were analyzed by gel electrophoresis using 2% agarose stained with ethidium bromide. Precautions were taken to avoid carryover. and one negative control (extraction buffer) for each processed sample was inserted at the extraction step and run to the end of the test. The amplified DNA products were sequenced using the Sequitherm Cycle Sequencing Kit (Epicentre Technologies, Madison, WI, USA). Sense and antisense oligonucleotide primers used for sequencing analyses were located. respectively, at genomic position 689996919 (5’-GGC CAG GGG GTC TCC CCC CTC-3’) and at position 71517131 (3’-GGA ACG GAT ACT TCC CTC TCA-5’) of the published nucleotide sequence of HCV-J (16).
al. (6-S) while others failed to find a correlation between ISDR variability and response to IFN-ar (9-13). A limitation of all the studies performed so far is the short follow-up after stopping therapy (usually 6 months) in patients considered long-term responders (LTR). The aim of our study was to examine the relationship between ISDR variability and response to IFN-a in a series of patients from different Italian centers who showed complete biochemical and virological responses for a mean time of 38 months.
Materials and Methods Twenty-four anti-HCV and serum HCV RNA positive patients infected with genotype lb, who had been treated with IFN-a, were retrospectively studied (Table 1). All patients had histologicallyproven mild or moderate chronic hepatitis or, in one case, cirrhosis. None of them were hepatitis B viral antigen (HBsAg) positive, HIV infected or alcohol abusers. The routes of infection were unknown in all patients. Recombinant IFN-a 2a or 2b was administered subcu-
TABLE
1
Clinical
and histological
characteristics
Cases
of study group
Sex
Age
Histology Pre-treatment
Post-treatment
M M M M M M
50 50 65 57 32 60
7
F
50
8
M
31
9 10 11
F F M
48 59 63
12 13 14 15 16
F M M M F
59 44 45 45 30
17 18
F F
42 49
19
M
50
20
M
52
21 22 23
M M M
65 65 38
24
F
65
CAH” CAH” CPH” CAH” CPH” Cirrhosis score 334b CAH score 322b CAH score 121b CAH” CAH” CAH” score 423b CAH” CAH” CAH” CAH” CAH score 332b CAH” CAH score 323b CAH score 322b CAH score 322b CAH” CAH” CAH score 331b CAH” hepatitis.
nd: not done. NL: normal
CAH: chronic active hepatitis. CPH: chronic a According to De Groote et al. (14). b According to Desmet et al. (15).
1024
persistent
Follow-up (months)
Total IFN dose (MU)
nd NL” nd CPH” NL” Cirrhosis score 224b NLb
30 30 24 25 18 58
432 1350 486 729 729 540
26
468
NLb
28
468
nd nd nd
48 51 25
432 432 432
nd NL” NL” nd NLb
48 36 65 24 51
288 288 216 216 504
nd CAH” score 112b CAH” score 10lb CAH score 222b nd nd CAH score lllb nd
24 64
432 468
28
468
46
504
36 28 61
432 480 468
35
216
liver.
ISDR
2209 HCVJ
2240
PSLKATCTTHHDSPDADLIEANLLWFtQEMGGNITFWESEN
CASES
1 2 3 4 5 6
___----__R-_________---------___________
7
--------_R__________------___--_________
8
---______~______________________________
9
____---_-R------_________------_-_______
10
-----____R__________--__________________
11
-________~______________________________
12
_------__R-_________-___________________
.13
_________R---------___---------_________
14
_______--R__________-___________________
15
_________R------____-------_____________
16
__-------R__T_______-__________________-
17
_________&----_____------_Q____________
18
_---g--__R_____-----_____----D-_-_________
19
----E____R__________-____L______________
20
_______--R______----______-----______C_D
21
-----____R__---S____-----L-_____________~-
22 23 24
--_______~_---_______-_---__________c_____D____P__R__S___-_________----_______
_-------_~_----S____p-*-r+--___________-
Fig. 1. Amino acid sequence in Nonstructural Protein 5A (NS5A2209_2248) in 24patients infected with HCVgenotype lb. The NS’.5AZZ09_2248 amino acid sequence of HCV-J is shown in standard single-letter codes at the top. The sequences of the 24 patients are shown consecutively; dashes indicate amino acid residues identical to those in HCV-J.
The resulting amino acid sequences of ISDR in the NSSA of HCV (AA 2209-2248) were aligned and compared with that of HCV-J. Statistical signiticances were evaluated by Student’s t-test.
