Immunological and antiviral responses after therapeutic DNA immunization in chronic hepatitis B patients efficiently treated by analogues

Abstract

A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ-producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ-producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4(+) T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.

Trial registration: ClinicalTrials.gov NCT00536627.

Figures

Figure 1

Treatment and sampling schedule. Antiviral treatments were stopped at W48 for all chronic hepatitis B (CHB) HBeAg negative patients except for 7 and restarted when hepatitis B virus (HBV) DNA level was >120 IU/ml in sera and this was confirmed by a second test 2 weeks later. Some patients were re-treated after the first positive viremia without waiting for the results of retesting according to the physician's decision. Dotted lines represent fluctuating periods of time. Blood samples were collected at W0, W18, W40, and W46 for immunological assessments.

Figure 2

Ex vivo interferon (IFN)-γ production by peripheral blood mononuclear cells (PBMCs) after stimulation with hepatitis B virus (HBV)-derived peptides. HBV-specific IFN-γ–secreting T cells were evaluated by the ELISpot assay at W0, W18, W40, and W46 in PBMCs from control (n = 28; left panels, empty circles) and vaccine (n = 31; right panels, black circles) groups after stimulation with preS2, S or Core peptide pools. Each symbol stands for one patient and only responders are shown. The number of IFN-γ–producing cells is expressed per 106 PBMCs and horizontal black bars represent medians.

Figure 3

Cytokine production by peripheral blood mononuclear cells (PBMCs) after stimulation with HBV peptides. PBMCs were stimulated as described in Figure 2 and the concentrations of IL-1β, IL-10, TNF-α, and of IL-6, IL-8, GM-CSF, IFN-γ cytokines released in the supernatants were determined at 48 and 96 hours, respectively. (a) The values of each axis represent the median concentration of cytokines (pg/ml) after stimulation of PBMCs from a group of healthy volunteers (n = 10; white circles) or from chronic hepatitis B (CHB) patients (n = 22; black circles) taken at W0. P values show statistical differences between patients and healthy volunteers (*P < 0.05; **P < 0.01;***P < 0.001). (b) The changes in cytokine profile after vaccination were calculated as the ratio of the cytokine concentrations found at W18, W40, or W46 and W0. The squares on each axis represent the calculated mean ratio for patients from the vaccine (n = 11; black squares) and control (n = 11; white squares) groups. Ratios greater than 2 (bold lines) are considered as positive.

Figure 4

Percentage of patients with T-cell responses and diversity of responses. Fresh peripheral blood mononuclear cells (PBMCs) from patients taken at different time points were stimulated with peptide pools corresponding to the hepatitis B virus (HBV) capsid (core) and envelope proteins for 10 days and interferon (IFN)-γ–secreting T cells were enumerated by an in vitro ELISpot assay. (a) Percentage of patients with positive IFN-γ ELISpot response (responders) among vaccine (n = 17; black bars) and control (n = 16; white bars) groups over time. (b) Pie charts represent the relative frequency of cells producing IFN-γ following re-stimulation with preS2 and S1-S5 envelope subpools.

Figure 5

Production of envelope-specific Th1 cytokines by short-term cell lines. Frozen peripheral blood mononuclear cells (PBMCs) of patients from vaccine (n = 12) and control (n = 10) groups were thawed and stimulated in vitro for 10 days with peptide pools corresponding to the envelope proteins encoded by the vaccine, and T-cell responses were measured by ICS assay. (a) Frequency of patients with envelope-specific IFN-γ–, IL-2–, or TNF-α–secreting CD4+ (left panel) and CD8+ (right panel) T cells analyzed from W0 to W46. Black and white bars represent % of patients with cytokine-producing cells from vaccine and control groups, respectively. (b) IFN-γ, IL-2, and TNF-α production by CD4+ (left panel) and CD8+ (right panel) T cells was evaluated from W0 to W46. Results are expressed as mean frequency of cytokine-positive T cells + SEM.

Figure 6

Evolution of cytokine expression patterns in T cells over time. Pie charts were created taking into account the total CD4+ T-cell responses of 13 and 11 patients from the vaccine and control groups, respectively. Pies stand for the mean fractions of CD4+ T cells able to produce single (white segment), double (gray segment), or triple (black segment) Th1 cytokines (IFN-γ, IL-2, and TNF-α) after stimulation with envelope-derived peptide pools at W0, W18, W40, and W46.

Figure 7

Virological responses after treatment discontinuation. (a) Time course of hepatitis B virus (HBV) DNA levels (log10 IU/ml) in sera of chronic hepatitis B (CHB) patients from the control (n = 20) and vaccine (n = 22) groups after treatment discontinuation at W48. As stated in the protocol, two and five patients from the control and vaccine groups did not stop therapy due to virological breakthroughs between W0 and W46. For 6 and 4 from the control and vaccine groups, HBV DNA level was available at only one time point during the off-therapy period because they resume their antiviral treatment without waiting for confirmation of viremia. Bold lines stand for virological responders (VR) defined as a decline of serum HBV DNA levels on two consecutive measurements. Dotted lines correspond to relapsers. (b) HBsAg titers (IU/ml) in relapsers (n = 29), VR (n = 13), and serological responders (SR; n = 4) at W46.