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Results: 5

1.
Figure 2.

Figure 2. From: Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

A single injection of TLR9-L into FL-treated animals at the time of initial DNA priming markedly enhances the magnitude of polyfunctional, antigen-specific CD8+ T cells after the MVA boost. Total PMBCs were stimulated with a pool of overlapping peptides spanning SIV Gag or HIV Env proteins at week 1 after a second rMVA boost vaccination. IFN-γ–producing T cells were detected by intracellular staining (A) or ELISPOT (B). Each symbol represents an individual animal in the experimental group; bars represent the mean frequencies ± SEM (n = 5). (C) Kinetics of Gag- (top) and Env- (bottom) specific IFN-γ–producing CD8+ T cells. Animals in each group were vaccinated as described in Table I and Fig. S1, and PBMCs were stimulated as described. Each line represents the mean of all individuals in the group ± SD (n = 5). (D) Frequencies of multicytokine IFN-γ–, TNF-α–, and IL-2–producing Gag-specific T cells after the second rMVA boost vaccination. Total PMBCs at weeks 1 (left) and 10 (right) after the second rMVA boost vaccination were stimulated with a pool of overlapping Gag peptides and stained for intracellular IFN-γ, TNF-α, and IL-2. Percentages of cells simultaneously producing all three cytokines of total Gag-specific CD8+ T cells are shown. Error bars represent the mean frequencies ± SEM. All vaccinated groups were compared with group 1 by a nonparametric Mann-Whitney test. P < 0.05 was considered statistically significant (n = 5). (E) Frequencies of IFN-γ–producing Gag-specific CD8+ T cells at week 1 after a second rMVA boost vaccination in Mamu-A*01–positive animals from groups 2, 3, and 4. Each symbol represents an individual animal in the experimental group. Error bars represent the mean frequencies ± SEM (n = 3 or 4). N.A., not available.

Marcin Kwissa, et al. J Exp Med. 2007 October 29;204(11):2733-2746.
2.
Figure 4.

Figure 4. From: Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

Control of the viremia in the vaccinated groups after SIV challenge. All animals in vaccinated groups 1–4, as well as naive animals in group 5, were challenged intrarectally with SIVmac251 virus at week 77 of the experiment. (A) Plasma was collected at weeks 1, 2, 3, 7, 10, 18, and 24 after challenge and tested for SIV viral loads (SIV RNA copies/ml). Symbols represent the dynamics of viremia for each animal in the group. In groups 1 and 3, single animals remained uninfected over the entire challenge phase and were not included in the statistical analyses. (B) Viral titers at the peak of viremia at week 2 after SIV challenge. Each symbol represents an individual animal in the experimental group; bars represent the geometric mean frequencies ± SEM. All vaccinated groups were compared by a nonparametric Mann-Whitney test. P < 0.05 was considered statistically significant (n = 4 or 5). (C and D) AUC from weeks 1 through 24 was calculated for each animal in all experimental groups (C), or Mamu-A*01–positive animals only in groups 2, 3, 4, and 5 (D). Log-transformed areas (AUC [log10]) were used in the analysis. Error bars represent the median with range (C, n = 5; D, n = 2 or 3). Analysis of variance was used for the comparisons among the groups and adjusted for multiple comparisons with the Bonferroni method. P < 0.05 was considered significant (n = 4 or 5). N.A., not available.

Marcin Kwissa, et al. J Exp Med. 2007 October 29;204(11):2733-2746.
3.
Figure 1.

Figure 1. From: Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

Expansion and activation of APCs in vivo. (A) Gating strategy for APC populations in FL-treated animals. Total PBMCs were isolated using standard procedures, and the mononuclear fractions were analyzed by flow cytometry to assess the frequencies of various APCs in the blood. Total DC population subsets were defined by a lack of lineage marker expression (CD3CD14CD20) and expression of HLA-DR. CD11c+ DCs were defined as CD11c+HLADR+Lin, and pDCs were defined as CD123++ HLA-DR+CD11cLin. Monocytes were defined as HLA-DR+CD14+ within the entire PBMC population. (B) Animals were injected subcutaneously with 100 μg/kg FL daily from days 0 to 14. The frequencies of specific APC subsets in the blood at the indicated time points are shown. Numbers in squares represent the mean fold increase between days 0 and 14. Error bars represent SD. Mean frequencies of specific APC subsets at days 0 and 14 were compared by a nonparametric Mann-Whitney test (n = 5). P < 0.05 was considered statistically significant (*). (C) Activation of CD11c+ DCs and monocytes was assessed by staining for CD80 and CD86 surface markers at days 14 (1 d before vaccination; black line) and 16 (1 d after vaccination; red line). Shading represents the isotype. (D) Mean fluorescent intensity (MFI) of CD80 and CD86 expression was measured on activated CD11c+ DCs and monocytes (four out of five animals are presented in group 4 [blue diamonds]) caused by problems with PBMC isolation. Error bars represent the mean frequencies ± SEM. Mean MFIs were compared by a nonparametric Mann-Whitney test (n = 4 or 5). P < 0.05 was considered statistically significant. FSC, forward scatter; SSC, side scatter.

Marcin Kwissa, et al. J Exp Med. 2007 October 29;204(11):2733-2746.
4.
Figure 3.

Figure 3. From: Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

Co-injection of TLR9-L and pDNA at priming markedly enhances the magnitude of polyfunctional antigen-specific CD8+ T cell response after SIV challenge. All animals in vaccinated groups 1–4, as well as naive animals in group 5, were challenged intrarectally with SIVmac251 virus at week 77 of the experiment. Total PMBCs were stimulated with a pool of overlapping Gag peptides at weeks 2, 10, and 24 after SIV challenge. (A) Frequencies of IFN-γ–producing CD8+ (top) and CD4+ (bottom) T cells were detected by ICC staining. Each symbol represents an individual animal in the experimental group; bars represent the means ± SEM (n = 4 or 5). (B) Correlates of Gag-specific CD8+ T cell responses after SIV challenge. Magnitude of expansion of Gag-specific CD8+ T cells after the second rMVA vaccination correlates with specific T cell responses at week 2 (left) or 24 (right) after SIV challenge. The p-values were calculated for correlation efficiency and were considered significant at P < 0.05. Spearman's rank correlation coefficient values are represented (r). Diagonal lines represent linear regression. (C) Frequencies of multicytokine IFN-γ–, TNF-α–, and IL-2–producing Gag-specific T cells after SIV infection. Total PMBCs at weeks 10 (top) and 24 (bottom) after the second vaccination were stimulated with a pool of overlapping Gag peptides and stained for intracellular IFN-γ, TNF-α, and IL-2. Percentages of all three cytokine-producing cells of total Gag-specific CD4+ T cells are shown. Each symbol represents an individual from a vaccinated group (represented by different colors). Error bars represent the mean frequencies ± SEM (n = 4 or 5). All vaccinated groups were compared with group 1 by a nonparametric Mann-Whitney test.

Marcin Kwissa, et al. J Exp Med. 2007 October 29;204(11):2733-2746.
5.
Figure 5.

Figure 5. From: Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

Correlates of immune responses and protection. Control of viral loads at the set point of challenge at week 24 was tested for correlation with Gag-specific T cell responses after the last rMVA boost (A and B) or after SIV infection (C–F). Magnitudes of expansion of Gag-specific CD8+IFN-γ+ T cells after the second rMVA vaccination at weeks 1 (A) and 10 (B), and at week 2 after SIV challenge (C) inversely correlated with the viremia level of experimental animals. Magnitudes of multicytokine IFN-γ–, TNF-α–, and IL-2–producing Gag-specific CD4+ (D) and CD8+ (E) T cells after SIV challenge also showed strong inverse correlation with the control of the viral load at week 24. (F) Frequencies of central memory CD4 T cells were measured in PMBCs at week 24 after challenge. Population of central memory CD4+ T cells were defined as CD95high CD28high CCR7high, and their frequency correlated inversely with the viral loads at week 24 after SIV, showing preservation of memory cells in vaccinated animals. (G and H) Rectal biopsies were collected at week 18 after challenge. Lamina propria lymphocyte CD4+ T cells are represented as the percentage of total CD3+ T cells extracted from the gut tissue. (G) Proportions of CD4+ T cells in mucosal tissue inversely correlated with the viral loads at week 18 after challenge. (H) Direct correlation between the magnitude of expansion of Gag-specific CD8+ T cells at week 2 after SIV challenge and control of mucosal CD4+ T cell by trial animals at week 18. Each symbol represents an individual animal from a vaccinated group (signified by different colors) or naive animals (asterisks). The p-values were calculated for correlation efficiency and were considered significant at P < 0.05. Spearman's rank correlation coefficient values are represented (r). Diagonal lines represent linear regression.

Marcin Kwissa, et al. J Exp Med. 2007 October 29;204(11):2733-2746.

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