Anti-idiotypic humoral and cellular responses to antibody to hepatitis B surface antigen in hepatitis B viral infections.

In order to investigate regulatory significance of humoral and cellular responses to the idiotypic (Id) determinants on the antibody to hepatitis B surface antigen (anti-HBs), they were studied in acute hepatitis B and in chronic HBV infection. The results were compared with humoral and cellular responses of the same patients to hepatitis B surface antigen (HBsAg). In acute hepatitis B, the responses to HBsAg, were delayed until 3-4 weeks after the onset of clinical symptoms. However, the leucocyte migration inhibition (LMI) and the lymphocyte transformation (LTT) responses to affinity purified anti-HBs were found to be evolved very early in the course of acute hepatitis B, though anti-Id antibodies were absent. The majority of chronic HBV carriers showed a poor humoral and cellular response to HBsAg. Ten out of 38 chronic carriers showed anti-Id antibodies which recognized a major cross-reactive idiotype (CRI) on the anti-HBs molecule. Twenty-five out of 38 chronic carriers also showed LMI response to the Id determinants on the anti-HBs. LMI response induced by anti-HBs could be blocked by a specific Balb/c anti-Id antibody which also recognized the CRI. Thus, in both acute and chronic HBV infections, the anti-Id humoral and cellular responses correlated with poor humoral and cellular responses to HBsAg, indicating regulatory significance.     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

Anti-hepatitis B surface antigen IgG1 subclass is predominant in individuals who have recovered from hepatitis B virus infection, chronic carriers and vaccinees

Patterns of each IgG-specific subclass for hepatitis B virus (HBV) core antigen (anti-HBc) are remarkably different among individuals with different infection status, i.e., completely recovered or chronic carrier. Each of the IgG-specific subclasses of HBV surface antigen (anti-HBs) was tested for ELISA sensitivity using four commercially available hepatitis B surface antigen (HBsAg) kits and one self-prepared plate. The specificity in 18 serum samples obtained from chronic HBV carriers, recovered individuals, vaccinees and non-infected individuals was investigated. Differences in absorbance values were obtained by comparing results from these different plates. Data on the absorbance values of anti-HBs IgG subclasses obtained indicated that one to four subjects had a false-negative or false-positive result using the four commercial plates. Only the self-prepared plate demonstrated 100% specificity and sensitivity for anti-HBs subclasses. Moreover, the results indicate that anti-HBs subclass IgG1 was predominant in cured patients, chronic carriers and vaccinees. The samples from both chronic carriers and vaccinees exhibited a significantly higher concentration of total IgG and IgG1 than samples in recovered individuals (P<0.05).     

Occult hepatitis B virus infection.

The detection of HBV DNA without HBsAg with or without the presence of HBV antibodies outside the acute phase window period defines occult HBV infection. This condition has been described in hepatocellular carcinoma (HCC), chronic hepatitis B, healthy HBV carriage and recovered infection, chronic hepatitis C and individuals without serological markers of HBV. The frequency of the diagnosis depends on the relative sensitivity of both HBsAg and HBV DNA assays. It also depends on the prevalence of HBV infection in the population. Occult HBV in blood donors has a wide range of potential origins within the natural history of the infection. It may originate from recovered infections with anti-HBs and persistent, low-level, viral replication, escape mutants undetected by the HBsAg assays or healthy chronic carriage. The last situation is mostly found with anti-HBc only. Over time, antibody markers may become undetectable leaving HBV DNA as the only marker of the infection. In all cases, the viral load is low, mostly below 10(4) IU/ml, often below 100 IU/ml. At these levels, nucleic acid testing (NAT) in pools is likely to be largely ineffective. Is occult HBV transmissible by transfusion? Carriers of anti-HBs or anti-HBc only were shown infectious in immunosuppressed organ or bone marrow transplant recipients. In immunocompetent recipients, there is no evidence that anti-HBs-containing components are infectious, even in low titre. Donations carrying anti-HBc only and HBV DNA can be infectious and this is a threat where anti-HBc is not screened. Anti-HBc screening identifies most occult HBV infection but not all. HBV NAT needs either extreme sensitivity or to be performed on individual donations to eliminate HBV DNA-containing units.     

Acute hepatitis B viral infection in a patient with anti-HBs.

