Detection of M1, NP, and M1 proteins in TIV by cytotoxic CD8+ and CD4+ T cell lines. A. The CD8+ T cell line, 1-1 specific to the NP _383-391 epitope was tested for recognition of autologous target cells infected with three TIV vaccines at concentrations from 5-.05 μg/ml at an E/T ratio =10. ISCOMATRIX was first incubated with vaccinefor 24 hours, added to autologous BLCL for 1 hour, washed and then incubated overnight at 37 °C. B. The CD8+ T cell line, 1–7K, specific to M1_58-66 epitope was tested for recognition of autologous target cells infected with three TIV vaccines at concentrations from 5-.05 μg/ml at an E/T ratio =10. ISCOMATRIX was first incubated with vaccinefor 24 hours, added to autologous BLCL for 1 hour, washed and then incubated overnight at 37 °C. C. The CD4⁺ T cell line, 3E-2 specific to HA _267-283 epitope was tested for recognition of autologous target cells infected with three TIV vaccines at concentrations from 5-.05 μg/ml at an E/T ratio =10. D. The CD4+ T cell line, 1–3, specific to M1 _17-31 epitope was tested for recognition of autologous target cells infected with three TIV vaccines at concentrations from 5-.05 μg/ml at an E/T ratio =10.

ELISPOT responses to three commercial TIV vaccines using prevaccination PBMC from 30 donors. Prevaccination PBMC from 30 healthy donors were tested against each of the TIV vaccines for the frequency of IFNγ producing cells using IFNγ ELISPOT assay. Responses with media background were subtracted from each value. All values were expressed as SFU/10⁶. The A/Wisconsin/67/2005X-161B (H3N2) virus was tested in each subject as a positive control with average SFU/10⁶ =1209 ± 816 (Data not shown).

CD4 and CD8 T cell IFNγ responses to three commercial inactivated influenza vaccines in Donor NFLU002. Intracellular cytokine staining assay was performed on PBMC obtained from a naturally infected individual NFLU 002. For CD4 and CD8 T cell subsets, dead cells were gated out using Live- Dead stain viability marker and then the lymphocyte gate was drawn using forward and side scatter populations and further gating was done on the CD3+/CD4+ or CD3/CD8+ cells for either IFNγ cytokine positive cells. Shown in the first row are the flow cytometry plots depicting % of IFNγ-producing CD3+/CD8+ cells after stimulation with each of the three vaccines. The second row shows the flow cytometry plots depicting % of IFNγ producing CD3+/CD4+ T cells. Medium and PMA controls are also shown for both CD4 and CD8 populations. Geometric mean fluorescence intensity for each population is represented as MFI.

Comparison of the amount of internal proteins in the vaccines. A. M1. 45 μl of three TIVs were separated in a sodium dodecyl sulfate-polyacrylamide gel electroporesis (SDS-PAGE), and a goat polyclonal antibody recognizing the N-terminus of influenza A M1 was used. NC (H1N1) and W (H3N2) are A/New Caledonia/20/99 IVR-166 (H1N1) and A/Wisconsin/67/2005X-161B (H3N2) used as positive controls, and allantoic fluid was used as a negative control. Relative intensity of the band is shown. B. NP. A rabbitpolyclonal antibody specific to influenza A NP was used to detect the NP. C. HA (H1) as a control, HA (H1) was detected by a mouse monoclonal antibody specific to influenza A H1 HA. D, HA (H3) as a control, HA (H3) was detected by a mouse monoclonal antibody specific to influenza A H3 HA.