Induction of a transgene-specific memory response.

<p>C57Bl/6 female mice were injected IV with PBS or LV-MHCII. 35 days later, mice were challenged by an intramuscular injection of a mixture of Dby and Uty peptides complexed with IFA or as control with PBS complexed with IFA. IFNγ secretion of splenocyte was assayed <b>A</b>. 5 days after the challenge following a 24 hour Dby restimulation. <b>B</b>. 14 days after the challenge following a 24 hour Dby restimulation or <b>C</b>. Uty restimulation. Each symbol represents IFN-γ spot-forming unit (duplicate measures) of each mouse from 2 independent experiments (n = 8 mice per group). Statistical analysis shows ** for p values<0.01 and *** for p values<0.001.</p

Evaluation of vector biodistribution and the vector-targeted cell population.

<p>C57Bl/6 female mice were injected intravenously with PBS, 50 ng RT of LV-VSVg or LV-MHCII. 3 days later, spleen, thymus, lymph nodes, kidney and liver from these mice were harvested. <b>A</b>. The vector copy number (VCN) per cell was assessed by qPCR on genomic DNA extracted from spleen, thymus, kidney, liver (n = 5 mice) and <b>B</b>. from sorted spleen population with GFP-HY specific Taqman probes (n = 2) <b>C</b>. GFP-HY expression in the thymic, splenic and lymph node immune cells was assessed by flow cytometry. Thymic, splenic and lymph node cells were marked by CD3, CD19, MHC-II, CD11b or CD11c to identify macrophages, DC, T and B cells. GFP-HY fluorescence for each population is shown (n = 2).</p

Characterization of the LV model.

<p>A. Schema of the transfer plasmid. The pRRL-PGK-GFP-HY transfer plasmid used for the production of LV-MHCII or LV-VSVg contains a heterologous RSV/HIV 5′ long-terminal repeat (LTR) and 3′ deleted LTR, psi encapsidation and Rev response element (RRE) sequences, a human phosphoglycerate kinase 1 gene (PGK) promoter, GFP-HY transgene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). BamH1 and Sal1 sites used for cloning are shown. B. Representative expression of GFP obtained from GFP-HY constructs. Result from 2 experiments showing expression of GFP by flow cytometry in 293T cells transfected with pRRL-PGK-GFP-HY (solid line, Mean Fluorescence intensity (MFI): 25683) or with the pRRL-PGK-GFP plasmid containing the native GFP (dotted line, MFI: 56779) or without plasmid. C. Production of LV-MHCII vector according to different concentration protocols: 50000 g 2 hours 4°C (n = 1), 2500 g 12 hours 4°C (n = 3) or 2500 g 24 hours 4°C centrifugation (n = 5) and measured as P24 concentration factor (left panel) or P24 yield (right panel) after P24 titers were measured on harvested stock and concentrated product. P24 concentration factor and yield were calculated for each condition tested n = 1 to 5. D. Transduction specificity <i>in vitro</i>. Freshly isolated C57Bl/6 mice splenocytes (5.10⁵ cells per well) pre-stimulated with IL2, IL4, IL7 overnight and the next day, LV-MHCII (50 ng RT/well) was added to the culture or not. Two days later, the expression of GFP-HY and MHC class II were measured by multi-color flow cytometry on the live population (7AAD- cells) and/or on the transduced cells in 3 separate experiments. Plots and histograms show additional CD3 and CD19 staining performed in 2 of these experiments.</p

Immune responses induced by IV administration of LV with different pseudotypes.

<p>C57Bl/6 female mice were injected IV in the tail vein with PBS, LV-VLP, LV-VSV or LV-MHCII vectors. Immune responses were measured by IFNγ ELISPOT following a 24-hour-Dby or Uty ex vivo stimulation of splenocytes (<b>A</b> to <b>D</b> and <b>F</b>) or a <i>in vivo</i> cytotoxic assay (<b>E</b>). A. and B. Dose response curves with LV-MHCII. Each mouse received IV 0, 10, 30, 50 or 100 ng RT and 14 days later, spleen cells were collected and IFNγ ELISPOT was performed following Dby (a) or Uty (b) stimulation. Each symbol represents IFN-γ spot-forming unit (duplicate measures) of each mouse (n = 3 mice per group). C. and D. Kinetics of response to 50 ng RT of vector. Immune response to LV-MHCII (C) or LV-VSVg-GFP-HY (D) was measured by IFNγ ELISPOT 3, 7, 14 and 21 days after injection and following Dby or Uty peptide stimulation as indicated (n = 2 separate experiments, 5 to 10 mice per group). E. <i>In vivo</i> cytotoxic assay. C57Bl/6 mice were injected with 50 ng RT of LV-VSVg or LV-MHCII. 13 days later, a mix of congenic C57Bl/6 CD45.1 male CFSE^low-labeled cells and female CFSE^high-labeled cells was injected to the previously immunized mice (10⁷ cells per mice) to measure the specific killing of male cells by flow cytometry (n = 2, 10 mice per group). Statistical analyses using Student’s t Test or ANOVA with Bonferroni multiple group comparison show significance indicated with ** for p values<0.01 and *** for p values<0.001. F. Lack of immunization from VLP. 50 ng RT of LV-VLP were injected per mice and 14 days later immune responses were measured by IFN-γ ELISPOT (n = 3 mice per group).</p

Cytokines secreted following immunization.

<p>C57Bl/6 female mice were injected IV with PBS, 50 ng RT of LV-VSVg or LV-MHCII vectors. 7 days later, spleen cells of these mice were harvested and assayed for IL-2, IL-4, IL-6, IL-10, IL-17A, IFNγ, TNFα and GM-CSF secretion after a 48 hour stimulation ex vivo with Dby (<b>A</b>) or Uty (<b>B</b>) using the cytokine bead array (n = 5 mice per group). Data and assay limits of detection are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101644#pone-0101644-t002" target="_blank">Table 2</a>.</p

LV-MHCII vector production titers.

<p>Virus particles were produced by transient transfection of 293T cells and the harvested stock was concentrated using the indicated conditions of centrifugation. The concentrated LV was 0.45 µm-sterile filtered, cryopreserved (−80°C) and subsequently titered for P24 or RT content by ELISA. n = number of independent productions.</p

Cytokine quantification by cytometric bead array technology following <i>ex vivo</i> spleen cell restimulation with peptides.

<p>The data presented here are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101644#pone-0101644-g003" target="_blank">Figure 3</a> and represent averaged values of cytokine levels measured by CBA in 5 mice ± standard deviation. The limits of detections for each cytokine are: IL-2: 0.2 pg/mL; IL-4: 0.3 pg/mL; IL-6: 1.4 pg/mL; IL-10: 9.6 pg/mL; IL-12p70: 1.9 pg/mL; IL-17A: 0.95 pg/mL; IFNγ: 0.5 pg/mL; TNFα: 2.8 pg/mL and GM-CSF: 1.5 pg/mL.</p

MOESM1 of Systemic epigenetic response to recombinant lentiviral vectors independent of proviral integration

Additional file 1. Characteristics of different batches of vector and particles tested on CD34+ cells