We report a patient with antibody to hepatitis B surface antigen (anti-HBs) but no antibodies to other hepatitis B virus components, who developed acute symptomatic type B hepatitis. The possible explanations for this unusual serological pattern are 1) the antibody-positive status, which developed against only a subdeterminant of hepatitis B surface antigen (HBsAg), arose naturally or as the result of cross-reaction with a variety of antigens; and 2) seroconversion to anti-HBs occurred in response to surface antigen of a mutant strain of hepatitis B virus (HBV). This anti-HBs positivity, in the absence of antibody to hepatitis B core antigen, does not provide natural immunization against HBV infection, and so is not protective. Individuals who are positive to anti-HBs antibody alone which is not elicited by HBV vaccine, should be vaccinated against possible HBV infection.     

Production and efficacy of a dendritic cell-based therapeutic vaccine for murine chronic hepatitis B virus carrierer

Recently a new field of immunological research and clinical application of vaccines for therapeutic purposes (vaccine therapy) has been developed for treating several chronic viral infections including chronic hepatitis B virus (HBV) infection. Administration of vaccine containing hepatitis B surface antigen (HBsAg) for 1 year has resulted in negative HBsAg and development of antibody to HBsAg (anti-HBs) in some, but not in all, HBV transgenic mouse (HBV-Tg). In order to develop more potent regimen of vaccine therapy for chronic HBV carrier, we prepared a dendritic cell (DC)-based therapeutic vaccine and evaluated their therapeutic potential in HBV-Tg. DCs were isolated from single cell suspensions of murine spleen cells by collagenase digestion, density centrifugation and depletion of lymphocytes. Spleen DCs were cultured with HBsAg (100 microg) for 24 h to produce HBsAg-pulsed DCs. HBV-Tg expressing HBsAg and HBV DNA in the sera were randomly assigned to receive either HBsAg-pulsed DCs (n = 20) or unpulsed DC (n = 20) or vaccine containing HBsAg (n = 39) or complete Freund's adjuvant (n = 20) or left untreated (n = 20). Only two intraperitoneal injections of HBsAg-pulsed DCs resulted in negative HBsAg and production of anti-HBs in the sera in all HBV-Tg (n = 20). However, administration of un-pulsed DCs (n = 20) or vaccine containing HBsAg (n = 39) or only complete Freund's adjuvant did not induce negative HBsAg or production of anti-HBs in any HBV-Tg within 6 months of therapy commencement. Taken together, this study showed that HBsAg-pulsed DCs represent a highly potent therapeutic vaccine for chronic HBV infection and inspire optimism of using this vaccine in clinical conditions. A clinical trial of HBsAg-pulsed DC in patients with chronic hepatitis B is warranted.     

Co-occurrence of HBsAg and anti-HBs: Two consecutive infections or a sign of advanced chronic liver disease?

Simultaneous presence of hepatitis B surface antigen (HBsAg) and antibodies to the surface antigen (anti-HBs) was detected in 32 out of 89 Dutch chronic hepatitis patients of Caucasian race. HBsAg was subtyped ad in 28 and ay in four cases. Anti-HBs could be subtyped in 25 cases using reference antigens discriminating between d, y, and w1-4 subdeterminants. In 20 patients HBsAg subtype ad (HBsAg/ ad) was accompanied by antibody to subdeterminant y (anti-y), whereas HBsAg/ ay and anti-d were simultaneously detected in the serum of one patient. The antibody pattern in sera from the remaining patients was complex. Eighteen anti-HBs-positive patients were matched for age, histology, and hepatitis B e antigen (HBeAg) status with 18 anti-HBs-negative patients. Differences in risk factors for acquiring a hepatitis B infection were not found. These results do not support the hypothesis that co-occurrence of HBsAg and anti-HBs is due to two consecutive infections with hepatitis B virus. The frequency of the co-occurrence of HBsAg and anti-HBs was found to be related to the degree of progressive liver disease, since anti-HBs was found in three out of 23 asymptomatic carriers, in four out of 20 chronic persistent hepatitis patients, in 20 out of 41 chronic active hepatitis patients, and in all five patients with chronic active hepatitis and cirrhosis. The high frequency of anti-HBs in advanced liver disease may be the result of a disturbed immunologic response mechanism.     

Monoclonal antibody recognizing pre-S(2) epitope of hepatitis B virus: characterization of pre-S(2) epitope and anti-pre-S(2) antibody

A hybrid cell line producing monoclonal antibodies recognizing an epitope encoded by the pre-(S)2 region of hepatitis B virus (HBV) genome was obtained by fusion of mouse myeloma cells with lymphocytes from mice immunized with HBV. The monoclonal antibody Mo-F124 secreted from the hybrid line reacted with the pre-S(2) epitope expressed on the surface of both viral and recombinant HBsAg particles--pre-S(2) and S gene product--localised on 34 kD glycoprotein of the viral envelope. The pre-S(2) epitope was sensitive to digestion with V8 protease from Staphylococcus aureus. The enzyme abolished reactivity with Mo-F124 and polymerized human serum albumin (pHSA) binding activity of recombinant particles. Mo-F124 antibody was used to develop highly sensitive radioimmunoassays for determination of pre-S(2) epitope and anti-pre-S(2) antibody in sera of hepatitis B patients. Detection of a pre-S(2) epitope by the monoclonal antibody-based assay in the early phase of acute HBV infection correlated well with the presence of markers of active viral replication (HBeAg, HBV DNA). The appearance of anti-pre-S(2) antibody, usually in the third month after onset of symptoms, was followed by elimination of circulating HBsAg and seroconversion to anti-HBs in all tested cases of uncomplicated acute hepatitis followed by recovery. Anti-pre-S(2) response was not observed in patients with chronic hepatitis B or acute HBV infection progressing to chronic disease. The observed correlation of anti-pre-S(2) response with recovery suggests that the pre-S(2) epitope may represent one of the epitopes inducing antibodies that neutralize the hepatitis B virus.     

Diagnosis of type B hepatitis

Since 1965, when Blumberg discovered the Australia antigen, the hepatitis B surface antigen (HBsAg), the research on viral hepatitis has rapidly progressed. The identification of specific hepatitis B associated antigens and antibodies in blood, and liver tissue, together with the improvement of detection systems, have enhanced our knowledge about the mechanism of liver injury and the natural history of hepatitis B virus (HBV) infection. Now it has been recognized that HBV has no direct cytopathic effect on hepatocytes and that hepatocyte necrosis is associated with the virus induced immunological reaction of the host. From the reaction, there are two types of HBV infection, i.e., transient (acute) and persistent (chronic) infection. In addition to the conventional measurements, such as HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe and anti-IgM HBc, recently pre S1, pre S2 antigen/antibody systems and polymerized human albumin receptor and antibody have been developed. The significance of the detection of these antigen/antibody systems was discussed. On the other hand, to determine the presence of HBV, the state of HBV replication or the infectivity directly, HBV associated DNA polymerase and HBVDNA should have been detected. (Very recently, the polymerase chain reaction method has been introduced to detect very small amounts of HBVDNA). In this presentation, the change of these viral markers in various cases was shown, and especially emphasized was anti-IgM HBc in acute hepatitis and HBeAg/Ab status in chronic liver disease. Lastly, the present state of Interferon therapy for type B chronic hepatitis was mentioned.     

Diagnostic value of anti-HBc IgM in high HBV prevalence areas

The diagnostic value of an anti-mu-capture immunoassay for the detection of IgM antibody against hepatitis B core antigen (anti-HBc) was evaluated. Strongly positive results were obtained from the acute phase sera of the 25 acute hepatitis B patients who were hepatitis B surface antigen (HBsAg) positive and of the 18 confirmed acute hepatitis B patients who had already cleared HBsAg when symptoms developed. Negative results were obtained in 5 hepatitis A patients, 20 non-A, non-B acute hepatitis patients serologically susceptible to HBV, 22 patients with chronic hepatitis B liver disease, 15 asymptomatic HBsAg carriers, and 10 healthy patients immune from past HBV infection. Fourteen of the acute hepatitis patients remained HBsAg positive for a follow-up period of at least 6 months, and 12 of these were found consistently anti-HBc IgM negative. These were considered as chronic HBsAg carriers with a superimposed form of acute liver injury. These data show that this assay can differentiate between acute from chronic (HBsAg positive) and recent from old (HBsAg negative) hepatitis B virus infection. Thus, it should be very useful in the complex diagnostic situations encountered commonly in areas with high prevalence of HBV infections.     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

Long-term persistence of hepatitis B surface antigen and antibody induced by DNA-mediated immunization results in liver and kidney lesions in mice

DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV) infection. However, the side effects of this approach have not been well described. Here we report that DNA-mediated immunization by intramuscular injection of plasmid DNA encoding HBV surface antigen (HBsAg) induced long-term persistence of HBsAg and HBsAg-specific antibody (anti-HBs) in the sera of the immunized BALB/c mice and resulted in liver and kidney lesions. The lesions persisted for 6 months after injection. Lesions were also found in normal mice injected with the sera from immunized mice, and in HBV-transgenic mice injected with anti-HBs antibody, or sera from immunized mice. Furthermore, lesions were accompanied by deposition of circulating immune complex (CIC) of HBsAg and anti-HBs antibody in the damaged organs. These results indicate that long-term persistence of HBsAg and anti-HBs in the immunized mice can result in deposited CIC in liver and kidney, and in development of lesions. The use of DNA containing mammalian replication origins, such as the plasmids used in this study, is not appropriate for human vaccines due to safety concerns relating to persistence of DNA; nevertheless, the safety of DNA-mediated immunization protocols still needs to be carefully evaluated before practical application.     

