Improved Serodiagnosis of Non-A, Non-B Hepatitis by an Assay Detecting Antibody to Hepatitis C Virus Core Antigen TOHRUKATAYAMA,' TOSHIOMAZDA,'SHU KIKUCHI,3 SHIZUKO HARJIDA,~ YOSHIHARU MATSUURA,4 JOECHIBA,5 HIROYOSHI OHBA,5 IZUMUSAIT04* AND TATSUO MIYAIvIURA4 'Tokyo National Chest Hospital, Kiyose-shi 204; 2Tokyo Red Cross Blood Center, Musashino-shi 180; 3Sendai National Hospital, Sendai 983; 4National Institute of Health, Tokyo 141; and 5Science University of Tokyo, Noda-shi 278, Japan
throughout the world (3-9). The assay is also useful for the diagnosis of chronic hepatitis C. We have isolated HCV cDNA fragments directly from human healthy HCV carriers and analyzed the HCV genome organization (10-12). The putative core region was then efficiently expressed in monkey COS cells, and a properly processed core protein with the expected size of 22 kD was identified by its specific reaction with sera of typical NANB hepatitis patients by Western blotting (13). The same protein was also efficiently synthesized by a baculovirus vector-expression system in insect cells, and an ELISA was developed t o detect the circulating antibody reacting with this protein (p22) (14). By using this assay to detect the antibody to the p22 protein expressed by a recombinant baculovirus (antip22), we examined sera from NANB hepatitis patients who had an acute, self-limited clinical course. We previously reported that the rate of detection of antiC100-3 in those patients was significantly lower than that in patients whose NANB hepatitis had developed to the chronic stage (4). Here we discuss a group of patients with NANB hepatitis caused by HCV infection that had not been demonstrated to be NANB hepatitis by the The isolation and analysis of the complementary DNA present anti-C100-3 assay but was proved to be so by the (cDNA) of the hepatitis C virus (HCV) genome and the new anti-p22 assay. subsequent development of an assay to detect specific PATIENTS AND METHODS antibody to HCV (anti-C100-3) was a breakthrough for Patients. A 61-yr-old woman had stomach cancer. On both basic and clinical research on non-A, non-B (NANB) hepatitis (1, 2). Because the presence of December 12, 1988,2%yr after initial resection of the cancer, anti-C100-3 was thought to be closely associated with she underwent surgery for metastatic retroperitoneal carthe presence of infectious HCV virions, the ELISA for cinoma. On this occasion, she was given a transfusion of 15 (1 unit = 200 ml) of donated blood (6 units of concenanti-C100-3 has been widely used for blood screening units trated red cells, 6 units of whole blood and 3 units of fresh
W e examined sequential serum samples from 12 patients with well-characterized posttransfusion non-A, non-B hepatitis who had an acute, resolving self-limited type of clinical course for the presence of antibody to the hepatitis C virus nucleocapsid (core) protein (p22) expressed by a recombinant baculovirus. These sera were simultaneously examined for antibody to the hepatitis C virus nonstructural protein (C100-3) that is presently used for blood screening worldwide. In three patients, both anti-p22 and anti-C100-3 antibodies were detected, but anti-p22was detected much earlier. In four patients, only anti-p22 was detected. Two other patients were considered to be hepatitis C virus carriers who had been already infected with hepatitis C virus. In one patient, only anti-(2100-3was detected, and it was transient. In two patients, neither antibody was detected. Anti-p22 was detected in at least one of eight samples of transfused blood. Of the nine samples of donated blood that were positive for anti-p22, only four were positive for anti-ClOO-3.This new assay detecting the antibody to the p22 protein is thus useful for the serodiagnosis of non-A, non-B hepatitis in the acute phase and for blood screening. (HEPATOLOGY 1992;1539 1-394.)
Received July 15, 1991; accepted October 11, 1991. This work was supported in part by grants from the Ministry of Health and Welfare, and the Ministry of Education, Science and Culture, Japan. A preliminary report of this work was presented at the VIIIth International Congress of Virology, Berlin, Germany, August 26-31, 1990. *Current address: Laboratory of Molemlar Genetics, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108, Japan. Address reprint requests to: Tatsuo Miyamura, Laboratory of Hepatitis Viruses 11, Department of Enteroviruses, National Institute of Health, 2-10-35, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan. 3111134987
frozen plasma). Eight weeks after the transfusion, her serum ALT and AST levels had increased to 88 IU and 102 IU, respectively. The enzyme levels kept increasing and finally reached maximum levels 10 wk after the operation (ALT = 226 IU; AST = 336 IU). Jaundice was not observed throughout the study. However, after this enzyme elevation peak, although she did not receive any particular therapy other than general liver protection treatment, the ALT and AST levels began to decrease. After 12 wk, they were 33 IU and 24 IU, respectively. After that the enzyme levels were under 20 IU until the patient died on April 23, 1990. Thus this case was considered to be a typical self-limited, resolving case of
39 1
KATAYAMA ET AL
392
HEPATOLOGY
ALT
+ + + + +
0
+ +
+ +
+ +
+
anti-p22
+
anti - ClOO - 3
12
6 months
FIG.1. ALT levels and Anti-HCV (anti-p22 and anti-C100-3) in patient A with acute, resolving type of NANB hepatitis. ALT is expressed in internationid units.
posttransfusion NANB hepatitis. Eleven other patients were one of them was found to be positive for anti-p22. selected from 83 patients with well-characterized posttrans- However, none were positive for anti-C100-3. fusion NANB hepatitis observed a t the Tokyo National Chest We then tested a series of sera including pretransHospital and Sendai National Hospital between 1982 and fusion sera from 11 other posttransfusion NANB hep1989. All 11patients met the following criteria: (a) They were atitis patients who had shown single-peak enzyme clinically diagnosed as having posttransfusion NANB hepatitis by the criteria proposed by the Japanese Society of Gastroen- elevations; the results are summarized in Table 1. terology (4); (b) Sera had been collected every week or every Patients B and C showed seroconversion to anti-C100-3 other week, and liver function was examined on each occasion; positive 14 and 24 wk after their respective transfusions. (c) They were observed up for at least 12 mo; (d) By clinical However, it was noted that anti-p22 was detected much findings and enzyme monitoring, their NANB hepatitis was earlier than anti-C100-3in both cases. Anti-p22 was also retrospectively diagnosed as the self-limited resolving type; detected in the blood of one donor for each of these and (e) During the time of abnormal ALT levels, no remission patients. For patient B, anti-(3100-3 was detected in the was observed (single-peak enzyme elevation). blood donor who was positive for anti-p22. In patient C, Assays for Antibody to HCV. The assay for anti-C100-3was none of the 23 blood samples were positive for antieither RIA or ELISA (Ortho Diagnostics, Raritan, NJ). The C100-3 at the time of the transfusion, but the follow-up assay for anti-p22 was described previously (14). The cut-off study of those donors revealed that one of them value was determined by assaying blood donor samples actually transfused into recipients whose outcomes were all seroconverted to anti-(2100-3 positive after 4 yr. The analyzed by clinical follow-up study and by enzyme monitoring donor blood was already positive for anti-p22 at the time (14). The cut-off value was validated by immunoblotting of the transfusion. Patients D and E received transfusions of blood analysis with cell lysates of Sf-9 cells infected with a recombinant baculovirus or monkey COS cells transiently expressing samples that were positive for anti-C100-3. Although the same HCV core region under the SRol promoter (15). typical NANB hepatitis developed, the patients’ sera
RESULTS We examined the sera of the 12 posttransfusion NANB hepatitis patients for anti-p22 and anti-C100-3. We also tested the respective blood samples actually transfused in each case for these antibodies. The timing of the appearance of anti-p22 and anti-(3100-3 in patient A, who had shown a typical clinical course, is illustrated in Figure 1.Anti-p22 was detected as early as 6 wk after the transfusion when the ALT level was still 15 IU/L. ALT elevation was observed 8 wk after the transfusion, and anti-C100-3 was detectable after 9 wk when the ALT level reached its peak. After that, both anti-p22 and anti-C100-3 were detectable until the patient died 13 mo later. This patient had received 15 units of blood, and
were negative for anti-C100-3 throughout the study (36 and 48 mo, respectively). Anti-p22, however, was detected in these patients 4 and 14 wk, respectively, after the transfusions, almost at the same time as their respective enzyme elevations. In both cases, one of the donor sera was shown to be positive for both anti-p22 and anti-C100-3. In patient F, anti-(2100-3 was detected only transiently 9 wk after the transfusion. By the recently developed recombinant immunoblot assay test, however, the reaction was not confirmed as positive. Anti-p22 was not detected throughout the course of the 4-yr follow-up study. In 20 donor blood samples transfused into this patient, none were positive for either anti-p22 or anti-(2100-3.
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393
TABLE1. Clinical findings and anti-p22 and anti-C100-3in 11 patients with posttransfusion NANB hepatitis
Patient
Agelsex
B C D E
70m 67iM 52/34 59rM 45iM 7l/M 49lF 57m 49iF 49lF
F G H I J K L
51iM
Incubation period (wk)”
peakb
Duration of abnormal ALT (wk)
7 6 4 10 3 13 9 7 2 2 5
596 602 252 225 280 468 317 218 501 881 170
6 7 4 15 2 48 13 4 6 11 21
ALT
Time of seroconversion to anti-p22 positive (wk)
Time of seroconversion to anti-C100-3 positive (wk)
8 10 4 14
14 24 9 -
-
24 192
-
-
-
No. of blood donors
No. of anti-p22 positive donors
No. of anti-C100-3 positive donors
8 23 5 4 20 4 7 31 3 7 7
1 1 1 1 0 1 1 2 0 0 0
1 0 1 1 0 0 0 0 0 0 0
”Interval between blood transfusion and ALT elevation. bALT value expressed in international units. ‘Antibody detected before transfusion.
Before this study, the conditions of patients G, H, I, J, K and L were not considered to be related to HCV infection because of the absence of anti-C100-3. Seroconversion t o anti-C100-3 positive occurred in none of those patients, and none of the blood samples transfused were positive for anti-C100-3. Among these six patients, patients G and H had seroconverted to anti-p22 positive 24 wk and 4 yr later, respectively. One of the four transfused blood samples for patient G and one of the seven samples for patient H contained anti-p22. Consequently, both patients were considered to be HCV infected, although the time of seroconversion in patient H seemed too late. Patients I and J had anti-p22 even before the transfusion, suggesting that they themselves had already been infected with HCV. In patients K and L, HCV infection was not proved by the two assays used in this study. Some surgically treated patients who have not received blood transfusions sustain liver cell damage after the operation from the original disease, a drug or anesthesia. Usually in these cases the ALT elevation occurs immediately and transiently after the operation. In some cases, however, it is rather difficult to distinguish this damage clinically from NANB viral hepatitis, particularly of the self-limited, resolving type described above. Therefore we assayed sera from such patients with single-peak enzyme elevation without a blood transfusion. None of these eight patients were positive for anti-C100-3 or anti-p22. DISCUSSION
An assay to detect anti-C100-3 has been used worldwide for screening blood for transfusions. It is also useful for the diagnosis of chronic hepatitis C. Application of the anti-C100-3 assay system, however, raised three important problems for a precise diagnosis of hepatitis C, which are as follows: (a) It takes at least several weeks to months for seroconversion to be detectable; (b) Cases of NANB hepatitis exist that are
negative for anti-(3100-3 even in long follow-up studies; and (c) The possibility of a false-positive reaction, particularly in cases of autoimmune hepatitis, has been pointed out (16). In patients having only one episode of liver cell enzyme elevation, the frequency of anti-C100-3 detection was much lower than in those having multiple peaks of enzyme elevation (4). It is not yet clear whether this is because of some other viral agent, a difference in host response or insufficiency of the test system. We induced expression of properly processed HCV core protein in insect and animal cells (13, 14) and established an antibody detection system using the synthesized protein (14). This study revealed the involvement of HCV infection in cases of acute, resolving NANB hepatitis in which viral involvement had not been shown by the currently available anti-C100-3 assay. Of course, we cannot rule out the possibility of the existence of other virus(es)that could induce this elusive hepatitis. As in patients K and L, undetermined posttransfusion NANB hepatitis still exists. However, it is interesting to note that in these two cases, in one possible HCV carrier (patient J) and in one cryptic case (patient F) the incubation periods were shorter than in other typical hepatitis C cases (Table 1).Such patients with a very short incubation period could have nonviral acute hepatitis. This study also shows that an assay for anti-p22 is useful for the early diagnosis of HCV infection (13, 14). Inclusion of this assay in blood screening should also be considered, Retrospective analysis of donor blood that had been actually transfused and of the consequences for the recipients is now in progress. Why could anti-p22 be detected earlier and with higher sensitivity than anti-C100-3 in some cases of NANB hepatitis, particularly the acute, self-limited type described here? One possible explanation is that a structural protein is somehow more immunogenic and that therefore anti-p22 would appear much earlier and more persistently than antibodies to the nonstructural
394
KATAYAMA ET AL.
components of HCV. Alternately, the higher detection rate may be caused by the conservation of sequence variations among different HCV isolates in the core domain of the HCV genome (16-20). Recently, antibody detection assays for the HCV core protein have been developed that use synthetic oligopeptides (21,221 and a protein expressed in Escherichia coli (E. coli) (23) or in yeast (24).We compared the ELISA derived from the p22 core protein described in this paper and other ELISA assays derived from the core protein expressed in E. coli or various synthetic oligopeptides deduced from the putative core gene using sera of patients with NANB hepatitis. The results showed that the anti-p22 ELISA assay was more sensitive than the ELISAs using synthetic oligopeptides derived from four different core regions (Chiba et al., Unpublished observations, 1991). p22 was expressed in insect cells, to which human beings generally do not have antibodies. Because of its relatively low background in immunoblotting (data not shown), the anti-p22 ELISA assay is expected to be more sensitive than the other two ELISAs based on proteins expressed in E. coli and in yeasts. New assays for the anti-HCV core protein antibody by themselves or in combination with the present anti-C100-3 assay will be useful for understanding the viral pathogenesis of HCV infection and for efficient diagnosis and prevention.
Acknowledgments: We are indebted to Dr. G. Kuo for anti-C100-3 RIA and advice; to Dr. H. Shimojo for a critical review of the manuscript; to Dr. W. J.Rutter, Dr. H. Thomas, Dr. T. Oda and Dr. K. Nishioka for discussions; to Ms. Y. Kaminuma, Ms. F. Sakurai and Ms. Y. Hirama for their laboratory assistance; and to Ms. T. Mizoguchi for the preparation of the manuscript. REFERENCES 1. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton
M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362. 2. Kuo G, Choo Q-L, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244:362-364. 3. Alter IIJ, Purcell RH, Shih JW,Melpolder JC, Houghton M, Choo &-I., Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321:14941500. 4. Miyamura.'l' Saito I, Katayama T, Kikuchi S, Tateda A, Houghton M, Choo Q-L, et al. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis. Proc Natl Acad Sci USA 1990;87:983-987. 5.Katayama T, Kikuchi S, TanakaY, Saito I, Miyamura T, Choo Q-L, Houghton M, et al. Blood screening for non-A, non-B hepatitis by hepatitis C virus antibody assay. Transfusion 1990;30:374-376. 6. Esteban JI, Esteban R, Viladomiu L, Ropez-Talavera JC, Gonzales A, Hernandez JM, Roget M, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;2:294-296. 7. Van der Poel CL, Reesink IIW, Lelie PN, Leentvaar-Kuypers A, Choo Q-I., Kuo G, Houghton M. Anti-hepatitis C antibodies and
HEPATOr.OGY
non-A, non-B post-transfusion hepatitis in The Netherlands. Lancet 1989;2:297-298. 8. Kuhnl P, Seidl S, Strange1 W, Beyer J , Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors. Lancet 1989;2:324. 9. Roggendorf M, Deinhardt F, Rasshofer R, Eberle J, Hopf U, Moller B, Zachoval R, et al. Antibody t o hepatitis C virus. Lancet 1989;2:324-325. 10. Kubo Y. Takeuchi K. Boonmar S. Katavama T. Choo 8 - L . Kuo G. Weiner AW, et al. A cDNA fragment OF hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucl Acids Res 1989;17:10367-10372. 11. Takeuchi K, Boonmar S, Kubo Y, Katayama T, Harada H, Ohbayashi A, Choo Q-L, et al. Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in posttransfusion non-A, non-B hepatitis. Gene 1990;91:287-291. 12. Takeuchi K, Kubo Y, Boonmar S, Watanabe Y, Katayama T, Choo Q-L, Kuo G, et al. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol 1990;71:3027-3033. 13. Harada S, Takeuchi K, Suzuki T, Katayama T, Watanabe Y, Takebe Y, Saito I, et al. Expression of processed core protein of hepatitis C virus in mammalian cells. J Virol 1991;65:3015-3021. 14. Chiba J, Ohba H, Matsuura Y, Watanabe Y, Katayama T, Kikuchi S, Saito I, et al. Serodiagnosis of hepatitis C virus (HCV) infection with an HCV core protein molecularly expressed by a recombinant baculovirus. Proc Natl Acad Sci USA 1991;88:4641-4645. 15. Takebe Y, Seiki M, Fujisawa J , Hoy P, Yokota K, Arai K, Yoshida M, et al.SR promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1long terminal repeat. Mol Cell Biol 1988;8:466-472. 16. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenic factor or false-positive result? Lancet 1990; 335:754-757. 17. Choo Q-L, Richman K, Han J , Berger K, Lee C, Dong C, Gallegos C, et al. The genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991;88:2451-2455. 18. Okamoto H, Okada S, Sugiyama S, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990;60:167-177. 19. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A non-B hepatitis. Proc Natl Acad Sci USA 1990;87:9524-9528. 20. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J , Onishi E, et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol1991;65: 11051113. 21. Okamoto H, Munekata E, Tsuda F, Takahashi K, Yotsumoto S, Tanaka T, Tachibana K, et al. Enzyme-linked immunosorbent assay for antibodies against the capsid protein of hepatitis C virus with a synthetic oligopeptide. Jpn J Exp Med 1990;60: 223-233. 22. Hosein B, Fang CT, Popovsky MA, Ye J , Zhang M, Wang CY. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Natl Acad Sci USA 1991;88:3647-3651. 23. Muraiso K, Hijikata M, Ohkoshi S, Cho M-J, Kikuchi M, Kato N, Shimotohno K. A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus. Biochem Biophys Res Commun 1990;172:511-516. 24. Van der Poel CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, Di Nello R, Van Boven JJP, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991;337:317-319,
throughout the world (3-9). The assay is also useful for the diagnosis of chronic hepatitis C. We have isolated HCV cDNA fragments directly from human healthy HCV carriers and analyzed the HCV genome organization (10-12). The putative core region was then efficiently expressed in monkey COS cells, and a properly processed core protein with the expected size of 22 kD was identified by its specific reaction with sera of typical NANB hepatitis patients by Western blotting (13). The same protein was also efficiently synthesized by a baculovirus vector-expression system in insect cells, and an ELISA was developed t o detect the circulating antibody reacting with this protein (p22) (14). By using this assay to detect the antibody to the p22 protein expressed by a recombinant baculovirus (antip22), we examined sera from NANB hepatitis patients who had an acute, self-limited clinical course. We previously reported that the rate of detection of antiC100-3 in those patients was significantly lower than that in patients whose NANB hepatitis had developed to the chronic stage (4). Here we discuss a group of patients with NANB hepatitis caused by HCV infection that had not been demonstrated to be NANB hepatitis by the The isolation and analysis of the complementary DNA present anti-C100-3 assay but was proved to be so by the (cDNA) of the hepatitis C virus (HCV) genome and the new anti-p22 assay. subsequent development of an assay to detect specific PATIENTS AND METHODS antibody to HCV (anti-C100-3) was a breakthrough for Patients. A 61-yr-old woman had stomach cancer. On both basic and clinical research on non-A, non-B (NANB) hepatitis (1, 2). Because the presence of December 12, 1988,2%yr after initial resection of the cancer, anti-C100-3 was thought to be closely associated with she underwent surgery for metastatic retroperitoneal carthe presence of infectious HCV virions, the ELISA for cinoma. On this occasion, she was given a transfusion of 15 (1 unit = 200 ml) of donated blood (6 units of concenanti-C100-3 has been widely used for blood screening units trated red cells, 6 units of whole blood and 3 units of fresh
W e examined sequential serum samples from 12 patients with well-characterized posttransfusion non-A, non-B hepatitis who had an acute, resolving self-limited type of clinical course for the presence of antibody to the hepatitis C virus nucleocapsid (core) protein (p22) expressed by a recombinant baculovirus. These sera were simultaneously examined for antibody to the hepatitis C virus nonstructural protein (C100-3) that is presently used for blood screening worldwide. In three patients, both anti-p22 and anti-C100-3 antibodies were detected, but anti-p22was detected much earlier. In four patients, only anti-p22 was detected. Two other patients were considered to be hepatitis C virus carriers who had been already infected with hepatitis C virus. In one patient, only anti-(2100-3was detected, and it was transient. In two patients, neither antibody was detected. Anti-p22 was detected in at least one of eight samples of transfused blood. Of the nine samples of donated blood that were positive for anti-p22, only four were positive for anti-ClOO-3.This new assay detecting the antibody to the p22 protein is thus useful for the serodiagnosis of non-A, non-B hepatitis in the acute phase and for blood screening. (HEPATOLOGY 1992;1539 1-394.)
Received July 15, 1991; accepted October 11, 1991. This work was supported in part by grants from the Ministry of Health and Welfare, and the Ministry of Education, Science and Culture, Japan. A preliminary report of this work was presented at the VIIIth International Congress of Virology, Berlin, Germany, August 26-31, 1990. *Current address: Laboratory of Molemlar Genetics, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108, Japan. Address reprint requests to: Tatsuo Miyamura, Laboratory of Hepatitis Viruses 11, Department of Enteroviruses, National Institute of Health, 2-10-35, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan. 3111134987
frozen plasma). Eight weeks after the transfusion, her serum ALT and AST levels had increased to 88 IU and 102 IU, respectively. The enzyme levels kept increasing and finally reached maximum levels 10 wk after the operation (ALT = 226 IU; AST = 336 IU). Jaundice was not observed throughout the study. However, after this enzyme elevation peak, although she did not receive any particular therapy other than general liver protection treatment, the ALT and AST levels began to decrease. After 12 wk, they were 33 IU and 24 IU, respectively. After that the enzyme levels were under 20 IU until the patient died on April 23, 1990. Thus this case was considered to be a typical self-limited, resolving case of
39 1
KATAYAMA ET AL
392
HEPATOLOGY
ALT
+ + + + +
0
+ +
+ +
+ +
+
anti-p22
+
anti - ClOO - 3
12
6 months
FIG.1. ALT levels and Anti-HCV (anti-p22 and anti-C100-3) in patient A with acute, resolving type of NANB hepatitis. ALT is expressed in internationid units.
posttransfusion NANB hepatitis. Eleven other patients were one of them was found to be positive for anti-p22. selected from 83 patients with well-characterized posttrans- However, none were positive for anti-C100-3. fusion NANB hepatitis observed a t the Tokyo National Chest We then tested a series of sera including pretransHospital and Sendai National Hospital between 1982 and fusion sera from 11 other posttransfusion NANB hep1989. All 11patients met the following criteria: (a) They were atitis patients who had shown single-peak enzyme clinically diagnosed as having posttransfusion NANB hepatitis by the criteria proposed by the Japanese Society of Gastroen- elevations; the results are summarized in Table 1. terology (4); (b) Sera had been collected every week or every Patients B and C showed seroconversion to anti-C100-3 other week, and liver function was examined on each occasion; positive 14 and 24 wk after their respective transfusions. (c) They were observed up for at least 12 mo; (d) By clinical However, it was noted that anti-p22 was detected much findings and enzyme monitoring, their NANB hepatitis was earlier than anti-C100-3in both cases. Anti-p22 was also retrospectively diagnosed as the self-limited resolving type; detected in the blood of one donor for each of these and (e) During the time of abnormal ALT levels, no remission patients. For patient B, anti-(3100-3 was detected in the was observed (single-peak enzyme elevation). blood donor who was positive for anti-p22. In patient C, Assays for Antibody to HCV. The assay for anti-C100-3was none of the 23 blood samples were positive for antieither RIA or ELISA (Ortho Diagnostics, Raritan, NJ). The C100-3 at the time of the transfusion, but the follow-up assay for anti-p22 was described previously (14). The cut-off study of those donors revealed that one of them value was determined by assaying blood donor samples actually transfused into recipients whose outcomes were all seroconverted to anti-(2100-3 positive after 4 yr. The analyzed by clinical follow-up study and by enzyme monitoring donor blood was already positive for anti-p22 at the time (14). The cut-off value was validated by immunoblotting of the transfusion. Patients D and E received transfusions of blood analysis with cell lysates of Sf-9 cells infected with a recombinant baculovirus or monkey COS cells transiently expressing samples that were positive for anti-C100-3. Although the same HCV core region under the SRol promoter (15). typical NANB hepatitis developed, the patients’ sera
RESULTS We examined the sera of the 12 posttransfusion NANB hepatitis patients for anti-p22 and anti-C100-3. We also tested the respective blood samples actually transfused in each case for these antibodies. The timing of the appearance of anti-p22 and anti-(3100-3 in patient A, who had shown a typical clinical course, is illustrated in Figure 1.Anti-p22 was detected as early as 6 wk after the transfusion when the ALT level was still 15 IU/L. ALT elevation was observed 8 wk after the transfusion, and anti-C100-3 was detectable after 9 wk when the ALT level reached its peak. After that, both anti-p22 and anti-C100-3 were detectable until the patient died 13 mo later. This patient had received 15 units of blood, and
were negative for anti-C100-3 throughout the study (36 and 48 mo, respectively). Anti-p22, however, was detected in these patients 4 and 14 wk, respectively, after the transfusions, almost at the same time as their respective enzyme elevations. In both cases, one of the donor sera was shown to be positive for both anti-p22 and anti-C100-3. In patient F, anti-(2100-3 was detected only transiently 9 wk after the transfusion. By the recently developed recombinant immunoblot assay test, however, the reaction was not confirmed as positive. Anti-p22 was not detected throughout the course of the 4-yr follow-up study. In 20 donor blood samples transfused into this patient, none were positive for either anti-p22 or anti-(2100-3.
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393
TABLE1. Clinical findings and anti-p22 and anti-C100-3in 11 patients with posttransfusion NANB hepatitis
Patient
Agelsex
B C D E
70m 67iM 52/34 59rM 45iM 7l/M 49lF 57m 49iF 49lF
F G H I J K L
51iM
Incubation period (wk)”
peakb
Duration of abnormal ALT (wk)
7 6 4 10 3 13 9 7 2 2 5
596 602 252 225 280 468 317 218 501 881 170
6 7 4 15 2 48 13 4 6 11 21
ALT
Time of seroconversion to anti-p22 positive (wk)
Time of seroconversion to anti-C100-3 positive (wk)
8 10 4 14
14 24 9 -
-
24 192
-
-
-
No. of blood donors
No. of anti-p22 positive donors
No. of anti-C100-3 positive donors
8 23 5 4 20 4 7 31 3 7 7
1 1 1 1 0 1 1 2 0 0 0
1 0 1 1 0 0 0 0 0 0 0
”Interval between blood transfusion and ALT elevation. bALT value expressed in international units. ‘Antibody detected before transfusion.
Before this study, the conditions of patients G, H, I, J, K and L were not considered to be related to HCV infection because of the absence of anti-C100-3. Seroconversion t o anti-C100-3 positive occurred in none of those patients, and none of the blood samples transfused were positive for anti-C100-3. Among these six patients, patients G and H had seroconverted to anti-p22 positive 24 wk and 4 yr later, respectively. One of the four transfused blood samples for patient G and one of the seven samples for patient H contained anti-p22. Consequently, both patients were considered to be HCV infected, although the time of seroconversion in patient H seemed too late. Patients I and J had anti-p22 even before the transfusion, suggesting that they themselves had already been infected with HCV. In patients K and L, HCV infection was not proved by the two assays used in this study. Some surgically treated patients who have not received blood transfusions sustain liver cell damage after the operation from the original disease, a drug or anesthesia. Usually in these cases the ALT elevation occurs immediately and transiently after the operation. In some cases, however, it is rather difficult to distinguish this damage clinically from NANB viral hepatitis, particularly of the self-limited, resolving type described above. Therefore we assayed sera from such patients with single-peak enzyme elevation without a blood transfusion. None of these eight patients were positive for anti-C100-3 or anti-p22. DISCUSSION
An assay to detect anti-C100-3 has been used worldwide for screening blood for transfusions. It is also useful for the diagnosis of chronic hepatitis C. Application of the anti-C100-3 assay system, however, raised three important problems for a precise diagnosis of hepatitis C, which are as follows: (a) It takes at least several weeks to months for seroconversion to be detectable; (b) Cases of NANB hepatitis exist that are
negative for anti-(3100-3 even in long follow-up studies; and (c) The possibility of a false-positive reaction, particularly in cases of autoimmune hepatitis, has been pointed out (16). In patients having only one episode of liver cell enzyme elevation, the frequency of anti-C100-3 detection was much lower than in those having multiple peaks of enzyme elevation (4). It is not yet clear whether this is because of some other viral agent, a difference in host response or insufficiency of the test system. We induced expression of properly processed HCV core protein in insect and animal cells (13, 14) and established an antibody detection system using the synthesized protein (14). This study revealed the involvement of HCV infection in cases of acute, resolving NANB hepatitis in which viral involvement had not been shown by the currently available anti-C100-3 assay. Of course, we cannot rule out the possibility of the existence of other virus(es)that could induce this elusive hepatitis. As in patients K and L, undetermined posttransfusion NANB hepatitis still exists. However, it is interesting to note that in these two cases, in one possible HCV carrier (patient J) and in one cryptic case (patient F) the incubation periods were shorter than in other typical hepatitis C cases (Table 1).Such patients with a very short incubation period could have nonviral acute hepatitis. This study also shows that an assay for anti-p22 is useful for the early diagnosis of HCV infection (13, 14). Inclusion of this assay in blood screening should also be considered, Retrospective analysis of donor blood that had been actually transfused and of the consequences for the recipients is now in progress. Why could anti-p22 be detected earlier and with higher sensitivity than anti-C100-3 in some cases of NANB hepatitis, particularly the acute, self-limited type described here? One possible explanation is that a structural protein is somehow more immunogenic and that therefore anti-p22 would appear much earlier and more persistently than antibodies to the nonstructural
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components of HCV. Alternately, the higher detection rate may be caused by the conservation of sequence variations among different HCV isolates in the core domain of the HCV genome (16-20). Recently, antibody detection assays for the HCV core protein have been developed that use synthetic oligopeptides (21,221 and a protein expressed in Escherichia coli (E. coli) (23) or in yeast (24).We compared the ELISA derived from the p22 core protein described in this paper and other ELISA assays derived from the core protein expressed in E. coli or various synthetic oligopeptides deduced from the putative core gene using sera of patients with NANB hepatitis. The results showed that the anti-p22 ELISA assay was more sensitive than the ELISAs using synthetic oligopeptides derived from four different core regions (Chiba et al., Unpublished observations, 1991). p22 was expressed in insect cells, to which human beings generally do not have antibodies. Because of its relatively low background in immunoblotting (data not shown), the anti-p22 ELISA assay is expected to be more sensitive than the other two ELISAs based on proteins expressed in E. coli and in yeasts. New assays for the anti-HCV core protein antibody by themselves or in combination with the present anti-C100-3 assay will be useful for understanding the viral pathogenesis of HCV infection and for efficient diagnosis and prevention.
Acknowledgments: We are indebted to Dr. G. Kuo for anti-C100-3 RIA and advice; to Dr. H. Shimojo for a critical review of the manuscript; to Dr. W. J.Rutter, Dr. H. Thomas, Dr. T. Oda and Dr. K. Nishioka for discussions; to Ms. Y. Kaminuma, Ms. F. Sakurai and Ms. Y. Hirama for their laboratory assistance; and to Ms. T. Mizoguchi for the preparation of the manuscript. REFERENCES 1. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton
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