Matrix M(TM) adjuvanted virosomal H5N1 vaccine induces balanced Th1/Th2 CD4(+) T cell responses in man

Abstract

T cellular responses play a significant role in mediating protective immune responses against influenza in humans. In the current study, we evaluated the ability of a candidate virosomal H5N1 vaccine adjuvanted with Matrix M(TM) to induce CD4(+) and CD8(+) T cell responses in a phase 1 clinical trial. We vaccinated 60 healthy adult volunteers (at days 0 and 21) with 30 μg haemagglutinin (HA) alone or 1.5, 7.5, or 30 μg HA formulated with Matrix M(TM). To evaluate the T cellular responses, lymphocytes were stimulated in vitro with homologous (A/Vietnam/1194/2004 [H5N1]) and heterologous H5N1 (A/Anhui/1/05 or A/Bar-headed Goose/Qinghai/1A/05) antigens. The antigen-specific cytokine responses were measured by intracellular cytokine staining and by multiplex (Luminex) assays. An increase in CD4(+) Th1 and Th2 cytokines was detected 21 days after the first vaccine dose. No increase in Th cytokine responses was observed after the second dose, although it is possible that the cytokine levels peaked earlier than sampling point at day 42. Formulation with the Matrix M(TM) adjuvant augmented both the homologous and cross-reactive cytokine response. Antigen-specific CD8(+) T cell responses were detected only in a few vaccinated individuals. The concentrations of Th1 and to a lesser extent, Th2 cytokines at 21 days post-vaccination correlated moderately with subsequent days 35 and 180 serological responses as measured by the microneutralisation, haemagglutination inhibition, and single radial hemolysis assays. Results presented here show that the virosomal H5N1 vaccine induced balanced Th1/Th2 cytokine responses and that Matrix M(TM) is a promising adjuvant for future development of candidate pandemic influenza vaccines.

Keywords: CD4; CD8; H5N1; Matrix M; Th1; Th2; cytokine; human; influenza; vaccine.

Figures

Figure 1. The Matrix MTM adjuvanted boosted the Th1 and Th2 cytokine responses. Four groups of 15 vaccinees were given 2 immunizations with 30 μg HA of H5N1 NIBRG-14 virosomal vaccine alone (30 μg-) or increasing doses of virosomal vaccine (1.5, 7.5, or 30 μg HA) in combination with Matrix MTM adjuvant (+). PBMCs were isolated at indicated time-points and stimulated for 72 h with H5N1 virosomes. The supernatant was frozen at –80 °C and later thawed for analysis of Th1 (IL-2, IFN-γ, TNF-α), Th2 (IL-4, IL-5, IL-10, IL-13), and Th17 (IL-17) cytokines and GM-CSF. The mean ± SEM is shown for each vaccine group. *, **, and *** indicate significant differences (P < 0.05, P < 0.01, and P < 0.001, respectively) from the 30 μg- group, One-way ANOVA with the Dunnet post hoc test.

Figure 2. The cross-reactivity of the Th1 cytokine responses measured by Luminex and correlation with homologous responses. Four groups of 15 vaccinees were given 2 immunizations with 30 μg HA of H5N1 NIBRG-14 virosomal vaccine alone (30 μg-) or increasing doses of virosomal vaccine (1.5, 7.5 or 30 μg HA) in combination with Matrix MTM adjuvant (+). PBMCs were isolated at day 42 into the study and stimulated for 72 h with influenza H5N1 antigen. The homologous response was evaluated toward the NIBRG-14 antigen, using the H5N1 virosomes. The cross-H5 clade response was measured against A/Anhui/1/05 (Anhui) and A/Bar-headed Goose/Qinghai/1A/05 (BHG). The supernatants were frozen at −80 °C and later thawed for analysis of Th1 (IL-2, IFN-γ, TNF-α) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokines. (A and B) Mean cytokine concentrations +SEM after Anhui or BHG stimulation (bars, y-axis) and response in percentage relative to that observed for homologous stimulation as shown in Figure 1 (circles, second y-axis) are shown. *Indicates significant difference (P < 0.05) from the 30 μg- group in a One-way ANOVA with Dunnet’s post hoc test. (C–H) Individual IL-2, IFN-γ, and TNF-α cytokine concentrations after NIBRG-14 stimulation plotted against those after Anhui or BHG stimulation as indicated on the y-axis. The correlation was tested by Spearman rank test and the correlation coefficient (r) and P value are shown on the graphs.

Figure 3. The Matrix MTM adjuvant boosted the frequencies of Th1 cells specific for the homologous strain, while lower frequencies of cells were cross-reactive toward HA of heterologous clades as measured by intracellular staining. Four groups of 15 vaccinees were given 2 immunizations with 30 μg HA of H5N1 NIBRG-14 virosomal vaccine alone (30 μg-) or increasing doses of virosomal vaccine (1.5, 7.5 or 30 μg HA) in combination with Matrix MTM adjuvant (+). PBMCs were isolated at day 42 into the study and stimulated for 16 h with influenza H5N1 antigen, co-stimulators, brefeldin A, and monensin. The homologous response was evaluated toward the NIBRG-14 antigen (clade1), using the H5N1 virosomes. The cross-H5 clade response was measured against A/Anhui/1/05 (Anhui) (clade 2.3,4) and A/Bar-headed Goose/Qinghai/1A/05 (BHG) (clade 2.2). (A) The mean percentage of influenza-specific CD4+ T cells ± SEM is shown for each vaccine group. *, **, and *** indicate significant differences (P < 0.05, P < 0.01, and P < 0.001, respectively) from the 30 μg- group, One-way ANOVA with Dunnet’s post hoc test. (B and C) Correlation between NIBRG-14 and Anhui/BHG reactive CD4+ T cells. The correlation was tested by Spearman rank test and the correlation coefficient (r) and P value are shown on the graphs.

Figure 4. A correlation was observed between cytokine responses measured by multiplex and intracellular cytokine staining. Four groups of 15 vaccinees were given 2 immunizations with 30 μg HA of H5N1 NIBRG-14 virosomal vaccine alone (30 μg-) or increasing doses of virosomal vaccine (1.5, 7.5, or 30 μg HA) in combination with Matrix MTM adjuvant (+). PBMCs were isolated at day 21 into the study and stimulated in vitro with influenza H5N1 virosomes for 16 h for intracellular cytokine staining (ICS) or 72 h for Luminex analysis. ICS was performed on fresh cells, gating on CD3+CD4+ T cells, while the supernatant was frozen at –80 °C and later thawed for analysis of IL-2, IFN-γ, and TNF-α. A to C) Graphs show the correlation between CD4+ T cells displaying the indicated cytokine after ICS (x-axis) plotted against cytokine concentrations measured by Luminex (y-axis). The correlation was tested by Spearman rank test and the correlation coefficient (r) and P value is shown in the graphs.