The clinical relevance of the antibody to hepatitis B core antigen (anti-HBc): A review

The antibody against the core component of the Dane particle (anti-HBc) is generally detected in the sera of individuals with acute type B hepatitis and in chronic HBsAg carriers. While the serological demonstration of HBsAg with or without anti-HBc indicates continued replication of viral antigens, the co-occurrence of anti-HBs and anti-HBc is considered a marker of recent HBV replication. The demonstration of anti-HBc in the absence of HBsAg and anti-HBs is in agreement with at least four different states of HBV infection. As this pattern indicates persistent HBV infection in some cases and recovery from an acute type B hepatitis in others, current efforts focus on further characterization of this pattern, using additional test methods such as anti-HBe and anti-HBc of the IgM class.     

Small vessel vasculitis caused by hepatitis B virus immune complexes: Small vessel vasculitis and HBsAg

In a comprehensive study of 80 patients with vasculitis, 4 had concurrent hepatitis B virus (HBV) infection. Polyarteritis nodosa was present in 2 and in the other 2, cutaneous vasculitis, presenting clinically as palpable or Henoch-Schönlein purpura. In one of these patients skin biopsies demonstrated granular deposits of IgM, C3, C4, and the hepatitis B surface antigen (HBsAg) and electron-dense deposits of aggregated 20-nm particles resembling HBsAg in postcapillary venules. Evidence for circulating HBsAg-immune complexes included increased serum C1q binding activity, decreased serum complement, and a cryoprecipitate containing both HBsAg and IgM anti-HBs. Aggregated 20-nm particles resembling intact HBsAg were also seen by negative staining electron microscopy of the serum cryoprecipitate. This patient fulfills all the criteria for a specific immune complex vasculitis caused by his immune response to a chronic HBV infection. These findings emphasize that HBV infection may be associated with small vessel vasculitis as well as polyarteritis nodosa, mixed cryoglobulinemia, and glomerulonephritis. A similar immune response to other viral infections may be expressed as palpable (Henoch-Schönlein) purpura also.     

Cell-mediated immunity to hepatitis B surface antigen in man.

An aqueous preparation of hepatitis B virus (HBV) vaccine was used as an intradermal skin test antigen to assess delayed hypersensitivity to hepatitis B surface antigen (HBsAg). Thirty-five persons were tested including 10 individuals seronegative for all HBV markers, 10 positive for HBsAg (chronic carriers) and 15 positive for antibody to HBsAg (anti-HBs), five of whom had received the HBV vaccine. All patients were also studied for lymphocyte blastogenic responses to phytohaemagglutinin, concanavalin A, pokeweed mitogen and purified HBsAg. Only one individual had a positive delayed skin test reaction to HBsAg. This person had received the HBV vaccine and had high titres of anti-HBs in serum. However, neither this individual nor any other subject exhibited a positive lymphocyte blastogenic response to HBsAg in vitro. Thus, delayed hypersensitivity skin test reactivity to HBsAg was not detected after natural infection with HBV and was rarely present in hyperimmunized individuals. In vitro assays of immune responsiveness failed to demonstrate cellular immunity to HBsAg even in hyperimmunized persons. These studies provide no evidence that cell-mediated immunity to HBsAg plays a role in the immunopathogenesis of acute or chronic type B hepatitis.     

Immunoglobulin- and Hepatitis B Surface Antigen-Specific Circulating Immune Complexes in Chronic Hepatitis B Virus Infection

For assessing the role of circulating immune complexes (CIC) in chronic hepatitis B virus (HBV) infection, CICs containing IgM, IgG, and HBsAg were determined by C1q and conglutinin (K) assays in 216 patients with chronic HBV infection and 54 healthy controls. The concentration of each type of CIC in patients is higher than in controls (P= 0.0001). CIC is a common feature of chronic HBV infection with 95.8% of cases having at least one abnormal test result. At least one type of HBsAg-CIC is positive in 54.2% of patients. HBsAg-CIC positivity is associated with HBeAg positivity (P= 0.0001), higher aminotransferase levels (P< 0.002), and younger age (P= 0.001). IgG-CIC or IgM-HBsAg-CIC correlates with higher aminotransferase activity (P= 0.001). In conclusion, HBsAg-CIC correlates with HBV replication. IgG-CIC and/or IgM-HBsAg-CIC correlate with disease activity. Immune-mediated injury may play a role in the pathogenesis of chronic HBV infection.     

Changes in serologic markers of hepatitis B following autologous hematopoietic stem cell transplantation.

Korea is an endemic area for hepatitis B virus (HBV) infection. Reactivation of HBV is a well-recognized complication in patients with chronic HBV infection undergoing cytotoxic or immunosuppressive therapy, and there are some reports of hepatitis B reverse seroconversion after HSCT. This study evaluated changes in HBV serology after HSCT. We reviewed the medical records of 141 patients who had available HBV serologic data after autologous HSCT. Patient information was retrospectively collected from the BMT database. Before transplantation, 12 patients were positive for hepatitis B surface antigen (HBsAg) and received lamivudine prophylaxis. There was 1 case of reactivation of HBV among these patients. One hundred twenty-nine patients were negative for HBsAg before HSCT, of whom 110 were positive and 19 were negative for hepatitis B surface antibody (anti-HBs). Sixty-two of the 110 patients who were positive for anti-HBs were also positive for hepatitis B core antibody (anti-HBc). Eight patients were negative for anti-HBs and anti-HBc. Seven patients who were initially negative for HBsAg were identified as positive after HSCT, and 5 of those 7 patients developed acute hepatitis, thus indicating reverse seroconversion. Univariate analysis showed that reverse seroconversions were observed more frequently with multiple myeloma than another disease (P = .005; relative risk, 11.854; 95% confidence interval, 1.381-101.770). Other factors, such as age, sex, and presence of HBcAb before HSCT, had no statistically significant affect on reverse seroconversion. In conclusion, reverse seroconversion of HBV is not a rare complication of autologous HSCT, and the risk of reverse seroconversion after treatment is a serious concern due to possible complications arising from patients' suppressed immune systems.     

Fatal liver failure with the emergence of hepatitis B surface antigen variants with multiple stop mutations after discontinuation of lamivudine therapy

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine is effective and well-tolerated. However, discontinuation of the treatment is associated frequently with acute exacerbation of liver diseases. A patient suffering from acute liver failure after discontinuation of lamivudine treatment is described. The patient was treated with lamivudine for 4 months and ceased the treatment without consulting. After receiving lamivudine, the patient developed anti-HBs and became negative for hepatitis B surface antigens (HBsAg). However, HBV DNA reappeared to a level of 6.47 x 10(5) copies/ml. The patient died due to acute liver failure. Sequencing of HBV isolates revealed that mutations including G145R and stop codons occurred within the HBsAg coding region. In conclusion, HBV replication resumed after the uncontrolled cessation of lamivudine treatment in this patient and may have triggered the process leading to liver failure. Anti-HBs antibody appeared and may be the selective force for the emergence of HBV mutants.     

In vitro immune responses to hepatitis B surface antigen (pre-S2 and S) following remote infection by hepatitis B virus in humans

In this report we evaluate the human immune response to hepatitis B surface antigen (HBsAg) following remote infection with hepatitis B virus (HBV). HBsAg-reactive lymphocytes can be readily demonstrated in the peripheral blood of individuals with established immunity following infection with HBV.In vitro stimulation with small doses of plasma-derived HBsAg, yeast-derived HBsAg (S region) or pre-S2 peptide will induce specific IgG to HBsAg (anti-HBs) in the absence of a polyclonal increase in total IgG. The pre-S2 peptide will stimulate, in a T cell-dependent fashion, thein vitro production of anti-HBs with specificity for the S domain. This anti-HBs production is mediated by pre-S2-stimulated soluble T-cell factors. Peripheral blood mononuclear cells from individuals with established immunity proliferate to the yeast-derived HBsAg but not to the plasma-derived HBsAg or pre-S2 peptide. The chronic HBsAg carriers do not produce anti-HBs following stimulation with HBsAg regardless of the source or component of antigen used. Different study protocols failed to demonstrate HBsAg-specific responses in the peripheral blood mononuclear cells of chronic carriers.