Results We found that all the cases had different ISDR nucleotide sequences and none of them were identical to the reference HCV J NSSA, although most of the mutations were silent (data not shown). The number of amino acid changes in the ISDR ranged from 0 to 5. Twenty-three of the 24 patients showed amino acid substitutions and all but one had the arginine for histidine substitution at codon 2218 which is common in genotype lb (Fig. 1). Among these 23 patients, 14 had
variability and cure
of chronic hepatitis C
only the 2218 mutation, two had 2 mutations, five had 3 mutations, one had 4 and one patient had 5 mutations. When we compared the ISDR sequences from the 24 long-term responders with that of our previous published series of 41 non-responders, we found no significant correlation between the number of mutations and the response to therapy (mean: 1.75? 1.22 vs 1.45 1.99 respectively, p=O.4 NS) (11). It should be pointed out that there are consensus data in the literature concerning the rate of ISDR mutations in NR (5-13). We further divided our patients into three categories according to the criteria used by Enomoto and colleagues: a “mutant” form, defined by HCV genomes with at least 4 ISDR mutations was shown in two patients; with the same criteria, the “intermediate” type (1 to 3 mutations) was found in 21 subjects, whereas one patient was infected with the “wild” type (no amino acid changes). It is worthy of note that both wild-type sequence and viral strains with a single mutation at codon 2218 have also been reported as being associated with IFN resistance in Japanese studies.
Discussion HCV lb is the most prevalent genotype in Japan and in European countries such as Italy. Several studies have indicated the lack of correlation between genotype lb and response to treatment. Promising data for a better understanding of the mechanisms of interferon resistance in patients infected with genotype lb have come from studies by Enomoto et al. (4,5). These authors showed that the finding of high variability before starting therapy (from 4 to 11 amino-acid substitutions) in a region (ISDR) of the amino-terminal part of the HCV NSSA gene could predict the response to IFN-ol in 100% of cases (5). However, several subsequent reports from eastern as well as western countries have provided contradictory results (613). In vitro studies performed by Gale et al. provided interesting insight regarding the resistance to therapy of HCV-lb (17,18). In fact, they have shown that the ISDR of HCV genotype lb interferes with the catalytic domain of the PKR kinase, a cellular protein with antiviral properties induced by interferon, confirming that this region of HCV may play a critical role in interfering with the therapeutic activity of IFN-(x. These authors hypothesized that the presence of multiple amino acid changes at the ISDR level inhibits the binding of the virus with the PKR. However, Pawlotsky et al., using SSCP analysis and sequencing of cloning products from the NSSA region, were unable to find an ISDR sequence intrinsically resistant or sensitive to IFN-cx (13).
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In light of the consensus data in the literature concerning the rate of ISDR mutations in NR, we concentrated our attention on the 40-amino-acid stretch of ISDR in 24 patients selected on the basis of the very long sustained response to IFN therapy (5513). These patients were followed up for a period of 38 months (range 18-85 months) and 13 of them had a histological recovery or marked improvement. It is worthy of note that this is the Grst report to date on HCV variability in a considerable number of patients infected by lb genotype with such a sustained response to therapy, considering also that in a recent report non-lb genotype was recognized as one of the main predictors for a 4-year virological response to IFN-(x (3). In our study we found that the mutation rate of ISDR amino-acid sequence cannot be taken before therapy as a predictive factor of response to IFN-a in patients with chronic hepatitis C-genotype 1b, since the mutant strain of ISDR (from 4 to 11 amino-acid substitutions) was found in only two of 24 LTR, while 15 cases showed no or a single 2218 mutation. In a previous report in which we investigated ISDR variability in a series of Italian and French patients, we failed to show a significant statistical correlation between the number of amino acid substitutions in the NSSA and response to therapy (11). Two major criticisms were made with respect to this study: firstly, a relatively small number of LTR’s (seven patients) was included; secondly, differences in the total IFN-a doses were thought to be the cause of the discrepancies among studies from the two different geographical areas. The present study extends and largely confirms our previous report since we investigated a new and highly informative series of 24 HCV-lb LTR to interferon who had been treated with a dose of IFN-a similar to that given in the Japanese studies (5,7). Our data suggest that contradictory results obtained so far may be, at least in part, related to the duration of follow up in patients infected with genotype lb who had an apparent benefit from IFN-a. With this view it would be interesting to revise the previous data regarding ISDR variability in patients with chronic hepatitis C due to genotype lb. As mentioned above, in vitro studies have shown that NSSA protein may repress the IFN-induced protein kinase PKR (17,18). However, our results demonstrate that in viva the response to IFN-a is not related to the number of ISDR amino acid substitutions. At present there is no explanation for these discrepancies. However, we cannot exclude that NSSA sequences outside the ISDR might be involved in the mechanisms of IFN resistance. In this context it should be noted that recent reports indicate that binding of NSSA to PKR includes IO26
the region adjacent to the C-terminal extremity of the ISDR, and its variability might be involved in resistance to interferon during HCV infection (18,19).
