Fig. 4: CAF08 enhances RSV-specific Th1 and CD8 T cell responses in newborn mice.

Neonatal CB6F1 mice were immunized with formulations as indicated at day of life (DOL) 4-7 and DOL 21-24. At DOL 35-38, spleen and lymph nodes were harvested, and cell suspensions prepared for flow cytometry staining with either a pre-F-specific MHC II tetramer, or a control tetramer containing the CLIP peptide, and stained for intracellular presence of Th1 transcription factor T-bet or Th2 transcription factor GATA3. a Representative histograms are shown. b Lymph node suspensions from all animals except saline-treated had a frequency of RSV tetramer-positive cells (closed circles) that was significantly higher than the frequency of control tetramer-positive cells (open circles,) Statistical comparisons to each corresponding control tetramer group employed two-tailed paired Student’s t test (*p < 0.05, **p < 0.01, ***P < 0.001).. No significant presence of RSV tetramer-specific T cells as compared to control tetramer-positive T cells was observed in the spleen. n = 5 newborn mice/group, shown as Box-whisker plots, center:mean, boxes:75th and 25th percentile, whiskers: minimum and maximum values. c Analysis of the relative percentage of T-bet+ Th1 cells and GATA3+ Th2 cells within the tetramer-positive gate revealed an increase in Th1 cells and a relatively lower abundance of Th2 cells in CAF08-immunized animals (n = 5 newborn mice/group) Statistical comparisons to SAL group employed two-tailed Mann–Whitney U test (*p < 0.05, **p < 0.01, ***P < 0.001. d Draining lymph node cultures were stimulated with 1 μg/ml pre-F antigen for 24 h in the presence of Brefeldin A. Quantification of CD4⁺CD44⁺Cytokine⁺ (IFN-γ) cells confirmed a statistically significant generation of Th1 cells in CAF08-immunized mice. Detection of CD8⁺CD44⁺ Cytokine⁺ (IFN-γ and/or TNF-α) cells indicates induction of RSV^-specific CD8⁺ T cells in CAF08-treated mice. Cytokine analysis indicates prominent production of Th2/anti-inflammatory cytokines (IL-5, −13, −10) in newborn mice as compared to adult mice. CAF08b reduced production of Th2/anti-inflammatory cytokines and increased that of IFN-γ, indicating enhancement of a Th1 response. Statistical comparisons to SAL group employed two-tailed Mann–Whitney U test (*p < 0.05, **p < 0.01, ***P < 0.001 s (n = 6 newborn mice (CD4 analysis), 10 newborn mice (CD8 analysis and cytokine secretion analysis). Source data are provided as a Source Data file.