Primary Occult Hepadnavirus Infection Induces Virus-Specific T-Cell and Aberrant Cytokine Responses in the Absence of Antiviral Antibody Reactivity in the Woodchuck Model of Hepatitis B Virus Infection

Abstract

Although the virological features of serologically silent hepadnaviral primary occult infection (POI) have been relatively well recognized in the woodchuck model of hepatitis B virus infection, the characteristics of accompanying immune responses remain unknown. In this study, the kinetics of woodchuck hepatitis virus (WHV)-specific and generalized (mitogen-induced) T-cell proliferative responses and cytokine expression profiles in circulating lymphoid cells and the liver, along with WHV-specific antibody responses, were investigated during experimentally induced POI and subsequent challenge with a liver-pathogenic dose (>10 3 virions) or liver-nonpathogenic dose (50 virions) of the same virus. The data revealed that POI, which does not prompt WHV surface antigenemia, antiviral antibody response, and hepatitis or protect from challenge with a liver-pathogenic virus dose, was accompanied by the appearance of a strong WHV-specific T-cell response directed against multiple viral epitopes that intermittently persisted at low levels for up to 10-months during follow-up. Furthermore, immediately after exposure to a liver-nonpathogenic dose of WHV, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic stimuli and displayed augmented expression of alpha interferon, interleukin-12 (IL-12), and IL-2, but not tumor necrosis factor alpha. Overall, the kinetics of WHV-specific and mitogen-induced T-cell proliferative and cytokine responses in POI were closely comparable to those seen in infection induced by liver-pathogenic viral doses. The data demonstrated that virus-specific T-cell proliferative reactivity is a very sensitive indicator of exposure to hepadnavirus, even to small amounts inducing serologically mute infection. They also showed that hepadnaviral POI is not only a molecularly but also an immunologically identifiable and distinctive entity.Hepatitis B virus (HBV) is a noncytopathic virus causing an infection having several distinctive clinical profiles ranging from acute hepatitis (AH) or chronic hepatitis (CH) to a serologically undetectable, seemingly asymptomatic infection, called an occult HBV infection (OBI) (67). Following exposure to HBV, more than 90% of immunocompetent adults developing AH resolve liver inflammation (4, 17), but they fail to eradicate the virus completely and persistent occult infection seems to invariably follow (52,57,67,68,77). The remaining ϳ10% of individuals develop CH, which is diagnosed when detection of hepatitis B surface antigen (HBsAg) in serum and biochemical and histological indicators of liver inflammation protract for more than 6 months. This form of hepatitis frequently advances to cirrhosis and hepatocellular carcinoma (HCC) (4, 9).In the last decade, it became apparent that HBV replication commonly persists at low levels after resolution of AH in the context of apparent absence of clinical symptoms. It is also expected that this form of HBV infection could be a consequence of resolution of a clinically asymptomatic, but serologically tran...

Repeated Exposure to Trace Amounts of Woodchuck Hepadnavirus Induces Molecularly Evident Infection and Virus-Specific T Cell Response in the Absence of Serological Infection Markers and Hepatitis

“…This form of silent WHV infection was subsequently reproduced in adult animals by intravenous (i.v.) injection with WHV doses containing less than 1,000 DNase digestion-protected virions (11,12). WHV replication was again restricted to the lymphatic system and progressed without histologically apparent liver injury.…”

“…This is accompanied by expression of type 2 cytokines, such as IL-4 and IL-10 (33,35,36). Other studies, including those from this laboratory, delineated the kinetics of antiviral T cell and cytokine responses in POI (11,16,37,38).…”

“…In general, serum WHV loads during POI do not exceed 100 to 200 virus genome equivalents (vge) or copy numbers/ml, the virus has the wild-type sequence, and it retains liver-pathogenic competence when administered to virus-naïve animals at doses greater than 10 3 virions (9, 14) (Mulrooney-Cousins and Michalak, unpublished). It was also established that POI induces a WHV-specific T cell response but not an antiviral antibody response and, importantly, that the animals are not protected from reinfection and hepatitis when challenged with liver-pathogenic doses of WHV (i.e., Ͼ10 3 virions) (11,12,15). …”

“…The data acquired from the woodchuck model of hepatitis B showed that exposure to a singular low dose (i.e., Ͻ1,000 virions) of woodchuck hepatitis virus (WHV), which is a close relative of HBV (7)(8)(9), establishes serologically undetectable infection in which the virus genome and its replication are detectable when nucleic acid amplification assays of enhanced sensitivity are applied (10)(11)(12)(13). This molecularly evident but immunovirologically silent infection was designated primary occult infection (POI) (12,14).…”

Reovirus Virotherapy Overrides Tumor Antigen Presentation Evasion and Promotes Protective Antitumor Immunity

“…Lymphocytes were isolated from single-cell suspensions of spleens, inguinal lymph nodes (LN), or tumors using Ficoll-Paque Plus (Amersham Pharmacia) density gradient as described (32). These lymphoid cells were incubated with negative selection MACS beads (Invitrogen) containing anti-CD3, anti-CD14, and anti-B220 antibodies and then passed through magnetic column to obtain enriched dendritic cells (DC) with a purity of ≥95% as determined by subsequent flow cytometry analysis.…”