Transduction of human genital epithelial stem cells by AAV-6-GFP. At 3 days after exposure to AAV-6-GFP, cells were stained with anti-Ck17 and anti-p63 antibodies.

<p>(A) The stained cells were analyzed by flow cytometry by gating first on CK17/p63 double positive cells (A) and then on the GFP-positive cells within this population (B). The AAV-6-GFP transduced cells were then sorted for further examination by fluorescence microscopy. A representative cluster of cells displaying all three distinct colors are shown: (C) blue (anti-ck17), (D) red (anti-p63) and (E) green (GFP). (F) Merged image of C, D and E. (G-I) Immunohistochemical staining of epithelial stem cells in vaginal, ectocervical and endocervical tissues. The p63 positively stained cells are mainly located in the basal epithelial cell layer. Note that in the endocervix (I), the epithelium is composed of a single cell thick layer under which the epithelial stem cells are located.</p

Inhibition of HIV-1 transfer and activity by b12 minibodies in the human VEC organotypic model tissues.

<p>After a 1 h pre-incubation of b12 minibodies or full-length b12 IgG (10 µg/ml) with HIV-1_bal (50 ng), medium from the basal chambers were collected at different time points and tested for inhibition of HIV-1_bal transfer by measuring p24 content by ELISA (A) and for inhibition of virus infectivity by incubation on TZM-bl target cells (B). Note that media collected at 3 and 6 h from tissue samples treated with HIV-1_bal and b12 IgG1 antibodies or with b12 minibodies had almost completely lost their ability to infect TZM-bl cells. Irrelevant 11 A minibodies served as negative controls.</p

Transduction of human endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.

<p>(A) Expressions of GFP protein by transduced cells were detected by FACS and presented as percentages of GFP positive cells. Note that AAV-2 and AAV-6 yielded the highest transduction rates. (B) A dose dilution of AAV-6-GFP vector. (C) AAV-8-GFP and AAV-9-GFP transduction of COS-1 cells. (D) Visual assessment of AAV-6-GFP transduction by fluorescence microscopy of vaginal, ectocervical and endocervicel cell lines.</p

Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.

<p>AAV-6-b12 minibodies or AAV-6-11A minibodies (control) at 5×10¹⁰ particles was applied to the upper layers of the VEC tissues for 24 h for transduction. Four days after the transduction, HIV-1_bal (50 ng) was applied to the upper layers of the tissues, and medium from the basal chambers were collected at various timepoints and tested for inhibition of viral transfer (A) and infectivity (B) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026473#pone-0026473-g005" target="_blank">Fig. 5</a>.</p

Functional comparison of b12 minibodies and full-length b12 IgG.

<p>Both b12 minibodies and full-length b12 IgG proteins were tested at equimolar concentrations for their capacity to (A) bind to HIV-1 gp120 by ELISA, and (B) to neutralize HIV-1_bal virus.</p

Selection of rare scFvFc expressing cells by a two-step magnetic bead and FACS sorting procedure.

<p>293T cells were transduced with either PS11-scFvFc-CD28-gp41-IRES-ZsGreen or 11A-scFvFc-CD28-gp41-IRES-ZsGreen encoding lentiviruses. These two transduced cell populations were mixed at different 11A- to PS11-scFvFc ratios with a total cell number of ∼10⁹. Mixed cells were incubated with the biotinylated GD03-Fc protein antigen and APC-streptavidin. APC-positive cells were subjected to an enrichment using anti-APC magnetic micro-beads followed by FACS sorting. Cells from either R2 gate (high APC expressing cells) or R3 gated (low APC expressing cells) were isolated, propagated, re-stained for biotin-GD03-Fc binding and also analyzed for their scFv gene content by PCR rescue and DNA sequencing. <i>Panel a</i>, the FACS dot-blot profiles of APC- and ZsGreen positive cells within the 11A∶PS11 scFvFc expressing cell population (initial mixing ratio at 1∶10⁶), before magnetic beads enrichment (left panel), following magnetic bead enrichment (middle panel), and the R2 gated cells after sorting and expansion (right panel). <i>Panel b</i>, DNA sequencing results of individual clones recovered from the R2 or R3 gated pools of cells sorted from different 11A∶PS11 scFvFc expressing cell ratios (1∶10³–1∶10⁶). Note that the irrelevant sequencing data are most likely originated from cloning background.</p

Neutralization of SARS-CoV TOR2 spike protein pseudotyped lentiviral infection of ACE2 expressing cells mediated by cell-surface displayed anti-TOR2 spike 80R-scFvFC antibodies.

<p>Single round, TOR2 spike protein pseudotyped luciferase expressing lentiviral particles were incubated with increasing concentrations of 293T cells expressing on their surface the 80R scFvFc (blue diamond) or the control PS11 scFvFc (pink circle). As a control for non-specific reporter virus absorption, a VSV-G pseudotyped luciferase reporter lentivirus was also incubated with 80R scFvFc (red triangle) or PS11 scFvFc expressing cells (brown square). Following incubation, the supernatant containing remaining lentivirus was used to infect permissive cells that express the ACE2 receptor for SARS CoV. At 48 hours post transduction, cells were harvested, luciferase activity was measured and relative inhibition of reporter virus infection was calculated. Asterisks in the designated points represent a statistical analysis that was performed to verify significant differences in % of inhibition between viral absorptions with the 80R- or PS11-scFvFc at a specific cell number point (P<0.05).</p

Expression of scFvFc on the surface of lentivirus transduced cells.

