Antibody responses to preS components after immunization of children with low doses of BioHepB.

BioHepB is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing HBs as well as preS1 and preS2 antigens, in their glycosylated and non-glycosylated forms. The vaccine was administered intramuscularly to 18 children aged 5 months to 11 years at 0, 1 and 6 months. One hundred percent seroconversion and seroprotection rates were achieved after primary and secondary immunization with the 2.5 microg doses of BioHepB. Ten out of the 18 children (56%) responded with the appearance of anti-preS1 and/or anti-preS2 antibodies in circulation, when analyzed 1, 2, 6, 7 and 12 months after the initiation of vaccination. In comparison with the emergence of the anti-HBs response, early (month 2, after two injections) or late (month 7, after three injections) peak responses were noted for the kinetics of anti-preS1 and anti-preS2 production during the course of immunization, demonstrating that the anti-preS1 and anti-preS2 responses are differently regulated, compared with the anti-HBs response. At month 6, just prior to the final injection, BioHepB caused significantly higher anti-HBs responses (GMT) in preS1-reactive children than in children without preS1 antibodies (P<0.005). Moreover, a significantly higher, anti-HBs response in GMT was also noted for anti-preS2-reactive children compared with anti-preS2-negative children (P<0.05). These findings demonstrated that recognition of the preS epitopes contained in the experimental preS1/preS2/S vaccine is accompanied by a more rapid onset and pronounced antibody response to the S-gene-derived protein in healthy children.

Impact of a vaccination campaign on adult immunity to diphtheria.

OBJECTIVES: to raise the level of immunity to diphtheria in the adult population of Stockholm by a vaccination campaign. The rationale behind the campaign, conducted during 1995-1996, was the re-emergence of epidemic diphtheria in the countries of the former Soviet Union and earlier surveys of immunity to diphtheria showing low levels of protection in adults. DESIGN AND MAIN OUTCOME MEASURES: the impact of the vaccination campaign was measured by recording the age and sex of vaccinees, the type and number of vaccine doses given and any side-effects. The effect on immunity was evaluated in 1998-1999 by measuring the neutralising antibodies in blood samples from 1863 inhabitants, chosen by random stratified sampling. Vaccines and vaccinations: three doses of diphtheria (D) or diphtheria-tetanus (DT) vaccine were given to those without documented previous vaccination; others received a booster dose. The DT vaccine, with the D component purified before toxoiding, contained 15 Lf of D and 7.5 Lf of T per ml, and was given in 0.5 ml doses for the two priming doses and 0.25 ml as booster. RESULTS: 184969 doses of D or DT vaccine were given to 99939 individuals. Of the vaccinees, 65% were 50 years of age or older and 60% were women. The highest rates of reported local reactions were 1.8-5.4% and of systemic reactions, such as fever, 0.2-0.8%. The campaign resulted in a significant increase in antitoxin concentrations in the age cohorts targeted, and especially in women, less well protected than men. CONCLUSIONS: a vaccination campaign, targeting the adult part of a population, can result in a major improvement in immunity to diphtheria with only a few and minor side-effects with a DT vaccine where the D component was purified prior to toxoiding. Extending national immunisation programmes to include adults would, however, seem preferable.

Quantitative monitoring of serum hepatitis B virus DNA and blood lymphocyte subsets during combined prednisolone and interferon-alpha therapy in patients with chronic hepatitis B.

Several investigators have reported a significantly reduced CD4/CD8 ratio, as defined by monoclonal antibodies, in the peripheral blood of Caucasian patients with chronic active hepatitis B (CAHB). In Asian patients with chronic hepatitis B, quantitative analyses of subpopulations of peripheral blood lymphocytes have not been able to confirm these findings. In this work, we analysed the frequency of peripheral blood lymphocyte subsets in 10 Chinese patients with histologically proven CAHB and seven healthy Chinese individuals. Four of the 10 CAHB patients received combined prednisolone/interferon-alpha2b (IFN-alpha2b) therapy. Peripheral blood samples were consecutively collected for analysis of lymphocyte subpopulations using an indirect immunofluorescence (IF) method, and hepatitis B virus (HBV) DNA was quantified by a chemiluminescent, molecular-hybridization assay. Peripheral blood mononuclear cells from seven Chinese control individuals comprised 63 +/- 3% CD3+ cells, of which 41 +/- 4% were of CD4+ and 23 +/- 2% of CD8+ subsets. The mean CD4/CD8 ratio in the healthy controls was 1.9 (95% confidence interval = 1.1-2.7). The CD4/CD8 ratios were significantly reduced (P < 0.01) in the 10 patients with chronic hepatitis B, compared with those of the controls, owing to a significant increase in the number of CD8+ cells (P < 0.005). During the treatment with prednisolone, a significant increase in the CD4/CD8 ratio was observed in all treated patients. This increase was mainly caused by a decrease in the number of CD8+ cells and was accompanied by an increase in serum HBV DNA levels, which peaked during the latter part of the prednisolone cycle. During the treatment with IFN-alpha2b, a second increase in the CD4/CD8 ratio was observed, which was caused by an increase in CD4+ cells. A marked decrease in viral load was observed, during treatment with IFN-alpha2b, in patients with HBV DNA levels below 10 000 pg ml-1. Our data indicate that the CD4/CD8 ratios in Chinese CAHB patients do not differ from those of Caucasian patients with CAHB, when analysed using similar methods for the enumeration of lymphocyte subsets. Profound effects on cellular distribution and viral replication were noted during the combined prednisolone/IFN-alpha2b therapy. Additional studies of the modulatory effect of the combined therapy on the distribution of lymphocyte subsets and cytokine profiles in relation to the therapeutic outcome of HBV infection are warranted.

Quantitative assessment of IgM antibodies towards an immunodominant B-cell epitope within the preS2 domain of HBV in the natural course and during combined prednisone/interferon alpha 2b treatment of chronic hepatitis B virus infection.

A direct binding enzyme-linked immunosorbent assay (ELISA) was established for quantitative determination of serum IgM antibodies towards a synthetic peptide corresponding to a selected segment (14-21) of the preS2-gene product containing an immunodominant linear B-cell epitope. The prevalence of IgM anti-preS2 (14-21) antibody titers > 1,000 for hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B virus (HBV) infection was 38% (22/58) and 10% (2/21) for HBeAg-negative subjects (P < 0.005). IgM anti-preS2 (14-21) reactivity was detected during the clinical course of chronic HBV infection and IgM anti-peptide antibody titers declined and disappeared before spontaneous HBe/anti-HBe seroconversion. Recombinant interferon (IFN)-alpha 2b with an antecedent short course of corticosteroids was administered to eight Chinese patients with chronic HBV infection. The IgM anti-preS2 (14-21) reactivity was monitored consecutively during treatment and patients were followed for more than 1 year. A close association between the presence of pretreatment IgM anti-preS2 (14-21) in serum and the capacity to respond favorably to the combined prednisone/IFN-alpha 2b therapy was detected. The IgM anti-preS2 (14-21) titers decreased during treatment with subsequent loss of detectable antibodies 8-16 weeks after the initiation of therapy. This decrease was concomitant with an alanine aminotransferase (ALT) augmentation preceding the disappearance of HBV-DNA and anti-HBe seroconversion. Long-term remission was not observed in treated patients who lacked detectable levels of pretreatment IgM anti-preS2 (14-21) in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin M reactivity towards the immunologically active region sp75 of the core protein of hepatitis C virus (HCV) in chronic HCV infection.

Serum samples from a cohort of patients with chronic hepatitis C virus (HCV) infection were assayed for IgM anti-HCV/core reactivity with a "site-specific" ELISA, in which the solid phase was charged with the synthetic polypeptide analogue corresponding to the first 75 amino acids of the HCV core antigen (sp75). Thirteen of 24 (54%) patients exhibited IgM anti-sp75 reactivity. Both high-titered (1/16,000-1/32,000) and low-titered (1/1,000-1/4,000) IgM anti-sp75 reactive sera were found. IgM anti-sp75 antibodies persisted in the circulation over a long period in patients with fluctuating abnormal ALT levels. There was a striking association between detection of specific IgM anti-sp75 reactivity and the presence of HCV RNA in serum. Thus 11 of 15 (73%) sera containing HCV RNA also contained IgM anti-sp75 antibodies, while none of the HCV RNA-negative sera were IgM anti-sp75 reactive. Five of 11 patients without detectable levels of specific IgM anti-sp75 antibodies had their ALT levels returned to normal within 8 months to 3 years. Furthermore, a significant correlation was noted between the specific IgM anti-sp75 titers and the concentration of total plasma IgM, indicating that the immunological active region sp75 within the capsid of HCV has the capacity to induce an IgM secretion, which constitutes a substantial portion of the total plasma IgM, in patients with chronic HCV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the immune response to hepatitis B virus and human serum albumin. III. Induction of anti-albumin antibody secretion in vitro by C-gene-derived proteins in peripheral B cells from chronic carriers of HBsAg.

