Endo grade ovalbumin protein

Intranasal procedures were performed in 30 μL volumes on ketamine/xylazine sedated mice. For single-dose assays, mice received either PBS or 5-A-RU/MG (MG; Sigma-Aldrich). For OVA-specific prime-boost assays, mice received either 5 nmol Endo Grade® Ovalbumin protein (Sigma-Aldrich) alone, or mixed with 5-A-RU/MG (75 nmol 5-A-RU mixed with 750 nmol MG). Three homologous immunisations were performed at two-week intervals. For HA-specific and RBD-specific prime-boost assays, mice received either PBS, 5-A-RU/MG alone, 10 μg of IAV HA (A/X-31) protein alone, 50 μg of SARS-CoV-2 RBD (dimerised, ancestral virus) protein alone, or protein mixed with 5-A-RU/MG. As a benchmark, proteins were mixed with AddaVax (InvivoGen) at 1:1 v/v and delivered either subcutaneously into the right and left flanks (HA vaccination) or intramuscularly into the left and right legs (RBD vaccination) in 50 μL volumes according to the same prime-boost regimen as above.

For detection of antigen-specific serum IgG, microtitre plates (Maxisorp, Nunc) were incubated overnight with either 20 μg/mL Endo Grade® Ovalbumin protein (Sigma-Aldrich), 10 μg/mL HA protein, or 2 μg/mL RBD protein in bicarbonate buffer. 10% FBS was applied to prevent non-specific binding, prior to application of serially diluted serum samples. A final incubation was performed with a variety of Ig isotypes; IgG_TOTAL (G21040), IgG1 (A10551), IgG2a/c (PA129288), IgG2b (M32407), IgG3 (M32707) conjugated to HRP (Life Technologies). Tetramethylbenzidine (TMB; BD Biosciences) and 2M H₂SO₄ (Sigma-Aldrich) was applied to develop and stop reactions, before immediately being read at 450 nm. Wash steps were performed 5x between each incubation with 10% PBS-Tween (Sigma-Aldrich).

BALF was collected by inserting a catheter into the trachea. 0.6 mL of 10% FCS PBS was injected and aspirated three times. Approximately 0.5 mL of BALF was recovered from each mouse and supernatant was collected following centrifugation. Aliquots of BALF were stored under -80°C. IgA antibodies in the BALF were measured using a mouse Total IgA ELISA kit (Thermo Fisher Scientific) according to manufacturer’s instructions with the exception of the coating antigen. For detection of antigen-specific IgA, microtitre plates (Maxisorp, Nunc) were incubated overnight with either 20 μg/mL Endo Grade® Ovalbumin protein (Sigma-Aldrich), 10 μg/mL HA protein or 2 μg/mL RBD protein in bicarbonate buffer.