<p><i>Panel a</i> 293T cells were transduced with increasing dilutions (different MOIs as indicated) of lentivirus encoding the PS11-scFvFc-CD28-gp41-IRES-ZsGreen. Transduced cells were harvested, stained for Fc-surface expression, and analyzed by FACS. Expression of ZsGreen was measured to monitor levels of transduction. The graphs depict the percentage of transduced cells that express ZsGreen (blue diamonds) and the percentage values of transduced cells that express PS11-scFvFc as monitored by staining with APC-conjugated anti-human Fc IgG (pink squares). <i>Panels b</i> and <i>c</i>, cell -surface expressed scFvFc proteins are functional. 293T cells were transduced with a lentivirus encoding PS11-scFv-Fc-CD28-gp41-IRES-ZsGreen (<i>Panel b</i>) or 11A-scFvFc-CD28-gp41-IRES-ZsGreen (<i>Panel c</i>). Cells were incubated with biotinylated GD03-Fc, a specific antigen for 11A-scFvFc, and stained for streptavidin-APC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#s4" target="_blank">Methods</a>, and then analyzed by FACS. Note that two clusters of cells in <i>Panel c</i> represent high (R2 gated) and low (R3 gated) levels of 11A-scFvFc on their surface as measured by APC staining. These could reflect variations in cell-surface expression levels resulting from multiple integration events of the scFvFc cassette following transduction, or the difference in transgene integration site, <i>i.e.</i>, its proximity to active transcriptional units. R2 = 1390 and R3 = 220 are MFI values of 11A scFvFc expressing cells, where percentage of positive cells in each gate is 31% and 49% respectively. <i>Panel d.</i> a summary of specific antigen binding by the scFvFc displayed on the lentivirus transduced cells. The table shows the percentage of transduced cells expressing X48-scFvFc-CD28-gp41 or PS11 scFvFc-CD28-gp41 that bind to their cognate or irrelevant biotinylated antigens as visualized by APC staining and their corresponding MFI values.</p

Functional scFvFc are incorporated into lentivirus particles.

<p><i>Panel a.</i> An HIV-1 gp41 incorporation motif enhances scFvFc incorporation into viral particles-equal loads of lentiviruses encoding ZsGreen (lane 1), PS11-scFvFc-CD28-gp41-IRES ZsGreen (lane 2) or PS11-scFvFc-CD28-IRES ZsGreen (lane 3) were subjected to SDS-PAGE analysis, followed by western blotting using either HRP-conjugated anti-human Fc (upper panel) or anti-HIV-1 p24 (lower panel) antibodies. <i>Panel b.</i> Immunostaining of viruses. Viral particles were attached to Hela cells for 2 hour at 4°C. Cells were then fixed and stained with anti-HIV p24 antibody followed by Cy2-conjugated anti-mouse IgG or a rhodamine-conjugated anti-human Fc for surface Fc staining. Following these procedures, cells were washed and analyzed by confocal microscope. Shown separately are viruses stained for detection of p24 (image a) and scFvFc (image b) along with a merged image (image c). <i>Panel c.</i> Antigen specific capture of lentiviruses displaying corresponding scFvFc antibodies. Equal amounts of lentivirus particles expressing on their surface either the PS11-scFvFc or the 11A-scFvFc were loaded on a 96-well plate that was coated with the following specific antigens: TRM-peptide (PS11 specific), GD03-Fc (11A specific) or BSA. Following incubation to allow capture of the viruses to the antigens, wells were washed extensively and viral particles were eluted and quantitated by RT assay. Presented are normalized RT counts, where RT counts of particles bound to their antigens were divided by the RT counts of virus bound to the BSA control. Values are the average of duplicated samples and data are representative of two separate experiments.</p

Optimization of scFvFc cell-surface expression using different transmembrane domains.

<p>293T cells were transfected with the pcDNA 3.1 based constructs encoding PS11-scFvFc antibodies of different configurations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#pone-0003181-g001" target="_blank">Figure 1</a> and labeled under each lane in Panels a and b. pcDNA3.1-CMV-GFP was co-transfected as an internal control for transfection efficiency. At 48 hours post transfection, cells were harvested and analyzed for GFP and scFv-Fc expression by FACS analysis. <i>Panels a</i> and <i>b</i>, represent results from FACS analysis of the percentage of cells that are positive for APC-anti-human Fc staining (<i>a</i>) and their respective MFI values (<i>b</i>). Error bars represent the standard deviation of the average of three experiments. <i>Panel c</i>. Cellular localization of the PS11-scFvFc-TM analyzed by confocal immunomicroscopy. 293T cells were transfected with either ZsGreen expression vector alone, or with a bicistronic vector expressing both the PS11 scFvFc-TM fusion proteins and ZsGreen. At 48 hours post transfection, cells were stained with a rhodamine-conjugated anti-human Fc for the detection of scFvFc expression as visualized by a confocal microscope. <i>Image a</i>, cells transfected with ZsGreen only vector; <i>Images b</i><i> and </i><i>c</i>, cells transfected with vectors expressing either PS11-scFvFc-gp41 (665–856)-IRES ZsGreen or PS11-scFvFc-CD28-gp41 (706–713)-IRES-ZsGreen, respectively. Absence of the ZsGreen fluorescence in some of the APC+ cells is likely the result of low level expression of ZsGreen from the second cassette of the bi-cistronic message. <i>Panel d</i>. PS11-scFv-CD28-gp41 is present as a dimer in transfected cells. 293T cells expressing pCDNA3.1-PS11-scFvFc-CD28-gp41 fusion protein were metabolically labeled with [³⁵S]-cysteine and [³⁵S]-methionine mixture. Cell lysates were immunoprecipitated with protein A sepharose beads, resuspended with 2× SDS non-reducing (lane 1) or reducing buffer (lane 2), and subjected to SDS-PAGE and autoradiogram.</p