Dose-dependent Toxicity of Panenza on Memory T cells specific for influenza vaccine.

Abstract

<p>A- PBMC from patients vaccinated with the seasonal flu vaccine (Mutagrip) and the pandemic 2009 H1N1 vaccine (Pandemrix) were collected 21 days after vaccine administration. Cells (5×10⁵ per well) were stimulated for 5 days with Mutagrip or the nonadjuvanted pandemic 2009 H1N1 Panenza vaccine at the indicated concentrations (vaccine concentration is expressed as the final concentration of HA). Cells were also stimulated with CMV or EBV peptides at 0.25 μg/ml, tetanus toxoid (TT) or tuberculin PPD at 5 μg/ml. Cultures were pulsed with 1 μCi per well of [3H] thymidine over the final 16 h of culture and cell proliferation expressed in cpm. All tests were done in triplicate and results show the mean values for three patients. The bars indicate the mean and standard deviation. Asterisks indicate significant <i>P-</i>values (p = 0.03) for comparison of cultures stimulated with Panenza at 0.02 to 0.5 μg/ml to cultures stimulated with 0.01 μg/ml of Panenza (ANOVA test). B- PBMC from Pandemrix vaccinated patients were incubated with soluble Panenza at indicated concentrations, or added to plates coated overnight at 4°C with indicated concentrations of Panenza. H3TdR (1μCi/well) was added at day 4 and T-cell proliferation was assessed at day 5. The bars indicate the mean and standard deviation. Asterisks indicate significant <i>P-</i>values (* <0.05, ** <0.01, ***<0.001) for comparison of cultures stimulated with coated Panenza to cultures stimulated with soluble Panenza. C- PBMC from a patient vaccinated with Mutagrip and then with Pandemrix were incubated overnight with culture medium, Mutagrip or Panenza at 0.25 μg/ml and the percentage of apoptotic cells was determined by flow cytometry combining 7-AAD staining with CD3 and CD4 membrane detection. Dot plots represent the co-expression of CD4 and 7-AAD on gated CD3⁺ T cells. D- PBMC from a patient vaccinated with Mutagrip and Panenza were incubated overnight with either vaccine at various concentrations and the percentage of apoptotic (7-AAD⁺) cells within indicated subsets was determined combining 7-AAD staining with CD19, CD3, CD8, CD4, CD56, CD16 or CD14. B cells were CD3⁻CD19⁺, T cells were CD3⁺CD8⁺ or CD3⁺ CD4⁺, NK cells were CD3⁻CD56⁺CD16⁺, monocytes were CD3⁻CD19⁻CD14⁺.</p