Deficiencies in the acute-phase cell-mediated immune response to viral antigens are associated with development of chronic woodchuck hepatitis virus infection following neonatal inoculation

Abstract

In vitro proliferation of peripheral blood mononuclear cells was used to measure virus-specific cell-mediated immunity (vCMI) following neonatal woodchuck hepatitis virus (WHV) infection. Fifteen neonates were inoculated with the W8 strain of WHV. In 11, infection was resolved, and 4 became chronic carriers. Nineteen neonates were inoculated with the W7 strain and all became chronic carriers. Seven age-matched uninfected woodchucks served as controls. Virologic and vCMI profiles among the W8 and W7 infections were compared and related to the outcome of infection. Resolving woodchucks had robust, acute-phase vCMI to WHV antigens (core, surface, and x) and to several nonoverlapping core peptides. The acute-phase vCMI was associated temporally with the clearance of viral DNA and of surface antigen from serum at 14 to 22 weeks postinfection. In contrast, in approximately half of the W8 and W7 infections that progressed to chronicity, no significant acute-phase vCMI was detected. In the remaining carriers, acute-phase vCMI was observed, but it was less frequent and incomplete compared to that of resolved woodchucks. Serum viral load developed less rapidly in those carriers that had evidence of acute-phase vCMI, but it was still increased compared to that of resolving woodchucks. Thus, vigorous and multispecific acute-phase vCMI was associated with resolution of neonatal WHV infection. Absent or incomplete acute-phase vCMI was associated with the progression to chronic infection. By analogy, these results suggest that the onset of chronic hepatitis B virus (HBV) infection in humans may be associated with deficiencies in the primary T-cell response to acute HBV infection.

FIG. 1.

WHV DNA in serum and vCMI to rWHcAg and WHc peptide C97-110 in woodchucks with different outcomes following experimental neonatal infection with cWHV8P1 (W8). (A) Woodchucks in which W8 infection was resolved. (B) Woodchucks in which W8 infection became chronic. (C) Uninfected control woodchucks. SI values of ≥3.1 (cutoff line) were considered positive for specific vCMI. See Materials and Methods for other details. Black bars, WHV DNA in serum; ▵, vCMI (SI) to rWHcAg; ○, vCMI (SI) to WHc peptide C97-110. The mean counts per minute ± SD of the unstimulated PBMC from resolved woodchucks, from chronic carriers, and from uninfected control woodchucks were 3,304 ± 2,117, 3,209 ± 1,884, and 3,016 ± 1,822, respectively. Levels of WHV DNA are expressed in genomic equivalents (ge) per milliliter.

FIG. 2.

Comparison of vCMI to rWHcAg and WHc peptide C97-110, and of WHV DNA and WHsAg in serum in chronic carriers with significant acute-phase vCMI (SI ≥ 3.1, vCMI⁺) and without significant acute-phase vCMI (vCMI⁻) following experimental neonatal infection with WHV7P1 (W7). (A) vCMI to rWHcAg (SI). (B) vCMI to WHc peptide C97-110 (SI). (C) Titers of WHV DNA in serum (geometric mean log viral genomic equivalents [ge] ≥ per milliliter ± SD). (D) Relative levels of WHsAg in serum (mean optical density units [ODU] ± SD). SI values of ≥3.1 (cutoff line in panels A and B) were considered positive for specific vCMI. The interval during which significant acute-phase vCMI was observed is shown by a bar and brackets. The asterisk identifies a late and isolated result above the positive cutoff line. The mean counts per minute ± SD of the unstimulated PBMC was 3,688 ± 2,239 for the vCMI⁺ carriers and 3,593 ± 2,511 for the vCMI⁻ carriers.

FIG. 3.

Summary of vCMI to WHc peptides in woodchucks with resolved WHV infection and in carriers with positive acute-phase vCMI. Values for the W8 and W7 outcome categories were expressed relative to the most frequently recognized WHc peptide, C97-110, in the resolved W8 infection group (i.e., 51% in Table 3 was set at 100%, underlined, here). Other values were calculated accordingly. Sites I to V were demarcated based on differential and similar response frequencies for the present groups of neonatally infected woodchucks and also on other published information (33, 34). In site IV, a and b represent at least 2 nonoverlapping CMI-related epitopes, one of which (C97-110) has been shown previously to be protective in experimental challenge studies (34). Sites I, II, and IV appeared to be preferred recognition sites for woodchucks with resolved infections (bold numbers). Site III appeared to be recognized similarly in both outcome groups. Site V may be recognized preferentially by neonatal carriers (numbers are marked with an asterisk). See the text for details and Table 3 for relevant statistical comparisons.