Ad5 gene transfer to AI-WAm cells is limited by deficiency in expression and/or cell surface localization of the Ad5 receptor CAR.

<p><b>A.</b> Gene transfer efficiency of a human Ad5 vector expressing Luc reporter (Ad5 (L)) to an AI patient-derived ameloblast-like cells (AI-WAm) at different multiplicities of infection (MOI) (MOI = 10, 50 and 250 TCID₅₀/cell) in comparison to CAR-positive A549 and CAR-negative RD cells by conventional Luc assay at 20 hours post infection. Results are presented in Relative Luc Units (RLU) per cell with mean values shown above each bar plus/minus standard deviation. All differences were statistically significant (<i>P</i><0.05) <b>B.</b> Expression levels of hCAR mRNA in AI-WAm and A549 cells relative to that in RD cells as determined by qRT-PCR and presented as “fold difference”. All differences were statistically significant (P<0.05). <i>P_{(A549/AI-WAm)} = 0.027</i>. <b>C.</b> Quantitative analysis of hCAR and hCD46 mRNA expression levels in RD, AI-WAm and A549 cells as determined by qRT-PCR and normalized to the housekeeping gene <i>GAPDH</i>. The data are presented as ΔΔCt values. For AI-WAm <i>P_(CAR/CD46)</i> = 0.44; for A549 <i>P_(CAR/CD46)</i> = 0.127; <i>for RD P_(CAR/CD46)</i> = 0.007; for CAR <i>P_{(AI-WAm/A549)} = 0.049</i>; <i>P_(RD/AI-WAm) = 0.033</i>; for CD46 <i>P_(RD/AI-WAm) = 0.0011</i>; <i>P_{(AI-WAm/A549)} = 0.0008</i>. <i>P</i>-values for all other differences were <0.05. <b>D.</b> Flow cytometry analysis of Ad5 receptor (CAR) expression in AI-WAm cell population in comparison with CAR-positive (A549) and CAR-negative (RD) control cells. Cells were incubated with primary anti-CAR (RmcB) monoclonal antibody (Ab) followed by labeling with Alexa 488-conjugated secondary antibodies. No primary Ab was used in negative control samples. The extent of shift in the fluorescent peak positions (color lines) relative to control peak(s) of unlabeled cells (black dotted lines) reflects the extent of cell labeling, corresponding to the receptor expression on each cell type and is expressed as Mean Fluorescence Intensity (MFI). Numbers above each peak correspond to percentage (%) of gated (M2) cells calculated using subjective gating. Fluorescence intensity (X-axis) is plotted as histograms on log scale (X-axis) using Flowjo 7.6.4 software (Tree Star Inc., Ashland OR). Y-axis depicts total events (cells) and expressed either as counts or % of maximal. <i>P_{(AI-WAm/A549)} = 0.0038</i>; <i>P_(AI-WAm/RD) = 0.39</i>; <i>P_(A549/RD) = 0.0018</i>; <i>P_{(A549/HEK293T)} = 0.018</i>; CAR <i>P_{(AI-WAm/HEK293)} = 0.0001</i>; <i>P_(HEK293/RD) = 0.001</i>; <b>E.</b> Comparison of hCAR expression in AI-WAm and control cells by IHC staining. CAR-specific (RmcB) primary antibody (same as used for FACS analysis, D) was used to stain AI-WAm cells. A549 and HEK293-T cells were used as positive and RD cells as negative controls for CAR expression. All cells were counter-stained for 5 min. with 300 nM DAPI to visualize nuclear DNA (blue). No primary antibody was used with control samples. Negative control (RD) cells show efficient nuclear staining but no discernible CAR staining (green), while A549 cells demonstrate a strong CAR-specific signal and AI-WAm cells display a moderate level of hCAR signal with diffuse pattern of cytosolic localization (white arrows) similar to that in A549 cells. In sharp contrast, CAR-overexpressing HEK293-T cells show a distinct localization of CAR protein in the cell membrane tight junctions (red arrows) with lesser cytosolic staining. Scale bars correspond to 100 µm.</p

Schematic representation of the Ad5 fiber proteins carrying intact and modified C-terminal knob domains.

<p>Fiber modifications are indicated in the corresponding vector names: Ad5 (G/L) has unmodified fiber knob and possesses the native CAR tropism; Ad5-RGD contains a peptide ligand with an “RGD motif” in the HI loop (red loop) of the fiber knob; Ad5-pK7 contains a stretch of seven lysine residues (green oval) fused to the C-terminus of the Ad5 knob via a (GS)₅ linker (green hook); Ad5-pK7/RGD incorporates both modifications in the corresponding locales of the same fiber molecule; Ad5/3 contains a chimera fiber with Ad5 fiber “knob” domain (gray) replaced with the Ad serotype 3 (Ad3) knob (blue), which retargets the vector to Ad3 receptor(s).</p

The infectivity enhancement effect of fiber-modified Ad5 vectors is mediated by α_vβ3/α_vβ5 integrins and/or HSPG molecules on AI-ameloblasts.

<p><b>A.</b> Differential blocking of gene transfer to AI-WAm cells by integrins. Ad5 RGD shows the highest sensitivity to integrin blocking, while transduction with Ad5-pK7/RGD (G/L) is only partially inhibited. Ad5-pK7 (G/L) gene transfer shows no statistically significant inhibition by integrins. <b>B.</b> Blocking of AI-WAm gene transfer by modified vectors with heparin. Heparin shows a profound dose-dependent blocking effect on transduction with pK7-modified Ads, as opposed to RGD-modified vector. Gray bars (with % values on the top) show percentage of the residual gene transfer level (RLU) resulting from blocking relative to that of unblocked controls (100%) shown by black bar for each fiber-modified vector. All bars represent mean values with standard deviations. All differences were statistically significant except where indicated by asterisk and <i>P</i> values (<i>P</i>>0.05) on the data bars.</p

Expression analyses of HSPG and integrin molecules as alternate receptors for fiber-modified Ads on AI-WAm cells.

<p>Expression of α_vβ3 and α_vβ5 integrins (<b>A</b> and <b>B</b>) and heparin sulfate proteoglycans (<b>C–E</b>) in AI-WAm cell population and control cells was analyzed by flow cytometry (<b>A</b>, <b>C</b>, <b>E</b>) and IHC staining (<b>B</b> and <b>D</b>). Cells were incubated with primary anti-α_vβ3 or anti-α_vβ5 monoclonal antibodies for detection of corresponding integrin molecules or 10E4 antibody for detection of HSPG side chains (GAG) or anti-human syndecan 4 monoclonal antibody, followed by Alexa 488-conjugated secondary antibody. <b>A</b> and <b>C</b>, top charts: AI-WAm cells; middle charts: RD cells; bottom charts: A549 cells. For AI-WAm cells: <i>P</i>_{(αvβ3/αvβ5)} = <i>0.75</i>; <i>P</i>_(Synd4/HSPG) = <i>0.29</i>; For RD cells: <i>P</i>_(Synd4/HSPG) = <i>0.67</i>; <i>for</i> α_vβ3: <i>P_(RD/A549) = 0.38</i>; <i>for</i> α_vβ5: <i>P_{(AI-WAm/A549)} = 0.23</i>; <i>for syndecan 4: P</i>_{(<i>RD</i>/A549)} = <i>0.2</i>; <i>for</i> all other differences <i>P</i><0.05; <b>E.</b> HSPG Ab (10E4) specificity control sample: A549 cells were treated with heparitinase (10 U/ml) for 1 hr at 37°C to remove GAG side chains. Green arrow shows shift of the fluorescence intensity peak resulting from reduction in cell labeling with 10E4 antibody (MFI decrease). Other details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024281#pone-0024281-g002" target="_blank"><b>Fig. 2D</b></a>. <b>B</b> and <b>D</b>, scale bars correspond to: 100 µm in top image panels (integrins/AI-WAm, 10× objective), 10 µm (insert, 60× objective) and 50 µm (40× objective) in all other panels. Insert shows syndecan 4 staining image (60×) of A549 cells, clearly demonstrating a polarized intracellular localization of the protein.</p

Characterization of the human AI-ameloblast cell population by immunohistochemistry, ALP in situ histochemistry and qRT-PCR analysis.

<p><b>A.</b> Image of a tooth extracted from an AI patient that was used to establish an EOE primary cell culture. <b>B.</b> Phase contrast image of AI-WAm cell monolayer. <b>C.</b> AI-WAm cells stained for ALP activity followed by immunostaining for the epithelial marker cytokeratin 14; a single, highly ALP-positive cell is evident (arrow). <b>D.</b> Mouse molar stained for ALP activity showing ALP-negative secretory ameloblasts (Am) with highest activity (dark purple) in the stratum intermedium (SI) followed by the stellate reticulum (SR). Low (<b>E</b>) and high (<b>F</b>) magnification of AI-WAm cells positively stained for the major enamel protein Amel. Arrowheads on panel <b>F</b> indicate cells that appear polarized with unidirectional orientation of the Golgi apparatus. Low (<b>G</b>) and high (<b>H</b>) magnification of AI-WAm cells positively stained for the largest enamel protein Enam. <b>I.</b> Quantitative expression levels of the enamel matrix protein genes <i>AMBN</i>, <i>AMELX</i>, <i>ENAM</i>, <i>AMTN</i> and <i>ODAM</i> in AI-WAm cells relative to dental pulp and normal EOE cells as determined by RT-qPCR following normalization to a housekeeping gene <i>GAPDH</i> and presented as ΔΔCt values. Statistical analysis was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024281#s2" target="_blank">Materials and Methods</a>. Scale bars are: 50 µm (<b>B</b>, <b>F</b>, <b>H</b>), 100 µm (<b>C</b>, <b>D</b>, <b>E</b>, <b>G</b>).</p

Capsid-modified vectors can induce a greater number of Gag-specific CD8 T cells and memory T cells than wild type vectors.

<p>Cohorts of BALB/c mice (n = 8) were immunized (prime) by injection of 10¹⁰ vp i.m. with of one of the following Ad vectors: AdCMVGag, Ad5/HVR2-MPER-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i>. Gag-specific T cells were detected in the peripheral blood of mice 2 weeks following the initial vaccination, and given a boost immunization in the same manner on day 40. Peripheral blood Gag-specific CD8 T cells were enumerated 26 d.p.p, 69 d.p.p, and 84 d.p.b. A) Flow cytometric analyses bivariate pseudocolor plots are shown for a single mouse from each group for each time point. B) The percent and total number of Gag-specific T cells per million lymphocytes are shown for each mouse and their level of T cells linked for prime and boost time points. C) Paired scatter plots show significant differences in the percent and number of Gag-specific CD8 T cells induced by either MPER-modified or AdCMVGag gene encoding vaccines. Statistical significance was determined by student's t-test, (two-tailed) P<0.03.</p

HIV envelope gp41 or Gag genes were genetically incorporated into hexon hypervariable region 2 or adenovirus Δ<i>E1</i> region.

<p>Rescued viruses were amplified and viral DNA analyzed to confirm stable modification of relevant genes. A) Hexon-specific PCR primers confirmed incorporation of coding regions for MPER epitope inserts at the hexon HVR2 site. Lane 1, AdCMVGag; lane 2, Ad5/HVR2-MPER-L15(Gag). B) Gag-specific primers confirmed the incorporation of coding regions for Gag inserts in the Δ<i>E1</i> region. Lane 1, AdCMVGag; lane 2, Ad5/HVR2-MPER-L15(Gag). C) Vectors used for this study are depicted in this figure.</p

HIV epitopes incorporated in HVR2 are exposed on the virion surface.

<p>A) In the assay, varying amounts of AdCMVGag, Ad5/HVR2-MPER24-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i> were immobilized in the wells of ELISA plates and incubated with anti-gp41 antibody. The binding was detected with an HRP-conjugated secondary antibody. B) In the assay 6×10⁸ VP of either AdCMVGag, Ad5/HVR2-MPER-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i> were immobilized on an ELISA plate followed by varying dilutions of gp41 antibody (1;6,000; 1∶3,000;1∶1,500; and 1∶750). The binding was detected with an HRP-conjugated secondary antibody.</p

Comparison of infectivity between unmodified and hexon-modified Ad5 vectors.

<p>A) 293AD cells were infected at 10 MOI with either AdCMVGag (top panel) or Ad5/HVR2-MPER-L15(Gag) (bottom panel) adenoviral vectors for 24 hours and then Gag expression was measured by flow cytometry and analyzed with FlowJo version 8.8.6 software. The numbers indicate the percentage of cells positive for Gag expression. B) The experiment was repeated as in A (one of three representative experiments is shown) in the presense or absense of serially-diluted neutralizing ascites. The percentage (%) of infectivity was calculated by normalizing Gag expression to the infectivity in the absense of neutralizing ascites.</p

Adenovirus expressing capsid-incorporated HIV antigens elicit an HIV humoral immune response.

<p>BALB/c mice (n = 8) were primed and boosted with 10¹⁰ VP of Ad vectors. Pre-immunization, post-prime, and post-boost sera was collected at various time points for ELISA binding assays. 10 µM of purified MPER (EKNEKELLELDKWASLWNWFDITN) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera and the binding was detected with HRP conjugated secondary antibody. OD absorbance at 405_nm represents MPER antibody levels in sera.</p