Volume 15, Issue 2, Pages 393-403 (February 2007) Type I Interferon Inhibits Antibody Responses Induced by a Chimpanzee Adenovirus Vector  Scott E Hensley, Ann S Cun, Wynetta Giles-Davis, Yan Li, Zhiquan Xiang, Marcio O Lasaro, Bryan R G Williams, Robert H Silverman, Hildegund C J Ertl  Molecular Therapy  Volume 15, Issue 2, Pages 393-403 (February 2007) DOI: 10.1038/sj.mt.6300024 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Ad vectors expressing rab.gp induce type I IFN and this promotes DC maturation. 129Sv/Ev and IFNAR−/− BM-DCs were transduced with AdC68rab.gp or AdHu5rab.gp or incubated with poly (I:C). (a) After 24 h, supernatant was collected and measured for IFNα by ELISA and (b) CD40 and MHC II expression on CD11c+ cells infected with 1,000 pt/cell of vector was determined by FACS analysis. ELISA data show mean±SD for quadruplicate wells. Student's t-tests were completed and statistical significance is indicated (*P<0.05). Data are representative of results from three independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Type I IFN is not required for the generation of transgene product-specific antibody responses following vaccination with AdHu5 vectors. 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1010 pts of AdHu5rab.gp. (a) At several time points after vaccination, ELISAs were performed to measure total rab.gp-specific antibodies in the serum. The serum samples were initially diluted 1:200 and then serially diluted thereafter. (b) At 56 days after vaccination, ELISAs were performed to determine the isotypes of the rab.gp-specific antibodies using a 1:200 serum dilution. Each data point shows mean±SD for serum from three individual mice. In all experiments, absorbance readings of serum from naïve IFNAR−/− mice were not statistically different from serum from naïve 129Sv/Ev mice (data not shown). Data are representative of results from three independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Type I IFN negatively affects the generation of transgene product-specific antibody responses following vaccination with AdC68 vectors. 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1010 pts of AdC68rab.gp. (a) Rab.gp-specific antibodies in the serum and (b) their isotypes were determined by ELISA as described in the legend of Figure 2. Student's t tests were completed and statistical significance between 129Sv/Ev and IFNAR−/− vaccinated mice is indicated (*P<0.05). Data are representative of results from three independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 IFNAR−/− mice generate stronger rabies neutralizing antibodies compared to 129Sv/Ev mice following vaccination with AdC68rab.gp. 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1010 pts of (a) AdHu5rab.gp or (b) AdC68rab.gp and sera were collected 4 and 8 weeks after vaccination and tested for viral neutralizing antibodies to rabies virus. A World Health Organization reference serum was used for comparison and data is expressed in IU. Each dot represents serum from one mouse and each line represents the mean of each group. Student's t-tests were completed and statistical significance between 129Sv/Ev and IFNAR−/− vaccinated mice is indicated. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Ad induced IgG1 does not correlate with Th polarization. 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1010 pts of (a) AdHu5 or (b) AdC68 vectors expressing HIV-1 gag. At 7, 14, and 21 days after vaccination, splenocytes were isolated and tested for their ability to produce IL-4, IFN-γ, and IL-2 by enzyme-linked immunospot assay after addition of a HIV-1 gag peptide that activates CD4+ T cells. Each data point shows mean±SD for results obtained using splenocytes from three individual mice. Student's t-tests were completed and statistical significance between 129Sv/Ev and IFNAR−/− vaccinated mice is indicated (*P<0.05). Data are representative of results from two independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Type I IFN does not affect anti-capsid responses. 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1010 pts of AdHu5rab.gp (a, c) or AdC68rab.gp (b, d). (a, b) At 56 days after vaccination, ELISAs were performed to measure total Ad-specific antibodies in the serum. The serum samples were initially diluted 1:200 and then serially diluted thereafter. (c, d) ELISAs were performed to determine the isotypes of Ad-specific antibodies using a 1:200 dilution. Each data point shows mean±SD for serum from three individual mice. In all experiments, absorbance readings of serum from naïve IFNAR−/− mice were not statistically different from serum from naïve 129Sv/Ev mice (data not shown). Data are representative of results from three independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Increased amounts of antigen promote stronger antibody responses with more diverse isotype profiles. (a) 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 1011 pts of Ad vectors expressing GFP and transgene product expression was visualized after illuminating the muscle with an Illumatool Lighting System. Shown are intensity maps of the images; the highest intensity is red and the lowest intensity is blue. (b, c) 129Sv/Ev and IFNAR−/− mice were vaccinated intramuscularly with 1 × 109, 1 × 1010, or 1 × 1011 pts of AdC68rab.gp and 56 days later, serum levels of rab.gp-specific (b) IgG2a and (c) IgG1 antibodies were determined by ELISA. Each data point shows mean±SD for serum from 3 to 6 individual mice. Data are representative of results from two independent experiments. Student's t-tests were completed and statistical significance between 129Sv/Ev and IFNAR−/− vaccinated mice is indicated (*P<0.05). Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Type I IFN suppresses levels of transgene RNA. 129Sv/Ev or IFNAR−/− BM-DCs were infected with Ad vectors at 4°C for 30 min and then incubated at 37°C for 24 h. (a) The amount of transgene-specific DNA in the nucleus and (b) the amount of transgene-specific RNA (cDNA) in infected cells were determined by quantitative PCR. Each data point shows mean±SD for three individual samples. Student's t-tests were completed and statistical significance between 129Sv/Ev and IFNAR−/− infected cells is indicated (*P<0.05). (c) C57B/6, PKR−/−, and RNase L−/− BM-DCs were transduced with GFP expressing Ad vectors (10,000 pt/cell) and GFP expression was determined by FACS analysis 24 h later. Data are representative of results from three independent experiments. Molecular Therapy 2007 15, 393-403DOI: (10.1038/sj.mt.6300024) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions