O r i ~ n a l Articles

Significance of IgG and IgM HCV Antibody Secretion I n Vitro in Patients with Chronic Hepatitis C: Correlation with

Disease Activity and Response to Interferon-a

H A " S LOHR,' CHRISTOPHER NAGEL,' HANS-PETER DIENES,2 BARRY SIMPSON,3 GERD MICHEL,3 BERND GOERGEN,' KARL-HERMANN MEYER ZUM BUSCHENFELDE~ AND GUIDO GERKEN'

lFirst Department of Internal Medicine and 2Znstitute of Pathology, Johannes-Gutenberg-University Mainz, 551 01 Mainz, and 3Abbott Diagnostics, European Research and Development, 65008 Wiesbaden, Germany

Hepatitis C virus antibodies are found in the serum of most patients with chronic hepatitis C. However, the significance of the humoral response is still uncertain. In this study, in uitro IgG and IgM anti-hepatitis C virus secretion by peripheral blood mononuclear cells of patients with chronic hepatitis C was analyzed. Periph- eral-blood mononuclear cells from 21 of 36 patients (58.3%) secreted IgG anti-hepatitis C virus in uitro, as demonstrated with anti-hepatitis C virus-specific en- zyme immunoassays and recombinant immunoblot assays. Ten of the 36 patients (27.8%) showed both IgG and IgM anti-hepatitis C virus core in U ~ ~ F O . In 9 of these 10 patients, IgM anti-hepatitis C virus was also detected in serum. Patients with in uitro IgM or IgG anti-hepatitis C virus secretion had higher ALT levels in serum than did patients without such secretion in vitro (99.5 2 22.1 and 85.6 & 34.4 vs. 38.1 37.4 UL; p < 0.0001, p < 0.001). Furthermore, with a histology activity score it was demonstrated that patients with in uitro IgM or IgG HCV antibodies (or both) had more severe chronic active hepatitis than did patients without in uitro hepatitis C virus antibody secretion (p < 0.01). To analyze the therapy outcome, we in- cluded in this study 18 patients who had received interferon-a previously. Seven of eight in uitro hepa- titis C virus antibody-positive patients were nonre- sponders, whereas the in uitro hepatitis C virus anti- body-negative patients were mostly complete therapy responders (8 of 10). The follow-up study of eight patients with chronic hepatitis C after the beginning of therapy revealed that interferon-a decreases the in uitro humoral response to hepatitis C virus in treat-

Received December 8, 1993; accepted June 10, 1994. Other abbreviations used in the text: anti-HCV, antibody to hepatitis C virus;

HCV, hepatitis C virus; IFN, interferon; PBMC, peripheral-blood mononuclear cell; PWM, pokeweed mitogen.

This work was supported by the Deutsches Bundesministerium fur Fors- chung und Entwicklung (BMFT), grant no. 01KJ891415, and by the Deutsche Forschungsgemeinschaft. SFB 311, projects A5, A13 and A14.

Address reprint requests to: P.D. Dr. Guido Gerken, I. Department of Internal Medicine, Johannes-GutenbergUniversity Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany.

Copyright 0 1994 by the American Association for the Study of Liver Diseases.

0270-9139/94 $3.00 + 0 31/1/59486

ment responders. In conclusion, this study demon- strates (a) in uitro secretion of IgG and IgM hepatitis C virus antibodies in patients with chronic hepatitis C, (b) that higher disease activity and persistent hepatitis C virus replication may be associated with ongoing antibody production in uitro and (c ) in uitro antibody production seems to correlate negatively with the response of patients to antiviral treatment with inter- feron-a (HEPATOLOGY 1994;20: 1383-1389.)

Transfusion-transmitted chronic non-A, non-B hepa- titis is generally characterized by the presence of circulating antibodies against structural and nonstruc- t u r d antigens of the HCV, fluctuating transaminase levels, high progression rate and development of cir- rhosis (1-3). Preliminary results have demonstrated that the inflammatory activity in chronic hepatitis C is related to the presence of detectable HCV RNA in serum (4,5). However, the existence of so-called asymptomatic HCV carriers with HCV replication in serum without clinical hepatitis suggests that immunological mecha- nisms are also important (6). The histological findings of intralobular lymphocytic infiltrations and the demon- stration of HLA class I-restricted CD8 + cytotoxic liver- infiltrating T lymphocytes led to the hypothesis that cellular immune mechanisms play a crucial role in the pathogenesis of chronic hepatitis (7-10). However, the significance of humoral immune responses in HCV infection is not completely understood. Although serum IgG anti-HCV is not correlated with inflammatory activity, viral clearance or tolerance, the occurrence of serum IgM anti-HCV core may be related to HCV activity and to the outcome of antiviral therapy (11-18).

Recently, anti-HCV secretion in serum and in uitro by PBMCs was demonstrated in patients with chronic hepatitis C (19). The detection of anti-HCV antibody secretion in uitro offers a suitable tool to measure viral activity indirectly and to study the regulation of the humoral response in uitro. The aims of this study were (a) to study in uitro IgG and IgM antibody production, (b) to correlate anti-HCV secretion with disease activity and viral replication and (c) to evaluate the effects of IFN-ol on anti-HCV production.

1383

1384 LOHRETAL HEPATOLOCY December 1994

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cutoff entl-c22-3, antl-c33c 0.3

FIG. 1. Blot diagram of anti-HCV secretion by PBMCs from 36 patients with chronic hepatitis C. IgG antibodies to the recombinant c22-3 core and c33c non-structural antigens of the HCV were detected with an anti-HCV EM. CPH, chronic persistent and minimal hepatitis. Cutoff > 0.3.

TABLE 1. Characteristics of patients with chronic hepatitis C ~~ - ~~

Serum Histological Sex Age Treated ALT y-Globulins protein activity HCV

In uitro antibody secretion (mM (yr) patients (U/L)--d (%I (gm/L) scorea-d RNA

IgG and IgM anti-HCV in urtro positive (n = 10) 515 59.3 3 99.5 ? 22.1 28.5 77.6 5.33 2 1.24 10 IgG anti-HCV in vitro positive (n = 11) 9/2 53.7 5 85.6 ? 34.4 20.6 79.4 4.37 2 1.32 11 Anti-HCV in uitro negative (n = 15) 9/6 46.7 10 38.1 2 37.4 16.8 76.8 3.16 2 0.79 7

Clinical, virological and histological data from patients with chronic hepatitis C with or without IgM and/or I& anti-HCV secretion in uitro. Patients with in uitro humoral immune response showed higher inflammatory activities, confirmed by histological appearance and ALT levels, than in uitro anti-HCV-negative patients.

“Data expressed as mean 2 S.D. Student’s t test: *p = 0.14, patients with IgM/IgG secretion vs. patients with IgG anti-HCV secretion in uitro; ‘p < 0.0001, patients with

IgMiIgG secretion vs. those negative for in uitro anti-HCV; dp < 0,001, patients positive for in uitro I& anti-HCV vs. those positive.

PATIENTS AND METHODS Patients. Thirty-six patients with chronic hepatitis C were

analyzed. Eighteen patients were untreated, and 12 of them subsequently received IFN-0. In addition, 18 were seen at least 6 mo after withdrawal of a 6- to 12-mo antiviral therapy with 3 or 5 mIU IFN-a,, three times a week subcutaneously.

Before therapy, all HCV patients had markedly increased transaminase levels (ALT normal range, <23 U/L), were positive for anti-HCV (second-generation assay) and HCV RNA and showed histologically proven chronic hepatitis. At the time the study was performed, 28 of 36 patients with chronic HCV infection were viremic (77.8%). However, 8 of the 36 patients (22.2%) showed complete responses to an- tiviral treatment as defined by serum HCV RNA negativity, sustained improvement of liver histology and long-term normalization of aminotransferase levels (Table 1). Patients with chronic hepatitis B (n = 6) or autoimmune liver diseases (n = 5) and healthy blood donors (n = 10) served as controls. All diagnoses were based on clinical, histological and sero- logical data. All patients gave informed consent to the experiments, in accordance with the Helsinki Declaration of ethical guidelines.

Detection of Vim1 and Autoantibody Markers. Markers of HBV or HCV infection were determined in sera by means of commercially available assays (Abbott Diagnostics, Wiesbaden, Germany). We performed indirect immunofluorescence or EIAs to detect antinuclear, anti-smooth muscle, antimitochon- drial, antimicrosomal and anticytoplasmatic antibodies, as described in detail elsewhere (20).

For the detection of HCV RNA in serum, nucleic acids were extracted according to the classical guanidine- thio-phenol/chloroform method. Reverse transcription and nested polymerase chain reaction were performed with primers of the 5’-noncoding region of the HCV, as described recently (21, 22). Each experiment was carried out twice, independently.

Histology Activity Znder. Liver biopsy specimens taken for diagnostic purposes were prepared according to standard protocols; histopathology was classified as CAH, chronic per- sistent hepatitis or minimal hepatitis (9, 23). The inflam- matory activity of the disease was evaluated in 29 of 36 biopsy specimens according to Scheuer’s scoring system (24). Af- terward, the inflammatory activity was correlated with the in vitro anti-HCV production.

HEPATOLOGY Vol. 20, NO. 6, 1994 LOHR ET AL. 1385

P N 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

FIG. 2. IgG anti-HCV secretion by PBMCs confirmed in uitro with a RIBA specific for structural (c22-3) and nonstructural proteins (c33c, c100-3, 5-1-1) of the HCV. Positive (P) and negative (N) controls. Lanes 1-5, 7,9, 10: Supernatants positive for c22-3 and c33c antibodies with EIA. Lanes 6 and 8: Supernatants positive for c22-3 HCV core antibodies with EM. Lanes 11 and 12: Supernatants positive for c33c antibodies with EM.

TABLE 2. In uitro anti-HCV secretion in relation to disease activity and therapeutic outcome

Group

IgM HCV RNA

patients treatment PBMCs (n) (U/L)- in serum No. of Response to anti-HCV-secreting ALT

Untreated 13

63.2 -r 37.6 5 10 8 2

7 Anti-HCV in uitro positive 13 - 0 Anti-HCV in uitro negative 5

Anti-HCV in uitro positive 8 7 NR/1 PR/O CR 3

- Treated 18 8 NRl2 PRI8 CR

Anti-HCV in uitro negative 10 1 NR/1 PR/8 CR 0 25.6 -t- 30.4

Eight patients with complete response (CR) to previous interferon treatment eliminated HCV RNA from serum. NR, nonresponders with HCV persistence, increased ALT levels and histological signs of CAH; PR, partial responders with reduced but not normalized ALT levels and HCV persistence.

Patients with anti-HCV-secreting PBMCs. both treated and untreated, showed higher ALT levels than in uitro anti-HCV-negative patients. "Data expressed as mean r ~ _ S.D. *p = 0.04. 'p = 0.0001.

Cell Culture. PBMCs were separated from heparinized blood by means of density-gradient centrifugation on Ficoll- Hypaque (Sigma, Deisenhofen, Germany). PBMCs were washed three times in RPMI-1640 medium and then resus- pended in culture medium supplemented with 5% fetal calf serum, 1% glutamine and 1% gentamicin. Cell culture was usually performed in duplicate with 1 x lo6 PBMCs/ml in 24-well culture plates for 8 days at 37" C in a 5% CO, atmosphere in the presence or absence of 1 pg/ml PWM. Supernatants were harvested, passed through a 0.2-pm filter to remove cellular contaminants and kept at -20" C until antibody assays were performed (19).

IgG Anti-HCV Assays. Supernatants of cell cultures were examined with a commercially available anti-HCV EIA (HCV Supplemental Assay; Abbott Diagnostics) specific for the recombinant HCV c22-3 core and c33c nonstructural polypep- tides. Diluent (100 p1) was added to 100-p1 aliquots of super- natant, and each was incubated, one with the structural and one with the nonstructural antigen-coated bead. The cutoff of the in uitro assay was calculated from the mean of superna- tants from a panel of patients without chronic hepatitis C plus 2 S.D. (optical density, > 0.3).

Anti-HCV secretion in cell culture supernatants was con- firmed by use of a RIBA using four recombinant HCV antigens derived from the structural and nonstructural regions of HCV (RIBA HCV test system 11; Ortho Diagnostics, Neckargemund, Germany). For the analysis of anti-HCV secretion, we per- formed RIBA with undiluted supernatants. Cell culture super- natants were regarded as anti-HCV-positive when EIA was positive and HCV antigen-specific bands were detectable on RIBA (25).

Detection of IgM Anti-HCV. Antibodies of the IgM subclass directed against the recombinant HCV core antigen have been assayed with a specific EL4 similar to that described before (26). In brief, 100-11.1 samples of supernatant or 10 pl of serum were added to 100 p1 of diluent containing goat anti-human IgG. Then HCV core antigen-coated beads (0.4 Fglml) were added and incubated for another hour. The beads were then washed and incubated together with 300 pl freshly prepared o-phenylenediamine substrate for 30 min. The absorbance of substrate blanks, controls and supernatant samples was determined at 492 nm within 2 hr of addition of acid. The cutoff in this assay was calculated from the mean of a panel of patients without hepatitis C plus 2 S.D. (optical density > 0.2).

1386 LoHR El ' AI,. HEPATOLOGY December 1994

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Fic:. 3. Occurrence of IgM anti-HCV core in PBMC supernatants and corresponding sera derived from 21 HCV-infected patients with i n uitro IgG anti-HCV secretion. CPH, chronic persistent hepatitis. The dashed line represents the cutoff in this assay.

Statistical Analysis. Data are expressed as mean * S.D. Correlation analysis was carried out with Student's t test for unpaired data. p Values less than 0.05 were considered statisticallv significant.

RESULTS

IgG Anti-HCV Secretion in Vitro. Anti-HCV was detected with the supplemental EIA in the supernatants harvested from the PBMCs of 21 of 36 patients with chronic HCV infection (58.3%). In 3 of the 21 patients the PBMCs secreted only c22-3 core antibodies, in another 3 patients the PBMCs secreted c33c antibodies alone and in 15 of the 21 patients (71.4%) the PBMCs secreted antibodies to structural and nonstructural HCV antigens (Fig. 1).

All the supernatants derived from the PBMCs of controls with HCV-unrelated chronic hepatitis and healthy blood donors were negative for anti-HCV.

For confirmation, we also studied the in uitro antibody secretion by use of RIBA. The pattern of anti-HCV detected on RIBA demonstrated secretion of anti-HCV directed against the recombinant c22-3, c33c, c100-3 and 5-1-1 polypeptides derived from the structural and nonstructural regions of the HCV (Fig. 2).

This HCV-specific humoral response in uitro was not influenced by the polyclonal activation of the PBMCs with PWM leading to increased immunoglobulin syn- thesis. This was ruled out in all patients and controls. Anti-HCV secretion by PBMCs observed in uitro was always spontaneous.

IgM Anti-HCV Core In Vitro und in Serum. Analysis of IPM anti-HCV core was Derformed with cell culture

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FIG. 4. Representative data showing the influence of interferon-a on the IgG anti-HCV secretion by PBMCs. Treatment, responder J.H., with decreasing anti-HCV i n uitro and normalizing ALT levels (top). Patient M.P., without therapy response, showed recurrent anti-HCV in uitro after initial disappearance (middle). Patient H.P. had recurrent i n uitro humoral immune response to HCV during treatment before reactivation of the disease (hoftnm 1

HEPATOLOGY Vol. 20, NO. 6, 1994 LOHR ET AL. 1387

supernatants and corresponding sera obtained from all patients with chronic hepatitis C.

In 10 of 21 patients with chronic HCV infection (27.8%), the PBMCs secreted IgM and IgG anti-HCV core in their supernatants. No IgM anti-HCV secretion was observed in the PBMC cultures of 15 patients without in uitro IgG anti-HCV production.

Corresponding serum analysis demonstrated IgM anti-HCV core antibodies in 9 of the 10 patients with IgM anti-HCV secretion in uitro (90%). One additional patient with CAH-C had IgM anti-HCV core in his serum but not in uitro (Fig. 3).

HCV-specific Humoral Response In Vitro and Disease Activity. The inflammatory activity of chronic hepatitis C was determined on the basis of the levels of ALT in sera and histological appearance.

Patients whose PBMCs secreted IgG or even IgM anti-HCV in uitro showed higher ALT levels in their sera than did patients without HCV-specific humoral re- sponse in uitro. The mean ALT levels were 99.5 * 22.1 U/L in the IgM/IgG anti-HCV in uitro-positive patient group, 85.6 2 34.4 U/L in the IgG anti-HCV in uitro- positive group and 38.1 f 37.4 U/L in the anti-HCV in uztro-negative group (Table 1).

Histological examination of HCV-infected patients with in uitro anti-HCV showed severe CAH, with partial transition to cirrhosis in 18 of 21 (85.7%) and persistent hepatitis in 3 of 21 (14.3%). Of 15 patients with chronic HCV infection who were in uitro anti-HCV antibody negative, 2 (13.3%) had active, 6 (40.0%) had persistent and 7 (46.6%) had signs of minimal hepatitis (Fig. 1).

Inflammatory activity was assessed in 29 of 36 patients with chronic hepatitis C according to the histology activity score of Scheuer (24). Patients with in uitro anti-HCV secretion (n = 17) showed significantly higher disease activity than did the 12 patients without in uitro anti-HCV secretion (4.9 k 0.8 vs. 3.1 k 0.7). In addition, patients with in uitro IgM and IgG anti-HCV secretion (n = 9) had an even higher score than patients without IgM but with IgG anti-HCV secretion (n = 8; 5.3 k 1.2 vs. 4.3 * 1.3) (Table 1).

Anti-HCV Secretion in Relation to Viremia. HCV RNA was demonstrated with reverse transcription- polymerase chain reaction in the sera of 28 of 36 patients with chronic HCV infection (77.8%). Eight additional patients (8 of 36; 22.2%) become HCV RNA seronegative through complete response after a 6-mo follow-up after withdrawal of IFN-a. These eight patients had improved liver histology, had normal serum transaminase levels and were actually negative for HCV RNA in serum and in uitro anti-HCV secretion (Table 2).

Anti-HCV Secretion In Vitro and Response to IFN-a To show the relationship between the humoral immune response in uitro and the effectiveness and outcome of treatment with IFN-a, we retrospectively studied 18 HCV patients after they completed IFN therapy. Ten of the 18 patients showed complete responses to therapy as defined by normalized serum aminotransferase levels and improvement of histological appearance. However, none of them had in uitro anti-HCV secretion. On the other hand, 8 of the 18 treated HCV patients, 7

nonresponders and 1 partial responder showed in uitro anti-HCV production (Table 2).

Twelve of the 18 untreated HCV patients received IFN-a subsequently. The influence of IFN-a on in uitro anti-HCV secretion was followed in 8 of these 12 patients with detectable in uitro IgM and IgG anti-HCV. During IFN-a therapy the virus-specific humoral response in uitro stopped within 1 to 4 mo in six of the eight patients (75%; Fig. 4). However, one of these eight patients was a complete nonresponder with persistent in uitro anti- HCV secretion (Fig. 4), and another patient was a par- tial responder with recurrent in uitro anti-HCV se- cretion (Fig. 4).

DISCUSSION It has been suggested that the liver injury in chronic

hepatitis C is correlated with the persistence of de- tectable HCVRNAin serum (5,15,27,28). However, the presence of HCV-RNA in asymptomatic individuals indicates that some immunological mechanisms may also be pathogenetically relevant (6, 29).

With virus-specific recombinant polypeptides, hu- moral responses against different HCV antigens derived from structural and nonstructural regions can be de- tected as important diagnostic markers in HCV patients. The biological significance of anti-HCV in serum, how- ever, is unclear because a specific antibody pattern cor- relating with inflammatory activity, tolerance or clinical features has not been established (15, 25, 30-33). Some immunocompromised patients lack anti-HCV in serum, and chronic hepatitis develops (6, 34). Preliminary data suggest that HCV mutants may escape neutralizing an- tibodies (35). Recently, specific IgM solid-phase EIAs for the diagnosis of HCV infection have been developed, and the occurrence of IgM anti-HCV antibodies has been correlated with disease activity and the outcome of anti- viral therapy (16-18,26,36).

For the first time we were able to detect anti-HCV secretion in the supernatants of PBMCs from patients with chronic hepatitis C but not from controls. As we have shown, the serological anti-HCV pattern did not differ from the in vitro response, suggesting that anti-HCV secretion in uitro is specific and may serve as an effective in uitro model to investigate virus-specific humoral immune responses (19). In this study, we confirmed the specificity of the antibody secretion by performing the RIBA with undiluted supernatants. However, in one patient c33c antibodies were detected only on RIBA and not on EIA, suggesting greater sensitivity for the immunoblot technique. However, the polyclonal activation of B and T cells with PWM never influenced the humoral response in uitro. These results give evidence that persistent antigeneic stimulation is responsible for the production of HCV-specific anti- bodies by B cells in uitro.

The presence of HCV antibody secretion in uitro (i.e., secretion of both IgG and IgM anti-HCV core) in patients with inflammatory active disease indicates that virus- specific humoral immune responses may have pathoge- netic relevance. Furthermore, HCV viremia as deter- mined by reverse transcription-polymerase chain re- action assay was found in all patients with untreated

1388 LOHRETAL

chronic hepatitis C, as well as in patients with negative outcome from IFN treatment. However, eight complete responders had normalized ALT levels, improvement of liver histology and loss of HCV RNA from serum; all were in uitro anti-HCV negative. Therefore our data confirm that the humoral immune response to HCV is closely related to viremia in patients who are known to have more severe hepatitis (5, 27, 32).

Recently it was demonstrated that HCV can infect PBMCs and that these cells may represent a virus reservoir (37-40). Because the anti-HCV secretion by PBMCs is due to persistent antigeneic stimulation of B cells by the ongoing production of viral antigens, the humoral immune response may indicate persisting HCV in PBMCs.

Only a few patients with chronic HCV infection had improvement of their hepatitis after treatment with IFN-a, although HCV viremia decreases (41,421. In our study, it seems likely that the presence of anti-HCV in uitru correlates with the negative outcome of antiviral therapy. Follow-up of patients with anti-HCV secretion in uitro clearly showed that treatment with IFN-a suppresses the humoral immune response. Otherwise, the humoral response persisted in patients with ongoing active disease or reactivation after treatment. The recurrence of anti-HCV in uitro and the correlation with the negative outcome from IFN-a therapy emphasize that immunological mechanisms may be responsible for the defective virus elimination leading to long-term persistence of HCV.

In conclusion it can be speculated that the humoral response to HCV has pathogenetic significance. More- over, in vitro anti-HCV antibody secretion seems to be a predictive factor for response to IFN-a therapy. How- ever, this must be further evaluated in prospective studies.

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