Journal of Medical Virology 301-6 (1990)
Comparison of Class and Subclass Distribution of Antibodies to the Hepatitis B Core and B e Antigens in Chronic Hepatitis B Matti Sallberg, Helene Norder, and Lars 0. Magnius Department of Virology, The National Bacteriological Laboratory, Stockholm, Sweden The IgG subclasses IgM and l g A l of antibodies t o hepatitis B core antigen (anti-HBc) and hepatitis B e antigen (anti-HBe) were assayed in sera from 82 patients with chronic hepatitis B utilising classhubclass-specific enzyme immunoassays (EIA). The solid-phase was either recombinant hepatitis B core antigen (rHBcAg) or rHBcAg converted t o HBeAg by addition of 0.1% SDS with remaining HBcAg antigenicity blocked with monoclonal anti-HBc. Anti-HBc lgGl was detected in 81 sera at a geometrical mean titre (GMT) of 296,110 2.9. Anti-HBc lgG2 was not detected in any of the sera, and anti-HBc lgG3 and lgG4 were detected in 50 and 37 sera, respectively. Anti-HBc IgM and l g A l were both significantly correlated t o the presence of HBV DNA. The predominant antibody to HBeAg was found t o be IgGl, being detected in 45 sera with a GMT of 1,035 5 3.3. Anti-HBe lgG2 was not detected in any serum, while anti-HBe lgG3 and lgG4 were found in 8 and 23 sera, respectively. Anti-HBe IgG1, lgG3, and lgG4 were mainly detected i n sera positive for anti-HBe in RIA (Abbott). No patient was found positive for anti-HBe l g A l or IgM. Thus, in contrast t o HBcAg, HBeAg does not trigger a persistent IgM and l g A l response in chronic hepatitis B. The levels of antiHBe lgGl and lgG3 were much lower than the levels of anti-HBc l g G l and lgG3. The presence of anti-HBe lgG4 was significantly correlated t o that of anti-HBc lgG4. The relative levels of antiHBc IgG subclasses were found to be l g G l >> lgG3 > lgG4, whereas the relative levels of antiHBe subclasses were l g G l 2 lgG4 2 lgG3. The anti-HBe IgGI:IgG4 ratio was much lower than that of anti-HBc, 2.7:l versus 890:l. Our findings support the suggestion of differences at the Bcell level in the regulation of the immunoresponse t o HBeAg and HBcAg, though both antigenic epitopes reside on the same polypeptide.
5
KEY WORDS: Hepatitis B Virus, anti-HBc isotypes, anti-HBe isotypes 0 1990 WILEY-LISS, INC.
INTRODUCTION Hepatitis B core antigen (HBcAg) of the hepatitis B virus (HBV) has been shown to elicit a homogeneous antibody response, directed to one single epitope [Ferns and Tedder, 19861. Treatment of native or recombinant HBcAg (rHBcAg1 with proteolytic enzymes, 2-mercaptoethanol, or sodium dodecyl-sulphate (SDS) reveals antigenic epitopes with hepatitis B e antigen (HBeAg) specificity [MacKay et al., 1981; Ohiro et al., 1980; Takahashi et al., 19793. HBeAg in serum of patients with chronic hepatitis B has been reported to exist complexed to plasma proteins, particularly IgG, or in a free form [Takahashi et al., 1978; Takekoshi et al., 19791. Anti-HBe has previously been shown to be of the IgG class [Mushahwar et al., 1978; Takahashi et al., 19781. IgGl has been described as the main antibody to HBcAg in chronic hepatitis B [Sallberg et al., 19881. HBcAg has also been demonstrated to elicit IgM [Gerlich et al., 19861, IgA [Nomura et al., 19851, and IgD [Brzosko et al., 19751 responses. HBcAg has been shown to function as a T cell-dependent as well as T cell-independent antigen, whereas HBeAg has been found to be strictly T cell-dependent [Milich and McLachlan, 1986; Milich et al., 19881.Further, the high levels of anti-HBc in chronic hepatitis B as opposed t o the slow development and rather low levels of anti-HBe suggest that the two antigens are dealt with differently by the immune system. Though they apparently elicit an individual epitope-specific humoral response in mice [Imai et al., 1982; Ferns and Tedder, 1984, 19861 they have been shown to be crossreactive on the T-cell level [Milich et al., 19871. We compared the immunoglobulin class and subclass distributions of anti-HBc and anti-HBe in chronic hepatitis B.
Accepted for publication August 29, 1989. Address reprint requests to Matti Sallberg, Department of Virology, The National Bacteriological Laboratory, S-105 21, Stockholm, Sweden.
Sallberg et al.
