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Immunoglobulin a antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus
GASTROENTEROLOGY
1985;89:1109-13
Immunoglobulin A Antibody Against Hepatitis B Core Antigen in the Acute and Persistent Infection With Hepatitis B Virus MOTOZUMI NOMURA, MITSUNOBU IMAI, FUMIO TSUDA, SEIICHI FURUTA, YOSHIHIRO AKAHANE, KATSUMI TACHIBANA, SADAKAZU USUDA, YUZO MIYAKAWA, and MAKOTO MAYUMI Immunology Division, Jichi Medical School; Hepatitis Division, Tokyo Metropolitan Institute of Medical Science; Second Department of Internal Medicine. Shinshu University: First Department of Internal Medicine, Yamanashi Medical College; Japanese Red Cross Blood Center at Saitama-Ken; and Institute of Immunology, Japan
Antibody to hepatitis B core antigen ofimmunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio >2.1, was detected in all of39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected carriers of the in only 2 (4%) of 46 asymptomatic virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean -+ SE titer of antibody in chronic persistent hepatitis (3.8 2 0.9) was significantly lower than those in chronic active hepatitis 113.8 * 3.2)and cirrhosis with or without carcinoma (25.6t 6.1)(p < 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection. The discovery of antibodies to hepatitis B core antigen (anti-HBc) by Almeida et al. 11) and their clinical (2) have
applications helped
introduced
by Hoofnagle
in the understanding
et al.
of hepatitis
B
Received February 8, 1985. Accepted May 10. 1985. Address requests for reprints to: Makoto Mayumi, M.D., Immunology Division. Jichi Medical School, Minamikawachi-Machi, Tochigi-Ken 329-04, Japan. Dr. Nomura’s present address is Second Department of Internal Medicine. Fukui Medical College, Fukui-Ken 910-11, Japan. 0 1985 by the American Gastroenterological Association 0016.5085/85/$3.30
virus (HBV) infection. Being a product of the earliest humoral immune response of hosts to HBV (3), anti-HBc provides a useful means for diagnosing acute HBV infection, especially when hepatitis B surface antigen (HBsAg) is no longer detectable in the serum. The diagnostic value of anti-HBc may be extended further by specifying its antibody classes in terms of “early” immunoglobulin M (IgM) and “late” immunoglobulin G (IgG) (4-6). Little is known, however, concerning the anti-HBc of the other major class, immunoglobulin A (IgA]. This may come as a surprise, indeed, in view of a well-recognized elevation in circulating levels of IgA in a variety of hepatic disorders, including type B viral hepatitis (7-g). We developed a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies for the determination of IgA anti-HBc in the serum, and sought the antibody in serum samples from patients who were infected with HBV acutely or persistently. Materials
and Methods
Serum Samples Serum samples were obtained from 39 consecutive patients with acute type B hepatitis. The diagnosis of acute type B hepatitis was contingent upon the following: (a) abnormally high serum glutamic pyruvic transaminase values (SGPT, >40 Karmen units/ml) in at least two determinations and (b) a high level of IgM anti-HBc, but not of IgM antibody against hepatitis A virus. Serial serum Abbreviations used in this paper: anti-&A/a, antibodies against immunoglobulin A: CAH, chronic active hepatitis; CPH, chronic persistent hepatitis: LCMCC, liver cirrhosis with or without hepatocellular carcinoma; PBS, phosphate-buffered saline.
lllu
NOMURA ET AL
CASTROE:h’TEROLO(;Y 1’01. 89. So. 5
samples were collected from 1 patient starting from the early phase of acute type B hepatitis. Serum samples were also obtained from a total of 177 individuals, identified consecutively, who were persistently infected with HBV with or without signs and symptoms of hepatic disorders. These individuals consisted of 46 asymptomatic carriers without clinical or biochemical evidence of hepatic disease, 37 patients with chronic persistent hepatitis (CPH), 48 patients with chronic active hepatitis (CAH), and 46 patients with liver cirrhosis with or without hepatocellular carcinoma (LC/HCC). Categories of chronic liver disease were determined by liver biopsy. Hepatitis B e antigen (HBeAg) was detected in the serum by radioimmunoassay (10) in 8 (17.4%) asymptomatic carriers, 9 (24.30/o) patients with CPH, 32 (66.7%) patients with CAH. and 16 (34.8%) patients with LCIHCC; the others were all positive for antibody to HBeAg (anti-HBe). Serologic
Testing
Hepatitis B surface antigen was determined by hemagglutination method (ll),and the result was pressed by the highest twofold dilution (2”) of the serum that induced hemagglutination. Immunoglobulin anti-HBc was determined by solid-phase radioimmunoassay in the serum diluted 200-fold after the method scribed previously (12), and the result was expressed the sample to normal ratio.
the extest M deas
unoccupied protein-binding sites. The plate was kept at room temperature for 4 h. washed, and then stored at 4°C. Serum to be tested was diluted 1000-fold with calf serum. The dilution was necessary because IgG or IgM anti-HBc. or both, which is contained in high levels in some serums, could induce false-positive results in the test of the diluted serum for IgA anti-HBc. A loo-p1 portion was delivered to a well coated with anti-IgAicu. The plate was incubated at 37°C for 3 h, and washed with saline five times. Then each well received 50 ~1 of PBS supplemented with 1% (wt/vol) bovine serum albumin and containing HBcAg (equivalent to an ODzso of 0.004) purified by the method described elsewhere (16).The plate was incubated at 37°C for 3 h, and washed with saline. Thereafter, each well received 100 ~1 of PBS supplemented with 25% (vol/vol) calf serum and containing 2 ng of monoclonal anti-HBc (15) that had been labeled with IZ51 at a specific activity of 5 &iipg by the chloramine-T method (17). The plate was incubated at 37°C for 3 h, and washed with saline. Wells were cut out, and the radioactivity of each individual well was counted. Serum samples from 30 normal controls without markers of HBV infection were tested for IgA anti-HBc by the solid-phase radioimmunoassay with monoclonal antibodies, and their mean counts per minute was calculated. The result of test serum was expressed by the sample to normal ratio, and values >2.1 were interpreted as being positive for IgA anti-HBc. Statistical
MonocJonaJ
Monoclonal antibodies against IgA (anti-IgAicu) were obtained by a modification by Oi and Herzenberg (13) of the method originally described by KGhler and Milstein (14). Briefly, BALBic mice were immunized with IgA purified from the serum of a patient with IgA myeloma. Immune spleen cells were hybridized with mouse myeloma (NS-1) cells. Clones secreting anti-IgAicu were grown in the peritoneal cavity of mice that had been made ascitic by means of 2,6,10,14-tetramethylpentadecane. Ascites fluid was harvested after 14 days of inoculation, and immunoglobulin fractions were isolated by precipitation with 1.33 M (NH4)$0, followed by gel filtration in Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, N.J.). Monoclonal anti-IgAla was equally reactive with both of the known subclasses of IgA, i.e., IgAl and IgAB, but not to ariy appreciable extent with IgG, IgM, IgE, or IgD. Monoclonal antibodies against hepatitis B core antigen (HBcAg) were obtained as described previously (15). Determination Hepatitis A Class
Analyses
Antibodies
of Antibodies J3 Core
Antigen
Against of lmmunoglobulin
Wells of a polyvinyl U-bottom microtiter plate (Dynatech Laboratories, Alexandria, Va.) received 100 ~1 of phosphate-buffered saline (PBS: phosphate 0.015 M, NaCl 0.15 M, pH 7.2) containing anti-IgAicu (ODzao 0.2), and were incubated at 4°C overnight. The plate was washed with saline and then exposed to PBS containing 2% (wt/vol) bovine serum albumin in order to saturate
Differences in the mean value of each group were evaluated by the Wilcoxon rank-sum test. The difference in the proportion of patients with abnormal values between two groups was evaluated by the J method.
Results Jmmunoglobulin
A Antibody
Hepatitis
Antigen
B Core
Against
in Patients
With
Acute Type B Hepatitis Figure 1 illustrates the courses of IgA and IgM anti-HBc, as well as that of HBsAg, and the duration of elevated SGPT levels in a patient followed prospectively since the early stage of acute type B hepatitis. It can be seen that IgM anti-HBc appeared early, before the elevation of SGPT, and stayed at high levels for more than 2 mo after the SGPT level had returned to normal. The pattern of IgA anti-HBc was different in that it appeared later than IgM anti-HBc, peaking at or slightly after the normalization of SGPT, and then dropped more rapidly. Figure 2 illustrates the titers of IgA and IgM anti-HBc for serum samples from 39 patients with acute type B hepatitis. They were all positive for IgM anti-HBc with high activities [sample to normal ratio 24.0 ? 0.8 (mean 2 SE), range 12.7-39.71. Also, they were all positive for IgA anti-HBc [39.2 5 5.7 (mean 2 SE), range
4.0-118.01.
Unlike
IgM anti-HBc,
how-
November
Figure
IgA ANTI-HBc
1985
IN HBV INFECTION
1111
of hepatitis B virus infection in a patient with acute type B hepatitis: 0-O represents immunoglobulin A (IgA) antibody to hepatitis B core antigen (anti-HBc), 0- -0 represents IgM antiHBc. and n---n represents hepatitis B surface antigen (HBsAg). The duration of elevated serum glutamic pyruvic transaminase (SGPT) levels is indicated by the shaded bar.
1. Markers
AUG.
ever, IgA anti-HBc was distributed widely so that there was no correlation between the level of IgA anti-HBc and that of IgM anti-HBc (r = 0.194). Immunoglobulin A Antibody Against Hepatitis I3 Core Antigen in Persons With Persistent Hepatitis B Virus Infection
SEP.
OCT.
NOV.
”
with CPH was between these two extremes (41%). The prevalences of IgA anti-HBc among these three carrier state [low], CPH groups, i.e., asymptomatic (intermediate), and CAH as well as LC/HCC (high), were significantly different from each other (p < 0.001).
Table 1 lists the prevalence of IgA anti-HBc in persons with persistent HBV infection of various clinical categories. Only 2 (4%) of 46 asymptomatic carriers possessed IgA anti-HBc. The low prevalence of IgA anti-HBc in asymptomatic carriers stood in marked contrast with the high prevalences in patients with CAH (94%) and those with LC/HCC (87%). The prevalence of IgA anti-HBc in patients
Figure 3 compares the mean -+ SE titer of IgA anti-HBc and that of IgM anti-HBc in persons with persistent HBV infection of various categories. The level of IgA anti-HBc sharply increased along the spectrum of persistent HBV infection, ranging from asymptomatic carriers to patients with LUHCC. Immunoglobulin M anti-HBc exhibited a similar tendency, but in a much less prominent manner than IgA anti-HBc. The level of IgA anti-HBc was significantly different (p < 0.001) between asymptomatic
loo-
Table % 3
6;
50r
0
I
I
i::i
,
,
,
,
!,
,
,I
1. Prevalence of Immunoglobulin A Antibody Against Hepatitis B Core Antigen in Persons With Persistent Hepatitis B Virus Infection
Categories of HBV infection
n
Asymptomatic
46
carrier CPH
IgA anti-HBc”
P
1
2 (4%)
state 37
15 (41%)1
CAH
48
45 (94%)1
Lcn-Icc
46
40 (87%)-
A Y
HBV, hepatitis B virus: IgA, immunoglobulin A; anti-HBc, antibody to hepatitis B core antigen; CPH, chronic persistent hepati1
2 IgA
Figure
ANTI-H&,
5
10
20
50
RAOIOIMMUNOASSAY
2. Antibodies to hepatitis B core antigen immunoglobulin A (IgA) and IgM classes with acute type B hepatitis.
100 N/S
200 RATIO
(anti-HBc) of in 39 patients
tis: CAH, chronic active hepatitis; LC/HCC, liver cirrhosis with or without hepatocellular carcinoma; NS, not significant. ’ Immunoglobulin A anti-HBc was determined by the sandwich-type solid-phase radioimmunoassay with monoclonal antibodies, and serum samples that showed a sample to normal ratio of >2.1 were considered
to be positive
for the antibody.
1112
NOMURA
ET AL.
CASTROENTEROLOGY
T T
CPH Figure
CAH
LC/HCC
3. Antibodies to hepatitis B core antigen (anti-HBc) in asymptomatic carriers (AC). patients with chronic persistent hepatitis (CPH), patients with chronic active hepatitis (CAH), and patients with liver cirrhosis with or without hepatocellular carcinoma (LCIHCC). The mean values k SE are given for anti-HBc of immunoglobulin A (IgA) class (closed bars) and for anti-HBc of IgM class (open bars).
carriers and patients with CPH, between patients with CPH and those with CAH, and between patients with CAH and those with LCIHCC. Also, the level of IgM anti-HBc was significantly different (p < 0.01) between each category, except between patients with CAH and those with LC/HCC. The mean value of IgA anti-HBc was significantly higher in HBeAg-positive serum samples than in anti-HBe-positive serum samples from patients with CPH (8.9 ? 3.2 vs. 2.2 k 0.4,p < 0.05).A similar tendency was observed for patients with CAH (16.5 ? 4.4 vs. 9.8 k 3.8),but the difference fell short of being significant. There were no appreciable differences in the level of IgA anti-HBc between HBeAgpositive and anti-HBe-positive asymptomatic carriers and patients with LC/HCC.
Discussion By means of a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies, anti-HBc of IgA class was evaluated in the serum of persons infected with HBV. Immunoglobulin A antiHBc was rarely detected in asymptomatic carriers (4%). In contrast, patients with CAH and those with LC/HCC displayed IgA anti-HBc much more frequently (94% and 87%, respectively]. Patients with CPH displayed IgA anti-HBc at a frequency between those of asymptomatic carriers and patients with advanced forms of liver disease. These observations
Vol. 89. No. !i
suggested that IgA anti-HBc in persons with persistent HBV infection of various clinical categories would reflect the hepatic dysfunction, but not the infection with HBV per se. The level of IgM anti-HBc has been reported to reflect hepatic injury in chronic We observed that the level of HBV infection (12.18). IgA anti-HBc increased in parallel with IgM antiHBc, in a much more exaggerated fashion, along different categories of persistent HBV infection covering the asymptomatic carrier state, CPH, CAH, and LCIHCC. The correlation between elevations in serum levels of IgA anti-HBc and hepatic injury in persistent HBV infection was the most salient finding in the present study. All patients with acute type B hepatitis displayed IgM anti-HBc in high activities in accordance with previous reports (4-6).All these patients were positive also for IgA anti-HBc, with widely distributed levels. This lack of parallelism between IgM and IgA anti-HBc levels in acute HBV infection might be attributable to the delay of IgA anti-HBc response and its rapid waning, as compared with IgM antiHBc, that were observed in a prospectively followed patient (Figure 1). As serum samplings were not synchronized with a stage of acute hepatitis for the 39 patients we studied, the level of IgA anti-HBc might or might not have been high in the presence of invariably high levels of IgM anti-HBc. Immunoglobulin A increases in the circulation in a variety of hepatic disorders (7-9). At least two different mechanisms have been proposed to explain the elevated serum levels of IgA in hepatic diseases, i.e., [a) increased production and (b) decreased clearance. The liver has excretory functions, with an outlet into the alimentary tract via the biliary system. Any antigens that are confined to the circulation or to the liver, if transferred into the biliary tract, may stimulate the local production of IgA by the gut immune system; the integrity of normal hepatic function might be required for the segregation of some immunogens from the intestine. The failure in secluding them on the part of the liver, either temporarily or persistently, would furnish the intestinal immune system with immunogens not normally accessible to the alimentary tract. Turning to its clearance, IgA in the serum, especially in polymeric forms, is excreted by the hepatobiliary system (19,20). Extensive liver damage and cholestasis, therefore, can contribute toward the elevation of serum IgA levels. The liver is the major target organ of HBV; the polyalbumin receptor of HBV most likely accounts for its hepatotropism (21~2). There has been no evidence for the replication of HBV in the alimentary tract so far. To initiate HBV infection, therefore, HBV has to be introduced parenterally. usually into the
November
1985
bloodstream. As a corollary to this, HBcAg, representing the n~ucleocapsid of HBV, would not activate the gut immune system so long as it is confined to the liver and circulation. The induction of an IgA anti-HBc response could be initiated only after the antigen was supplied to the mucosal surface of the intestine, presumably as a consequence of hepatic dysfunction. Indeed, the extremely low prevalence of IgA anti-HBc (4%) in asymptomatic carriers might represent the failure of inducing an IgA anti-HBc response. The validity of this assumption may be tested by prospectively following the asymptomatic carriers who contract HBV infection through the mother-to-baby transmission, to see if IgA anti-HBc develops in the absence of any hepatic damage. Once IgA anti-HBc develops in the circulation, the decreased clearance, if any, would help elevate its serum levels. This may explain why the level of IgA anti-HBc in patients with LC/HCC was significantly higher than that in patients with CAH (Figure 3). In contrast, the level of IgM anti-HBc was comparable between patients with LCEICC and those with CAH; the serum level of IgM was not as dramatically increased as that of IgA in patients with cirrhosis (8,9), presumably because of the lack of its excretion by the hepatobiliary system. Measurement of IgA anti-HBc by radioimmunoassay may have applications to clinical medicine, inasmuch as high levels of this antibody may reflect hepatic injury. Subpopulations of IgA anti-HBc in terms of monomeric or polymeric forms, as well as the association or lack of association with secretory component or J chain, or both, deserve analysis in order to establish the intestinal origin of IgA antiHBc in acute and persistent HBV infection.
References 1. Almeida
JD. Rubinstein D. Stott EJ. New antigen-antibody system in Australia-antigen-positive hepatitis. Lancet 1971; ii:1225-7. 2. Hoofnagle JH, Gerety RJ, Barker LF. Antibody to hepatitis-Bvirus core in man. Lancet 1973;ii:869-73. 3. Aikawa T, Sairenji H, Furuta S, et al. Seroconversion from hepatitis B e antigen to anti-HBe in acute hepatitis B virus infection. N Engl J Med 1978;298:439-41. 4. Gerlich WH. Liier W, Thomssen R, et al. Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J Infect Dis 1980;142:95-101.
IgA ANTI-HBc
IN HBV INFECTION
1113
5. Lemon SM. Gates NL. Simms TE, Bancroft WH. IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 1981:143:803-9. 6. Shimizu M. Ohyama M, Takahashi Y. et al. Immunoglobulin M antibody against hepatitis B core antigen for the diagnosis of fulminant type B hepatitis. Gastroenterology 1983;84:60410. 7. Tomasi TB. Tisdale WA. Serum gamma-globulins in acute and chronic liver diseases. Nature 1964:201:834-5. 8. Lee FI, Lond MB. Immunoglobulins in viral hepatitis and active alcoholic liver-disease. Lancet 1965:ii:1043-6. 9. Thompson RA, Carter R, Stokes RP, Geddes AM, Goodall JAD. Serum immunoglobulins. complement levels and autoantibodies in liver disease. Clin Exp Immunol 1973;14:335-46. 10. Arakawa K. Tsuda F, Takahashi K, et al. Maternofetal transmission of IgG-bound hepatitis B e antigen. Pediatr Res 1981;16:247-50. 11. Vyas GN. Shulman NR. Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 1970; 170:332-3. 12. Tsuda F. Naito S, Takai E, et al. Low molecular weight (7s) immunoglobulin M antibody against hepatitis B core antigen in the serum for differentiating acute from persistent hepatitis B virus infection. Gastroenterology 1984;87:159-64. 13. Oi VT, Herzenberg LA. Immunoglobulin producing hybrid cell lines. In: Mishell BB, Shiigi SM, eds. Selected methods in cellular immunology. San Francisco: WH Freeman, 1980: 351-72. 14. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256: 495-7. 15. Takahashi K. Machida A. Funatsu G, et al. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol 1983;130:2903-7. 16. Takahashi T. Nakagawa S. Hashimoto T, et al. Large-scale isolation of Dane particles from plasma containing hepatitis B antigen and demonstration of a circular double-stranded DNA molecule extruding directly from their cores. J Immunol 1976:117:1392-7. 17. Greenwood FC, Hunter WM. Glover JS. The preparation of ‘“‘I-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89:114-23. 18. Popper H. Summary of workshop on pathology. In: Vyas GN, Dienstag JL. Hoofnagle JH, eds. Viral hepatitis and liver disease. Orlando: Grune & Stratton, 1984:515-g. 19. Nagura H, Smith PD, Nakane PK, Brown WR. IgA in human bile and liver. J Immunol 1981;126:587-95. 20. Kutteh WH. Prince SJ, Phillips JO, Spenney JG, Mestecky J. Properties of immunoglobulin A in serum of individuals with liver diseases and in hepatic bile. Gastroenterology 1982;82: 184-93. 21. Imai M, Yanase Y. Nojiri T. Miyakawa Y. Mayumi M. A receptor for polymerized human and chimpanzee albumins on hepatitis B virus particles co-occurring with HBeAg. Gastroenterology 1979:76:242-7. 22. Thung SN, Gerber MA. Polyalbumin receptors: their role in the attachment of hepatitis B virus to hepatocytes. Sem Liver Dis 1984:4:69-75.
1985;89:1109-13
Immunoglobulin A Antibody Against Hepatitis B Core Antigen in the Acute and Persistent Infection With Hepatitis B Virus MOTOZUMI NOMURA, MITSUNOBU IMAI, FUMIO TSUDA, SEIICHI FURUTA, YOSHIHIRO AKAHANE, KATSUMI TACHIBANA, SADAKAZU USUDA, YUZO MIYAKAWA, and MAKOTO MAYUMI Immunology Division, Jichi Medical School; Hepatitis Division, Tokyo Metropolitan Institute of Medical Science; Second Department of Internal Medicine. Shinshu University: First Department of Internal Medicine, Yamanashi Medical College; Japanese Red Cross Blood Center at Saitama-Ken; and Institute of Immunology, Japan
Antibody to hepatitis B core antigen ofimmunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio >2.1, was detected in all of39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected carriers of the in only 2 (4%) of 46 asymptomatic virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean -+ SE titer of antibody in chronic persistent hepatitis (3.8 2 0.9) was significantly lower than those in chronic active hepatitis 113.8 * 3.2)and cirrhosis with or without carcinoma (25.6t 6.1)(p < 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection. The discovery of antibodies to hepatitis B core antigen (anti-HBc) by Almeida et al. 11) and their clinical (2) have
applications helped
introduced
by Hoofnagle
in the understanding
et al.
of hepatitis
B
Received February 8, 1985. Accepted May 10. 1985. Address requests for reprints to: Makoto Mayumi, M.D., Immunology Division. Jichi Medical School, Minamikawachi-Machi, Tochigi-Ken 329-04, Japan. Dr. Nomura’s present address is Second Department of Internal Medicine. Fukui Medical College, Fukui-Ken 910-11, Japan. 0 1985 by the American Gastroenterological Association 0016.5085/85/$3.30
virus (HBV) infection. Being a product of the earliest humoral immune response of hosts to HBV (3), anti-HBc provides a useful means for diagnosing acute HBV infection, especially when hepatitis B surface antigen (HBsAg) is no longer detectable in the serum. The diagnostic value of anti-HBc may be extended further by specifying its antibody classes in terms of “early” immunoglobulin M (IgM) and “late” immunoglobulin G (IgG) (4-6). Little is known, however, concerning the anti-HBc of the other major class, immunoglobulin A (IgA]. This may come as a surprise, indeed, in view of a well-recognized elevation in circulating levels of IgA in a variety of hepatic disorders, including type B viral hepatitis (7-g). We developed a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies for the determination of IgA anti-HBc in the serum, and sought the antibody in serum samples from patients who were infected with HBV acutely or persistently. Materials
and Methods
Serum Samples Serum samples were obtained from 39 consecutive patients with acute type B hepatitis. The diagnosis of acute type B hepatitis was contingent upon the following: (a) abnormally high serum glutamic pyruvic transaminase values (SGPT, >40 Karmen units/ml) in at least two determinations and (b) a high level of IgM anti-HBc, but not of IgM antibody against hepatitis A virus. Serial serum Abbreviations used in this paper: anti-&A/a, antibodies against immunoglobulin A: CAH, chronic active hepatitis; CPH, chronic persistent hepatitis: LCMCC, liver cirrhosis with or without hepatocellular carcinoma; PBS, phosphate-buffered saline.
lllu
NOMURA ET AL
CASTROE:h’TEROLO(;Y 1’01. 89. So. 5
samples were collected from 1 patient starting from the early phase of acute type B hepatitis. Serum samples were also obtained from a total of 177 individuals, identified consecutively, who were persistently infected with HBV with or without signs and symptoms of hepatic disorders. These individuals consisted of 46 asymptomatic carriers without clinical or biochemical evidence of hepatic disease, 37 patients with chronic persistent hepatitis (CPH), 48 patients with chronic active hepatitis (CAH), and 46 patients with liver cirrhosis with or without hepatocellular carcinoma (LC/HCC). Categories of chronic liver disease were determined by liver biopsy. Hepatitis B e antigen (HBeAg) was detected in the serum by radioimmunoassay (10) in 8 (17.4%) asymptomatic carriers, 9 (24.30/o) patients with CPH, 32 (66.7%) patients with CAH. and 16 (34.8%) patients with LCIHCC; the others were all positive for antibody to HBeAg (anti-HBe). Serologic
Testing
Hepatitis B surface antigen was determined by hemagglutination method (ll),and the result was pressed by the highest twofold dilution (2”) of the serum that induced hemagglutination. Immunoglobulin anti-HBc was determined by solid-phase radioimmunoassay in the serum diluted 200-fold after the method scribed previously (12), and the result was expressed the sample to normal ratio.
the extest M deas
unoccupied protein-binding sites. The plate was kept at room temperature for 4 h. washed, and then stored at 4°C. Serum to be tested was diluted 1000-fold with calf serum. The dilution was necessary because IgG or IgM anti-HBc. or both, which is contained in high levels in some serums, could induce false-positive results in the test of the diluted serum for IgA anti-HBc. A loo-p1 portion was delivered to a well coated with anti-IgAicu. The plate was incubated at 37°C for 3 h, and washed with saline five times. Then each well received 50 ~1 of PBS supplemented with 1% (wt/vol) bovine serum albumin and containing HBcAg (equivalent to an ODzso of 0.004) purified by the method described elsewhere (16).The plate was incubated at 37°C for 3 h, and washed with saline. Thereafter, each well received 100 ~1 of PBS supplemented with 25% (vol/vol) calf serum and containing 2 ng of monoclonal anti-HBc (15) that had been labeled with IZ51 at a specific activity of 5 &iipg by the chloramine-T method (17). The plate was incubated at 37°C for 3 h, and washed with saline. Wells were cut out, and the radioactivity of each individual well was counted. Serum samples from 30 normal controls without markers of HBV infection were tested for IgA anti-HBc by the solid-phase radioimmunoassay with monoclonal antibodies, and their mean counts per minute was calculated. The result of test serum was expressed by the sample to normal ratio, and values >2.1 were interpreted as being positive for IgA anti-HBc. Statistical
MonocJonaJ
Monoclonal antibodies against IgA (anti-IgAicu) were obtained by a modification by Oi and Herzenberg (13) of the method originally described by KGhler and Milstein (14). Briefly, BALBic mice were immunized with IgA purified from the serum of a patient with IgA myeloma. Immune spleen cells were hybridized with mouse myeloma (NS-1) cells. Clones secreting anti-IgAicu were grown in the peritoneal cavity of mice that had been made ascitic by means of 2,6,10,14-tetramethylpentadecane. Ascites fluid was harvested after 14 days of inoculation, and immunoglobulin fractions were isolated by precipitation with 1.33 M (NH4)$0, followed by gel filtration in Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, N.J.). Monoclonal anti-IgAla was equally reactive with both of the known subclasses of IgA, i.e., IgAl and IgAB, but not to ariy appreciable extent with IgG, IgM, IgE, or IgD. Monoclonal antibodies against hepatitis B core antigen (HBcAg) were obtained as described previously (15). Determination Hepatitis A Class
Analyses
Antibodies
of Antibodies J3 Core
Antigen
Against of lmmunoglobulin
Wells of a polyvinyl U-bottom microtiter plate (Dynatech Laboratories, Alexandria, Va.) received 100 ~1 of phosphate-buffered saline (PBS: phosphate 0.015 M, NaCl 0.15 M, pH 7.2) containing anti-IgAicu (ODzao 0.2), and were incubated at 4°C overnight. The plate was washed with saline and then exposed to PBS containing 2% (wt/vol) bovine serum albumin in order to saturate
Differences in the mean value of each group were evaluated by the Wilcoxon rank-sum test. The difference in the proportion of patients with abnormal values between two groups was evaluated by the J method.
Results Jmmunoglobulin
A Antibody
Hepatitis
Antigen
B Core
Against
in Patients
With
Acute Type B Hepatitis Figure 1 illustrates the courses of IgA and IgM anti-HBc, as well as that of HBsAg, and the duration of elevated SGPT levels in a patient followed prospectively since the early stage of acute type B hepatitis. It can be seen that IgM anti-HBc appeared early, before the elevation of SGPT, and stayed at high levels for more than 2 mo after the SGPT level had returned to normal. The pattern of IgA anti-HBc was different in that it appeared later than IgM anti-HBc, peaking at or slightly after the normalization of SGPT, and then dropped more rapidly. Figure 2 illustrates the titers of IgA and IgM anti-HBc for serum samples from 39 patients with acute type B hepatitis. They were all positive for IgM anti-HBc with high activities [sample to normal ratio 24.0 ? 0.8 (mean 2 SE), range 12.7-39.71. Also, they were all positive for IgA anti-HBc [39.2 5 5.7 (mean 2 SE), range
4.0-118.01.
Unlike
IgM anti-HBc,
how-
November
Figure
IgA ANTI-HBc
1985
IN HBV INFECTION
1111
of hepatitis B virus infection in a patient with acute type B hepatitis: 0-O represents immunoglobulin A (IgA) antibody to hepatitis B core antigen (anti-HBc), 0- -0 represents IgM antiHBc. and n---n represents hepatitis B surface antigen (HBsAg). The duration of elevated serum glutamic pyruvic transaminase (SGPT) levels is indicated by the shaded bar.
1. Markers
AUG.
ever, IgA anti-HBc was distributed widely so that there was no correlation between the level of IgA anti-HBc and that of IgM anti-HBc (r = 0.194). Immunoglobulin A Antibody Against Hepatitis I3 Core Antigen in Persons With Persistent Hepatitis B Virus Infection
SEP.
OCT.
NOV.
”
with CPH was between these two extremes (41%). The prevalences of IgA anti-HBc among these three carrier state [low], CPH groups, i.e., asymptomatic (intermediate), and CAH as well as LC/HCC (high), were significantly different from each other (p < 0.001).
Table 1 lists the prevalence of IgA anti-HBc in persons with persistent HBV infection of various clinical categories. Only 2 (4%) of 46 asymptomatic carriers possessed IgA anti-HBc. The low prevalence of IgA anti-HBc in asymptomatic carriers stood in marked contrast with the high prevalences in patients with CAH (94%) and those with LC/HCC (87%). The prevalence of IgA anti-HBc in patients
Figure 3 compares the mean -+ SE titer of IgA anti-HBc and that of IgM anti-HBc in persons with persistent HBV infection of various categories. The level of IgA anti-HBc sharply increased along the spectrum of persistent HBV infection, ranging from asymptomatic carriers to patients with LUHCC. Immunoglobulin M anti-HBc exhibited a similar tendency, but in a much less prominent manner than IgA anti-HBc. The level of IgA anti-HBc was significantly different (p < 0.001) between asymptomatic
loo-
Table % 3
6;
50r
0
I
I
i::i
,
,
,
,
!,
,
,I
1. Prevalence of Immunoglobulin A Antibody Against Hepatitis B Core Antigen in Persons With Persistent Hepatitis B Virus Infection
Categories of HBV infection
n
Asymptomatic
46
carrier CPH
IgA anti-HBc”
P
1
2 (4%)
state 37
15 (41%)1
CAH
48
45 (94%)1
Lcn-Icc
46
40 (87%)-
A Y
HBV, hepatitis B virus: IgA, immunoglobulin A; anti-HBc, antibody to hepatitis B core antigen; CPH, chronic persistent hepati1
2 IgA
Figure
ANTI-H&,
5
10
20
50
RAOIOIMMUNOASSAY
2. Antibodies to hepatitis B core antigen immunoglobulin A (IgA) and IgM classes with acute type B hepatitis.
100 N/S
200 RATIO
(anti-HBc) of in 39 patients
tis: CAH, chronic active hepatitis; LC/HCC, liver cirrhosis with or without hepatocellular carcinoma; NS, not significant. ’ Immunoglobulin A anti-HBc was determined by the sandwich-type solid-phase radioimmunoassay with monoclonal antibodies, and serum samples that showed a sample to normal ratio of >2.1 were considered
to be positive
for the antibody.
1112
NOMURA
ET AL.
CASTROENTEROLOGY
T T
CPH Figure
CAH
LC/HCC
3. Antibodies to hepatitis B core antigen (anti-HBc) in asymptomatic carriers (AC). patients with chronic persistent hepatitis (CPH), patients with chronic active hepatitis (CAH), and patients with liver cirrhosis with or without hepatocellular carcinoma (LCIHCC). The mean values k SE are given for anti-HBc of immunoglobulin A (IgA) class (closed bars) and for anti-HBc of IgM class (open bars).
carriers and patients with CPH, between patients with CPH and those with CAH, and between patients with CAH and those with LCIHCC. Also, the level of IgM anti-HBc was significantly different (p < 0.01) between each category, except between patients with CAH and those with LC/HCC. The mean value of IgA anti-HBc was significantly higher in HBeAg-positive serum samples than in anti-HBe-positive serum samples from patients with CPH (8.9 ? 3.2 vs. 2.2 k 0.4,p < 0.05).A similar tendency was observed for patients with CAH (16.5 ? 4.4 vs. 9.8 k 3.8),but the difference fell short of being significant. There were no appreciable differences in the level of IgA anti-HBc between HBeAgpositive and anti-HBe-positive asymptomatic carriers and patients with LC/HCC.
Discussion By means of a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies, anti-HBc of IgA class was evaluated in the serum of persons infected with HBV. Immunoglobulin A antiHBc was rarely detected in asymptomatic carriers (4%). In contrast, patients with CAH and those with LC/HCC displayed IgA anti-HBc much more frequently (94% and 87%, respectively]. Patients with CPH displayed IgA anti-HBc at a frequency between those of asymptomatic carriers and patients with advanced forms of liver disease. These observations
Vol. 89. No. !i
suggested that IgA anti-HBc in persons with persistent HBV infection of various clinical categories would reflect the hepatic dysfunction, but not the infection with HBV per se. The level of IgM anti-HBc has been reported to reflect hepatic injury in chronic We observed that the level of HBV infection (12.18). IgA anti-HBc increased in parallel with IgM antiHBc, in a much more exaggerated fashion, along different categories of persistent HBV infection covering the asymptomatic carrier state, CPH, CAH, and LCIHCC. The correlation between elevations in serum levels of IgA anti-HBc and hepatic injury in persistent HBV infection was the most salient finding in the present study. All patients with acute type B hepatitis displayed IgM anti-HBc in high activities in accordance with previous reports (4-6).All these patients were positive also for IgA anti-HBc, with widely distributed levels. This lack of parallelism between IgM and IgA anti-HBc levels in acute HBV infection might be attributable to the delay of IgA anti-HBc response and its rapid waning, as compared with IgM antiHBc, that were observed in a prospectively followed patient (Figure 1). As serum samplings were not synchronized with a stage of acute hepatitis for the 39 patients we studied, the level of IgA anti-HBc might or might not have been high in the presence of invariably high levels of IgM anti-HBc. Immunoglobulin A increases in the circulation in a variety of hepatic disorders (7-9). At least two different mechanisms have been proposed to explain the elevated serum levels of IgA in hepatic diseases, i.e., [a) increased production and (b) decreased clearance. The liver has excretory functions, with an outlet into the alimentary tract via the biliary system. Any antigens that are confined to the circulation or to the liver, if transferred into the biliary tract, may stimulate the local production of IgA by the gut immune system; the integrity of normal hepatic function might be required for the segregation of some immunogens from the intestine. The failure in secluding them on the part of the liver, either temporarily or persistently, would furnish the intestinal immune system with immunogens not normally accessible to the alimentary tract. Turning to its clearance, IgA in the serum, especially in polymeric forms, is excreted by the hepatobiliary system (19,20). Extensive liver damage and cholestasis, therefore, can contribute toward the elevation of serum IgA levels. The liver is the major target organ of HBV; the polyalbumin receptor of HBV most likely accounts for its hepatotropism (21~2). There has been no evidence for the replication of HBV in the alimentary tract so far. To initiate HBV infection, therefore, HBV has to be introduced parenterally. usually into the
November
1985
bloodstream. As a corollary to this, HBcAg, representing the n~ucleocapsid of HBV, would not activate the gut immune system so long as it is confined to the liver and circulation. The induction of an IgA anti-HBc response could be initiated only after the antigen was supplied to the mucosal surface of the intestine, presumably as a consequence of hepatic dysfunction. Indeed, the extremely low prevalence of IgA anti-HBc (4%) in asymptomatic carriers might represent the failure of inducing an IgA anti-HBc response. The validity of this assumption may be tested by prospectively following the asymptomatic carriers who contract HBV infection through the mother-to-baby transmission, to see if IgA anti-HBc develops in the absence of any hepatic damage. Once IgA anti-HBc develops in the circulation, the decreased clearance, if any, would help elevate its serum levels. This may explain why the level of IgA anti-HBc in patients with LC/HCC was significantly higher than that in patients with CAH (Figure 3). In contrast, the level of IgM anti-HBc was comparable between patients with LCEICC and those with CAH; the serum level of IgM was not as dramatically increased as that of IgA in patients with cirrhosis (8,9), presumably because of the lack of its excretion by the hepatobiliary system. Measurement of IgA anti-HBc by radioimmunoassay may have applications to clinical medicine, inasmuch as high levels of this antibody may reflect hepatic injury. Subpopulations of IgA anti-HBc in terms of monomeric or polymeric forms, as well as the association or lack of association with secretory component or J chain, or both, deserve analysis in order to establish the intestinal origin of IgA antiHBc in acute and persistent HBV infection.
References 1. Almeida
JD. Rubinstein D. Stott EJ. New antigen-antibody system in Australia-antigen-positive hepatitis. Lancet 1971; ii:1225-7. 2. Hoofnagle JH, Gerety RJ, Barker LF. Antibody to hepatitis-Bvirus core in man. Lancet 1973;ii:869-73. 3. Aikawa T, Sairenji H, Furuta S, et al. Seroconversion from hepatitis B e antigen to anti-HBe in acute hepatitis B virus infection. N Engl J Med 1978;298:439-41. 4. Gerlich WH. Liier W, Thomssen R, et al. Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J Infect Dis 1980;142:95-101.
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5. Lemon SM. Gates NL. Simms TE, Bancroft WH. IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 1981:143:803-9. 6. Shimizu M. Ohyama M, Takahashi Y. et al. Immunoglobulin M antibody against hepatitis B core antigen for the diagnosis of fulminant type B hepatitis. Gastroenterology 1983;84:60410. 7. Tomasi TB. Tisdale WA. Serum gamma-globulins in acute and chronic liver diseases. Nature 1964:201:834-5. 8. Lee FI, Lond MB. Immunoglobulins in viral hepatitis and active alcoholic liver-disease. Lancet 1965:ii:1043-6. 9. Thompson RA, Carter R, Stokes RP, Geddes AM, Goodall JAD. Serum immunoglobulins. complement levels and autoantibodies in liver disease. Clin Exp Immunol 1973;14:335-46. 10. Arakawa K. Tsuda F, Takahashi K, et al. Maternofetal transmission of IgG-bound hepatitis B e antigen. Pediatr Res 1981;16:247-50. 11. Vyas GN. Shulman NR. Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 1970; 170:332-3. 12. Tsuda F. Naito S, Takai E, et al. Low molecular weight (7s) immunoglobulin M antibody against hepatitis B core antigen in the serum for differentiating acute from persistent hepatitis B virus infection. Gastroenterology 1984;87:159-64. 13. Oi VT, Herzenberg LA. Immunoglobulin producing hybrid cell lines. In: Mishell BB, Shiigi SM, eds. Selected methods in cellular immunology. San Francisco: WH Freeman, 1980: 351-72. 14. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256: 495-7. 15. Takahashi K. Machida A. Funatsu G, et al. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol 1983;130:2903-7. 16. Takahashi T. Nakagawa S. Hashimoto T, et al. Large-scale isolation of Dane particles from plasma containing hepatitis B antigen and demonstration of a circular double-stranded DNA molecule extruding directly from their cores. J Immunol 1976:117:1392-7. 17. Greenwood FC, Hunter WM. Glover JS. The preparation of ‘“‘I-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89:114-23. 18. Popper H. Summary of workshop on pathology. In: Vyas GN, Dienstag JL. Hoofnagle JH, eds. Viral hepatitis and liver disease. Orlando: Grune & Stratton, 1984:515-g. 19. Nagura H, Smith PD, Nakane PK, Brown WR. IgA in human bile and liver. J Immunol 1981;126:587-95. 20. Kutteh WH. Prince SJ, Phillips JO, Spenney JG, Mestecky J. Properties of immunoglobulin A in serum of individuals with liver diseases and in hepatic bile. Gastroenterology 1982;82: 184-93. 21. Imai M, Yanase Y. Nojiri T. Miyakawa Y. Mayumi M. A receptor for polymerized human and chimpanzee albumins on hepatitis B virus particles co-occurring with HBeAg. Gastroenterology 1979:76:242-7. 22. Thung SN, Gerber MA. Polyalbumin receptors: their role in the attachment of hepatitis B virus to hepatocytes. Sem Liver Dis 1984:4:69-75.