All immunizations were performed under anesthesia using isoflurane vaporizer. Immunizations were administered intramuscularly (i.m.) in 50 μl total volume. All immunizations contained either 5 μg EndoFit Ovalbumin (OVA) protein (Invivogen) or 1 μg hemagglutinin (HA) recombinant protein from A/California/07/2009 H1N1 (pdm09) (International Reagent Resource, Manassas, VA, USA), or 1 μg HA protein from A/Puerto Rico/8/34 (Sino Biological Inc., Beijing, China), as indicated. In addition to antigen, mice received LMW or HMW CS at 4 or 40 μg. Low dose, 1 μg antigen per mouse vaccinations was chosen to allow for modest CS MW effects to be observed, as well as to investigate potential antigen dose sparing effects of CS adjuvantation (Lampe et al., 2020 (link)). As a negative control, mice were immunized with antigen protein alone. Low dose PR8 immunizations were delivered intranasally (i.n.) at 500 egg infective dose (EID)50 in 30 μl PBS as a positive control for protection against viral challenge and antibody production. Antigen combined with 20 μg MPLA delivered i.m. was also used as a positive control for antibody production (Lampe et al., 2020 (link)). Mice were weighed for up to 7 days after immunization to assess adverse effects caused by adjuvantation.