Since a high level of protection against influenza virus only lasts for 3 days (Fig. 1) , prolonged protection would require repetitive dosing. To test whether tachyphylaxis to the protective effects of the aerosolized NTHi lysate occurs, we challenged mice with influenza in three different conditions: no lysate treatment, a single lysate treatment, or repetitive lysate treatment. We again observed 100% mortality in the untreated group, but found identical protection by NTHi lysate treatment whether given once one day prior to infection, or given three times (seven, four and one days prior to infection, Fig. 3A ). In both NTHi lysate treated groups, protection was associated with recovery of early weight loss by the fifth day after infection (Fig. 3B ). Observation for 21 days after the challenge showed no subsequent mortality.