Fig. 4: CVP-containing influenza vaccines activate CTLs.

A Mice were intranasally or intramuscularly administered saline (SA), split influenza vaccine (SV) alone, or SV with each CVP or existing adjuvants twice, with a 4-week interval. Ten days after the boost, spleens or lungs were harvested, and lymphocytes were purified. Purified lymphocytes were incubated with SV antigen, and IFNγ-producing cells were measured by ELISPOT following overnight antigen stimulation. B Mice vaccinated with the same protocol above were intravenously injected with CFSE-labeled splenocytes pulsed with antigen from untreated mice. Twenty-four hours post-transfer, spleens or lungs were collected, and the frequency of CFSE-labeled transferred cells was measured by FACS to assess specific killing. C Lungs from mice immunized with OVA antigen using the same protocol were collected, and lymphocytes were purified. Purified lymphocytes were stained with antibodies against surface antigens and antibodies recognizing the OVA antigen peptide–H2Kb complex. The frequency of antigen-presenting cells was analyzed by FACS. Each dot represents an individual animal. Bar graphs indicate mean values and error bars represent the SEM (n = 5) of data from two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Dunnett’s multiple comparison test following the Kruskal–Wallis test (compared with the SA or OVA group).