MATERIALS AND METHODS Materials Eighty-two sera, negative for antibodies to hepatitis D virus (anti-HD), from the same number of adult chronic HBsAg carriers (mean age, 30.4 years; range, 18 t o 58 years) were selected from consecutive samples sent to the Department of Virology of the National Bacteriological Laboratory (Stockholm, Sweden) during three periods, January to July 1986, 1987, and 1988, when 21, 22, and 39 sera, respectively, were selected. Prior to analysis for classes and subclasses, sera were tested for HBsAg, HBeAgianti-HBe, anti-HBc, antiHBc IgM, antiHBs, and anti-HD using commercially available assays (Abbott Laboratories, Ill.). HBV DNA was determined in all sera using a dot-blot procedure according to the method previously described 1 Norder et al., 19891. Determination of Classes and Subclasses of Anti-HBc Enzyme immunoassays (EIAs) for the determination of the IgG subclasses of anti-HBc were performed a s follows. A 96 well polystyrene plate (Nunc 96F, Kopenhagen, Denmark), was coated overnight a t + 4°C with 50 pl rHBcAg (Biogene, Geneva, Switzerland) diluted to a final concentration of 100 ngiml in coating buffer (0.05M Na HCO,, pH 9.6). The sera to be tested were diluted in 100-fold steps from 1 : l O O to 1:1,000,000 in phosphate-buffered saline (PBS) with 0.05% Tween 20 and 1%bovine serum albumine (PBS-TA). After washing 10 times in PBS with 0.05% Tween 20 (PBS-T), the serum dilutions were added. After incubation over night a t + 4°C and washing, monoclonal antibodies (mAbs) to human IgG subclasses were added and the plate was incubated for 90 min a t + 37°C. The specificities and designations of the mAbs were as follows: anti-IgG1 (clone NL16), diluted 1:3,000; anti-IgG2 (clone HP6014, CDC, Atlanta), diluted I: 2,000; anti-IgG3 (clone ZG41, diluted 1:3,000; and antiIgG4 (clone M 4 ) , diluted 1:1,000 in PBS with 10%fetal calf sera (FCS).The clones NL16, ZG4, and RJ4 were purchased from Seward Laboratory (London, England). Excess mAbs were removed by washing, and 50 pl of biotinylated rabbit antibody to mouse IgGl (Zymed, Calif.), diluted 1:3,000 in PBS-TA, was added. After incubation for 90 min a t + 37°C the plate was washed and horseradish peroxidase (HRP0)-conjugated streptavidin (HRPO-SA; Zymed), diluted 1:6,000 in PBS-TA, was added. After another incubation for 30 min a t ambient temperature and washing, the plate was developed by the addition of 50 p1orthophenylenediamine (Sigma Chemicals, St. Louis, Mo.). After 30 min incubation a t ambient temperature the reaction was terminated with 50 p1 0.5 M H,SO,, and the absorbances were read a t 492 nm in a Kontron SLT 210 spectrophotometer. The EIAs for anti-HBc of IgM and IgAl classes were performed as follows. A microtitre plate (Nunc 96F)
was coated overnight a t +4"C with 50 pl of mAb specific for human IgM (clone AF-6; Seward Laboratories) diluted 1:1,000 or for human IgAl (clone NlF2; Seward Laboratories) diluted 1:1,000 in coating buffer. Unbound capture antibodies were removed by washing with PBS-T. The sera were diluted in 100-fold steps from 1 : l O O to 1:1,000,000 in PBS-TA. Fifty microlitres of each dilution was used for each well. After incubation overnight a t + 4°C the plate was washed, and 2 ng of rHBcAg in 50 pl of PBS-TA was added to each well. After another overnight incubation at + 4°C and washing, 50 pl of HRPO-conjugated monoclonal anti-HBc (clone 231; Behringwerke A.G, Marburg, FRG), diluted 1:500 in PBS-TA, was added. After incubation for 3 hr at + 37°C and washing, the plates were developed and read a s described above.
Conversion of rHBcAg to HBeAg rHBcAg (Biogen) was treated with 0.1% sodium dodecyl-sulphate (SDS) for 120 min a t 37°C and immediately stored a t -20°C. The SDS-treated rHBcAg was tested for HBeAg specificity by HRPO-conjugated mAb to the p epitope of HBeAg (51, clone 5718 (Behringwerke). Determination of Classes and Subclasses of Anti-HBe The assay was performed as described for subclasses of anti-HBc IgG with the following differences: 1)Microtitre plates (Nunc 96F) were coated overnight a t + 4°C with 50 ~1 SDS-treated rHBcAg diluted to a final concentration of 100 ng1ml in coating buffer. The remaining HBcAg activity was blocked with monoclonal anti-HBc of mouse IgG2 isotype, diluted 1:1,000 in PBS-TA for 90 min at 37°C (clone 371205; Behringwerke). After being washed, the plates were stabilised by drying for 60 min a t 37°C. 2) The mAbs used were as follows: anti-IgG1, clone HP6069, diluted 1:1,000; antiIgG2, clone HP6002, diluted 1:2,000; anti-IgG3, clone HB6047, diluted 1:4,000;and anti-IgG4, clone HP6025, diluted 1:1,000 in PBS-TA. All clones were biotinylated and purchased from Zymed. After incubation for 90 rnin a t +37"C, HRPO-SA was added and the plates were developed and read a s described above. EIAs for anti-HBe IgM and IgAl were performed as described for subclasses of anti-HBc IgG with the following differences: 1)The plate was coated with SDStreated rHBcAg and blocked with mAb anti-HBc. 2) Sera were diluted 1:lOO and 1:1,000 in PBS-TA. 3 ) Anti-HBe IgM and IgAl were indicated with monoclonal anti-IgM (clone AF-6), diluted 1:32,000, or antiIgAl (clone NlF2), diluted 1:2,000, in PBS with 10%) FCS and incubated for 60 min a t + 37°C. Standardisation of Results and Calculation of End-Point Titres For standardisation in all the class- and subclassspecific assays a serum, positive in the assay, was diluted in five- or two-fold steps. The logarithms of ab-
Subclasses of Anti-HBe IgG in Chronic Hepatitis B
3
TABLE I. Background Data on 82 Patients With Chronic Hepatitis B Providing Sera for the Study
Epidemiology Immigrants Homosexuals Drug addicts Renal recipients Other Totals
Mean age (years) 29.9 24.0 31.9 29.3 37.0 30.4
No. of patients 60 3 10 3 6 82
Male 36 3 8 3 6 56
Female 24 0 2 0 0 26
No. of patients with HBeAg HBV DNA 18" 17 2 2 7b
0 4 31
7
0 4 30
~'HBVDNA was found in 16 sera positive for HBeAg and in two sera positive for anti-HBe. hHBV DNA was found in six sera positive for HBeAg and in one serum with low-level anti-HBe (55% inhibition).
sorbances within the slope of the dose-response curve from the dilutions were used to construct a standard curve. This was linearised by least-squares analysis. Only sample dilutions with absorbances within the slope of the standard curve were used for relative endpoint titre determinations, which were calculated from the intersection of the absorbancy with the standard curve. In each assay a t least 15 different sera, negative for all HBV markers, were used for calculation of the mean (x) and standard deviation (SD) of the absorbances for negative samples. In the anti-HBc and antiHBe IgG subclass assays the positive cut-off was mean ? 3 and 7 SD, respectively. In the antibody capture assays the positive cut-off was mean 7 SD.
*
Statistical Methods Any significance deemed significant was found so, if not otherwise stated, by using Student's t test or Fisher's exact test when appropriate. RESULTS Epidemiological background data, sex, and mean age of the 82 patients with chronic hepatitis B are given in Table I. Anti-HBc Classes and Subclasses The outcomes of testing the patient sera for classes and subclasses of anti-HBc are shown in Table 11. The positive rate for anti-HBc IgM was significantly higher for HBV DNA-positive sera as compared with HBV DNA-negative sera ( P < 0,001). The geometric mean titre (GMT) of anti-HBc IgM was, however, not significantly higher in sera positive for HBV DNA than in sera negative for the marker. The GMT of anti-HBc IgAl was significantly higher in HBV DNA-positive sera compared with HBV DNA-negative sera ( P < 0.01). Fifteen of 31 HBV DNA-positive sera had high levels (>10,000) of anti-HBc IgAl compared with 6 of 51 sera negative for HBV DNA ( P < 0.001). High levels of anti-HBc IgAl were significantly correlated with high levels of anti-HBc IgM (Table 111, A). IgGl was found to be the main subclass of anti-HBc IgG and was detected in 81 of 82 patients. Only one (from a male immigrant) positive for HBeAg but negative for HBV DNA was also negative for anti-HBc IgGl. Anti-HBc
IgG2 was not detected in any of the tested sera. Frequencies and GMTs of anti-HBc IgG3 and IgG4 did not differ significantly between HBV DNA-positive sera and HBV DNA-negative sera. High levels of anti-HBc IgG3 were, however, significantly correlated to high levels of anti-HBc IgAl (Table 111, B; P < 0.001). Such a relation was not found between anti-HBc IgG3 and anti-HBc IgM. Anti-HBe Classes and Subclasses The performance of the EIAs for subclasses of antiHBe IgG in relation to the commercial RIA for antiHBe (Abbott) is shown in Table IV. Forty-three of 51 sera positive in RIA were also positive by the EIA for anti-HBe IgGl, giving a sensitivity of 84%.Of the eight sera not detected by the EIA for anti-HBe IgG1, five gave low-level inhibition (85% inhibition, one was found positive for anti-HBe IgG4 and two were not detected by any of the anti-HBe class- or IgG subclass-specific EIAs. Of these two sera, one was positive for HBV DNA. The result of testing the patient sera for subclasses of anti-HBe IgG is shown in Table V. No serum was found repeatedly positive for antiHBe IgM, IgA1, or IgG2. Anti-HBe IgGl was detected in two of the HBV DNA-positive sera, of which one was also HBeAg positive. Comparison of Anti-HBc and Anti-HBe Classes and Subclasses The relative levels of anti-HBc IgG subclasses were IgGl >> IgG3 > IgG4 and for anti-HBe IgG, IgGl 2 IgG4 2 IgG3. GMT of anti-HBc IgGl and IgG3 were much higher than the corresponding anti-HBe levels. The relation between anti-HBe IgG4 and anti-HBc IgG4 in the 51 anti-HBe-positive sera are shown in Table 111, C. Anti-HBe IgG3 was not correlated to antiHBc IgG3, whereas the correlation between anti-HBe IgG4 and anti-HBc IgG4 was found to be highly significant ( P < 0.001). The ratio between anti-HBc IgGl and IgG3 titres was 520:1, and the corresponding ratio for anti-HBe was 11.O:l. The ratios between IgGl and IgG4 titres for anti-HBc and for anti-HBe were 890:l and 2.7:1, respectively.
Sallberg et al.
4
TABLE 11. Prevalence and Titres of IgM and IgAl Anti-HBc and Subclasses of Anti-HBc IgG in Relation to HBV DNA Results in Sera From 82 Patients With Chronic HeDatitis B No. of sera with indicated HBV DNA result 31 + 51 Totals ~
IgM G M T ? SD 12,102"14 1,306"7.4 5,112"14
n 19 12 31
Number ( n ) and geometric mean titre (GMT) of sera positive for anti-HBc of indicated class and subclass IgAl IgGl IgG3 n GMT? SD n GMT"SD n G M T " SD 21 8,236' 17 30 260,010?3.4 20 27,309"12 29 5,707"24 51 319,644T2.6 30 769T7.8 50 6,658"20 81 296,110?2.9 50 3,208"17
IgG4 G M T * SD 1,572'16 2,760"lO 2,299"12
n 12 25 37
TABLE 111. Relation Between A: Presence of High Levels* of Anti-HBc IgM and High Levels of IgAl in 82 Patient Sera; B: Presence of High Levels of Anti-HBc IgG3 and High Levels of Anti-HBc IgAl in 82 Patient Sera; and C: Presence Anti-HBc IpG4 and Anti-HBe IeG4 in 51 Patient Sera Positive for Anti-HBe in RIA Anti-HBc IeA 1
+ ~
Totals
A: high levels of Anti-HBc IgM
+
-
14
6 60 66
2 16
Anti-HBc IeA1
B: high levels of Anti-HBc IgG3
+ ~
P> IgG3 > IgG4, whereas for anti-HBe the relative levels were IgGl 2 IgG4 2 IgG3. Thus the IgG4 to IgGl ratio was proportionately higher for anti-HBe than for anti-HBc. A prolonged antigenic stimulation has been suggested to result in a more IgG4-restricted response [Aalberse et al., 1983; Skvaril, 19863, which may explain the relatively higher IgG4 response to HBeAg, since in general patients with chronic hepatitis B and anti-HBe have been carriers for a longer period than patients with HBeAg. This may also explain the higher frequency and levels of anti-HBc IgG4 in HBV DNA-negative sera. The differences in humoral responses to HBcAg and HBeAg might be due to the latter antigen being strictly T celldependent, thereby limiting the pathways for triggering the immune system. Our finding of a n IgGrestricted response to HBeAg in chronic hepatitis B, together with the inability to demonstrate anti-HBe IgM or IgA despite a persistent antigenemia, further strengthens the strict T cell-dependence of HBeAg. Further, immune complexes with T cell-dependent antigens have been reported to have a n inhibitory effect on IgM production [Bruderer and Heusser, 19881. If so, a weaker anti-HBe IgM response would be expected, since HBeAg complexed to immunoglobulins has been reported in patients with hepatitis B [Takahashi et al., 1978; Takekoshi et al., 19791.
REFERENCES Aalberse RC, van Der Gaag R, van Leeuwen J (1983): Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4 restricted response. Journal of Immunology 130:722-726. Bruderer U, Heusser C (1988):Regulation of immunoglobulin isotype expression in mice by antibodies in immune complexes. Immunology Letters 17235-240. Brzosko WJ, Mikulska B, Cianciara J , Babiuch L (1975): Immunoglo-
bulin classes of antibody to hepatitis B core antigen. Journal of Infectious Diseases 132:l-5. Coleman RM, Nahimas AJ, Williams SC, Phillips DJ, Black CM, Reimer CB (1985): IgG subclass antibodies to herpes simplex virus. Journal of Infectious Diseases 151:929-936. Ferns RB, Tedder RS (1984): Monoclonal antibodies to hepatitis B e antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): Their use in characterization and detection of HBeAg. Journal of General Virology 65:899-908. Ferns RB, Tedder RS (1986): Human and monoclonal antibodies to hepatitis B core antigen recognise a single immunodominant epitope. Journal of Medical Virology 19:193-203. Gerlich WH, Uy A, Lambrecht F, Thomssen R (1986): Cutoff levels of immunoglobulin M antibody against viral core antigen for differentiation of acute, chronic, and past hepatitis B virus infections. Journal of Clinical Microbiology 24:288-293. Imai M, Nomura M, Gotanda T, Sano T, Tachibana K, Miyamoto H, Takahashi K, Toyama S, Miyakawa Y, Mayumi M (1982): Demonstration of two distinct determinants on hepatitis B e antigen by monoclonal antibodies. Journal of Immunology 128:69-72. Kiyosawa K, Sodeyama T, Franca STM, Yoda H, Ohike Y, Imai H, Imai Y, Furuta S (1988):Serial assay for IgM anti-HBc in patients with anti-HBe positive chronic hepatitis and its significance for longterm prognosis. Journal of Medical Virology 24241-250. Linde A, Sundqvist V-A, Mathiesen T, Wahren B (1988): IgG subclasses to subviral components. Monographs in Allergy 23:27-32. MacKay P, Lees J , Murray K (1981): The conversion of hepatitis B core antigen synthesized in E coli into e antigen. Journal of Medical Virology 8:237-243. Milich DR, McLachan A (1986):The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-celldependent antigen. Science 12:1398-1401. Milich DR, McLachlan A, Thornton GB, Hughes J L (1987): Immune response to hepatitis B virus core antigen (HBcAg):Localization of T cell recognition sites within HBcAgiHBeAg. Journal of Immunology 139:1223-1231. Milich DR, McLachlan A, Stahl S, Wingfield P, Thornton GB, Hughes JL, Jones JE (1988): Comparative immunogenicity of hepatitis B virus core and e antigens. Journal of Immunology 141:3617-3624. Mushahwar IK, Overby LR, Frosner G, Deinhardt F, Ling CM (1978): Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays. Journal of Medical Virology 2:77-87. Nomura M, Imai M, Tsuda F, Furuta S, Akahane Y, Tachibana K, Usuda S, Miyakawa Y, Mayumi M (1985): Immunoglobulin A antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus. Gastroenterology 89:1109-1113. Norder H, Brattstrom C, Magnius LO (1989):High frequency of HBV DNA in anti-HBe positive sera on longitudinal follow-up of patients with renal transplants and chronic hepatitis B. Journal of Medical Virology 27:322-328. Ohiro H, Yamaki M, Onodera S, Yamada E, Ishida N (1980): Antigenic conversion from HBcAg to HBeAg by degradation of hepatitis B core particles. Intervirology 13:74-82. Sallberg M, Norder H, Linde A, Magnius LO (1988) Subclass pattern of anti-HBc IgG in chronic hepatitis B: Relation to HBeAgiantiHBe and HBV-DNA. In Zuckerman AJ (ed): “Viral Hepatitis and Liver Disease.” New York: Alan R. Liss, Inc., pp 244-247. Sallberg M, Magnius LO (1989): An enzyme immunoassay for anti-
(i HBc IgG1: Significance of low level results in competitive assays for anti-HBc. Journal of Clinical Microbiology 272349-853. Sarov I, Insler V, Sarov B, Cevenini R, Rumpianesi F, Donati M, Kleinman D, Piura B, Lieberman J, Kimmel N, Friedman M, La Placa M 1984):Specific serum IgA antibodies in the diagnosis of active viral and chlamydia infection. In Sanna A, Morace J (eds): “New Horizons in Microbiology.” Amsterdam: Elsevier Biomedical Press, pp 157-158. Skvaril F ( 1986): IgG subclasses in viral infections. Monographs in Allergy 19:134-143. Takahashi KM, Imai M, Miyakawa Y, Iwakiri S, Mayumi M (1978): Duality of hepatitis B e antigen in serum of persons infected with hepatitis B virus: Evidence for the nonidentity with immunoglo-
Sallberg et al. bulins. Proceedings of the National Academy of Sciences USA 75: 1952-1956. Takahashi K, Akahane Y, Gotanda T, Mishiro T, Imai M, Miyakawa Y, Mayumi M (1979):Demonstration of hepatitis B e antigen in the core of Dane particles. Journal of Immunology 122:275-279. Takekoshi Y, Tanaka M, Miyakawa H, Yoshizawa K, Takahashi K , Mayumi M (1979): Free “small” and IgG associated “large” hepatitis B e antigen in the serum and glomerular capillary walls of two patients with membranous glomerulonephritis. New England Journal of Medicine 300:814-819. van Loon AM, Heessen FWA, van der Logt JTM (1987): Antibody isotype response after human cytomegalovirus infection. Journal of Virological Methods 15:lOl-107.
Comparison of Class and Subclass Distribution of Antibodies to the Hepatitis B Core and B e Antigens in Chronic Hepatitis B Matti Sallberg, Helene Norder, and Lars 0. Magnius Department of Virology, The National Bacteriological Laboratory, Stockholm, Sweden The IgG subclasses IgM and l g A l of antibodies t o hepatitis B core antigen (anti-HBc) and hepatitis B e antigen (anti-HBe) were assayed in sera from 82 patients with chronic hepatitis B utilising classhubclass-specific enzyme immunoassays (EIA). The solid-phase was either recombinant hepatitis B core antigen (rHBcAg) or rHBcAg converted t o HBeAg by addition of 0.1% SDS with remaining HBcAg antigenicity blocked with monoclonal anti-HBc. Anti-HBc lgGl was detected in 81 sera at a geometrical mean titre (GMT) of 296,110 2.9. Anti-HBc lgG2 was not detected in any of the sera, and anti-HBc lgG3 and lgG4 were detected in 50 and 37 sera, respectively. Anti-HBc IgM and l g A l were both significantly correlated t o the presence of HBV DNA. The predominant antibody to HBeAg was found t o be IgGl, being detected in 45 sera with a GMT of 1,035 5 3.3. Anti-HBe lgG2 was not detected in any serum, while anti-HBe lgG3 and lgG4 were found in 8 and 23 sera, respectively. Anti-HBe IgG1, lgG3, and lgG4 were mainly detected i n sera positive for anti-HBe in RIA (Abbott). No patient was found positive for anti-HBe l g A l or IgM. Thus, in contrast t o HBcAg, HBeAg does not trigger a persistent IgM and l g A l response in chronic hepatitis B. The levels of antiHBe lgGl and lgG3 were much lower than the levels of anti-HBc l g G l and lgG3. The presence of anti-HBe lgG4 was significantly correlated t o that of anti-HBc lgG4. The relative levels of antiHBc IgG subclasses were found to be l g G l >> lgG3 > lgG4, whereas the relative levels of antiHBe subclasses were l g G l 2 lgG4 2 lgG3. The anti-HBe IgGI:IgG4 ratio was much lower than that of anti-HBc, 2.7:l versus 890:l. Our findings support the suggestion of differences at the Bcell level in the regulation of the immunoresponse t o HBeAg and HBcAg, though both antigenic epitopes reside on the same polypeptide.
5
KEY WORDS: Hepatitis B Virus, anti-HBc isotypes, anti-HBe isotypes 0 1990 WILEY-LISS, INC.
INTRODUCTION Hepatitis B core antigen (HBcAg) of the hepatitis B virus (HBV) has been shown to elicit a homogeneous antibody response, directed to one single epitope [Ferns and Tedder, 19861. Treatment of native or recombinant HBcAg (rHBcAg1 with proteolytic enzymes, 2-mercaptoethanol, or sodium dodecyl-sulphate (SDS) reveals antigenic epitopes with hepatitis B e antigen (HBeAg) specificity [MacKay et al., 1981; Ohiro et al., 1980; Takahashi et al., 19793. HBeAg in serum of patients with chronic hepatitis B has been reported to exist complexed to plasma proteins, particularly IgG, or in a free form [Takahashi et al., 1978; Takekoshi et al., 19791. Anti-HBe has previously been shown to be of the IgG class [Mushahwar et al., 1978; Takahashi et al., 19781. IgGl has been described as the main antibody to HBcAg in chronic hepatitis B [Sallberg et al., 19881. HBcAg has also been demonstrated to elicit IgM [Gerlich et al., 19861, IgA [Nomura et al., 19851, and IgD [Brzosko et al., 19751 responses. HBcAg has been shown to function as a T cell-dependent as well as T cell-independent antigen, whereas HBeAg has been found to be strictly T cell-dependent [Milich and McLachlan, 1986; Milich et al., 19881.Further, the high levels of anti-HBc in chronic hepatitis B as opposed t o the slow development and rather low levels of anti-HBe suggest that the two antigens are dealt with differently by the immune system. Though they apparently elicit an individual epitope-specific humoral response in mice [Imai et al., 1982; Ferns and Tedder, 1984, 19861 they have been shown to be crossreactive on the T-cell level [Milich et al., 19871. We compared the immunoglobulin class and subclass distributions of anti-HBc and anti-HBe in chronic hepatitis B.
Accepted for publication August 29, 1989. Address reprint requests to Matti Sallberg, Department of Virology, The National Bacteriological Laboratory, S-105 21, Stockholm, Sweden.
Sallberg et al.
MATERIALS AND METHODS Materials Eighty-two sera, negative for antibodies to hepatitis D virus (anti-HD), from the same number of adult chronic HBsAg carriers (mean age, 30.4 years; range, 18 t o 58 years) were selected from consecutive samples sent to the Department of Virology of the National Bacteriological Laboratory (Stockholm, Sweden) during three periods, January to July 1986, 1987, and 1988, when 21, 22, and 39 sera, respectively, were selected. Prior to analysis for classes and subclasses, sera were tested for HBsAg, HBeAgianti-HBe, anti-HBc, antiHBc IgM, antiHBs, and anti-HD using commercially available assays (Abbott Laboratories, Ill.). HBV DNA was determined in all sera using a dot-blot procedure according to the method previously described 1 Norder et al., 19891. Determination of Classes and Subclasses of Anti-HBc Enzyme immunoassays (EIAs) for the determination of the IgG subclasses of anti-HBc were performed a s follows. A 96 well polystyrene plate (Nunc 96F, Kopenhagen, Denmark), was coated overnight a t + 4°C with 50 pl rHBcAg (Biogene, Geneva, Switzerland) diluted to a final concentration of 100 ngiml in coating buffer (0.05M Na HCO,, pH 9.6). The sera to be tested were diluted in 100-fold steps from 1 : l O O to 1:1,000,000 in phosphate-buffered saline (PBS) with 0.05% Tween 20 and 1%bovine serum albumine (PBS-TA). After washing 10 times in PBS with 0.05% Tween 20 (PBS-T), the serum dilutions were added. After incubation over night a t + 4°C and washing, monoclonal antibodies (mAbs) to human IgG subclasses were added and the plate was incubated for 90 min a t + 37°C. The specificities and designations of the mAbs were as follows: anti-IgG1 (clone NL16), diluted 1:3,000; anti-IgG2 (clone HP6014, CDC, Atlanta), diluted I: 2,000; anti-IgG3 (clone ZG41, diluted 1:3,000; and antiIgG4 (clone M 4 ) , diluted 1:1,000 in PBS with 10%fetal calf sera (FCS).The clones NL16, ZG4, and RJ4 were purchased from Seward Laboratory (London, England). Excess mAbs were removed by washing, and 50 pl of biotinylated rabbit antibody to mouse IgGl (Zymed, Calif.), diluted 1:3,000 in PBS-TA, was added. After incubation for 90 min a t + 37°C the plate was washed and horseradish peroxidase (HRP0)-conjugated streptavidin (HRPO-SA; Zymed), diluted 1:6,000 in PBS-TA, was added. After another incubation for 30 min a t ambient temperature and washing, the plate was developed by the addition of 50 p1orthophenylenediamine (Sigma Chemicals, St. Louis, Mo.). After 30 min incubation a t ambient temperature the reaction was terminated with 50 p1 0.5 M H,SO,, and the absorbances were read a t 492 nm in a Kontron SLT 210 spectrophotometer. The EIAs for anti-HBc of IgM and IgAl classes were performed as follows. A microtitre plate (Nunc 96F)
was coated overnight a t +4"C with 50 pl of mAb specific for human IgM (clone AF-6; Seward Laboratories) diluted 1:1,000 or for human IgAl (clone NlF2; Seward Laboratories) diluted 1:1,000 in coating buffer. Unbound capture antibodies were removed by washing with PBS-T. The sera were diluted in 100-fold steps from 1 : l O O to 1:1,000,000 in PBS-TA. Fifty microlitres of each dilution was used for each well. After incubation overnight a t + 4°C the plate was washed, and 2 ng of rHBcAg in 50 pl of PBS-TA was added to each well. After another overnight incubation at + 4°C and washing, 50 pl of HRPO-conjugated monoclonal anti-HBc (clone 231; Behringwerke A.G, Marburg, FRG), diluted 1:500 in PBS-TA, was added. After incubation for 3 hr at + 37°C and washing, the plates were developed and read a s described above.
