Cellular response against PR and RT induced by ID and IM DNA immunization assessed by FluoroSpot.

<p>Immune recognition of the peptides representing CD4⁺ and CD8⁺ epitopes of PR (A, B) and RT (C, D) by FluoroSpot test assessing in vitro production of IFN-γ (A, C) and IL-2 (B, D) in mice immunized with the plasmid encoding inactivated PR of HIV-1 HXB2 in pVax1 vector mixed with pVaxLuc (A, B); plasmid encoding inactivated RT of HIV-1 HXB2 in pVax1 vector mixed with pVaxLuc (C, D). Cytokine response to the immunodominant CTL epitope of Luc (LucP) in PR/Luc, RT/Luc and control empty vector/Luc DNA immunized mice (CTRL) is presented everywhere for comparison. Mice (n = 5 per group) were immunized as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197902#pone.0197902.g001" target="_blank">Fig 1</a> and their responses were assessed on experimental end-point at day 21 by INF-γ/IL-2 FluoroSpot. Data represent the average number of spot forming cells registered per million splenocyte per group (n = 5) with SE. Statistical comparison was done using Mann-Whitney U-test; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001; ****<i>p</i> < 0.0001.</p

In vivo registration of the volume of tissues emitting bioluminescence.

<p>Volume of tissues emitting bioluminescence in mice immunized intradermally (A, B) or intramuscularly (C, D) with PR/Luc (A, C) or RT/Luc (B, D) with sequential imaging done 20-24 hours post injection (day 1) and by the experimental end-point (day 21) (as indicated by the text box over the panels). Images were acquired by combined 3D BLI and micro-CT on Spectrum CT with data analysis using Living Image 4.5 software (both Perkin Elmer). Panels A-D demonstrate one representative mouse in a group of five. Images of the upper row of panels A-D represent combined micro-CT and 3D BLI in the coronal section, and of the lower row, external illumination surface reconstruction. Signal intensity in photons/sec is represented as a color scale to the right of each image.</p

Correlation between bioluminescence of the reporter gene and specific cellular responses to co-injected DNA immunogens.

<p>Correlation of the fraction of luminescence signal retained at injection sites on days 1 to 21 post immunization to the number of splenocytes expressing INF-γ, IL-2 and co-expressing INF-γ/IL-2 in mice co-immunized with PR/Luc and RT/Luc (as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197902#pone.0197902.g001" target="_blank">Fig 1</a>). Strength of correlation is depicted by a scheme relating <i>r</i> values to color (red for direct, and blue for inverse) and its intensity (weak from 0 to 0.5, moderate 0.6-0.8, or strong, <i>r</i> > 0.8) on the right to the heatmap. Correlation values represent spearman’s rank correlation coefficients; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001; ****<i>p</i> < 0.0001.</p

Antibody response against protease and reverse transcriptase assayed by ELISA.

<p>Antibody response in mice receiving PR and RT DNA by intradermal (skin) and intramuscular (muscle) delivery. Titer of IgG against PR (A), IgG, IgG1 and IgG2a against RT (B), and the ratio of anti-RT IgG2a/IgG1 (C). Mice (n = 5 per group) were immunized as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197902#pone.0197902.g001" target="_blank">Fig 1</a> and their responses were assessed on experimental end-point at day 21 by indirect ELISA on plates coated with recombinant PR (A), or RT (B, C) of HIV-1 HXB2 (NIH AIDS Reagent Program, Germantown, MD). Titer values represent average per group with SE. Statistical comparison was done using Mann Whitney U-test; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001; ****<i>p</i> < 0.0001.</p

List of HIV-1 protease- (PR) and reverse transcriptase- (RT) derived peptides used for <i>in vitro</i> test of immune response.

<p>Experimentally-verified peptides present in the Los Alamos National Laboratory database are demarcated by an asterisk following their amino acid position.</p

Analysis of protease and reverse transcriptase-specific immune response by flow cytometry.

