News and Publications

Immunobiology of Hepatitis B and C Virus Infections

D.R. Milich, J.L. Hughes, J.E. Jones, M. Sällberg,* M. Chen,* A. Birkett,** T. Maruyama***

* Huddinge University Hospital, Huddinge, Sweden
** Immune Complex Corp., San Diego, CA
*** University of Tokyo, Tokyo, Japan

HEPATITIS B

Our recent focus has been the question of why some patients infected with hepatitis B virus (HBV) clear the infection relatively efficiently, whereas others cannot clear it and become chronically infected. In a series of studies with human sera and in mouse experimental systems (i.e., transgenic mice), we have developed evidence suggesting that the characteristics of the patient's helper T cell (T_H cell) response may determine the outcome of the infection. Two major types of T_H cells have been described: T_H1 cells mediate cellular immunity (i.e., graft rejection, tissue injury, inflammation), and T_H2 cells mediate humoral immunity (i.e., antibody production). Theoretically, a correct balance between HBV-specific T_H1 and T_H2 cells would lead to viral clearance and a minimum of liver disease.

In previous studies, we examined 2 structural forms of the viral nucleoprotein: the particulate HBV core (HBcAg) and the nonparticulate or secretory HBeAg. The results suggested that 1 function of HBeAg is to establish T-cell tolerance to HBeAg and HBcAg in utero. This mechanism is relevant to vertical transmission of HBV and may explain the high rates of chronic infection that occur after perinatal infection in the offspring of HBeAg-positive mothers. Further studies indicated that secretion of HBeAg may have additional effects that promote chronic infection. We found that the continuous presence of HBeAg in the serum caused the preferential depletion of HBeAg- and HBcAg-specific proinflammatory T_H1 cells via Fas-mediated apoptosis and spared antiinflammatory T_H2 cells.

Because HBeAg in the serum preferentially depleted T_H1 cells, we examined the effect of secreted HBeAg on HBcAg-specific T_H1 cells by transferring HBeAg- and HBcAg-specific T_H cells into transgenic mice that expressed both HBV antigens. The presence of HBeAg in the serum eliminated the expected T_H1 cell--mediated antibody response to HBcAg and shifted the response toward a T_H2 phenotype. These results suggest that in the context of an HBV infection, circulating HBeAg can preferentially deplete inflammatory HBeAg- and HBcAg-specific T_H1 cells necessary for viral clearance and thereby promote persistence of HBV.

To pursue and extend these observations, we recently produced mice transgenic for the T-cell receptor in which 70--80% of the CD4⁺ T_H cells are specific for HBeAg. Of importance, the genes for the receptor were derived from transgenic mice expressing HBeAg. Therefore, the CD4⁺ T cells present in the mice represent an HBeAg-specific population of cells that was selected in the presence of circulating HBeAg, as may occur during chronic HBV infection. Breeding mice transgenic for the T-cell receptor with mice transgenic for HBeAg indicated that this selected HBeAg-specific T-cell population was not deleted in the thymus or the periphery, in contrast to what normally occurs in doubly transgenic mice. Therefore, the HBeAg-specific T_H cells can coexist with circulating HBeAg. Characterization of this HBeAg-specific population of T_H cells will provide insight into the unique repertoire of nucleocapsid-specific T_H cells that survive deletion processes and are present during a chronic HBV infection.

HBcAg is extremely immunogenic during HBV infection in humans and after immunization in animals. We found that when HBcAg binds to its specific membrane immunoglobulin antigen receptors on HBcAg-specific B cells, the B cells become activated and express so-called costimulatory molecules such as B7.1 and B7.2. This B-cell activation allows the HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive T_H cells in vivo and to T-cell hybridoma cells in vitro approximately 100,000 times more efficiently than classical macrophage or dendritic antigen-presenting cells can. These results may explain why HBcAg is such a good immunogen, and this information can be exploited to use HBcAg as a carrier or platform for the design of vaccines against HBV and non-HBV organisms.

HEPATITIS C

Most patients infected with hepatitis C virus (HCV) cannot clear the virus and become chronically infected. To better understand the immune mechanisms that may influence HCV clearance, we are doing serologic studies of chronically infected patients and immunogenicity studies in a mouse model.

The IgG antibody responses to the HCV structural and nonstructural proteins in patients with chronic HCV infection are relatively low and are defective in terms of IgG subclass switching. These data suggest a suboptimal activation of T_H cells in chronic HCV infection rather than a skewing of the T_H cell subsets as observed in chronic HBV infection. To determine if HCV proteins are intrinsically poor immunogens, we immunized mice with recombinant and peptide versions of HCV proteins and analyzed the T- and B-cell responses. Among other findings, it was clear that HCV proteins were not intrinsically poor immunogens when injected into mice in sufficient dose and with adjuvant. Therefore, it will be necessary to examine other characteristics of the HCV-specific immune response to explain the defective humoral responses observed during chronic HCV infection.

PUBLICATIONS

Chen, M., Sällberg, M., Sönnerborg, A., Jin, L., Birkett, A., Peterson, D., Milich, D.R. Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus non-structural 3 protein. Hepatology, in press.

Hultgren, C., Milich, D.R., Sällberg, M. Antibodies to hepatitis B e (HBeAg) can be induced in HBeAg transgenic mice by adoptive transfer of a specific T-helper 2 cell clone. Clin. Diagn. Lab. Immunol. 4:630, 1997.

Maruyama, T., Kuwata, S., Koike, K., Iino, S., Yasuda, K., Yotsuyanagi, H., Moriya, K., Maekawa, H., Shibata, Y., Milich, D.R. Pre-core wild type DNA and immune complexes persist in chronic hepatitis B after seroconversion: No association between genome conversion and seroconversion. Hepatology 27:245, 1998.

Milich, D.R. The immune response to the hepatitis B virus: Infection, vaccination, animal models. Viral Hepatitis Rev. 3:63, 1997.

Milich, D.R. Influence of T helper cell subsets and crossregulation in HBV infection. J. Viral Hepatitis 4:(Suppl. 2):48, 1997.

Milich, D.R. Pathobiology of acute and chronic hepatitis B virus infection: An introduction. J. Viral Hepatitis 4:(Suppl. 2):25, 1997.

Milich, D.R., Chen, M., Hughes, J.L., Jones, J.E. The secreted hepatitis B e antigen can modulate the immune response to the nucleocapsid: A mechanism for persistence. J. Immunol. 160:2013, 1998.

Milich, D.R., Chen, M., Schödel, F., Peterson, D.L., Jones, J.E., Hughes, J.L. Role of B cells in antigen presentation of the hepatitis B core. Proc. Natl. Acad. Sci. U.S.A. 94:14648, 1997.

Sällberg, M., Hughes, J., Javadian, A., Ronlov, G., Hultgren, C., Townsend, K., Anderson, C.-G., O'Dea, J., Alfonso, J., Eason, R., Murthy, K., Jolly, D., Chang, S., Mento, S., Milich, D.R., Lee, W. Genetic immunization of chimpanzees chronically infected with the hepatitis B virus (HBV) using a recombinant retroviral vector encoding the HBV core antigen. Hum. Gene Ther., in press.

Zhang, Z.-X., Milich, D.R., Peterson, D.L., Birkett, A., Schwarcz, R., Weiland, O., Sällberg, M. Interferon-alpha treatment induces delayed CD4⁺ proliferative responses to the hepatitis C virus non-structural 3 protein regardless of the outcome of therapy. J. Infect. Dis. 175:1294, 1997.