Volume 13, Issue 4, Pages 766-775 (April 2006) Development of Cell-Based Tuberculosis Vaccines: Genetically Modified Dendritic Cell Vaccine Is a Much More Potent Activator of CD4 and CD8 T Cells Than Peptide- or Protein-Loaded Counterparts  Janet I. Malowany, Sarah McCormick, Michael Santosuosso, Xizhong Zhang, Naoko Aoki, Patricia Ngai, Jun Wang, Jaina Leitch, Jonathan Bramson, Yonghong Wan, Zhou Xing  Molecular Therapy  Volume 13, Issue 4, Pages 766-775 (April 2006) DOI: 10.1016/j.ymthe.2005.10.018 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Immune phenotypes of dendritic cell TB vaccines. In vitro-differentiated dendritic cells were washed 5 h after being incubated without (DC) or with AdAg85A (AdAg85/DC), Ag85 complex proteins (Pro/DC), T cell peptides (Pep/DC), or LPS (LPS/DC). The cells were then incubated for an additional 19 h before immunostaining, FACS analysis, and/or ICCS. FACS analysis was carried out by gating on MHC class II- and CD11c-positive cells. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Percentage of IFN-γ-producing, Ag85A-sepcific CD8 and CD4 T cells in the spleen following intramuscular immunization with pep/DC, pro/DC, or AdAg85/DC. Mice were im immunized with various DC vaccines and sacrificed at weeks 2, 6, and 12. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % ± SEM (IFN-γ-positive cells of all splenocytes) from three or four mice/DC vaccine/time. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Percentage of IFN-γ-producing, Ag85A-specific CD8 and CD4 T cells in the spleen following intramuscular immunization with pep/DC, pepAddl/DC, pro/DC, proAddl/DC, or AdAg85/DC. Mice were im immunized with various DC vaccines and sacrificed at week 2. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % of two mice/vaccine (IFN-γ-positive cells of all splenocytes). Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Quantitation of IFN-γ production by Ag85A-specific CD8 or CD4 T cells in the spleen following intramuscular immunization with pep/DC, pro/DC, or AdAg85/DC. Mice were im immunized with various DC vaccines and sacrificed at weeks 2, 6, and 12. The whole splenocytes pooled from three or four mice/vaccine/time were cultured for 24 h with or without (A) CD8 or (B) CD4 T cell peptide stimulation. The amount of IFN-γ protein released into the culture supernatant was quantified by ELISA. Results are expressed as the means ± SEM of triplicate cultures. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 In vivo CD8 and CD4 T-cell-mediated cytotoxic activity following intramuscular immunization with pep/DC, pro/DC, or AdAg85/DC. Mice were im immunized with pep/DC, pro/DC, or AdAg85/DC vaccine. (A) At weeks 2, 6, and 12 postimmunization, CFSE-labeled Ag85A-specific CD8 T cell peptide-pulsed target cells were transferred intravenously into the immunized mice and the splenocytes were isolated 5 h after target cell transfer and examined by FACS analysis for the measurement of CD8 T-cell-mediated cytotoxicity (CTL), with the representative histogram of FACS analysis presented (B; week 2). (C) In separate experiments Ag85A-specific CD4 T cell peptide-pulsed target cells were transferred intravenously into the immunized mice and the splenocytes were isolated and examined by FACS analysis for the measurement of CD4 T-cell-mediated CTL. Results represent % loss of peptide-pulsed target cells and are expressed as the means ± SEM of three or four mice/DC vaccine/time. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Percentage of IFN-γ-producing, Ag85A-sepcific CD8 and CD4 T cells in the spleen following intramuscular or intravenous immunization with AdAg85/DC. Mice were im or iv immunized with AdAg85/DC and sacrificed at weeks 2 and 6. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % ± SEM (IFN-γ-positive cells of all splenocytes) from three or four mice/vaccination route/time. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Frequencies of Ag85A-specific CD8 and CD4 T cells in the spleen following intramuscular or intravenous immunization with pep/DC, pro/DC, or AdAg85/DC. Mice were immunized iv with pep/DC, pro/DC, AdAg85/DC, or Addl/DC as a control or immunized im with AdAg85/DC. Mice were then sacrificed at (A) week 2 or (B) week 6. The whole splenocytes were pooled from three or four mice/group and cultured for 24 h with or without CD8 or CD4 T cell peptide. The cells were then subjected to Elispot assay. The number of IFN-γ-positive spots/million cells was enumerated. The data are expressed as the means ± SEM from triplicate wells/DC vaccine/time. (C) In separate experiments, mice were immunized iv or im with AdAg85/DC. At weeks 2 and 6 postimmunization, CFSE-labeled Ag85A-specific CD8 T cell peptide-pulsed target cells were transferred intravenously to the immunized mice and the splenocytes were isolated 5 h after the target cell transfer and examined by FACS analysis for the measurement of CD8 T-cell-mediated CTL. Results represent % loss of peptide-pulsed target cells and are expressed as the means ± SEM of three or four mice/DC vaccine/time. Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 8 Immune protection from M.tb challenge by DC vaccination. Mice were immunized iv or im with pep/DC, pro/DC, or AdAg85/DC or sc with BCG as a control. At 6 weeks postimmunization, mice were ip infected with M.tb and the level of M.tb infection in the spleen was assessed by colony formation assay 4 weeks after M.tb challenge. Results (colony-forming units/spleen) are expressed as the means ± SEM of seven or eight mice/group. The difference between im AdAg85/DC and im pep/DC, im pro/DC, iv AdAg85/DC, iv pep/DC, or iv pro/DC is statistically significant (P = 0.008, P = 0.01, P = 0.0002, P = 0.04, or P = 0.04, respectively). Molecular Therapy 2006 13, 766-775DOI: (10.1016/j.ymthe.2005.10.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions