Six adult rhesus macaques (Macaca mulatta) were housed at BioQual and maintained in accordance with the Association for Accreditation of Laboratory Animal Care guidelines at the NIH. Three animals were infected with H3N2 A/Aichi/2/1968 influenza virus at 2.36 × 106 plaque-forming units per animal in a divided dose; for each animal, half of the infectious dose was given in 1 mL intranasally and the other half as 1 mL intratracheally. Animals were monitored for signs of infection and no intervention was needed. For all immunizations, animals were immunized intramuscularly with 100 μg of H3 A/Aichi/2/1968 recombinant protein per animal per time point given as 500-μL total injection volume divided into two sites—250 μL each at right and left quadriceps. The final immunization mixture contained 15% STS+R848+oCpG adjuvant (same reference as above) (14) with the remaining volume being sterile saline. Blood was collected in EDTA tubes per the schedule shown in Fig. 1 and processed for plasma and cells, which were cryopreserved.

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