Acknowledgement This work was supported Schering Plough.
in part by a grant from
References I. Hoofnagle JH. Therapy of acute and chronic viral hepatitis 1994. Adv Intern Med 1994; 39: 241-75. 2. Martinot-Peignoux M, Marcellin P Pouteau M, Castelnau C, Boyer N, Poliquin M, et al. Pretreatment serum HCV RNA levels and HCV genotype are the main and independent prognostic factors of sustained response to alpha interferon therapy in chronic hepatitis C. Hepatology 199s; 22: 1050-6. 3. Manesis EK. Papaioannou C, Gioustozi A. Kaliri G, Koskinas J, Hadziyannis S. Biochemical and virological outcome of patients with chronic hepatitis C treated with interferon alfa-2b for 6 or 12 months: a 4-year follow-up of 211 patients. Hepatology 1997; 26: 7349. 4. Enomoto N. Sakuma I, Asahina Y. Kurosaki M, Murakami T, Yamamoto C. et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b sensitivity to interferon is conferred by amino acid substitutions in the NSSA region. J Clin Inves 1995: 96: 22430. 5. Enomoto N, Sakuma I, Asahina Y, Kurosaki M. Murakami T, Yamamoto C. et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b interferon. N Engl J Med 1996; 334: 77-81. 6. Kurosaky M, Enomoto N, Murakami T, Sakuma 1, Asahina Y, Yamamoto C. et al. Analysis of genotypes and amino acid residues 2209 to 224X of the NSSA region of hepatitis C virus in relation to response to interferon-/i’therapy. Hepatology 1997; 25: 750-3. 7. Chayama K, Tsubota A, Kobayashi M. Okamoto K, Hashimoto M, Miyano Y, et al. Pretreatment virus load and multiple amino acid substitutions in the interferon sensitivjity-determining region predict the outcome of interferon treatment in patients with chronic genotype lb hepatitis C virus infection. Hepatology 1997: 25: 7459. 8. S&z J-C, Lopez-Labrador F-X, Ampurdants S, Dopazo J. Form X. Sanchez-Tapias J-M, et al. The prognostic relevance of the nonstructural 5A gene interferon sensitivity determing region is different in infections with genotype lb and 3a isolates of hepatitis C virus, J Infect Dis 1997: 177: 839-47. 9. Khorsi H, Castelain S. Wyseur A. Izopet J, Canva A, Rombout D, et al. Mutations of hepatitis C virus lb NSSA 2209-2248 amino acid sequence do not predict the response to recombinant interferon alfa therapy in French patients. J Hepatol 1997; 27: 72.-7 10. Zeuzem S, Lee J-H, Roth WK. Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa. Hepatology 1997: 25: 74011. 11. Squadrito G. Leone F, Sartori M, Nalpas B, Berthelot P Raimondo G, et al. Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology 1997; 113: 567--l?: 12. Hofgartner W. Polyak SJ. Sullivan D, Carithers RL Jr.. Gretch DR. Mutations in the NSSA gene of hepatitis C virus in north american patients infected with HCV genotype la or lb. J Med Viral 1997; 53: 1IX. 13. Pawlotsky J-M, Germanidis G. Neumann AU, Pellerin M, Frainais P-O, Dhumeaux D. Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations. J Virol 1998: 72: 27955805. 14. De Groote J. Desmet VJ. Gedigk P Korb G. Popper H, Poulsen
ISDR H, et al. A classification of chronic hepatitis. Lancet 1968; ii: 6268. 15. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 1994; 19: 1513-20. 16. Kato N, Hijikata M, Oostuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Nat1 Acad Sci USA 1990; 87: 95248. 17. Gale MJ, Korth MJ, Tang SL, Hopkins DA, Dever TE, Polyak SJ, et al. Evidence that hepatitis C virus resistence to interferon
variability and cure of chronic hepatitis C
is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997; 230: 217-27. 18. Gale MJ, Blakely CM, Kwieciszewski B, Tang SL, Dosset M, Tang N, et al. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 1998; 18: 243143. 19. Duverlie G, Khorsi H, Castelain S, Jaillon 0, Izopet J, Lunel F, et al. Sequence analysis of the NSSA protein of European hepatitis C virus lb isolates and relation to interferon sensitivity. J Gen Virol 1998; 79: 1373-81.
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