Humoral immunoreactivity to HCV core antigen in patients with chronic hepatitis C

Journal ofHepatology 1997; 26: 446452 Printed in Denmark. All rights reserved Munksganni Copenhagen

Correspondence

Humoral immunoreactivity to HCV core antigen in patients with chronic hepatitis C To the Editor:

HCV core antigen is immunogenic to B-cells in man. Both the current enzyme immunoassay (EIA) and recombinant immunoblot assays (RIBA HCV Strip Imnumoblot Assays versions 2.0 and 3.0) use recombinant HCV core antigen (RIBA 2.0, c-22, amino acid 2-120 of the 1-191 of the core antigen) or core peptide (RIBA 3.0) as one of the capture antigens for the detection of host HCV-specific humoral response. Machida et al. described the use of genotype-specific peptides derived from the HCV core region (a.a. 68-78, genotype 1: ARRPEGRTWAQ, genotype 2: ARRSTGKSWGK) for serologic genotyping of HCV isolates (1). Recently, it was also suggested that in patients with non-type 1 infection, there is a lack of immunoreactivity to the recombinant 5-l-l peptide (a.a. 1694-1735) (2). To clarify these issues and to further understand the host’s humoral immunoreactivity to HCV core, 27 consecutive US patients (M:F 17:lO; mean age 51+12 years; all patients were positive for

TABLE Humoral

serum HCV RNA by RT-“nested” PCR based on the 5’ UTR; liver histology: chronic persistent hepatitis 6, chronic active hepatitis 9, active cirrhosis 12) with chronic HCV infection were evaluated for their seroreactivity to the antigens used in RIBA version 2.0 (5-1-1, c-100, c33c, ~22). RIBA version 3.0 (combined band with cl00 peptide and 5-l-l peptide, c33c, ~22, NS5), the genotype-specific peptides within core as described by Machida et al. (1). and two peptides within core (a.a. lo-55 and a.a. 72-89) which were predicted to be antigenic based on the method of Jameson & Wolf (3). Humoral reactivity was assessed by RIBA strip technology, and HCV genotypes were determined by a commercial line probe assay which we have previously shown to be very reliable in US patients with chronic HCV infection (4). Table 1 summarizes the results. All reactivity was graded visually according to the RIBA assay guidelines (range from -, +/-, 1 to 4+). There were a few interesting observations. First, 7/8 patients with non-type 1 in-

1 immunoreactivity

Patient no.

to the various

HCV polypeptides

RIBA 2.0

Genotype

RIBA 3.0

HCV core

C-term.

aa

aa

5-l-l

cl00

c33c

c22

cl00 5-l-l

c33c

c22

NS5

lo-55

72-89

+I+l+I-

4+ l+

4+ 4+

4+

4+ 4+

4+ 4+

4+

4+ 4+ 4+ 4+ 4+

4+ 4+ 4+ 4+

+I4+

2+ 4+

4+ 4+ 4+

4+ 4+ 4+ 4+

+I2+ 4+ -

4+ 4+ 4+ 4+

3+ _

4+ 4+ +l4+ 4+ 4+ 4+ 4+ 4+

2+ 4+ +l4+ 2+ +l4+ +I2+

4+ 4+

+I4+

+l+I+I+I4+ il3+ 3+ l+ l+ +I4+

1 2

2 3a

3 4 5

3a 2b lb

+I_

3+ +l2+

2+ 4+ 4+

6 7 8

2b 1 la

+I4+ 4+

+I2+ 4+

4+ 4+

9 10 11 12 13

lb la la lb la

4+ 4+ 4+

4+ 4+ 4+ l+ 4+

14 15

la lb

16 17 18 19 20 21 22 23

la la 2a lb 2a+2b la lb 2a

-

24

la

4+

4+

4+ 4+

4+ 4+ 4+ 4+ 4+ 4+

25

la

4+

4+

4+

la lb

4+

4+

4+

4+

4+

4+

26 27

446

Machida

+/4+ 4+ 4+ _ 4+ 2+ 4+ 4+ _

2+ 4+ 4+ 4+ 2+ 4+ 4+ -

4+ 4+ 4+ 4+ 4+ 4+ 4+ +I+I4+ 4+ 4+ 4+ 4+ 4+

4+ 2+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ +I+l4+ 4+

+I+I4+ 4+ 4+ 4+ 4+ 2+ 4+ 4+ +I+I4+ 2+ 4+ 4+ 4+ 4+

4+ 4+ 4+ 4+

+l-

+I4+ l+ 4+ 4+ 4+

4+ 4+ 4+ 4+

4+ 4+ 4+ 4+

4+ 4+ 4+

4+ 4+ 4+

+l4+ +I-

+I+I4+ 4+

+I+I4+ 4+ 4+

+I+l4+ 4+ 4+

4+ 4+ 4+ 4+ 4+

+l4+ 2+ +l4+

4+ 4+ 4+

3+ -

4+ 2+

et al.

Spel

5pe2

+lil2+ -

+lil2+ -

+l-

+l3+ il+I-

+I-

4+ +I3+ +l4+ +I-

+I+I+I+I+I+I+l+I+l4+ +l+I+l+l+l+I-

2+ 4+

4+ 4+ 4+ 4+ 4+ 4+

4+

4+

4+

4+

4+

4+

+I-

+I-

+I-

4+

4+

4+

4+

4+

4+

+I-

l+

+I-

4+

4+

4+

4+

4+

4+

+l-

+I-

+I-

Correspondence

fection did not react to recombinant antigen 5-l-l compared to 3/19 patients infected with HCV type 1 &O.OOl). However, all seven patients with non-type 1 infection were reactive to the c-terminus of cl00 and S-l1 peptide band of RIBA version 3.0. One of the three patients infected with type 1 non-reactive to recombinant 5-l-l was also reactive to the cterminus of cl00 and 5-l-l peptide combined band on RIBA version 3.0. These data suggest that the use of c-terminus of cl00 and 5-l-l combined peptides of RIBA version 3.0 as capture antigens is much better than the ful-length recombinant 5-l-l alone. Second, c-22 is highly reactive in 251 27 samples. All samples that showed reactivity to c-22 (25/27) had strong reactivity to the peptide 10-55 (25/27), but only 14/27 had reactivity to the peptide a.a. 72-89, indicating that region a.a. lo-55 is highly immunogenic in man when compared to 72-89 Q~0.02). This may be related to lower antigenicity or antigenic variation due to sequence heterogeneity of that region (a.a. 72-89). The B-cell reactivity in mouse based on monoclonai antibody mapping showed a similar profile. Nine mouse monoclonal antibodies that we have generated were all mapped to within a.a. 10-55 by synthetic peptides (Mizokami et al., unpublished data). Third, the peptides described by Machida et al. have limited sensitivity (only 13/ 27 were positive). However, for those that were positive, lU13 had their serologic defined genotypes concordant with the genotype defined by LiPA. One patient with HCV type 3 was reactive to both type 1 and 2 peptides. These results indicate that: 1) the 5-l-l pepride used in RIBA version 2.0 has limited reactivity in patients with HCV non-type 1 infection and thec-tcrminusofclOOand5-1-1pe&decombinationbandusedinRIBA version 3.0 has a better reactivity across genotypes 1 to 3; 2) the region within a.a. 10-55 is highly immunogenic in man; and 3) the genotypespecific peptides described by Machida et al. have limited sensitivity in US patients with chronic HCV infection. However, for those patients who showed reactivity to these peptides, the concordance with HCV genotypes determined by LiPA was good.

Constantine C. Marousis, Stella Quau’, Gary L. Davis, Robert DiNello’, Alan Polito’, Masashi Mizokami’, and Johnson Y.N. Lau Section of Hepatobiliary Diseases, Division of Gastmentemlogy, Heparology, and Nutrition, Department of Medicine, Vniversiiy of Florida, Gainesville, Florida, USA; ‘Chimn CorporationI, Emeryville, California, USA; and zSecond Department of Internal Medicine, Nagoya City University, Nagoya, Japan

References Machida A, Ohnuma H, Tsuda F, Munekata E, Tanaka T, Akahane Y, Okamoto H, Mishiro S. wo distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core protein. Hepatology 1992; 16: 886-91. Prati D, Capelli C, Zanella A, Mozzi F, Bosoni P, Pappalettera M, Zanuso F, Vianello L, Locatelli E, De Fazio C, Ronchi G, Del Ninno E, Colombo M, Sirchia G. Influence of different hepatitis C virus genotypes on the course of asymptomatic hepatitis C virus infection. Gastroenterology 19%; 110: 178-83. Jameson BA, Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci 1988; 4: 181-6. Lau JYN, Davis GL, Prescott LE, Maertens G, Lindsay KL, Qian KP, Mizokami M, Simmonds P, Hepatitis Interventional Therapy Group. Distribution of hepatitis C virus genotypes determined by a line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Ann Intern Med 19%; 124: 868-76.

Long-term interferon alfa-2b retreatment of relapsing patients with chronic hepatitis C To the E&or: Ak!10~gb prolonged interfemn (FN) treatment significantly increases the rate of sustained responses, relapse still occurs in a large number of patients (1.2). Retreatmen of these patients remains an open issue. They usually respond to a second course of IFN on &ndard schedule but all of them relapse again soon after stopping hzatment (3,4). The aim of our study was to evaluate the biochemical and virological efficacy of 2-year retreatment in patients with biopsy-proven chronic hepatitis C with modemte to seven activity who normalized alanine aminotransfcrase (ALT) after a first cycle of FN and then relapsed after the end of treatment. After a previous cycle of IPN alfa-2b at the dose of 3 MU three times a week for 6 months, 20 consecutive patients (15 males and 5 females, aged 2&55 years) wexe enrolled to receive subsequent treatment with the same IFN and at the same dose for 24 months. All of them relapsed 1 month after the first cycle of therapy. Alanine aminotransfemse (ALT) values were tested before the 6rst cycle of therapy and then every month until the end of follow-up; qualitative (‘bested” pdymerase chain reaction) and quantitative (Quantiplex, Chimn Corp., Emeryville, CA, USA) HCV-RNA were detected both at the beginning and at the end of the two cycles of treatment, and at the end of follow-

up. HCV genotyping was determined by Inno-Lipa HCV kit (Inno Genetics, Belgium) in the baseline serum samples of 17 HCV-RNA positive patients out of the 18 patients who completed the 24-month treatment. HCV genotypes la, lb, 2a, 2c and 3a were found in 2,5,2,2 and 6 patients rcspectively. Autoimmunity and thyroid fun&m were checked at baseline and then every 6 months. Six patients agreed to undergo a second liver biopsy 3 months after the second treatment. Liver biopsies of these patients were coded and submitted for the pathologist’s examination as blind samples. All biopsies were scored using the histological grading and staging score according to Ishak et al. Statistical analysis was performed by the Wilcoxon signed ranks test. Informed consent was obtained from all patients and the trial was approved by the local ethical committee. ‘lXvo patients discontinued treatment due to non-compliance after 6 months of therapy, at which time ALT was normal. Biochemical and virological data relating to the 18 patients who completed the trial are reported in detail in Table 1. Seventeen of these patients (94.5%) had normal ALT both during and at the end of treatment, and were considered as biochemical responders. However, we considered as complete responders the 14 patients (77.8%) who had normal ALT values and were HCV-RNA negative. One patient (no. 15). whose. HCV-RNA was persistently positive, experienced a breakthrough in the 18th month and for the last 6

447