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

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

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

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

Western blotting confirmed the presence of HIV genes within Ad vectors.

<p>A) In the assay, HEK293 cells were infected with various vectors at 100 IFU per cell. Cell lysates were collected after 24 hours and equal protein amounts were separated on a 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinylidene fluoride membrane and then stained with Gag antibody. Lane 1, Ad5 negative control; lane 2, Ad5/HVR2-MPER-L15(Gag); lane 3, AdCMVGag. The arrow indicates Gag protein. B) 10¹⁰ VP of Ad5 (lane 1), Ad5/HVR2-MPER-L15(Gag) (lane 2), and Ad5/HVR2-MPER-L15Δ<i>E1</i> (lane 3) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinylidene fluoride membrane then stained with anti-gp41 antibody. The arrow indicates MPER protein genetically incorporated into the hexon protein.</p

Generation of Sam68-Deficient Mice

<div><p>(A) The genomic organizations of the wild-type and targeted <i>sam68</i> alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted <i>sam68</i> alleles. The targeted allele replaces exon 4 and part of exon 5 of <i>sam68</i> with a PGK-neomycin cassette.</p><p>(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.</p><p>(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.</p></div

Thermostability of modified Ad vectors.

<p>In these assays various viruses were heat inactivated in PBS at 45°C for 0, 5, 10, 20 or 45 minutes. After heat inactivation, the virus were serially diluted and the viral infectious titers were re-determined by TCID₅₀ assays.</p

Ex Vivo Activity of Sam68^+/+ and Sam68^−/− Osteoblasts and Osteoclasts

<div><p>Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.</p><p>(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68^−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent <i>p</i> < 0.01.</p><p>(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.</p></div

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

Ex Vivo Adipogenesis Analysis of Sam68^−/− Mouse Embryonic Fibroblasts

<div><p>MEFs were isolated from mouse embryos at embryonic day 14.5. Equal number of MEFs from Sam68^+/+ and Sam68^−/− was plated on glass cover slips in 24 well-plates. Adipocyte differentiation was carried out at indicated times by the addition of complete media containing the pioglitazone.</p><p>(A) Cultures were fixed in 4% paraformaldehyde and stained with Oil Red O to detect the fat droplets stored in adipocytes and photographed (top). The cell images were magnified ×10 and ×20 as indicated.</p><p>(B) RT-PCR was carried out on total cellular RNA isolated after differentiation of the MEFs for day 0, 2, 4, 6, and 12. The DNA fragments were visualized on agarose gels stained with ethidium bromide. The expression of adipogenic markers C/EBPβ, C/EBPδ, PPARα, and KLF5 was examined as well as the expression of controls including Sam68, β-actin, and GAPDH.</p></div