Conversion of rHBcAg to HBeAg rHBcAg (Biogen) was treated with 0.1% sodium dodecyl-sulphate (SDS) for 120 min a t 37°C and immediately stored a t -20°C. The SDS-treated rHBcAg was tested for HBeAg specificity by HRPO-conjugated mAb to the p epitope of HBeAg (51, clone 5718 (Behringwerke). Determination of Classes and Subclasses of Anti-HBe The assay was performed as described for subclasses of anti-HBc IgG with the following differences: 1)Microtitre plates (Nunc 96F) were coated overnight a t + 4°C with 50 ~1 SDS-treated rHBcAg diluted to a final concentration of 100 ng1ml in coating buffer. The remaining HBcAg activity was blocked with monoclonal anti-HBc of mouse IgG2 isotype, diluted 1:1,000 in PBS-TA for 90 min at 37°C (clone 371205; Behringwerke). After being washed, the plates were stabilised by drying for 60 min a t 37°C. 2) The mAbs used were as follows: anti-IgG1, clone HP6069, diluted 1:1,000; antiIgG2, clone HP6002, diluted 1:2,000; anti-IgG3, clone HB6047, diluted 1:4,000;and anti-IgG4, clone HP6025, diluted 1:1,000 in PBS-TA. All clones were biotinylated and purchased from Zymed. After incubation for 90 rnin a t +37"C, HRPO-SA was added and the plates were developed and read a s described above. EIAs for anti-HBe IgM and IgAl were performed as described for subclasses of anti-HBc IgG with the following differences: 1)The plate was coated with SDStreated rHBcAg and blocked with mAb anti-HBc. 2) Sera were diluted 1:lOO and 1:1,000 in PBS-TA. 3 ) Anti-HBe IgM and IgAl were indicated with monoclonal anti-IgM (clone AF-6), diluted 1:32,000, or antiIgAl (clone NlF2), diluted 1:2,000, in PBS with 10%) FCS and incubated for 60 min a t + 37°C. Standardisation of Results and Calculation of End-Point Titres For standardisation in all the class- and subclassspecific assays a serum, positive in the assay, was diluted in five- or two-fold steps. The logarithms of ab-
Subclasses of Anti-HBe IgG in Chronic Hepatitis B
3
TABLE I. Background Data on 82 Patients With Chronic Hepatitis B Providing Sera for the Study
Epidemiology Immigrants Homosexuals Drug addicts Renal recipients Other Totals
Mean age (years) 29.9 24.0 31.9 29.3 37.0 30.4
No. of patients 60 3 10 3 6 82
Male 36 3 8 3 6 56
Female 24 0 2 0 0 26
No. of patients with HBeAg HBV DNA 18" 17 2 2 7b
0 4 31
7
0 4 30
~'HBVDNA was found in 16 sera positive for HBeAg and in two sera positive for anti-HBe. hHBV DNA was found in six sera positive for HBeAg and in one serum with low-level anti-HBe (55% inhibition).
sorbances within the slope of the dose-response curve from the dilutions were used to construct a standard curve. This was linearised by least-squares analysis. Only sample dilutions with absorbances within the slope of the standard curve were used for relative endpoint titre determinations, which were calculated from the intersection of the absorbancy with the standard curve. In each assay a t least 15 different sera, negative for all HBV markers, were used for calculation of the mean (x) and standard deviation (SD) of the absorbances for negative samples. In the anti-HBc and antiHBe IgG subclass assays the positive cut-off was mean ? 3 and 7 SD, respectively. In the antibody capture assays the positive cut-off was mean 7 SD.
*
Statistical Methods Any significance deemed significant was found so, if not otherwise stated, by using Student's t test or Fisher's exact test when appropriate. RESULTS Epidemiological background data, sex, and mean age of the 82 patients with chronic hepatitis B are given in Table I. Anti-HBc Classes and Subclasses The outcomes of testing the patient sera for classes and subclasses of anti-HBc are shown in Table 11. The positive rate for anti-HBc IgM was significantly higher for HBV DNA-positive sera as compared with HBV DNA-negative sera ( P < 0,001). The geometric mean titre (GMT) of anti-HBc IgM was, however, not significantly higher in sera positive for HBV DNA than in sera negative for the marker. The GMT of anti-HBc IgAl was significantly higher in HBV DNA-positive sera compared with HBV DNA-negative sera ( P < 0.01). Fifteen of 31 HBV DNA-positive sera had high levels (>10,000) of anti-HBc IgAl compared with 6 of 51 sera negative for HBV DNA ( P < 0.001). High levels of anti-HBc IgAl were significantly correlated with high levels of anti-HBc IgM (Table 111, A). IgGl was found to be the main subclass of anti-HBc IgG and was detected in 81 of 82 patients. Only one (from a male immigrant) positive for HBeAg but negative for HBV DNA was also negative for anti-HBc IgGl. Anti-HBc
IgG2 was not detected in any of the tested sera. Frequencies and GMTs of anti-HBc IgG3 and IgG4 did not differ significantly between HBV DNA-positive sera and HBV DNA-negative sera. High levels of anti-HBc IgG3 were, however, significantly correlated to high levels of anti-HBc IgAl (Table 111, B; P < 0.001). Such a relation was not found between anti-HBc IgG3 and anti-HBc IgM. Anti-HBe Classes and Subclasses The performance of the EIAs for subclasses of antiHBe IgG in relation to the commercial RIA for antiHBe (Abbott) is shown in Table IV. Forty-three of 51 sera positive in RIA were also positive by the EIA for anti-HBe IgGl, giving a sensitivity of 84%.Of the eight sera not detected by the EIA for anti-HBe IgG1, five gave low-level inhibition (85% inhibition, one was found positive for anti-HBe IgG4 and two were not detected by any of the anti-HBe class- or IgG subclass-specific EIAs. Of these two sera, one was positive for HBV DNA. The result of testing the patient sera for subclasses of anti-HBe IgG is shown in Table V. No serum was found repeatedly positive for antiHBe IgM, IgA1, or IgG2. Anti-HBe IgGl was detected in two of the HBV DNA-positive sera, of which one was also HBeAg positive. Comparison of Anti-HBc and Anti-HBe Classes and Subclasses The relative levels of anti-HBc IgG subclasses were IgGl >> IgG3 > IgG4 and for anti-HBe IgG, IgGl 2 IgG4 2 IgG3. GMT of anti-HBc IgGl and IgG3 were much higher than the corresponding anti-HBe levels. The relation between anti-HBe IgG4 and anti-HBc IgG4 in the 51 anti-HBe-positive sera are shown in Table 111, C. Anti-HBe IgG3 was not correlated to antiHBc IgG3, whereas the correlation between anti-HBe IgG4 and anti-HBc IgG4 was found to be highly significant ( P < 0.001). The ratio between anti-HBc IgGl and IgG3 titres was 520:1, and the corresponding ratio for anti-HBe was 11.O:l. The ratios between IgGl and IgG4 titres for anti-HBc and for anti-HBe were 890:l and 2.7:1, respectively.
Sallberg et al.
4
TABLE 11. Prevalence and Titres of IgM and IgAl Anti-HBc and Subclasses of Anti-HBc IgG in Relation to HBV DNA Results in Sera From 82 Patients With Chronic HeDatitis B No. of sera with indicated HBV DNA result 31 + 51 Totals ~
IgM G M T ? SD 12,102"14 1,306"7.4 5,112"14
n 19 12 31
Number ( n ) and geometric mean titre (GMT) of sera positive for anti-HBc of indicated class and subclass IgAl IgGl IgG3 n GMT? SD n GMT"SD n G M T " SD 21 8,236' 17 30 260,010?3.4 20 27,309"12 29 5,707"24 51 319,644T2.6 30 769T7.8 50 6,658"20 81 296,110?2.9 50 3,208"17
IgG4 G M T * SD 1,572'16 2,760"lO 2,299"12
n 12 25 37
TABLE 111. Relation Between A: Presence of High Levels* of Anti-HBc IgM and High Levels of IgAl in 82 Patient Sera; B: Presence of High Levels of Anti-HBc IgG3 and High Levels of Anti-HBc IgAl in 82 Patient Sera; and C: Presence Anti-HBc IpG4 and Anti-HBe IeG4 in 51 Patient Sera Positive for Anti-HBe in RIA Anti-HBc IeA 1
+ ~
Totals
A: high levels of Anti-HBc IgM
+
-
14
6 60 66
2 16
Anti-HBc IeA1
B: high levels of Anti-HBc IgG3
+ ~
P> IgG3 > IgG4, whereas for anti-HBe the relative levels were IgGl 2 IgG4 2 IgG3. Thus the IgG4 to IgGl ratio was proportionately higher for anti-HBe than for anti-HBc. A prolonged antigenic stimulation has been suggested to result in a more IgG4-restricted response [Aalberse et al., 1983; Skvaril, 19863, which may explain the relatively higher IgG4 response to HBeAg, since in general patients with chronic hepatitis B and anti-HBe have been carriers for a longer period than patients with HBeAg. This may also explain the higher frequency and levels of anti-HBc IgG4 in HBV DNA-negative sera. The differences in humoral responses to HBcAg and HBeAg might be due to the latter antigen being strictly T celldependent, thereby limiting the pathways for triggering the immune system. Our finding of a n IgGrestricted response to HBeAg in chronic hepatitis B, together with the inability to demonstrate anti-HBe IgM or IgA despite a persistent antigenemia, further strengthens the strict T cell-dependence of HBeAg. Further, immune complexes with T cell-dependent antigens have been reported to have a n inhibitory effect on IgM production [Bruderer and Heusser, 19881. If so, a weaker anti-HBe IgM response would be expected, since HBeAg complexed to immunoglobulins has been reported in patients with hepatitis B [Takahashi et al., 1978; Takekoshi et al., 19791.
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