Fig. 2. cHA-LAIV study design and sample collection.

a Animals were randomly assigned into seven groups (n = 35). Groups 1-5 were intranasally infected with a sub-lethal dose of purified cH9/1-IBV (2 × 10⁵ PFU). Mice were immunized in 4-week intervals. Group 1 received cH8/1-N1-ΔNS1 and then cH11/1-N1-ΔNS1, group 2 received the vaccine doses in reverse order (10⁵ PFU per dose). Groups 3 and 4 followed the regimens of groups 1 and 2 except that the strains had a cold-adapted temperature-sensitive LAIV backbone (with a full length, functional NS1). Group 5 and 6 were mock immunized with identical volumes of sterile 1x PBS. Group 7 was intramuscularly given 50 µl of non-adjuvanted QIV per dose (~1.5 µg per dose). We used Group 7 as a “standard-of-care” vaccine comparator. Although this group does not include a cH9/1-IBV, previous experiments demonstrated that even in the presence of priming, QIV vaccination did not confer good protection against heterologous challenges²⁶. Animals were intranasally challenged 4 weeks post final-boost either with IVR-180 (BSL-2; 100x LD₅₀) or influenza A/Vietnam/1203/04 virus (BSL-3 + ; 20x LD₅₀) or 13 weeks post final-boost for a separate BSL-3+ challenge (1000x LD₅₀). b Animals were bled (submandibular), and nasal washes were taken at D0 (naïve) and D98 animals. Mice were bled between immunization doses at approximately 28-day intervals up until D98 since the first dose. c Sample collection for BSL2 100 LD₅₀ challenge included submandibular bleeds at D1, D5 and D10 (n = 5) for pooled circulating tetramer specific CD8⁺ T-cell analysis. Lungs (left lobes) and nasal turbinates were harvested on D3 and D5 for viral titration. Right lobes of lungs on D3 were used for analysis of tissue-resident memory CD8⁺ T-cell analysis and on D5 for histopathology. Spleens were harvested on D5 for enzyme-linked immunosorbent spot (ELISpot) and intracellular staining assays. For BSL-3+ studies, whole-lung and nasal turbinates were harvested on D3 and D5 for viral titration. Image was created using BioRender.