Pietro Pala

Hepatitis B virus-specific cytotoxic T lymphocyte responses in patients with acute and chronic hepatitis B virus infection

An in vitro model was developed that allowed the analysis of hepatitis B virus-specific cytotoxic T lymphocyte responses in patients suffering from acute and chronic hepatitis B virus infections. Since virus-specific cytotoxic T lymphocytes recognize endogenously synthesized and processed antigen only when it is presented in the context of autologous HLA class I molecules and since hepatitis B virus does not infect human cells in vitro, a panel of recombinant vaccinia viruses was constructed to induce the expression of hepatitis B virus envelope and nucleocapsid proteins in cultured primary cells or cell lines derived from the patients to be studied. In order for a cytotoxic T lymphocyte response to be detectable with the currently available techniques, a sufficient number of activated cytotoxic T lymphocytes is required. To meet this requirement, lymphocytes freshly isolated from venous blood were stimulated in vitro with recombinant vaccinia-infected and formaldehyde-fixed autologous T lymphoblasts. The presence of hepatitis B virus-specific cytotoxic T lymphocytes, amplified and activated during this induction culture, was demonstrated in a microcytotoxicity assay using 51Cr-labeled, recombinant vaccinia-infected Epstein-Barr virus-immortalized, autologous B lymphocytes as target cells. Using this in vitro model, we were able to demonstrate the presence of hepatitis B virus envelope- and nucleocapsid-specific cytotoxic T lymphocytes in venous blood from one subject who had recently recovered from an acute hepatitis B virus infection and in three patients suffering from chronic hepatitis B virus infections. No hepatitis B virus-specific cytotoxic T lymphocytes activity was discernible in the venous blood from two vaccine recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

A Method to Detect Hepatitis B Virus-specific Cytotoxic T Lymphocytes in Patients with Acute and Chronic HBV Infection

Since virus-specific cytotoxic T lymphocytes (CTL) recognize endogenously synthesized and processed antigen only when it is presented in the context of autologous HLA class I molecules and since hepatitis B virus (HBV) does not infect human cells in vitro, a panel of recombinant vaccinia (rec-vaccinia) viruses was constructed to induce the expression of HBV envelope and nucleocapsid proteins in cells from the patients to be studied. In order for a CTL response to be detectable, a sufficient number of activated CTL is required. Therefore, lymphocytes freshly isolated from venous blood were stimulated in vitro with rec-vaccinia-infected and formaldehyde-fixed autologous T lymphoblasts. The presence of HBV-specific CTL, amplified and activated during this induction culture, was demonstrated in a microcytotoxicity assay using 51Cr-labeled, rec-vaccinia-infected Epstein-Barr virus-immortalized, autologous B lymphocytes (EBV-LCL) as target cells. Using this in vitro model, we could demonstrate the presence of HBV envelope- and nucleocapsid-specific CTL in venous blood from one patient that had recently recovered from an acute HBV infection and in three patients with chronic HBV infections.