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. 2016 Jun 30;6:29131. doi: 10.1038/srep29131

Figure 2. Tat DNA vaccination induces Th cell responses and humoral immunity.

Figure 2

Mice (n = 7 per group) were vaccinated ID with 3 doses of 50 μg of pVAX, pVAX-Tat, pVAX-Tat-IMX313, pVAX-sTat or pVAX-sTat-IMX313 at 2 week intervals. Splenocytes were collected 14 days after the last dose. Blood was collected one day before each vaccination and on the day of euthanasia. (A) IFN-γ ELIspot assay to examine Tat-specific CMI in splenocytes stimulated with Tat peptides for 36 h. (B) FTA assay to examine Th cell responses from pVAX-Tat, pVAX-Tat-IMX313, pVAX-sTat or pVAX-sTat-IMX313 vaccinated mice above the GMFI of CD69 on FTA B220+ cells from naïve mice. The graph is representative of the FTA with cells pulsed with Tat peptide pools 1, 2 or 3. (C) AUC values for Th cell responses depicted in (B). (D) The results of an indirect ELISA to detect Tat-specific serum IgG. Titres are expressed as the reciprocal of the serum dilution and plotted as log10 IgG endpoint titre. The data are representative of 2 independent experiments in which each serum sample was analysed in duplicate. (E) Tat transactivation activity in Cf2-Luc cells after the addition of Tat pre-incubated in PBS or a 1/25 dilution of serum samples from mice vaccinated with the respective vaccines. Graphs show mean RLUs (±SEM) relative to untransfected control cells and are representative of 3 independent experiments in which each sample was analysed in triplicate. (F) Percentage decline in luminescence (an indicator of the neutralisation of Tat transactivation activity) depicted in (E). Data shown in the entire figure depict the mean (n = 7) ± SEM; an unpaired non-parametric Mann–Whitney U test was used to analyse the statistical significance of the data; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 and p ≥ 0.005 = non-significant (ns).