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Summary Persistence of hepatitis B virus (HBV) infection is associated with reducedanti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers. Thus, we compared myeloid (mDC) and plasmacytoid DC (pDC) from chronic HBV carriers and controls. Frequency and phenotype of isolated DC were analysed by fluorescence activated cell sorter staining, DC function by mixed lymphocyte reaction, cytokine bead array, intracellular cytokine staining, enzyme-linked immunosorbent assay and enzyme-linked immunospot. Expression of HBV DNA and mRNA was studied by polymerase chain reaction (PCR). Circulating total DC, mDC or pDC were not reduced in chronic HBV carriers. Isolated mDC and pDC from chronic HBV carriers exhibited similar expression of co-stimulatory molecules and alloreactive T helper cell stimulation as control DC, whether tested directly ex vivo or after in vitro maturation. Secretion of pro-and anti-inflammatory cytokines by CD40 or Toll-like receptor ligand-stimulated patient DC was intact, as was human leucocyte antigen A2-restricted HBV-specific cytotoxic lymphocyte stimulation. Although both DC populations contained viral DNA, viral mRNA was undetectable by reverse transcription-PCR, arguing against viral replication in DC. We found no quantitative, phenotypic or functional impairment of mDC or pDC in chronic hepatitis B, whether studied ex vivo or after in vitro maturation.

SUMMARYAntibodies 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 HBsspecific 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. HBVimmunized 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.

To evaluate therapeutic immunostimulation nine chronic hepatitis B patients received six monthly intradermal vaccinations with HBsAg in combination with daily lamivudine. Another five patients received six doses of the vaccine and daily lamivudine together with daily Interleukin-2 (IL-2) s.c. within 3 months in an open-labeled trial. Clinical efficacy was assessed by alanine transaminase levels and HBV serology. The induction of specific T and B cell responses was analyzed serially by 3H-thymidine uptake, ELISA and ELISPOT assays. After the therapy was stopped, seven of nine vaccine/lamivudine and two of five vaccine/lamivudine/IL-2 recipients did not have detectable HBV DNA. Four complete responders cleared the virus and had normalized ALT levels, however, one of these patients showed transient disease reactivation followed by spontaneous viral clearance and normal ALT five months later. Low frequencies of anti-HBs producing B cells and HBV specific T helper cells secreting predominantly interferon-gamma were induced by i.d. vaccine therapy. The ELISPOT technique demonstrated transient induction of HBV peptide specific cytotoxic T cells in seven HLA-A2 positive chronic HBV carriers. The preliminary data from this study demonstrate that the HBV surface antigen vaccine in combination with antiviral or immunomodulating drugs induced antiviral immune responses and consequently viral elimination may be achieved in patients with unfavorable prognosis.

Because hepatitis B virus (HBV) is a noncytopathic virus and T cells dominate the lymphocyte infiltrations of the liver, it is widely assumed that the inflammatory activity is mediated by the host' s cellular immune responses. In acute self-limited HBV infection, CD8-positive cytotoxic T cells were observed that recognized epitopes on the core, surface, and polymerase antigens in an HLA class I-restricted manner. 1-4 Furthermore, strong HLA class II-restricted T-helper cell (TH cell) responses against recombinant HBV core antigens and derived peptides have been demonstrated in acute infection and during exacerbations of chronic HBV infection. 5-9 Chronic HBV carriers widely recognized the same antigens, although the cellular immune responses were significantly weaker. 8-10 Thus, insufficient T-cell responses are thought to contribute to viral persistence and chronic liver disease.In addition to HBV-specific cellular immune reactions, the humoral immune response might be critical in the pathogenesis of HBV infection by binding extracellular viral particles and therefore limiting the spread of the infection. The occurrence of neutralizing surface antigen-specific antibodies indicates complete recovery from acute HBV infection, whereas these antibodies are usually undetectable in patients with viral persistence using conventional assays. Because hepatitis B surface antigen (HBsAg) is expressed on hepatocellular surfaces, 11 antibody-mediated cytotoxicity might also contribute to hepatocellular damage. 12,13 Moreover, it has been shown extensively in vitro and in vivo that anti-HBs antibodies can neutralize extracellular viral particles 14,15 and that active immunization with HBsAg protects from HBV infection. 16 Furthermore, passive immunoprophylaxis with anti-HBs immunoglobulins prevents HBV carriers from reinfection after liver transplantation. 17 Thus, regulation and modulation of the humoral immune response might be relevant to improving vaccine response in primary nonresponders or for treatment of patients with chronic hepatitis B (CHB).Little is known about the kinetics and regulation of the HBs-specific B-cell response in the clinical course of HBV infection and in vaccine recipients. Thus, the aims of this study were: 1) the quantification and characterization of the cellular and humoral immune responses in the follow-up of patients with acute hepatitis B (AHB) or CHB and in vaccine recipients; 2) the comparison of anti-HBs-secreting B cells in the peripheral blood and the bone marrow; and 3) the