PMC

BALB/c mice were immunized with 1x10⁸pfu AdHu5rab.gp to induce neutralizing antibodies to AdHu5. Then they were immunized orally with 5x10¹⁰vp of either AdHu5gag or AdC68gag. Ten days later, splenocytes were collected and stimulated with a CD8⁺-specific gag epitope and frequencies of IFN-γ⁺ cells were determined by intracellular cytokine staining. Frequencies shown are averages calculated from individual mice minus background (between 0.01–0.2%), and error bars shown here represent standard deviations.

BALB/c mice were immunized intramuscularly with 5x10¹¹vp AdC68gag. Lymphocytes from spleen, blood, popliteal lymph nodes (LN), peritoneal lavage (PerLav), MLN, and PP and were isolated after day 10 and month 18 and stimulated with a CD8⁺-specific gag epitope and frequencies of IFN-γ⁺ cells were determined by intracellular cytokine staining. Background levels (between 0.1–0.8%) were subtracted from each sample before plotting the frequencies here. Spleens were analyzed from individual mice, while the other tissues were pooled from 5 mice per group.

BALB/c mice were immunized orally and intramuscularly with 5x10¹⁰vp AdC68gag, and lymphocytes from tissues were isolated 10 days and 3 months later and stimulated with a CD8⁺-specific gag epitope and frequencies of IFN-γ⁺ cells were determined by intracellular cytokine staining. Lymphocytes from spleens were analyzed from individual mice, while blood, MLN, and PP were pooled from groups of 5 mice for this assay. Frequencies shown are averages (spleens) or values (blood, MLN, PP) with background frequencies (between 0.1–0.5%) subtracted, and error bars shown here represent standard deviations.

Groups of 5 BALB/c mice were orally immunized with 5x10¹⁰vp AdC68gag and i.m. immunized 2 months later with 1x10⁹vp AdHu5gag, as shown in the timeline (A). Lymphocytes from spleen, MLN, and PP were isolated from sacrificed animals at day 10 and month 2 after the boost, and were stimulated for gag-specific IFN-γ ELISpot (B). Data for spleen represent mean + SD of five individual mice, whereas data for MLN and PP represent mean + SD of pooled cells in triplicate wells. Unstimulated cells from each sample were used as background control, and the numbers of background spots, which were similar for each tissue (<100 SFU/1E6), was subtracted from each sample before plotting the data. (C) Spleen and MLN cells were harvested at the Month 2 timepoint post boost and stained with a gag tetramer. Percentage of tetramer⁺ CD8⁺ cells are shown in the upper right corner of each dot plot. The plots for spleen are representative samples from individual mice, while the plots for MLN show pooled lymphocytes from 5 mice per group.