Fig 7. CTL Recall Responses and Protective Heterosubtypic Immunity to Influenza Virus after Prime-boost IN Vaccination.

C57BL/6 mice were immunized intranasally with 1, 3, or 10 μg of HA-equivalent of BPL-inactivated influenza strain PR8xTexas/H3N2 in 50 μl PBS with or without 10% ADJ. Note that the vaccine preparation also contains other viral proteins including NP, PA, neuraminidase and matrix proteins. Vaccines were repeated 3 weeks later, and 3 weeks after the second immunization, mice were challenged by IN administration of 200 PFU of influenza A/PR/8/34/H1N1. 6 days after challenge 5 mice/group were sacrificed and lungs were collected for CTL analysis and viral titration. D^b/NP366-specific CD8 T cells in lung were quantified by MHC I tetramers or intracellular cytokine staining for IFN-γ. (A) Absolute numbers SIINFEKL-specific IFN-γ⁺ CTLs in lung. (B) Absolute numbers of CD103⁺ CD69⁺ CXCR3⁺ NP366-tetramer⁺ CTLs in lungs. (C) Frequencies of IFN-γ-producing CTLs specific for influenza NP366 and PA224 epitopes among all CD8⁺ cells in the lungs. (D) Total number of IFN-γ-producing CTLs specific for influenza NP366 epitope in the lungs. (E) Frequencies of CTLs double positive for IFN-γ and CD107a (LAMP-1) among all CD8⁺ cells in the lungs. (F) Lung viral titers expressed as plaque-forming-units (PFU) per gram of lung. * indicates p<0.05, ** indicates p<0.01, **** indicates p<0.0001. In panels A-E only, asterisks over a specific bar indicate significance over other groups, unless otherwise indicated. ** indicates significant difference of 3μg HA+ ADJ group with other groups excluding 10μg HA + ADJ. T-cell data are representative of two independent experiments. Viral titer data is pooled from two separate experiments.