Influenza a h1n1

The procedures used to measure HA-specific antibodies were previously described^{53 (link)}. Briefly, 96-well plates (Nunc) were coated with 1 μg/mL HA protein (Influenza A H1N1 (A/Puerto Rico/8/1934) Haemagglutinin, SinoBiological) in Coating Buffer (0.1 M Na₂CO₃, 0.1 M NaHCO₃, pH 9.5) at 4 °C overnight. HRP-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates), and HRP-conjugated anti-mouse IgG (R&D Systems) were used at 1:2000 to detect antigen-specific antibodies in serum. The procedures used in the microneutralization assay were previously described^{53 (link)}. Briefly, MDCK cells were seeded into 96-well plates on day –1. Then, these cells were washed twice with PBS and incubated in DMEM with 2 μg/mL trypsin (T1426, Sigma-Aldrich) at Day 0. Serum samples were serially diluted 2-fold in 50 μL of DMEM and then mixed with 100 TCID50 of PR8 influenza virus in 50 μL of DMEM for 1 h at 37 °C. 1 h later, the virus-serum mixture was transferred to MDCK cells and incubated for 24 h. After 24 h of incubation, the supernatant was removed, the cells were washed twice with PBS and fixed in 80% acetone for 30 min, and viral antigen was detected by ELISAs with a polyclonal antibody against NP protein. The OD₄₅₀ was recorded. Viral plaque assays were performed as previously described^{57 (link)}.

The influenza vaccine contained a 5 μg dose of Influenza A H1N1 (A/California/04/2009) hemagglutinin (HA) (Sino Biological) and varied amounts of TLR7/8a, HPMC‐C12, and NPs. Consistent between all gel groups unless specifically altered: 2 wt% HPMC‐C₁₂: 10 wt% NP, conjugated NPs have 10% TLR7/8a conjugated PEG–PLA and 90% unconjugated PEG–PLA, 20 μg TLR7/8a total dose. For the PNP hydrogels, the vaccine cargo was added at the appropriate concentration into the PBS component of the hydrogel before adding the polymer and NP solutions, as described above. For Alum (Invivogen) vaccines, the formulation was prepared according to the manufacturer's instructions with a 5 μg dose of HA.