Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

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.     

Immune response to hepatitis B surface antigen.

A total of 69 persons were investigated for assessment of cell-mediated and humoral immunity to hepatitis B surface antigen (HBsAg). Three groups, each consisting of 20 normal persons, 20HBsAg carriers, and 20 convalescent hepatitis B patients, were studied for HBsAg, anti-HBs, and leukocyte migration inhibition with purified HBsAg. Sequential sampling if an additional group of nine acute hepatitis B patients defined the cellular and humoral immune response to HBsAg. The antigen was eliminated rapidly by mounting of cell-mediated immune response detectable for a limited period, followed by antibody response in relatively few patients moore than 3 months after clearance of circulating HBsAg.     

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.     

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.     

Lack of genetic restriction by a potential anti-idiotype vaccine for type B viral hepatitis

Anti-idiotype (anti-Id) reagents that bear an internal image capable of mimicking hepatitis B surface antigen (HBsAg) were used to induce an antibody to HBsAg (anti-HBs) response in both rabbits and chimpanzees. The anti-idiotype induced antibody response produced in rabbits recognized HBsAg determinants associated with the induction of protective immunity against hepatitis B virus (HBV). Attesting further to the specificity was the binding of the rabbit anti-idiotype to the anti-idiotype induced anti-HBs containing sera. Our findings suggest that genetic restrictions associated with the induction of an interspecies immune response may not be a limitation of anti-idiotype based vaccines. In addition, anti-idiotype immunization also produced an anti-HBs in chimpanzees, a species susceptible to infection with human HBV. These data demonstrate that internal-image-bearing anti-idiotype reagents can induce an immune response across species barriers. Additionally, the reagents represent a viable alternative approach to vaccination against agents such as hepatitis B virus that cause human disease.     

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.

     

The effect of intradermal administration of corynebacterium parvum on the immune response to hepatitis Bs antigen

The immunopotentiating effect of the intradermal administration of a course of four doses (0.25 ml) of a standard suspension of killed C. parvum (2 mg/ml) was studied in a group of 10 asymptomatic chronic hepatitis B surface antigen (HBsAg) carriers, as well as in 11 persons with antibodies to HBsAg (anti-HBs) and six without HBsAg or anti-HBs. HBsAg, anti-HBs, and leukocyte migration inhibition (LMI) studies were performed in pre- and post-inoculation blood samples. C. parvum produced a substantial increase of anti-HBs titre in persons with preexisting anti-HBs. However, anti-HBs responses were not induced in carriers. HBsAg was not eliminated and its titre remained practically unchanged in carriers. These results support the hypothesis that in carriers the specific defect in the immune response to HBsAg possibly exists at the B cell level.     

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.     

Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches

The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.     

Antigen-antibody complex as therapeutic vaccine for viral hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-gamma. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the titer of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

Response to hepatitis B vaccine in family members of HBsAg carriers

The family members of HBsAg carriers have an increased risk of hepatitis B virus (HBV) infection. 214 subjects from 98 families with no HBV markers were randomized to receive hepatitis B vaccine: HEVAC B (Institute Pasteur) or GCC VAC (Green Cross Corporation) at 0, 1, and 5 months. Of those who completed the course, 87.8% had an anti-HBs response of greater than 10 mIU/ml at 6 months. The response rate was similar for both sexes. There was a decrease in response rate and anti-HBs titre with age. The response rate for HEVAC B was 92.5% and GCC VAC 84.3%. The offspring had comparable response to the spouses who were not blood relatives of the index carriers, but this could be related to their younger age. Discriminant analysis showed that a higher anti-HBs titre was associated with HEVAC B, younger age, and less direct relationship with the index carrier.     

Diagnostic significance of anti-HBcIgM prevalence related to symptoms in Canadian patients acutely or chronically infected with hepatitis B virus

A total of 362 sera from 295 Canadian patients were examined for HBsAg, anti-HBs, anti-HBc, anti-HBcIgM, HBeAg, and anti-HBe using commercial immunoassays. Serial samples from 70 acutely infected patients demonstrated that anti-HBcIgM may detect 10% more positives than HBsAg within 4 months after the onset of clinical symptoms, and all except two were negative for anti-HBcIgM after the fourth month. None of 66 asymptomatic (HBeAg rate 18.2%) and two of 14 (14.3%) symptomatic (HBeAg rate 64.3%) carriers of HBsAg were positive for anti-HBcIgM (P = 0.029). Elevated marker responses were measured in two symptomatic carriers for a 20-month period. Anti-HBcIgM was not detected in either 100 asymptomatic patients positive for total anti-HBc, negative for HBsAg and negative for or possessing low levels of anti-HBs, 25 patients with liver disorders not caused by HBV, or 20 healthy milk donors. In diagnostic laboratory practice this anti-HBcIgM test may be useful in the following situations: to supplement HBsAg testing, providing a theoretical 10% increase in positives within 4 months following onset of acute viral hepatitis; to replace testing for anti-HBc and anti-HBs in symptomatic HBsAg-negative patients; to confirm whether a patient is experiencing acute or chronic HBV infection or symptoms superimposed upon asymptomatic HBsAg carriage by another cause, such as nonA-nonB viral hepatitis.     

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.     

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs-specific T and B cell responses were observed.     

Dental care and spread of hepatitis B virus infection.

Sera from 576 healthy adults were tested for the hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) to evaluate the role of routine dental care as a factor in the spread of hepatitis B virus (HBV) infection. Serological evidence of prior HBV infection, manifested by acquisition of anti-HBs, was detected in 97 (16.8%) individuals, and 6 (1.0%) were identified to be asymptomatic HBsAg carriers. The anticipated correlations of HBsAg and anti-HBs with age, country of birth, and socioeconomic status were observed in the study population. However, prevalences of both HBsAg and anti-HBs were inversely related to the lifetime total of dental care visits. These findings indicated that, in a region in which the HBsAg carrier state and hepatitis B are prevalent, routine dental care is not identified as an important factor in the spread of HBV infection. While the results do not exclude the obvious possibility that cross-infections with HBV may occur during dental care in specific situations, they indicate that this mode of infection is exceptional.     

In VivoInduction of Specific Cytotoxic T Lymphocytes in Mice and Rhesus Macaques Immunized with DNA Vector Encoding an HIV Epitope Fused with Hepatitis B Surface Antigen

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.     

Immunoglobulin M antibodies against hepatitis B core antigen in patients with chronic hepatitis B infection

A batch of sera obtained from subjects with acute hepatitis B virus (HBV) infection, chronic carriers of hepatitis B surface antigen (HBsAg) who were either asymptomatic or who had chronic active hepatitis, and 32 sera from patients with HBsAg negative chronic active hepatitis were examined for the presence of antibodies against hepatitis B core antigen (anti-HBc) by radioimmunoassay (RIA). Sera containing anti-HBc were fractionated on sucrose density gradients to separate immunoglobulin M (IgM) and the titre of anti-HBc IgM was determined. In patients with acute HBV infection, anti-HBc IgM was detected during the acute phase of the illness with titres ranging from 1:128 to 1:4,096 (geometric mean titre 1:709). The titre of anti-HBc IgM fell rapidly over the following months and in most patients persisted at low levels for several years. Anti-HBc IgM was also detected in subjects with chronic HBV infection but with significantly lower titres. In asymptomatic carriers, anti-HBc IgM titres ranged from 1:4 to 1:32 (geometric mean titre 1:12), whilst carriers with chronic active hepatitis had titres ranging from 1:4 to 1:128 (geometric mean titre 1:35). By using a standardized assay procedure, the titre of anti-HBc IgM in a patient's serum may be of value in differentiating between acute and chronic HBV infection.     

Cellular immunity to nucleocapsid and pre-S determinants in asymptomatic carriers of hepatitis B virus.

Previous studies of cellular immunity in asymptomatic HBV carriers have been limited to evaluation of responses to plasma-derived HBsAg preparations. We have explored the specificity of cellular immune responses to HBV antigens in these subjects using an indirect T-lymphocyte migration inhibitory factor assay and three antigen preparations (recombinant nucleocapsid antigen (HBcAg), plasma-derived HBsAg with or without pre-S2, and Saccharomyces cerevisiae-synthesized HBsAg without pre-S2 region). T cells from 10 asymptomatic chronic HBV carriers with normal liver function tests were responsive to nucleocapside determinants (mean migration index = 0.55 +/- SD 0.07) and to pre-S2-positive plasma-derived HBsAg (MI = 0.62 +/- 0.05). However, none responded to HBsAg devoid of pre-S2 sequences (MI = 0.98 +/- 0.04). In further experiments, T cells from three HBV carriers, cultured with six different HBsAg preparations, exhibited responsiveness only to those preparations containing significant pre-S2 activities. Our results show that T-cell immunity to nucleocapsid determinants of the virus and HBsAg in present in asymptomatic HBV carriers; the latter is restricted to antigenic preparations containing significant pre-S2 activities. Hence, T-cell immunity to pre-S determinants may not always be associated with HBV clearance.     

Fulminant hepatitis in asymptomatic hepatitis B surface antigen carriers in Greece

Eleven male fulminant hepatitis (FH) patients (mean age: 47.7 +/- 16 years) positive for hepatitis B surface antigen (HBsAg) but negative for IgM antibody to hepatitis B core antigen (IgM anti-HBc) were admitted consecutively to the Athens Hospital for Infectious Diseases between May 1981 and November 1983. Because of the absence of IgM anti-HBc, determined by an enzyme immunoassay, these patients were considered to be HBsAg carriers with a superimposed acute hepatitis. Three of the 11 patients received immunosuppressive chemotherapy during the six months before the onset of the acute hepatitis. None of the patients was homosexual or a drug addict. Infection with hepatitis A virus (HAV), hepatitis B virus (HBV), or hepatitis delta virus (HDV) was detected with serologic markers and/or molecular hybridization techniques. Fulminant hepatitis was attributed to spontaneous reactivation of chronic hepatitis B in four patients, chemotherapy-induced reactivation of chronic hepatitis B in three patients, HDV superinfection in one patient and possible superinfection by non-A, non-B agent(s), HDV, or HDV-like agents in three patients. Reactivation of chronic hepatitis B was an important cause of apparent acute hepatitis in heterosexual male HBsAg carriers from an area with a high prevalence of HBV infection.