CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees

Abstract

Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals.

Keywords: CD4+ T cell; class II epitope; hepatitis B virus; persistence; sterilizing immunity; vaccine.

Figure 1.

Patr class II restriction and location of epitopes. A, Recognition of Patr class II partially matched B-lymphoblastoid cell lines pulsed with HBsAg and pol epitopes by CD4+ T cells lines derived from blood of both animals is shown. Data are presented as interferon γ (IFN-γ) spot-forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs) detected by an enzyme-linked immunospot assay. B, The location of epitopes and lamivudine-associated mutations in hepatitis B virus (HBV) surface antigen (HBsAg; sE164D and sI195M) and polymerase (rtV173L, rtL180M, and rtM204V) are indicated.

Figure 2.

CD4⁺ T-cell cross-reactivity for epitopes altered by lamivudine selection pressure. CD4⁺ T-cell lines expanded from blood after vaccination or infection were assessed for recognition of target cells pulsed with varying concentrations of the indicated wild-type or mutant epitopes. Data are presented as the number of spot-forming cells (SFCs)/10⁶ antigen-specific CD4⁺ T cells. CD4⁺ T cells primed by vaccination of animal CH10369 (epitope s4; A) or animal CH10364 (epitope s2; B) were assessed for recognition of the wild-type and mutant epitopes. A CD4⁺ T-cell line established from animal CH10364 after mutant hepatitis B virus (HBVmt) challenge (C) was assessed for recognition of pol p1 expressed by HBVmt and wild-type HBV.

Figure 3.

T-cell responses to HBsAg after vaccination and infection. A, Peripheral blood mononuclear cells (PBMCs) collected from CH10364 at day 42 after wild-type HBV (HBVwt) cross-challenge were stimulated with the s1, s2, and s3 epitopes, and the HBsAg peptide pool with (HBs) or without (HBs –s1, s2, s3) the s1, s2, and s3 peptides. The mutant HBV (HBVmt) variant of peptide s2 was also included in the assay. B, Analysis of PBMCs from animal CH10369, incorporating the HBsAg peptide pool, the s4 HBVwt (sI195) and HBVmt (sI195M) epitopes, and the HBsAg pool with the s4 epitope removed (HBs −s4). Data are presented as interferon γ (IFN-γ) spot-forming cells (SFCs)/10⁶ PBMCs detected by an enzyme-linked immunospot assay.

Figure 4.

CD4⁺ T-cell depletion and rechallenge with wild-type hepatitis B virus (HBVwt). Anti-CD4 antibodies (cmT412) and HBVwt were delivered at the time points indicated by arrows. The absolute number of CD4⁺ T cells/mm³ of blood before and after cmT412 treatment is indicated. On the day of inoculation, serum anti–HBV surface antigen antibody (anti-HBsAg) titers were 18 300 and 6900 IU/L for animals CH10364 (top panel) and CH10369 (bottom panel). T-cell responses to the HBsAg (HBs T cells) and pol (pol T cells) peptide pools are presented as interferon γ (IFN-γ) spot-forming cells (SFCs)/10⁶ peripheral blood mononuclear cells (PBMCs).

Figure 5.

Detection of hepatitis B virus (HBV) genomes. Liver samples collected at the indicated time points were analyzed for the presence of HBV DNA by Southern blotting. A sample collected from the animals before hepatitis B vaccination and virus challenge (day −1204) is included for comparison. The positive control well shows a sample from chimpanzee liver infected with wild-type HBV from a previous experiment. The negative control well is a sample from a liver biopsy specimen obtained from a naive chimpanzee.