Lentivectors induce gradual increase in antigen load in expressor cells.

<p>(<b>A</b>) Mice were immunized with Lv-Luc vector (encoding also eGFP) and luciferase expression was tracked <i>in vivo</i>. (<b>B</b>) Part of the Lv-Luc primed mice were euthanized on days 1, 5, 7 and 14 and their ears were collected and processed to obtain genomic DNA. DNA samples were subjected to quantitative real-time PCR analysis in order to calculate the relative amount of lentiviral DNA in each day tested. Lentiviral DNA was quantified using eGFP specific primers and was standardized according to the levels of endogenous 18S DNA. (<b>C</b>) The ears of Lv-Luc immunized mice were removed on days 1 and 11 post priming and were then subjected to immunofluorescence analysis. Images of confocal microscopy of the ear pinna are shown with a 5-µm-thick section using a 10×0.6 objective and 25× zoom. Control image represents staining with secondary antibody only. (Blue, nuclei stained with the DNA intercalating dye DAPI; green, anti-GFP antibody). Arrow heads indicate GFP-expressing cells. Dotted line was added to define the auricular cartilage (AC), D-dermis, E-epidermis. One representative out of two independent experiments is depicted. *, <i>P</i><0.005, compared to all immunized groups. #, <i>P</i><0.001, compared to DNA copies measured on days 1, 5 and 7 post-immunization.</p

Kinetics of antigen expression control lentivector-induced secondary CD8⁺ T-cell response.

<p>(<b>A</b>) B6 mice were primed, or primed and boosted intradermally with 5×10⁶ TU of Lv-OVA/Luc, and luciferase expression was determined <i>in vivo</i> using whole body imagining. The mean relative light unit (RLU) values expressed by a group of 4 mice ± SE are presented. (<b>B</b>) Immunization site of Lv-OVA (5×10⁶ TU) homologously boosted mice was removed on days 2, 4 or 6 following immunization, and the magnitude of OVA-specific CD8⁺ T cells was analyzed. Data are showed as the mean percentage ± SE of CD8⁺ tetramer⁺ T cells. n  = 3 mice per group for each time point. (<b>C</b>) Mice were primed with Lv-OVA and 7 weeks later were boosted with Lv-OVA (Lv-Lv) or Ad-OVA (Lv-Ad). In parallel, other groups of naïve mice were primed with Lv-OVA (Lv prime) or Ad-OVA (Ad prime) using the same viral vector employed for boosting to allow adequate comparison. Two weeks after immunization tetramer analysis was performed on blood samples obtained from the mice (n  = 4 to 5 mice for each group). The graph represents the fold increase in the magnitude of OVA-specific CD8⁺ T cells, Lv-Lv versus Lv prime, and Lv-Ad versus Ad prime. Results described in this figure are representative of two independent experiments. *, <i>P</i><0.01, prime versus boost response at the indicated time points. #, <i>P</i><0.05, compared to control uncut boosted group.</p

Shortened kinetics of antigen presentation <i>in vivo</i> in lentivector-boosted mice.

<p>B6 mice were primed, or primed and boosted with Lv-OVA, and then were adoptively transferred i.v. with 2×10⁶ CFSE-labeled OT-I (<b>A</b>) or OT-II (<b>B</b>) splenocytes at the indicated days. Three days later the LNs were harvested and the CFSE dilution was assessed by flow cytometry to analyze the proliferation of the transferred CD8⁺ or CD4⁺ T cells, respectively. Results are shown as representative flow plots gating on dividing CD8⁺ or CD4⁺ lymphocytes; numbers indicate the percentages of dividing cells and represent the mean of three mice per group for each time point ± SE. One representative out of 2 independent experiments is depicted. *, <i>P</i><0.01, primed mice versus boosted mice at the time points indicated.</p