GASTROENTEROLOGY 1992:103:622-629

Hepatitis C Virus Antigen in Hepatocytes: Immunomorphologic Detection and Identification KRZYSZTOF

KRAWCZYNSKI,

MICHAEL

J. BEACH,

DANIEL

W. BRADLEY,

GEORGE KUO, ADRIAN M. DI BISCEGLIE, MICHAEL HOUGHTON, GREGORY R. REYES, JUNGSUH P. KIM, QUI-LIM CHOO, and MIRIAM J. ALTER Hepatitis Branch, Centers for Disease Control, Atlanta, Georgia; Chiron Corporation, Emeryville, California; Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and Genelabs Inc., Redwood City, California

Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and nonstructural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.

M

olecular cloning of hepatitis C virus (HCV),* the main etiologic agent of transfusion and blood-related non-A, non-B hepatitis in humans and experimentally infected chimpanzees, was soon followed by extensive molecular studies of the virus.‘-” Recombinant proteins of HCV were expressed in procaryotic and eucaryotic vectors enabling the development of immunologic assays to detect specific humoral immune responses to structural and nonstructural antigens of HCV. Antibody responses in humans and chimpanzees to a portion of the HCV NS4 protein (Cl00-3, a recombinant protein pro-

duced in yeast) were previously reported.7*8 HCV RNA has been identified in sera of patients and experimentally infected animals by a polymerase chain reaction (PCR) method.Q-‘l However, progress in studies of the pathogenesis of liver disease during HCV infection was hampered by lack of the identification of HCV antigen(s) (HCVAg) in liver tissue specimens, although the immunohistochemical detection of non-A, non-B hepatitis-associated antigens was previously claimed, and equivocally defined antigens were found either in nuclei or in cytoplasm of liver cells.‘2-16 In this study, we present immunomorphologic evidence in support of the HCV viral specificity of HCVAg detected in hepatocytes of HCV infected patients with chronic hepatitis C and experimentally inoculated chimpanzees. Further application of the specific immunohistochemical methods for the identification of HCVAg may be used to obtain a better understanding of HCV replication, the pathogenesis of hepatitis C, and natural history of HCV infection. Materials and Methods Liver biopsy specimens (needle or wedge) were obtained from chimpanzees during the acute phase of HCV infection experimentally induced by antihemophilic factor (FVIII) inocu1ation.a Biopsies were also obtained from animals with chronic HCV infection and from some that had apparently recovered from acute HCV infection. Chimpanzees liver samples used as negative controls included (a) preinoculation liver biopsy specimens; (b) liver biopsy specimens from chimpanzees infected with hepatitis B virus (HBV), hepatitis A virus (HAV), or with hepatitis delta virus (HDV); (c) kidney, spleen, and lymph node specimens from either uninfected or HBV infected chimpanzees. Animals used in this study were cared for and This is a U.S. government work. There are no restrictions on its use. 0016-5065/92/$0.00

August 1992

IMMUNOMORI’HOI.OClC IDENTIFI 20%; intensity was evaluated on the arbitrary scale from 1 t to 3+. Risk factors (presumed source of hepatitis): TX, transfusion; OCC, occupational; IVDA, intravenous drug abuse. ALT, alanine aminotransferase; CPH, chronic persistent hepatitis: CAH, chronic active (aggressive) hepatitis; CIR, liver cirrhosis; nt, not tested.

changed when stained with FITC-anti-HCVAg adsorbed with normal chimpanzee liver, mouse liver, human IgG, fibrin, or chimpanzee red blood cells. The efficiency of the absorption of FITC-anti-HCVAg with recombinant HCV structural and nonstructural proteins expressed in yeast was documented by RIA. The assays showed a significant decrease of reactivity of the absorbed FA probe with RIA target proteins of immunologic specificities homologous to those used for absorption (Table 3). The efficiency of the absorption with the F5 (NS4) recombinant HCV nonstructural protein was documented by EIA, which showed a proportional reduction of the PRC.2126 IgG fraction reactivity with the Cl00 protein after absorption with increasing amounts of the F5 protein (data not included).

liver cells in both HCV-infected patients and chimpanzees are specific for HCV. First, FITC-labeled immunoglobulins, used for the detection of HCVAg, reacted with structural and nonstructural recombinant HCV proteins expressed in yeast or in E. coli when tested by RIA or EIA. Second, blocking studies with paired serum samples obtained from uninfected and infected chimpanzees or during the acute and chronic phases of the HCV infection from patients further suggested HCV specificity of HCVAg in liver sections. Third, absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Finally, absorption experiments with recombinant HCV nonstructural and structural proteins on the PRC.2126 FITC-labeled probe established a direct linkage between hepatocellular HCVAg and HCV. Our studies were based on stringent criteria de-

Discussion Several lines of evidence presented in this report indicate that the deposits of HCVAg detected in

Table 2. Reactivity of Chronic Hepatitis C Serum Samples (PRC.2126, JEN.A) Used for FA Detection With Recombinant HCV Structural and Nonstructural Proteins

of HCVAg in Liver

Controls Recombinant HCV proteins” C22 (C, capsid) S2 (El, envelope) NSl (E2, envelope) C33c (NS3, nonstructural) Cl00 (NS4,nonstructural) NS5 (NS5, nonstructural)

PRC.2126 + + t + +

(39169) (1748) (18664) (49903) (42847) (35441)

JEN.A (counts per minute)b + + + + + t

(34244) (3312) (8014) (44113) (5309) (19144)

“Putative equivalents of flavivirus proteins. bTested in duplicate, counts per minute are average of two results. “RIA negative cut-off values calculated from 9 normal healthy control samples (mean + 3SD).

Neg. cutor 1309 2172 1342 2674 2898 3525

Positive cutoff 45810 13574 31108 37965 35517 54303

IMMUNOMORPHOLOGIC

August 1992

Table

3. Specificity

of HCVAg

IDENTIFICATION

OF HCV ANTIGEN

627

in Liver: Absorption Experiments With Recombinant HCV Structural and Nonstructural

Proteins Reactivitv With HCVAg in hepatocytes by FAb (dilutions of FA reagent)

Immunoabsorbents” HCV proteins expressed

(1:lOO -/+ ++

(Cl',, ++ ++ ++ +t ++ tt +t

C22 (capsid) SOD El NSl (E2) SOD C22 + NSl Unabsorbed

F3 (NS3) F5 (NS4) F6 (NS4/NS5) F3, F5, F6 G30 (control) Unabsorbed

With HCV proteins by radioimmunoassay

(cpm)

c33c

Cl00

c22

NSl

403 20784

479 4250

7045 7107

1082 1008

31727 33463

14174 16272

5102 18019

4147 4430

32620 32928

18544 19427

18705 18067

3621 5723

in yeast

C200(NS3 + NS4) SOD (control) Unabsorbed

HCV proteins expressed

of FITC-IeG PRC.2126

1200) -

++ t+ 1:320) +t ++ t+ +t +t t+ t+

in E. coli (1:120 +/+ + + t +

1:240) -/t +/-;+ + +

FITC-IgG PRC.2126, fluorescein isothiocyanate-labeled polyclonal antibody for identification HCVAg in hepatocytes. “Recombinant HCV proteins (putative equivalents of flavivirus proteins). SOD, control protein: insoluble superoxide dismutase expressed in yeast; G30, control protein: insoluble yeast protein. bFluorescent antibody (FA) assay on liver sections from chimpanzee with acute (CH.1220, CH.1411) and chronic (CH.771) HCV infection.

signed to rule out detection of nonviral antigens since at least two host antigens are known to be induced during HCV infection. The interferon-induced host antigen in liver cells of chimpanzees infected with HCV and HDV23*24and anti-GOR antibodies in serum of HCV infected patients and chimpanzees were recently described.25 The specific identification of HCV antigen in hepatocytes by immunohistochemical methods was not feasible before molecular cloning of HCV and the identification of several immunoreactive epitopes. Therefore, previous reports of the detection of immunofluorescent granules in hepatocyte nuclei’2-‘5 or cytoplasmic deposits in hepatocytes of experimentally infected chimpanzees by immunoperoxidase staining16 remain unverified. The results of our absorption studies have provided preliminary but convincing evidence for the viral origin of HCVAg in hepatocytes of chimpanzees with acute or chronic HCV infection. The C200 nonstructural fusion protein expressed in yeast and encoded by a fragment of the HCV genome corresponding to portions of the putative NS3 and NS4 domains removed most of the anti-HCVAg activity from the FITC-labeled polyclonal probe. Less effective were absorptions with the NS3 (F3) protein

expressed in E. coli, which may reflect differences in the antigenicity of viral prateins expressed in eucaryotic (yeast) and procaryotic (E. coli) vectors.26 Absorption experiments with HCV structural proteins, including the putative capsid and envelope (E2) proteins, did not change the immunoreactivity of the FITC-labeled PRC.2126 probe with HCVAg in chimpanzee liver specimens. This may be explained by either the paucity of structural proteins in HCVAg deposits or by the reduced immunologic reactivity of particular recombinant fusion proteins due to conformational irregularities. Antibodies obtained after immunization with native influenza hemagglutinin, for example, did not react with any of several synthetic peptides. Absorption of the polyclonal FITClabeled probes with HCV envelope antigens expressed in yeast were ineffective, possibly due to antigenic differences between the presumed linear epitopes of the viral envelope protein expressed in yeast and conformational, “native” HCV envelope epitopes recognized by antibodies contained in the FITC-labeled probe. Conformational epitopes are characteristic for the viral envelope of flaviviruses2* whose genomic organization is similar to that of HCV.3*“*2gThe differences in antibody reactivity,

628

KRAWCZYNSKI ET AL.

which depend on the type of viral antigen used for immunization (native, recombinant, or synthetic), seem to advocate the use of polyclonal, naturally occurring antibodies from HCV-infected patients or chimpanzees for identification of HCVAg. The majority of HCVAg in livers of chimpanzees appears to consist of the putative NS3 protein of HCV, as indicated by inhibition of anti-HCVAg reactivity of the FITC-labeled probe by C2OO protein. Although C200 is a fusion protein composed of NS3 and NS4 proteins, its effectiveness in absorption seemed to primarily depend on the presence of the NS3 protein rather than the NS4 protein. This was inferred from the strong immunohistochemical reactivity with HCVAg of the JEN.A probe that was only weakly reactive for anti-ClOO-3 (NS4) protein by RIA and nonreactive by EIA. Also absorption with the NS4 (F5) recombinant protein only slightly decreased HCVAg fluorescence. The prevalence of NS3 protein in HCVAg in hepatocytes of HCV infected chimpanzees is consistent with the reported accumulation of NS3 proteins of flaviviruses in infected tissue culture cells.3o The immunohistochemical identification of HCV viral proteins in hepatocytes documented in this study and future experiments involving identification of individual structural and nonstructural proteins of HCV in liver cells will facilitate further studies of the pathogenesis of hepatitis C and natural history of HCV infection. References 1.

2.

3.

4.

5.

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Received August 5, 1991. Accepted February 18, 1992. Address requests for reprints to: K. Krawczynski, M.D., Ph.D., Hepatitis Branch, Building 6, Room 192, Centers for Disease Control, Atlanta, Georgia 30333. The authors thank D. Brazzeal and J. Spelbring for technical assistance.