T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge

Abstract

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.

FIG. 1

Humoral and cellular immune responses of woodchucks NW7029, NW7030, NW7031, and NW7032 during an acute, self-limited WHV infection. T-cell responses to WHsAg, rWHcAg, peptide 97–110, and peptide 129–140 were monitored during WHV infection with 10⁵ woodchuck ID₅₀. WHsAg, anti-WHs, and anti- WHc in the sera were detected by ELISA. SDH levels were assessed by a commercial enzyme assay. Viremia was detected by DNA dot blot (boldface +) and PCR (lightface +). T-cell responses (5 × 10⁴ PBMC) after stimulation with 2 μg of WHsAg per ml or 1 μg of rWHcAg or peptides per ml were analyzed weekly after infection by BrdU and [2-³H]adenine incorporation. Results are presented as mean SI of triplicate determinations. The mean values (optical density at 450 nm) for the controls were 0.22 ± 0.08. The mean cpm for the controls was 3,213 ± 1,722.

FIG. 2

T-cell responses of PBMC 6 weeks after experimental WHV infection and of rWHcAg-specific T-cell lines established at 6, 12, 18, and 24 weeks postinfection to 1 μg of rWHcAg or WHcAg peptides per ml were analyzed by [2-³H]adenine incorporation. PBMC (5 × 10⁴) were obtained from woodchucks NW7029, NW7030, NW7031, and NW7032 6 weeks after infection. T cells (5 × 10⁴) were derived from polyclonal T-cell lines established at weeks 6, 12, 18, and 24 by continuous stimulation with rWHcAg. Results are presented as mean SI of triplicate determinations. The mean cpm for the controls was 7,043 ± 3,368.

FIG. 3

T-cell responses of PBMC 1 year after experimental WHV infection. PBMC (5 × 10⁴) were obtained from woodchucks NW7030 and NW7031 and stimulated with 1 μg of rWHcAg or WHcAg peptides per ml. Proliferation was determined by [2-³H]adenine incorporation. Results are presented as mean SI of triplicate determinations. The mean cpm for the controls was 3,015 ± 1,968.

FIG. 4

Humoral and cellular immune responses of woodchucks NW7030 and NW7031 after viral challenge. T-cell responses to WHsAg, rWHcAg, peptide 97–110, and peptide 129–140 were analyzed after challenge with 10⁹ woodchuck ID₅₀. WHsAg, anti-WHs, and anti-WHc in the sera was detected by ELISA. Viremia was tested by nested PCR (top). T-cell responses (5 × 10⁴ PBMC) to stimulation with 2 μg of WHsAg per ml or 1 μg of rWHcAg or peptides per ml were assayed each week postchallenge (ch) by [2-³H]adenine incorporation (bottom). The mean cpm for the controls was 2,976 ± 1,711.

FIG. 5

T-cell responses of PBMC (5 × 10⁴) and rWHcAg-specific T-cell lines (5 × 10⁴) 1 week after viral challenge to 1 μg of rWHcAg or WHcAg peptides per ml were analyzed by [2-³H]adenine incorporation. PBMC were obtained from woodchucks NW7030 and NW7031 1 week postchallenge. T cells were derived from polyclonal T-cell lines established 1 week after viral challenge by continuous stimulation with rWHcAg. Results are presented as mean SI of triplicate determinations. The mean cpm for the controls was 4,691 ± 2,352.