The circulatory pool of B cells from the majority (11/13) of chronic hepatitis B surface antigen (HBsAg) carriers contained sensitized B cells with the capacity to secrete IgG antibodies with specificity for human serum albumin (HSA), when stimulated with E. coli-derived core protein at low concentrations in vitro. The IgG anti-HSA secretion was dependent upon and regulated by T cells, and optimal secretion was obtained at T/B-cell ratios of 1.0-4.0, varying for different individuals. The level of anti-HSA secretion was higher for patients with on-going viral replication as assessed by hepatitis B virus (HBV)-DNA in serum. Culture supernatants containing anti-HSA antibodies also contained anti-HBc antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) where the solid phase was charged with E. coli-derived core protein, or the synthetic peptides corresponding to the 75-84 and 132-147 sequences in the C region of HBV. In contrast, IgG anti-HBc (E. coli-derived), but no anti-HSA or anti-HBc 75-84, 132-147 antibodies, were detected at similar T/B-cell ratios in cell cultures from 5/6 individuals with naturally acquired immunity to hepatitis B. These data indicate that peripheral B cells from the majority of HB-immune donors are sensitized to unique (e.g. non-albumin associated) structures in the nucleocapsid of HBV, while B cells in the majority of chronic HBsAg carriers are sensitized to linear C-gene-derived structures in association with the host 'self'-component HSA.

HBcAg induced T-cell independent anti-HBc production in chronic HBsAg carriers.

The capacity of the nucleocapsid protein of HBV to function as a T-cell independent antigen in man was studied. When T-cell depleted B-cell cultures were challenged with E coli-derived HBcAg, anti-HBc production was registered in culture supernatants from the majority of chronic HBsAg carriers in a quiescent stage of disease. In contrast, similarly prepared and stimulated cultures from donors with natural acquired immunity to hepatitis B or HB-susceptible controls were non-responsive. Addition of autologous T-cells effectively restored anti-HBc responsiveness in T-cell depleted B-cell cultures from HB-immune donors, demonstrating the T-cell dependency for anti-HBc induction in natural HBV-infection.

Divergent anti-HBc reactivities in HB-immune and chronic HBsAg carriers.

We have monitored titers of anti-HBc antibodies in sera from acutely HBV-infected and chronic HBsAg carriers. Our data show that there is a divergence in the specificity of the antibodies in these two populations. We also present preliminary results showing that serum from HB-immune carriers contain antibodies that are multispecific and display autoimmune characteristics, reacting with human serum albumin.

IgM anti-preS2 monitoring during combined corticosteroid/interferon-alpha 2b therapy in chronic hepatitis B.

The biological significance of antibody reactivities towards the preS2 gene encoded proteins of HBV is not yet well known. We investigated the pretreatment IgM anti-preS2 (1-55) ad reactivity in 22 Chinese and 11 Swedish patients with active HBV disease. Significantly enhanced IgM anti-preS2 levels (titers greater than 1/1000) were observed in 48% (16/33) of these patients. The OD405 values for sera from patients with indolent HBsAg carriership were within the range of that obtained for "normal" control-sera when tested at dilutions from 1/1000 to 1/128,000. Five Chinese patients were treated with a short course of corticosteroids, followed by alpha-interferon 2b (IFN-alpha 2b) treatment for 16 weeks. The IgM anti-preS2 response was consecutively monitored during treatment. A beneficial effect on the outcome of the combined treatment was associated with rising titers of IgM anti-preS2 during the prednisolone cycle. The IgM anti-preS2 levels fell dramatically upon steroid withdrawal and were followed by a second peak response of IgM anti-preS2 reactivity during the IFN-alpha 2b treatment. No sustained loss of HBV-DNA or DNA polymerase with concomitant HBe/anti-HBe seroconversion was observed in treated patients, who lacked detectable pretreatment levels of IgM anti-preS2 in circulation.

HBsAg-induced interferon-gamma secretion in T cells from asymptomatic HBsAg carriers and HB-immune donors in vitro.

Peripheral T lymphocytes obtained from asymptomatic carriers of hepatitis B surface antigen (HBsAg) and donors immune to hepatitis B (HB) through natural infection or vaccination were induced by the envelope protein (HBsAg) of the hepatitis B virus (HBV) into secretion of interferon-gamma (IFN-gamma) in vitro. The kinetics of the IFN-gamma response varied between individuals, but was constantly found to be biphasic, with an early peak attained after 12 hr-4 days and a late peak after 5-8 days of antigen stimulation. The early release of IFN-gamma activity was antigen-specifically induced, as it was in T cells from HB-immune and asymptomatic carriers of HBsAg but not HB-susceptible controls. The second peak of HBsAg-induced IFN-gamma secretion was induced in all three donor groups and the kinetics of IFN-gamma release were similar to that of the mitogen phytohemagglutinin(PHA)-induced IFN-gamma production in similarly prepared T-cell cultures. Thus the late burst of IFN-gamma activity seems to result from a mitogenic property contained within the envelope material of HBV. The mitogenic response was three- to fivefold higher for 4/7 asymptomatic carriers of HBsAg compared to HB-immune donors and HB-susceptible controls, indicating that some patients with chronic asymptomatic carriership of HBsAg exhibit enhanced mitogenic responses to HBsAg.

Elimination of circulatory IgM anti-HSA precedes anti-HBe seroconversion in patients with CAH type B.

The presence of IgM and IgA antibodies with specificity for human serum albumin (HSA) were consecutively analyzed in serum samples from four patients with biopsy verified CAH type B during seroconversion in the HBe/anti-HBe antigen system. Circulatory IgM anti-HSA antibodies were present during HBe antigenemia. The antibody titers fluctuated, decreased, and were finally lost from the circulation. After the disappearance of IgM anti-HSA antibodies, seroconversion to anti-HBe reactivity occurred and a quiescent phase of the disease was established, as judged by normalization of transaminases and absence of circulatory HBV-DNA. IgA anti-HSA antibodies persisted in the circulation after the elimination of IgM anti-HSA and seroconversion to anti-HBe reactivity. For one of the patients, a dramatic increase in titers was followed by elimination of IgA anti-HSA and seroconversion to anti-HBs. The data indicate that the host "self"-component HSA, when associated with "foreign" HBe or HBs antigenic structures, elicit immune responses to HSA, preventing the adequate development of anti-HBe and anti-HBs. The cessation of anti-HSA reactivity, however, seemed to permit subsequent sensitization to HBe and HBs antigenic determinants, as detected by the presence of circulatory antibodies.

Regulation of the immune response to hepatitis B virus and human serum albumin. II. Spontaneous secretion of antibodies with specificity for albumin in patients with chronic hepatitis B virus infection.

Peripheral B and T cells sensitized to human serum albumin (HSA) were found in a cohort of patients chronically infected with hepatitis B virus (HBV). Throughout this study, two groups of symptomatic chronic hepatitis B surface antigen (HBsAg) carriers could be distinguished, characterized by divergent T-cell regulation of the spontaneous IgG anti-HSA secretion. Patients in a quiescent phase of the disease [patients with chronic persistent hepatitis B (CPH), anti-HBe reactivity and absence of viral replication, group A] had circulatory in vivo HBsAg-HSA preactivated B cells with the capacity to secrete spontaneously IgG antibodies with specificity for HSA when isolated and cultured. The addition of autologous T lymphocytes at a T/B-cell ratio of 8.0 suppressed the spontaneous anti-HSA secretion. In contrast, patients in an active stage of the disease exhibited in vivo preactivated T cells exerting helper cell functions on the spontaneous IgG anti-HSA secretion. Memory T cells, sensitized to low concentrations of HBsAg-HSA with disparate regulatory functions, were also detectable in the two groups of patients.

