Volume 15, Issue 9, Pages 1724-1733 (September 2007) A New Multi-clade DNA Prime/Recombinant MVA Boost Vaccine Induces Broad and High Levels of HIV-1-specific CD8+ T-cell and Humoral Responses in Mice  Andreas Bråve, Andreas Boberg, Lindvi Gudmundsdotter, Erik Rollman, Kristian Hallermalm, Karl Ljungberg, Pontus Blomberg, Richard Stout, Staffan Paulie, Eric Sandström, Gunnel Biberfeld, Patricia Earl, Bernard Moss, Josephine H. Cox, Britta Wahren  Molecular Therapy  Volume 15, Issue 9, Pages 1724-1733 (September 2007) DOI: 10.1038/sj.mt.6300235 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Vaccine constructs and immunization protocol. (a) Immunogens and amount injected (amount/inj.) at each immunization. (b) Immunization protocol. CMDR, Chiang Mai double recombinant cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; pfu, plaque forming units; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; *1 μg of recombinant granulocyte macrophage colony-stimulating factor delivered 15 minutes before DNA at the site of injection. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Cellular responses in blood. HIV-1-specific enzyme-linked immunospot assay responses in pooled peripheral blood mononuclear cells (PBMCs) from all animals in each group after the three first DNA injections and then after each of the subsequent modified vaccinia virus Ankara (MVA) injections. Cellular responses as measured by (a, b) interferon-γ (IFN-γ) and (c, d) interleukin-2 (IL-2) secretion by PBMCs stimulated with peptides covering p24gag subtype B or gp120env subtype B.cMVA, control MVA; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; SFC, spot-forming cell. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Envelope-specific cellular responses in spleen. Cellular responses against envelope of different subtypes as measured by enzyme-linked immunospot assay. (a) Interferon-γ(IFN-γ) secretion. Bars show mean values of the number of cytokine secreting splenocytes from each individual mouse, and error bars represent the standard deviation. (b) Interleukin-2 (IL-2) secretion by splenocytes pooled from all animals in each group. Note that, in contrast to the responses shown in Figure 2, the results here are presented on a logarithmic scale. The peptides covering gp160 subtype E include peptides covering gp120 and gp41; the other pools contain peptides covering only gp120. The horizontal broken line indicates the cut-off level for responses. No error bar is shown for some of the antigen responses in group A because the strong reactivity meant it was not possible to quantify the responses properly. cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; SFC, spot-forming cell. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Gag- and pol-specific cellular responses in spleen. Cellular responses against pol and multiple subtypes of gag as measured by enzyme-linked immunospot assay. (a) Interferon-γ(IFN-γ) secretion. Bars show mean values of the number of cytokine secreting splenocytes from each individual mouse, and error bars represent the SD. (b) Interleukin-2 (IL-2) secretion by splenocytes. No error bar is shown for the antigen responses in group A because the strong reactivity meant it was not possible to quantify the responses properly. cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; SFC, spot-forming cell. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Intracellular cytokine staining and subsequent fluorescence-activated cell sorting analysis. Splenocytes stained for CD4, CD8, interferon-γ (IFN-γ), and interleukin-2 (IL-2) after stimulation with pools of peptides. (a) Example, CD8+ splenocytes from immunized and control animals stimulated with peptides covering p55gagA or peptides from tick borne encephalitis (TBE) virus and stained for intracellular IFN-γ and IL-2. (b) Cumulative percentage of reactive CD8+ (left panel) or CD4+ splenocytes (right panel) responding with IFN-γ and/or IL-2 when stimulated ex vivo with peptides spanning either p55gagA, gp160envE, or polA. cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Mapping of the cellular response against gp160 subtype E by interferon-γ(IFN-γ) enzyme-linked immunospot assay. All analyses were carried out by pooling cells from three animals in each group. (a) Splenocytes stimulated with 21 different pools of peptides (9–10 peptides per pool) covering the indicated stretch of amino acids of gp160 subtype E. (b) Splenocytes stimulated with the indicated single peptide from the reactive pools of peptides shown in a. None of the peptides or pools of peptides gave rise to unspecific reactivity when cells from animals immunized with empty vector were stimulated (data not shown). cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; SFC, spot-forming cell. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Vaccine-specific humoral responses. Vaccine-specific antibodies measured by enzyme-linked immunosorbent assay over time directed against (a) p55gag and (b) gp160env. cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Anti–vaccinia virus reactivity. (a) Interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretion, as measured by enzyme-linked immunospot assay, by splenocytes stimulated with inactivated vaccinia (106particles/ml). The black bars show mean values of the number of IFN-γ-secreting splenocytes from each individual mouse and error bars represent the SD. Gray bars show responses from stimulated splenocytes pooled from each animal in the group. (b) Neutralization of vaccinia virus. cMVA, control modified vaccinia virus Ankara; mockDNA, empty plasmid; rDNA, recombinant plasmid DNA; rMVA, recombinant MVA; SFC, spot-forming cell. Molecular Therapy 2007 15, 1724-1733DOI: (10.1038/sj.mt.6300235) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions