Detection of antigen-specific CD4 T cells against influenza viruses by flow cytometry after in vitro expansion of effector cells. Peripheral blood mononuclear cells were expanded in vitro with interleukin-2 (IL-2) for 9 days in the presence or absence of specific influenza antigens, as indicated, then analyzed by flow cytometry by using the intracellular staining assay. The effector T-cell response was analyzed for interferon-gamma (IFN-γ) or IL-2 cytokine expression. Unstimulated cultures (A), CD4 T-cell response against human influenza vaccine strain preparation (B), inactivated avian influenza (H5N1) (C), and H5/N1 peptides (D) are shown in a representative donor.

H5 versus N1 specificity of the cell-mediated response. Profiling of influenza (H5N1)–specific CD4 T-cell response in a representative study participant is shown. Peripheral blood mononuclear cells (PBMC) were expanded in vitro in the presence of interleukin-2 (IL-2) and stimulated with inactivated influenza (H5N1) virus (B), peptide pool composed by 4 peptides from H5 and N1 (C), H5 peptides (D) and N1 peptides (E). Panel A shows unstimulated cultures. Dot plots showed the presence, at similar frequency, of specific CD4 T cells when PBMC were stimulated with inactivated influenza (H5N1) virus (panel B, 1.82%), influenza (H5N1) peptides (panel C, 1.52%), and N1 peptides (panel E, 1.49%). No specific CD4 T cells producing interferon-gamma (IFN-γ) were observed after challenge with H5 peptides (D). As negative control, either mock-infected culture supernatants or irrelevant peptides were used, giving results very similar to unstimulated cultures (not shown). A similar pattern was observed in 4 other study participants, supporting the hypothesis that the actual target of cross-subtype immunity could be N1.

Humoral response against vaccine preparation and influenza virus (H5N1) before (t0) and after (t1) seasonal influenza vaccination. Hemagglutination inhibition (HI) test was used to calculate the antibody (Ab) titer against vaccine preparation (top panel), whereas a neutralization test was used to calculate the antibody titer against influenza (H5N1) (bottom panel) in healthy donors enrolled in the study at baseline (t0) and 1 month after seasonal influenza vaccination (t1). At baseline (white bars), all donors had a detectable level of human influenza antibodies. At t1 (black bars), 28 donors (73.6%) (indicated by *) showed a >4 fold increase of Ab titer against vaccine preparation (HI) over t0. After seasonal influenza vaccination, 13 serum samples (33.3%) (indicated by *) from the study population showed a 20-fold increase of neutralizing Abs against influenza (H5N1) over t0..