<p>Analysis of CD4⁺ and CD8⁺ T cell responses in mice immunized with PR (A, C) and RT (B, D) presented as the percentage of the reactive CD8⁺ and CD4⁺ T cells (A, B) and as the percentage of cells producing INF-γ, IL-2, TNF-α, INF-γ/IL-2, INF-γ/TNF-α, IL-2/TNF-α, or INF-γ/IL-2/TNF-α with all reacting T cells taken for 100% (C, D). Peptides used for stimulation of murine splenocytes are designated by the number of first and last amino acid residues in the amino acid sequences of the respective proteins. The results of T cell response assays are from two to three independent DNA immunization experiments each done in 5 mice; all assays were done in duplicates. Frequency of T cell responses represents mean values ±SE. Statistical comparisons were done using multiple t-tests; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001; ****<i>p</i> < 0.0001.</p

In vivo kinetics of reporter signal after co-delivery of luciferase, and HIV PR and RT genes.

<p>Delivery and expression of HIV-1 protease (PR) and reverse transcriptase (RT) encoded by their expression-optimized genes in pVax1 vector (PR, RT) monitored indirectly by their co-administration with a plasmid directing the expression of firefly luciferase pVaxLuc (Luc). Mice (n = 5 per group) were immunized by ID or IM injections of PR/Luc, or RT/Luc, or pVax1/Luc, 10<i>μ</i>g each plasmid, administered in PBS at two sites to the left and to the right from the base of the tail. Injections were followed by electroporation performed as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197902#pone.0197902.ref058" target="_blank">58</a>] using a DermaVax electroporator equipped with multi-needle electrodes (Cellectis, Paris, France). Total photon flux from the injection sites was assessed by BLI on days 1, 3, 9, 15 and 21 as described in the Materials and methods. Data represent individual values for each injection site, and mean values (A). Luminescence kinetics measured by 2D BLI after delivery of PR and RT by ID (B) and IM routes (C). Luminescence kinetics was also registered by 3D BLI demonstrating the volume of expressing tissues after PR/Luc (D), RT/Luc (E) and vector/Luc administration (F). Data represent average photon flux (photons/sq cm/sec) and expression volume (mm³) for 4 to 5 mice per group and time point, with two simultaneous measurements per mouse. Statistical comparison was done using Mann Whitney U-test; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001; ****<i>p</i> < 0.0001.</p

Expression of IN variants in eukaryotic cells.

<p>Western blotting of lysates of HeLa cells transfected with the pVaxIN_a (lane 1), pVaxIN_a_e3 (lane 2), pVaxIN_in (lane 3), pVaxIN_in_e3 (lane 4), or empty vector pVax1 (lane 5); recombinant IN of HXB2 carrying 6His-tag (34 kDa) loaded in the amounts of 0.5, 2.5, 5 and 10 ng/well (lanes 7 to 10, respectively). Blot was stained with the rabbit polyclonal anti-IN antibodies, stripped, and re-stained with the monoclonal anti-actin antibodies. Molecular mass markers as defined by the protein ladder (Page Ruler Prestained Protein Ladder, Fermentas; lane 6) are given to the left (<b>A</b>). Average amount of the IN variants expressed per transfected human (HeLa, HEK293) or mouse (NIH3T3) cell (results of two independent runs, each done in duplicate) (<b>B</b>).</p

Catalytic activities of the recombinant integrases.

<p>Catalytic activities of the recombinant integrases.</p

Expression of integrase variants in <i>E.coli</i> BL21(DE3).

<p>SDS-PAGE analysis of the purified consensus HIV-1 clade A integrase (IN_a, lane 1), its inactivated variant (IN_in, lane 2) and the inactivated IN variant with elvitegravir resistance mutations (IN_in_e3, lane 3) eluted from the Ni-NTA-agarose column with 500 mM imidazole, followed by staining with Coomassie Blue (<b>A</b>). Western blotting of integrase preparations (diluted 1∶50) after SDS-PAGE and transfer, using polyclonal rabbit anti-IN antibodies (<b>B</b>). Data are representative of three independent experiments.</p