Circulating cholera antitoxin memory cells in the blood one year after oral cholera vaccination in humans.

Oral vaccination with the combined B subunit/whole cell cholera vaccine generates antitoxin memory cells that could be isolated from peripheral blood for at least 1 year after immunization. These memory cells were triggered by antigen in vitro to produce antitoxin in the presence of autologous T cells and monocytes. Antitoxin-producing peripheral blood lymphocytes (PBL) were found in 4 out of 5 previously vaccinated subjects. IgA and IgM isotypes dominated the memory response. The antigen-dose dependency and requirements for a specific ratio of T to B cells for activation of the memory cells in vitro implies T-cell control of antitoxin responses. These antitoxin memory cells in peripheral blood (and corresponding antibacterial memory cells) might represent a pool of circulating cells that on renewed exposure to cholera rapidly produce protective antibody in the gut and thus might have a central role in the long-term protection against reinfection and disease seen in convalescents from cholera.

Characterization of cell mediated immune responses to the hepatitis B core protein in man.

Eight donors immune to hepatitis B (HB) after natural infection were studied for their cell-mediated immune response to hepatitis B core (HBc) protein in vitro. Significant specific activation was observed in highly purified peripheral blood lymphocytes and T cells from these donors after 5-8 days of incubation with HBc protein. This in vitro response was modulated by monocytic cells and maximal 3H-thymidine incorporation was elicited with low concentrations of the antigen (0.1-10 ng/ml). The cell-mediated immune reactivity towards HBc-protein was significantly (P less than 0.005) elevated compared to the envelope material of the virus (HBsAg) when analysed in the same donor population. In contrast, donors with plasma-derived hepatitis B vaccine induced immunity to HB exhibited only specific lymphocyte activation to HBsAg. These data indicate that the composition of immune responses conferring protection against HBV after natural infection is far more complex than after immunization with the hepatitis B vaccine. Further characterization of the cell-mediated immune response to HBc protein and its relation to protection against HBV seems warranted when strategies for new HB-vaccines are being designed.

The immune response against pre-S2-encoded peptides and human serum albumin during hepatitis B virus infection.

Serum antibodies to human serum albumin (HSA) and its receptor structures (pre-S2) on HBV/HBsAg particles of IgG class were measured in patients with naturally acquired immunity to hepatitis B, HBsAg carriers with chronic liver disease, 'healthy' symptomless HBsAg carriers and controls. ELISA systems in which serum samples were diluted in 0.05 M PBS to give low protein concentrations followed by incubation at 4 degrees C were used. Anti-HSA and anti-pre-S2 were produced in patients during the acute phase of hepatitis B infection but were short-lived. The antibodies were absent in 'healthy' symptomless HBsAg carriers but present in the majority of HBsAg carriers with major liver disease. These results indicate that humoral immune responses of IgG class, normally initiated during the acute phase of HBV infection against the host 'self'-component HSA and/or its receptor structures, are down-regulated by immunological mechanisms while continuous production of these antibodies is associated with the pathogenesis of chronic HBV-induced liver disease.

Absence of pre-S2 antibodies in natural hepatitis B virus infection.

Antibodies to the host "self" component human serum albumin (HSA) and to its receptor are induced by the pre-S2 encoded aminoacid sequence (120-145) of the middle surface protein on the hepatitis B virus (HBV) particle and are produced as part of the immune response during acute HBV-infection. The antibodies disappeared rapidly during the convalescence phase and were not detectable in patients with naturally acquired immunity to HB. These data raise questions about the importance of anti-pre-S2 antibodies for long-term protection against HBV-infection. The coexistence of HBV-DNA with antibodies to native HSA (nHSA) in 13/16 patients with biopsy proven liver disease and of HBV-DNA with anti-pre-S2 in 10/13 patients argues against the attribution of virus eliminating properties to antibodies directed against pre-S2 determinants. In fact circulating anti-nHSA and anti-pre-S2 persisted in 16/17 and 13/17 patients, respectively, with major HBV-induced liver disease but not in symptomless HBsAg carriers. This finding suggests that continuous production of antibodies against the "self" component HSA and/or its receptor structures are associated with the pathogenesis of chronic HBV-induced liver disease. Pre-S2 coded peptide sequences should thus not be incorporated into HB vaccines.