1. Volume 25, Issue 11, Pages 2533-2545 (November 2017)
Recombinant LCMV Vectors Induce Protective Immunity following Homologous and Heterologous Vaccinations 
Jessica Wingerath, Dmitrij Ostroumov, Norman Woller, Michael P. Manns, Daniel D. Pinschewer, Klaus Orlinger, Ursula Berka, Florian Kühnel, Thomas C. Wirth 
Molecular Therapy 
Volume 25, Issue 11, Pages (November 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1
CD8 T Cell Kinetics following rLCMV-OVA Infection with Different Doses
(A) Experimental setup. In two separate experiments, mice (n = 5) were immunized with different doses of rLCMV-OVA. (B) Representative dot plot of SIINFEKL-tetramer-reactive CD8 T cells of the group with 2 × 105 ffu/mouse at day 7 after infection. (C) Percentage of SIINFEKL-specific CD8 T cells in total white blood (WBC) cells measured in peripheral blood. Data are from two separate experiments with different doses of rLCMV-OVA and represent the mean ± SD of five different mice in each group. (D) Frequency of SIINFEKL-specific CD8 T cells in individual mice from the same experiments. Differences between individual groups were calculated using the unpaired Student’s t test. (E) Primary memory phenotype of SIINFEKL-specific CD8 T cells in pooled blood samples (day 39 after priming). Numbers indicate the percentage of marker-positive CD8 T cells in total SIINFEKL-specific CD8 T cells. *p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2
Homologous rLCMV Booster Infections after Primary Infections with Different Doses
(A) Experimental setup. Mice (n = 5) were primed with different doses of rLCMV-OVA and boosted after 40 days with 2 × 105 ffu/mouse. (B) Percentage of SIINFEKL-tetramer-reactive CD8 T cells measured in peripheral blood after boosting. Data represent the mean ± SD of five different mice in each group. (C) Frequency of SIINFEKL-tetramer-reactive CD8 T cells in individual mice from the same experiment. Differences between individual groups were calculated using the unpaired Student’s t test. (D) Secondary memory phenotype of SIINFEKL-specific CD8 T cells in pooled blood samples (day 40 after boosting). Numbers indicate the percentage of marker-positive CD8 T cells in total SIINFEKL-specific CD8 T cells. (E) Total cell count of IFN-γ-producing CD8 T cells detected via ICS in the spleens on day 50 after boosting. (F) Percentage of IFN-γ/IL-2 and IFN-γ/TNF-α double-positive CD8 T cells in total IFN-γ-producing splenocytes of the same assay. **p ≤ 0.01, ***p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
Comparison of T Cell Kinetics following LCMV-WT and rLCMV-OVA Infection
(A) In three different experiments, mice (n = 3) were infected with 2 × 105 ffu LCMV-WT or rLCMV-OVA, respectively. (B) Kinetics of NP396-specific CD8 T cells, detected by intracellular cytokine staining (ICS), and total antigen-experienced CD8 T cells (CD8int CD11ahi) in peripheral blood samples of infected mice. Data represent the mean ± SD. (C) Individual numbers of T cells specific for NP396 or GP33 detected by ICS for IFN-γ, total antigen-experienced CD8 T cells, and total spleen cells (from left to right) on day 8 after vaccination. Differences between individual groups were calculated using the unpaired Student’s t test. Numbers indicate the x-fold difference between the WT and OVA group. (D) Individual numbers of T cells specific for NP396 or GP33 detected by ICS, total antigen-experienced CD8 T cells, and total spleen cells (from left to right) on day 40 after vaccination. Differences between individual groups were calculated using the unpaired Student’s t test. (E) Phenotype kinetics of splenic CD8 T cells. Numbers indicate the percentage of marker-positive CD8 T cells in NP396-specific CD8 T cells. LOD, limit of detection. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
CD8 T Cell Kinetics following Homologous Infections with rLCMV-OVA, rAd-OVA, rVV-OVA, and attLM-OVA
(A) Mice (n = 4) were primed on day 0 and boosted on day 40 with the indicated vectors. Kinetics of SIINFEKL-tetramer-reactive and total antigen-experienced CD8 T cells in peripheral blood samples. Data represent the mean ± SD. (B) Individual numbers of SIINFEKL-specific primary (1°) and secondary (2°) memory CD8 T cells (39 days after priming and 40 days after boosting, respectively). Numbers indicate the x-fold increase between both time points. Differences between individual groups were calculated using the unpaired Student’s t test. (C) Total number of SIINFEKL-specific secondary memory CD8 T cells (43 days after boosting) in the spleen. Numbers on the y-axis indicate the cell count in millions (×106). (D) Phenotype kinetics of CD8 T cells in pooled blood samples of the same experiment. Numbers indicate the percentage of marker-positive CD8 T cells in SIINFEKL-specific CD8 T cells. For a more detailed primary memory phenotype from the spleen, see also Figure S3. (E) In a separate experiment, 150 μL serum or 2 × 105 primary memory CD8 T cells from mice infected with LCMV-WT or Ad-WT were transferred into naive mice (n = 5) prior to infection with rLCMV-OVA or rAd-OVA, respectively. Control groups received no adoptive transfer prior to rLCMV-OVA or rAd-OVA vaccination. Numbers indicate the percentage of SIINFEKL-tetramer-reactive CD8 T cells in total white blood cells for each group. Data represent the mean ± SD of each group. **p ≤ 0.01, ***p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5
CD8 T Cell Kinetics following Heterologous rLCMV-OVA Infections with rLCMV-OVA Priming
(A) Experimental setup. Mice (n = 5) were either primed with rLCMV-OVA or left naive and were subsequently boosted with rLCMV-OVA, rVV-OVA, rAd-OVA, or attLM-OVA. (B) Kinetics of SIINFEKL-tetramer-reactive CD8 T cells in peripheral blood samples. Data represent the mean ± SD of each group. (C) SIINFEKL-specific CD8 T cells in memory (day 61). Memory CD8 T cell numbers of mice with or without rLCMV-OVA priming. Numbers indicate the x-fold difference in the percentage of SIINFEKL-specific CD8 T cells. Differences between individual groups were calculated using the unpaired Student’s t test. *p ≤ 0.05, **p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 6
CD8 T Cell Kinetics following Heterologous rLCMV-OVA Infections with rLCMV-OVA Boosting
(A) Experimental setup. Mice (n = 5) were infected with rLCMV-OVA, rVV-OVA, rAd-OVA, or attLM-OVA and boosted with rLCMV-OVA. (B) Kinetics of SIINFEKL-tetramer-reactive CD8 T cells in peripheral blood samples. Data represent the mean ± SD. (C) Memory CD8 T cell numbers of individual mice before (day −1) and after (day 42) rLCMV-OVA priming. Numbers indicate the x-fold difference in the percentage of SIINFEKL-specific primary and secondary memory CD8 T cells. Differences between both time points were calculated using the unpaired Student’s t test. (D) Total number of SIINFEKL-specific secondary memory CD8 T cells (64 days after boosting) in the spleen. *p ≤ 0.05, **p ≤ ns, not significant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 7
Functional Characterization of Memory CD8 T Cells Induced by rLCMV Vaccinations
(A) Experimental setup for (B) to (D). 5 × 103 naive Thy1.1+ (CD90.1+) CD8 OT-I cells were adoptively transferred into Thy1.2+ (CD90.2+) recipient mice (day −62). 1 day later, the mice were infected with rLCMV-OVA, rAd-OVA, or rVV-OVA. On day −1, memory OT-I cells were isolated and 5 × 103 primary memory OT-I T cells were adoptively transferred into naive Thy1.2+ recipient mice (n = 4). 1 day later, the mice were challenged with 5 × 106 CFU attLM-OVA. (B) Representative dot plots of adoptively transferred CD8+ CD90.1+ OT-I cells in recipient mice. (C) Phenotype of memory OT-I cells on day −1 before the second adoptive transfer. Numbers indicate the percentage of marker-positive OT-I cells in total OT-I T cells. (D) Individual frequencies of OT-I cells in peripheral blood after attLM-OVA challenge. Differences between individual groups were calculated using the unpaired Student’s t test. (E) At the same time points, phenotype markers on OT-I cells were analyzed. Numbers indicate the percentage of marker-positive cells in total OT-I cells. (F) Mice (n = 3) were immunized with rLCMV-OVA, rAd-OVA, or rVV-OVA or left naive. 44 days later, mice were infected with a lethal dose of 5 × 105 CFU virLM-OVA. 3 days after infection, bacterial burden was measured in the spleen and liver. Differences between individual groups were calculated using the unpaired Student’s t test. The experiment was repeated twice with similar results. (G) In vivo CTL (cytotoxic T lymphocyte) assay. Mice (n = 5) were immunized with rLCMV-OVA or rVV-OVA, respectively. 55 days later, splenocytes of naive donor mice were pulsed with SIINFEKL peptide or GP33 peptide as the irrelevant control peptide, labeled with CFSE, and injected into the infected mice. 19 hr later, CFSE-labeled splenocytes were quantified in peripheral blood and target cell survival was calculated. Differences between individual groups were calculated using the unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ ns, not signficant.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions