IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.     

Chronic liver disease: the detection and characterization of circulating immune complexes.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.     

Immunoglobulin isotypes of anti-HBc and anti-HBe and hepatitis B virus (HBV) DNA elimination in acute hepatitis B

Antibodies to hepatitis B core antigen (anti-HBc) and e antigen (anti-HBe) were assayed in 46 sera from ten patients with acute hepatitis B utilizing immunoglobulin class- and subclass-specific enzyme immunoassays (EIAs). The sera were sampled 1 to 512 days after onset of hepatic symptoms. Four patients cleared HBsAg rapidly, within 24 days, and six patients cleared HBsAg slowly, within 27-74 days after the onset of symptoms. In three of the patients with rapid clearance of HBsAg, hepatitis B virus (HBV) DNA was not detected in sera tested during the first week after onset. The fourth patient was not tested until 12 days after onset and was then found to be negative for HBV DNA. In four of the patients with slow clearance of HBsAg, HBV DNA was present during the first week of illness. In the other two patients, HBV DNA was not detected in the first serum, 11 and 17 days after the onset of illness. Anti-HBc IgM and IgA1 were detected in all patients, with maximum titers shortly after onset. Anti-HBc IgG1 was present in all sera tested. Anti-HBc IgG2 was not detected in any of the sera. Anti-HBc IgG3 and IgG4 were detected in all patient sera, with IgG3 paralleling IgG1, and IgG4 mainly in sera long after onset. Anti-HBe IgG1, IgG3, and IgG4 were detected in three, two, and two patients, respectively. Anti-HBe IgG2, IgM, IgA1, or IgA2 was not found in any patient. The time required for maximum titer of anti-HBc IgG1 was shorter in the patients with rapid clearance of HBsAg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of antigen moiety of HBsAg in the complement fixing immune complexes of hepatitis B virus positive chronic active hepatitis.

Circulating immune complexes (CIC) are frequently found in hepatitis B virus-induced chronic active hepatitis. Since antigen and antibody moieties of complexes are critical in determining many of its pathogenic factors, the constituents of these complexes were investigated with particular attention to the quantity and nature of the HBs antigen moiety of the complexes. Complement fixing immune complexes were isolated from sera of 14 patients with chronic active hepatitis by utilizing conglutinin's unique property to bind C3-fixed complexes. Low pH (2.6) was used to dissociate the complexes. Components of dissociated complexes were separated into antigen and antibody fractions using immobilized Protein A. Both fractions were analysed by electrophoresis in polyacrylamide gel with SDS. The antibody fractions showed heavy and light chains of IgG and IgM. The antigen fractions demonstrated six to 10 protein bands with mol. wt ranging between 17,000 and 120,000 daltons. To define precisely the polypeptide antigen moiety involved in the immune complex formation, a transfer blotting technique was used employing human anti-HBs globulin as probe. Polypeptides with mol. wt 97,000 and 49,000 reacted as antigen moieties of HBsAg. In addition, the levels of HBsAg in the antigen fractions were significantly greater (P less than 0.005) compared to sera from patients with acute hepatitis. Implications of these findings are discussed.     

Serum immunoglobulins in viral hepatitis

The serum levels of IgA, IgG and IgM were determined by single radial immuno-diffusion technique in 104 male patients of acute viral hepatitis. HBsAg was detected by counterimmuno-electrophoresis (CIEP) and 51% were HBsAg + ve. IgA, IgG and IgM levels were found to be higher than normal levels. Mean IgA and IgG levels were same in HBsAg + ve and HBsAg--ve patients. Mean IgM levels were however, higher in HBsAg--ve patients.     

Hepatitis B surface antigen containing immune complexes occur in seronegative hepatocellular carcinoma patients

IgG, IgM and hepatitis B surface antigen (HBsAg) containing immune complexes (IC) were detected by the Clq and conglutinin solid phase assays in both HBsAg+ and HBsAg- groups of patients with primary hepatocellular carcinoma (HCC). No differences were observed between the two patient groups either in the levels of antigen non-specific and HBsAg specific complexes or in the immunoglobulin isotype in the complexes. The results show that HBsAg can occur in an IC form in the sera of patients classified as HBsAg- by sensitive commercial assays and provides evidence of a further association of hepatitis B virus (HBV) and HCC in antigen negative patients. Furthermore, the HBsAg IC in HCC patients differ from those in other HBV infected subjects in that they are preferentially detected by the Clq assay.     

Immune complex myositis associated with viral hepatitis

Sarcolemmal osmiophilic deposits were demonstrated by electron microscopy in the striated muscle of a patient who developed viral hepatitis associated myositis. HBsAg, IgG, and C3 were found in the same location, suggesting that the deposits represent immune complexes and the primary cause of myositis.     

Prognostic value of HBsAg/IgM complexes in hepatitis B patients: nature of the proteins involved

Hepatitis B surface antigen (HBsAg)/IgM complexes were measured using a solid phase radioimmunoassay in sera of patients with acute or chronic hepatitis B. These complexes were found in 6 out of 18 patients four weeks after the onset of the disease and only one of them developed chronic hepatitis. HBsAg/IgM complexes correlated with HBsAg, hepatitis B e antigen (HBeAg), and pre-S2 concentration. The precipitation of HBsAg/IgM reactivity by polyethylene-glycol (PEG) and the binding of this activity to the surface of certain uncoated enzyme linked immunosorbent assay (ELISA) plates indicates that HBsAg/IgM positivity may reflect the presence of circulating complexes in serum. HBsAg and pre-S2 were found as components of the complexes but anti-HBs, anti-pre-S2, and polymerized human serum albumin (pHSA) were not. An immune binding between HBsAg and IgM is still questionable. Whatever the nature of the HBsAg/IgM complexes, their detection does not seem to be an earlier indicator of prognosis than HBeAg and/or pre-S2.     

Association of increased immune complexes, platelet IgG and serum IgG in chronic active hepatitis

Platelet associated IgG (PA-IgG), serum IgG and immune complexes (IC) were measured and correlated with the degree of thrombocytopenia in 32 patients with chronic active hepatitis (CAH), classified as of autoimmune type in 24 and cryptogenic in eight. Of the 32 patients, 24 had raised PA-IgG and 18 of these were thrombocytopenic (greater than 150 X 10(9) platelets/1); in only two thrombocytopenic patients was the level of PA-IgG not increased. Platelet counts showed a significant inverse logarithmic correlation with PA-IgG levels (r = 0.456, P less than 0.01). Twelve patients had raised levels in serum of ICs, 11 of these also had raised PA-IgG and 10 of these 11 were thrombocytopenic. Serum IgG was raised in 10 patients and correlated with PA-IgG (r = 0.489, P less than 0.01). Of the 24 patients with autoimmune CAH, 13 had low platelet counts and 10 had raised levels of PA-IgG whereas of the eight patients with cryptogenic CAH, seven had raised PA-IgG and all were thrombocytopenic. These results for patients with CAH show that raised levels of circulating ICs are associated both with thrombocytopenia and with raised PA-IgG, and support the hypothesis that serum IgG and/or ICs may influence amounts of PA-IgG.     

HBsAg immune complexes in the course of infection with hepatitis B virus

Serial serum samples from 113 patients with different forms of HBV-related liver disease and HBsAg carriership were tested for the presence of HBsAg, anti-HBc, anti-HBs, and HBsAg-anti-HBs immune complexes (IC). Eight patients with acute type B hepatitis had the irmultiple serum samples tested in an average period of time from 68 days before the appearance of clinical symptoms up to 277 days after the onset of clinical symptoms. In the remaining cases serum samples were obtained during the period after the appearance of clinical symptoms.

The highest frequency of immune complexes of HBsAg was observed in acute hepatitis (twenty-eight out of thirty examined cases—93·3%). The patients showing high level of anti-HBs response eliminated HBsAg from the circulation earlier than the patients showing low level of anti-HBs response. In chronic aggressive hepatitis the frequency of HBsAg complexes was higher (ten out of twenty-five cases—40%) than in chronic persistent hepatitis (two out of nine cases—22%); HBsAg complexes were found in four out of twenty-two symptomless carriers of HBsAg (18%).

The obtained results are in agreement with the hypothesis that an optimal humoral immune response at the acute stage of hepatitis type B results in rapid elimination of HBV antigens. Conversely, an inadequate response at this stage favours replication of the virus in hepatocytes, prolongation of HBs antigenaemia, and the appearance of chronic forms of hepatitis.

     

Immunoglobulin and hepatitis B surface antigen-specific circulating immune complexes in chronic hepatitis with hepatitis delta virus infection

IgM, IgG, and HBsAg containing circulating immune complexes (CIC) were determined, by conglutinin (K) and C1q assays, for assessing the role of CIC in hepatitis delta virus (HDV) infection in 54 HBsAg-negative controls and 85 HBsAg-positive patients with chronic hepatitis. The prevalence of HDV markers (HDV antigen and anti-HD) was 24.70% (21/85). CIC were a common feature of HDV infection with 95.24% of patients having at least one abnormal test resutlt. The prevalence of elevated IgM-K, IgG-K, IgM-C1q, and IgG-C1q CIC were 85.71, 85.71, 57.14, and 85.71%, respectively. The prevalence of IgM class CIC were statistically higher in patients with HDV infection than in those without (P = .001 for the K assay and P = .023 for the C1q assay). There was no difference in the prevalence of IgG class CIC. Patients with HDV infection also have significantly higher median levels of IgM K-CIC (P = .002), IgG K-CIC (P = .049), and IgG C1q-CIC (P = .008). In patients with HDV infection, there was positive correlation between IgM C1q-CIC and transminase levels (r = .519, P = .016 for AST; r = .500, P = .021 for ALT). There was no difference in the prevalence of HBsAg containing CIC between patients with HDV infection (76.19%) and those without (74.60%). In conclusion, IgM class CIC are the major CIC and correlate with disease activity in HDV infection. CIC may play a role in the pathogenesis of HDV infection.     

Excretion of hepatitis A virus in the stools of hospitalized hepatitis patients

A study was carried out to determine whether hepatitis A virus (HAV) can be detected in the stools of patients hospitalized for HAV infection. Acute phase samples of whole blood and stool, as well as completed questionnaires, were obtained from 31 patients hospitalized at any of 13 hospitals in the Phoenix metropolitan area. Blood specimens were tested for hepatitis B surface antigen (HBsAg), IgG antibody to HAV (IgG anti-HAV), and IgM antibody to HAV (IgM anti-HAV). Stools were tested for HAV by radioimmunoassay. Five patients (16.1%) had acute hepatitis B, five (16.1%) had acute non-A/non-B hepatitis, and 21 (67.7%) had acute hepatitis A. Of these 21 patients with acute hepatitis A, 11 (52.4%) were found to have HAV in their stools. These results confirm the potential for infectivity of stools of patients hospitalized for hepatitis A and emphasizes the need for caution when dealing with such stools.     

Kidney glomerular pathology in various forms of acute and chronic hepatitis.

Kidney tissue from 99 unselected necropsy cases of various forms of hepatitis and liver cirrhosis was examined by histology and direct immunofluorescence. Glomerular deposits of hepatitis B surface antigen (HBsAg), IgG, IgM, and complement were found in nine of 59 cases (15%) of acute and subacute hepatitis and in seven of 40 cases (17%) of chronic aggressive hepatitis and liver cirrhosis. Different amounts of granular hepatitis B surface antigen immune deposits were distributed along glomerular capillary walls and/or in mesangial areas. Glomerular lesions found in these cases consisted of thickening of glomerular capillary walls, a slight increase in glomerular cellularity, and an increase of mesangial matrix. These glomerular lesions are considered to result from the humoral immune elimination of circulating viral surface antigen immune complexes.     

Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes

Antigen-antibody complexes have been shown to enhance immune responses against several antigens given by parenteral immunization. Herein, we have evaluated the potential of administering such immunostimulatory complexes by a mucosal route. Hepatitis B surface antigen (HBsAg) complexed with antibodies against HBsAg (anti-HBs) (HBsAg/Ab) was administered to BALB/c mice by intranasal inhalation. HBsAg by itself did not induce immune responses, whereas with HBsAg/Ab complexes, both systemic and mucosal immune responses were observed and these could be modulated by adjuvants. With HBsAg/Ab (1 or 10 microg), anti-HBs antibodies induced were predominantly of the IgG1 isotype (Th2-like). In contrast, anti-HBs induced by HBsAg/Ab plus cholera toxin (CT) or oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) (1 microg each) were predominantly IgG2a (Th1-like). Results from this study indicate that HBsAg/Ab complexes can induce strong humoral immune responses when delivered by a noninvasive route, whether used alone or in combination with other mucosal adjuvants.     

Nuclear fluorescence of liver cells for IgG in viral hepatitis B: Significance and relation to hepatitis B-core and anti-hepatitis B-core formation

Summary The occurrence of anti-HBcAg antibodies in the blood as determined by indirect immunofluorescence and its relation to the occurrence of HBsAg in the cytoplasm and of HBcAg and IgG in the nuclei of hepatocytes were studied in the following groups of patients (total of 123 biopsies): I. 64 HBAg-negative patients with various liver diseases; II. 51 HBAg-positive patients without therapeutical immunosuppression (6 acute hepatitis, 10 nonspecific reactive and 10 chronic persistent hepatitis, 19 chronic aggressive hepatitis, 6 Hippie-hepatitis); III. 8 kidney transplant recipients.It could be shown that nuclear IgG is found only if both parameters can be demonstrated at the same time: HBcAg in liver cell nuclei and anti-HBcAg antibodies in the serum in titers higher than 1:64. Accordingly, all types of hepatitis with excess formation of nuclear HBcAg (early phase of acute hepatitis, chronic aggressive hepatitis and chronic non-aggressive forms with generalized core formation, i.e. carrier state or chronic persistent hepatitis of the HBc type) may show nuclear fluorescence for IgG. All forms of hepatitis B without detectable core formation (acute hepatitis in the elimination phase, chronic nonaggressive hepatitis with isolated HBsAg expression, i.e. carrier state or chronic persistent hepatitis of the HBs type, posthepatitic phase) do not present nuclear IgG despite eventual anti-HBcAg formation. Finally, lack of anti-HBcAg or very low titers associated with lack of IgG in hepatocytic nuclei do not exclude generalized core formation in liver cell nuclei in chronic persistent hepatitis of effectively immunosuppressed patients.Although the demonstration of nuclear IgG has several diagnostic and prognostic consequences in common with the demonstration of HBcAg, a specific search for the core antigen in the tissue is needed for the correct appraisal of the HBcAg- and HBsAg tissue expression pattern and the associated disease.     

Liver cell surface localization of hepatitis B antigen and of immunoglobulins in acute and chronic hepatitis and in liver cirrhosis.

This paper describes immunofluorescence studies on liver cell surface localization of hepatitis B surface antigen (HBsAg) and of IgG in acute and chronic hepatitis and in cirrhosis. In acute hepatitis B, HBsAg was found at the surface of hepatocytes in an early phase of the disease, but not during the recovery. This finding is consistent with the hypothesis that immune reactions to HBsAg may be responsible for the liver cell lysis. In HBsAg-positive chronic hepatitis and cirrhosis the antigen was found in the cytoplasm, but not on the surface of the hepatocytes, while in HBsAg-negative cases the antigen could not be detected in the liver cells. Both in HBsAg-positive and in HBsAg-negative chronic active hepatitis (CAH) and cryptogenic cirrhosis IgG bound to the membrane of the hepatocytes could be detected, suggesting a role of antibodies in the pathogenesis of the disease.     

Hepatitis B surface antigen/IgM complexes: relation to receptors for polymerized human serum albumin, hepatitis B virus (HBV) DNA polymerase activity and HBV markers

The presence and the level of hepatitis B surface antigen (HBsAg)/IgM complexes were determined in 54 chronic HBsAg carriers in relation to receptors for polymerized human serum albumin (pHSA-R) tested by specific radioimmunoassay, and to hepatitis B virus-DNA polymerase (HBV-DNAp). HBsAg/IgM complexes, correlated significantly with the HBsAg concentration but, at a similar HBsAg concentration, significant highest values of HBsAg/IgM complexes were found among HBeAg positive patients. In addition, a significant correlation was found between HBsAg/IgM complex levels, HBeAg titres and HBV-DNAp activity (r = 0.628, p less than 0.001 and r = 0.559, p less than 0.001, respectively). Moreover, a positive linear correlation was found when comparing HBsAg/IgM complexes and pHSA-R levels (r = 0.848, p less than 0.001). Patients who were positive for HBsAg/IgM complexes had a significantly higher glutamate-pyruvate transaminase (GPT) level than those who did not show any complexes. In conclusion, HBsAg/IgM complexes seemed to be indirectly related to HBV replication.     

Detection and characterization of circulating immune complexes during acute exacerbation of chronic viral hepatitis

For the detection and characterization of circulating immune complexes (CIC) in various liver diseases, a Clq binding test was used. Though the CIC level was almost normal in HB surface antigen (HBsAg) positive asymptomatic carriers, the level increased in patients with liver diseases. During acute exacerbation of chronic viral hepatitis, the CIC level reached peaks 1 to 3 weeks before and after the hepatic cell necrosis. Study of the sedimentation rates of CIC in various liver diseases showed CIC in the 19s-22s region and in the 7s-19s region. In acid buffer, CIC was dissociated into 5 to 6 components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In one case of HBsAg positive severe chronic aggressive hepatitis, CIC was composed of HBsAg, IgG and another three or four undetermined components. During acute exacerbation of chronic hepatitis, minor changes of these dissociation patterns of CIC were observed.     

Human IgG Rheumatoid Factors and RF-Like Immune Complexes Induce IgGl Rheumatoid Factor Production in Mice

The synovial fluid of patients with rheumatoid arthritis (RA) was found to contain IgG and /or IgG-containing immune complexes (ICs) that stimulated an intense antibody formation when injected into mice of certain strains, notably of NZ background. The response was characterized by high and sustained levels of IgG1 antibodies with rheumatoid factor (RF) activity. In the study described, we investigated whether it is the antibodies with RF activity in the synovial fluid, that are responsible for stimulation of mouse RF in vivo. Different mouse strains were injected with synovial fluid from a seropositive RA patient (RA-SF), with human monoclonal antibodies with RF activity, with a human non-RF monoclonal antibody or with different preformed RF-like antibody antibody (Ab-Ab) ICs. The experimental mice were monitored subsequently for IgGl RF production. IgGl RF antibodies were found in all strains (NZB, BALB/c and CBA) injected with Ab-Ab ICs formed at equivalence, but only in NZB using RA-SF or human monoclonal antibodies with RF activity. Optimal production of IgG I RF by Ab-Ab ICs required the integrity of Fc and F(ab)'₂ portions respectively of the antibodies; soluble and truncated ICs were less effective. Further studies demonstrated that the IgGl RF response was not simply the result of a specific immune response against human IgG, since humoral immunity against human IgG was induced only when combined with an efficient adjuvant. During a typical adjuvant-associated primary response specific antibodies of IgM, IgG1 and IgG2a isotypes were found, i. e. quite different from the selective IgGl response induced by RF-like containing immune complexes. This conclusion is substantiated further by the clear differences in responses to IgG containing fraction obtained from RA-SF in NZ mice compared to other strains. Our findings argue for a different type of reaction leading to the selective IgGl response and might aid in elucidating the mechanisms for chronic production of antibodies with RF activity in patients with RA.     

Isolation and partial characterization of circulating immune complexes in sera of children with HBV-mediated glomerulonephritis.

Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.