Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels

Abstract

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4(+) T cells broadly reactive with drifted H5. The CD4(+) response was dominated by IL-2(+) IFN-gamma(-) IL-13(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Conflict of interest statement

Conflict of interest statement: G.G., D.M., L.Z., M.B., S.N., S.T., C.S., A.K.H., V.B., A.B., R.R., G.D.G., and F.C. are employees of Novartis Vaccines, the sponsor of the study; and E.B. and C.M. have a PhD grant from Novartis Vaccines.

Figures

Fig. 1.

One dose of MF59-H5N1 induces the expansion of H5-specific CD4+ T cells, reacting to antigenically distinct H5 proteins. (A) Mean frequency of cytokine+ CD4+ T lymphocytes following in vitro stimulation of PBMC with a library of peptides spanning the whole H5 A/Vietnam/1194/2004. (*, significantly, P < 0.05, different from baseline; Wilcoxon's test for dependent variables). (B and C) Mean frequency (with 95% confidence interval, CI) of cytokine+ CD4+ T lymphocytes after in vitro stimulation of PBMC with a pool of peptides spanning the regions (B) nonconserved or (C) conserved between the indicated H5 strains or the whole H5 A/Vietnam/1194/2004. (*, significant, P < 0.05, differences compared to the Non-Adj-15 group; 1-factor ANOVA with least significant difference post hoc). PBMC were taken at the indicated time point from subjects vaccinated with nonadjuvanted H5N1 at 15 μg/dose (solid triangles or open bars), MF59-adjuvanted H5N1 at 7.5 μg/dose (solid squares or solid bars), and MF59-adjuvanted H5N1 at 15 μg/dose (solid circles or shaded bars).

Fig. 2.

Functional characterization of CD4+ T cells induced by vaccination with H5N1 A/Vietnam/1194/2004. Frequency of IL-13+ (open bars), IL2+IFN-γ (shaded bars), IL2+IFN-γ+ (solid bars), and IL2−IFN-γ+ (hatched bars) CD4+ T lymphocytes after a short in vitro stimulation of PBMC with (A and B) a library of peptides spanning H5 A/Vietnam/1194/2004 (H5-CD4+ T) or (B) the H5N1 subunit, the antigen preparation of the vaccine (H5N1-CD4+ T). (B) The pie charts represent the relative contribution (percentage) of each CD4+ T subset to the total response to H5 and H5N1. No significant variation in the proportion of IL-13+/IL-2+IFN-γ−/IL-2+IFN-γ+/IL-2−IFN-γ+ was associated with the plain or the MF59-adjuvanted vaccine formulations (P value >0.1, Kruskal–Wallis H-test, followed by Wilcoxon's test for pairwise comparisons). An asterisk marks the only significant variation observed (Wilcoxon's test, P = 0.049).

Fig. 3.

Two doses of MF59-H5N1 are required to expand a large and stable pool of H5N1-IgG memory B cells. Mean frequency (with 95% CI) of circulating H5N1-IgG memory B cells (MBC) as percentage of total circulating IgG MBC (*, significant, P < 0.01, different from baseline; Wilcoxon's test for dependent variables).

Fig. 4.

Two doses of MF59-H5N1 are required to induce sustained neutralizing antibodies. (A) Geometric mean titers (GMT) with 95% CI of circulating antibodies neutralizing the homologous A/Vietnam/1194/2004 NIBRG-14 recombinant virus in subjects vaccinated with Non-Adj-15 (triangles), MF59–7.5 (squares), and MF59–15 (circles). (*, significant, P < 0.01, different from baseline; Wilcoxon's test for dependent variables). (B) Percentage of subjects displaying MN antibody titers above the potentially protective threshold of 1:80 in Non-Adj-15 (open bars), MF59–7.5 (solid bars), and MF59–15 (shaded bars) groups.

Fig. 5.

Association between expansion of H5-CD4+ T cells after the first dose and MN titers at later time points. For each subject, the MN titer at day 223 (A) or 382 (B) is plotted vs. the H5-CD4+ T cells fold increase at day 22 over the preimmune time point. Horizontal dashed lines indicate the value of MN titer = 80, the proposed threshold of protective antibodies. Vertical dashed lines indicate the value of H5-CD4+ T cells 3-fold increase.