Western blotting analysis of coAdpIXPTM#2.

<p>(A) Constructs of the modified pIX genes in genomes of three parental viruses: AdLucIXpK, Ad-dE1-IX-sr39tk, and Ad-IX-mRFP1. Modified pIX genes (tagged with the Flag) were driven by the native pIX promoter. (B) Structural diagram of triple pIX-modified Ad (coAdpIXPTM#2) generated by co-infection strategy. (C) 5×10⁹ VPs of CsCl-purified control Ad5 (lane 1) and the triple pIX-modified coAdpIXPTM#2 (lane 2) were subjected to SDS-PAGE. The separated proteins were stained with Gelcode Blue (on the left) or probed with anti-Flag, anti-RFP, or anti-TK antibody. A long-exposure of the anti-Flag blot was included to show pIX-pK protein, which is incorporated into Ad particles at a very low level. The “*” asterisks indicate degradation products of pIX-mRFP1 fusion protein.</p

Schema of Ad pIX modifications.

<p>(A) Constructs of the modified pIX genes in genomes of three parental viruses: Ad-IX-Flag-pK, Ad-IX-H6-TK, and Ad-IX-myc-mRFP1. Modified pIX genes (tagged with the Flag, His₆, and c-myc, respectively) were driven by the native pIX promoter. (B) Structural diagram of triple pIX-modified Ad (coAdpIXPTM#1) generated by co-infection strategy. pK, TK, and mRFP1 peptides/proteins were incorporated in the C termini of pIX. (C) Western blotting analysis of Ad vector containing triple pIX modifications. 5×10⁹ VPs of CsCl-purified control Ad5 (lane 1) and the triple pIX-modified coAdpIXPTM#1 (lane 2) were subjected to SDS-PAGE. The separated proteins were stained with Gelcode Blue (Pierce, Rockford, IL.) to detect the total viral proteins (left panel) or probed with anti-Flag, anti-RFP, or anti-TK antibody (right panel).</p

Incorporation and presentation of modified IX proteins on the Ad capsid.

<p>ELISA analysis of Ad vector containing triple pIX modifications. 10⁹ VPs of CsCl-purified Ad5 (negative control) or coAdpIXPTM#1 were subjected to a serial dilution (1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128) and immobilized on an ELISA plate. The viruses were probed with anti-Flag (1∶2000), anti-His₆ (1∶1000), and anti-c-myc antibodies (1∶1000), followed by incubation with alkaline phosphatase-conjugated secondary antibodies (1∶1000). After color development, the light absorbance was measured and plotted on the Y-axis against the viral concentrations. Each point represents the mean and standard deviation (SD) of triplicate determinations. Some error bars standing for SD are smaller than their symbols.</p

Summary of immunogold staining of coAdpIXPTM#1.

<p>N/A  =  not available.</p><p>*Provided by the manufacturer.</p><p>The counting was performed in eleven electron microscopy images that totally contain 214 viral particles.</p

Fluorescence microscopy of cells infected with the triple pIX mosaic Ad vector.

<p>A549 cells were infected with wild type Ad5, Ad-wt-IX-mRFP1 (containg wild type E1 promoter), or coAdpIXPTM#1 at an MOI of 10,000 VP/cell, and the fluorescent signal of pIX-mRFP1 was monitored 1 hour post infection (nuclei were counterstained with Hoechst 33342). Fluorescent images were captured using an Olympus IX-70 microscope with a 100X objective (oil lens). The enlarged image of each rectangle area was presented underneath.</p

Cell Binding of the triple pIX-modified Ad vector.

<p>AU-565 cells were incubated with Ad5, AdLucIXpK, or coAdpIXPTM#2 at an MOI of 500 VP/cell in the presence of 500 µg/ml heparin or 200 µg/ml sCAR protein at 4°C. Bound Ad particles were quantified by quantitative real-time PCR and normalized with total cellular DNA. Each bar represents the mean and SD of triplicate determinations, and asterisks indicate significant difference between specified groups.</p

Multivalent vectors elicit an <i>in vivo</i> anti-His₆ immune response.

<p>BALB/c mice (n = 8) were primed, boosted, and reboosted with 1×10¹⁰ VP of Ad vectors. A) Immunization timeline showing when immunizations were performed (solid arrows); serum was collected (dashed arrows). B–C) Post-prime and post-reboost serum was collected for ELISA binding assays. 1 µM of purified His₆ (LGSHHHHHHLGS) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with varying concentrations of immunized mice serum and the binding was detected with HRP conjugated secondary antibody. OD absorbance at 450 nm represents His₆ antibody levels in serum. The values are expressed as the mean ± standard deviation. The asterisks (***) indicate a <i>P</i> value ≤ 0.001, ** indicate a <i>P</i> value ≤ 0.01, and * indicate a <i>P</i> value ≤ 0.05.</p

Multivalent vectors elicit an <i>in vivo</i> anti-KWAS immune response.

<p>BALB/c mice (n = 8) were primed, boosted, and reboosted with 1×10¹⁰ VP of Ad vectors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060347#pone-0060347-g004" target="_blank">Figure 4A</a>). A–B) Post-prime and post-reboost serum was collected at various time points for ELISA binding assays. 1 µM of purified KWAS (PCEWDEAELDKWASNLEEEDDDNE) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice serum and the binding was detected with HRP conjugated secondary antibody. OD absorbance at 450 nm represents KWAS antibody levels in sera. The values are expressed as the mean ± standard deviation. The *** indicate a <i>P</i> value ≤ 0.001, and ** indicate a <i>P</i> value ≤ 0.01.</p

Western blotting confirmed the presence of His₆, KWAS, and fiber on multivalent vaccine vectors.

<p>A) Western blotting confirmed the presence of His₆ incorporation within the dual modified vectors. In this assay, 5×10⁹ VP of Ad5 (lane 1), Ad/H5-HVR1-His₆ (lane 2), Ad5/H5-HVR1-KWAS-HVR2-His₆ (lane 3), and Ad5/H5-HVR1-KWAS-HVR5-His₆ (lane 4) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinylidene fluoride (PVDF) membrane then incubated with anti-His antibody. The arrow indicates the His tag is genetically incorporated into the hexon protein. B) Western blotting confirmed the presence of KWAS incorporation within the dual modified vectors. In this assay, 5×10⁹ VP of Ad5 (lane 1), Ad5/HVR2-MPER-L15ΔE1 (lane 2), Ad5/H5-HVR1-KWAS-HVR2-His₆ (lane 3), and Ad5/H5-HVR1-KWAS-HVR5-His₆ (lane 4) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to PVDF membrane then incubated with anti-KWAS antibody. The arrow indicates the KWAS epitope is genetically incorporated into the hexon protein. C) Western blotting confirmed the presence of fiber incorporation within the dual modified vectors. In this assay, 5×10⁹ VP of Ad5 (lane 1), Ad5/HVR2-MPER-L15ΔE1 (lane 2), Ad/H5-HVR1-His₆ (lane 3), Ad5/H5-HVR1-KWAS-HVR2-His₆ (lane 4), and Ad5/H5-HVR1-KWAS-HVR5-His₆ (lane 5) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to PVDF membrane then incubated with anti-Fiber antibody. The arrow indicates detection of the fiber protein.</p

HIV and His₆ epitopes in the HVRs are exposed on the virion surface.

<p>A) In the assay, varying amounts of Ad5, Ad/H5-HVR1-His₆, Ad5/H5-HVR1-KWAS-HVR2-His₆, or Ad5/H5-HVR1-KWAS-HVR5-His₆ were immobilized in the wells of ELISA plates and incubated with anti-His₆ antibody. The binding was detected with an HRP-conjugated secondary antibody. B) In the assay, varying amounts of Ad5, Ad5/HVR2-MPER-L15ΔE1, Ad5/H5-HVR1-KWAS-HVR2-His₆, or Ad5/H5-HVR1-KWAS-HVR5-His₆ were immobilized in the wells of ELISA plates and incubated with anti-HIV antibody. The binding was detected with an HRP-conjugated secondary antibody. C) In the assay 6×10⁸ VP of Ad5, Ad/H5-HVR1-His₆, Ad5/H5-HVR1-KWAS-HVR2-His₆, or Ad5/H5-HVR1-KWAS-HVR5-His₆ were immobilized on an ELISA plate followed by varying dilutions of His₆ antibody. The binding was detected with an HRP-conjugated secondary antibody. D) In the assay 6 x 10⁸ VP of Ad5, Ad5/HVR2-MPER-L15ΔE1, Ad5/H5-HVR1-KWAS-HVR2-His₆, or Ad5/H5-HVR1-KWAS-HVR5-His₆ were immobilized on an ELISA plate followed by varying dilutions of HIV antibody. The binding was detected with an HRP-conjugated secondary antibody.</p