Volume 19, Issue 2, Pages 417-426 (February 2011) Vaccine-induced T cells Provide Partial Protection Against High-dose Rectal SIVmac239 Challenge of Rhesus Macaques  Marcio O Lasaro, Larissa H Haut, Xiangyang Zhou, Zhiquan Xiang, Dongming Zhou, Yan Li, Wynetta Giles-Davis, Hua Li, Jessica C Engram, Lauren J DiMenna, Ang Bian, Marina Sazanovich, Elizabeth M Parzych, Raj Kurupati, Juliana C Small, Te-Lang Wu, Rachel M Leskowitz, Nicole R Klatt, Jason M Brenchley, David A Garber, Mark Lewis, Sarah J Ratcliffe, Michael R Betts, Guido Silvestri, Hildegund C Ertl  Molecular Therapy  Volume 19, Issue 2, Pages 417-426 (February 2011) DOI: 10.1038/mt.2010.238 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Scheme of experimental design. Times for vaccinations, challenge, and necropsy are indicated. AdC6, AdC vector derived from serotype 6; AdC7, AdC vector derived from serotype 7; MVA, modified vaccinia Ankara; SIV, simian immunodeficiency virus. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Kinetics of Gag-specific CD8+ T-cell responses. NHPs were vaccinated as described in Table 1 and challenged with SIVmac239. (a) PBMCs from animals from group 1 (top), and the two control groups (bottom) were stimulated with SIVgag peptide pools and tested for production of IFN-γ, IL-2, and TNF-α at various time points postimmunization. Lines show the percentage of the sum of CD8+ T cells producing the tested cytokines alone or in combinations, over all CD8+ T cells for individual animals. Gray filled lines represent means of the two groups. (b) Gag-specific CD8+ T-cell responses from blood of the two vaccinated Mamu-A01+ animals (#4327 and #4592) and the Mamu-A01+ control animal (#4607) were tested over time by staining of T cells with a Gag-specific tetramer and antibodies to CD3 and CD8. Graph shows tetramer+CD8+ T cells over all CD8+ T cells. (c) Production of IFN-γ, IL-2, and TNF-α by CD4+ T cells isolated from blood of vaccinated (upper graph) and control (lower graph) animals at various time points postimmunization was measured by ICS. Lines show the percentage of the sum of CD4+ T cells producing the tested cytokines alone or in combinations, over all CD4+ T cells for individual animals. Gray filled lines represent means of each group. AdC6, AdC vector derived from serotype 6; AdC7, AdC vector of serotype 7; IFN-γ, interferon-γ; IL-2, interleukin; MVA, modified vaccinia Ankara; NHP, nonhuman primate; PBMC, peripheral blood mononuclear cell; SIV, simian immunodeficiency virus; TNF-α, tumor necrosis factor-α. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Cytokines profiles of T cell subsets. Percentages of all possible combinations of IFN-γ (g), IL-2 (2), and TNF-α (T) production by effector (Effector), effector memory (EM), and central memory (CM) T cells isolated from blood shortly after each immunization and at necropsy were determined. (a) Graphs show average CD8+ T-cell responses over time for vaccinated animals on the left and for control animals on the right. (b) Graphs show responses for individual vaccinated animals tested 2 weeks before challenge. (c) Graph shows average relative distribution of the different CD8+ T cell subsets in blood tested just 2 weeks before challenge. (d) Graphs show average CD4+ T-cell responses over time for vaccinated animals on the left and for control animals on the right. AdC6, AdC vector derived from serotype 6; AdC7, AdC vector of serotype 7; IFN-γ, interferon-γ; IL-2, interleukin; MVA, modified vaccinia Ankara; SIV, simian immunodeficiency virus; TNF-α, tumor necrosis factor-α. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Levels of PD-1 expression by Mamu-A*01 Tet+CD8+ T cells. Geometric mean fluorescence intensity (gMFI) of PD-1 expression by effector and memory (CD28+CD95+) Mamu-A*01 Tet+CD8+ T cells isolated from blood of NHP # 4327, 4592, and 4607 were analyzed at various time points after immunization. PD-1, programmed death-1. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Prime-boost vaccine regiment increases breadth of Gag-specific IFN-γ T-cell responses. Breadths of specific IFN-γ responses were analyzed with PBMCs isolated from animals after AdC6 (20 weeks, open bars) and after AdC7 (10 weeks, black bars) immunizations by ELISpot assays using peptide matrices. AdC6, AdC vector derived from serotype 6; AdC7, AdC vector of serotype 7; IFN-γ, interferon-γ; PBMC, peripheral blood mononuclear cell. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Vaccine efficacy against SIV challenge. NHPs were challenged rectally with SIVmac239 and SIV RNA levels and CD4+ T counts were monitored. (a) Data show log of SIV RNA copies/ml of plasma of individual animals from vaccinated (top), and control (bottom) animals. (b) Percentages of CD4+ T lymphocytes in peripheral blood were analyzed from vaccinated (top), and control (bottom) animals. Gray filled lines represent means of each group. NHP, nonhuman primate; SIV, simian immunodeficiency virus. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 T-cell responses in tissues. (a) At necropsy, frequencies of all combined cytokine-producing CD8+ T cells were determined in lymphocytes isolated from spleen and MLN of vaccinated and control animals. Graphs show average frequencies of CD8+ T cells producing the different cytokine combinations in response to Gag peptides. (b) At necropsy Gag-specific CD8+ T cell subsets were identified on lymphocytes from the indicated tissues by staining of lymphocytes with the Gag-specific tetramer and antibodies to various T cell markers. CM, central memory; EM, effector memory; GT, genital tract; IEL, intestinal epithelium; ILN, ileocolical lymph nodes; MLN, mesenteric lymph nodes. Molecular Therapy 2011 19, 417-426DOI: (10.1038/mt.2010.238) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions