*2.5. Animal Immunizations*

For HIV dosing studies, C57BL/6 mice were immunized three times at three-week intervals with either 2.5 μg of HIV Env DNA only or 2.5 μg of HIV Env DNA and 11, 20, or 30 μg of opt-36βt in a total volume of 30 μL of water. Mice were injected using the intramuscular (IM) route in the shaved tibialis anterior muscle followed by electroporation (EP) using the CELLECTRA® 3P (Inovio Pharmaceuticals, Plymouth Meeting, PA, USA) as previously described [44]. For HIV plasmid comparison studies, C57BL/6 mice were immunized three times at three-week intervals with either 2.5 μg of HIV Env DNA only or 2.5 μg of HIV Env DNA and 11 μg of opt-36αt, opt-36βt, or opt-36γt in a total volume of 30 μL of water. For influenza studies, BALB/c mice were immunized two times at two-week intervals with 1 μg of HA1 DNA plasmid alone or 1 μg of HA1 DNA plasmid and 11 μg of opt-36αt, opt-36βt, or opt-36γt in a total volume of 30 μL of water delivered intramuscularly as described above. For Zika studies, IFNAR−/<sup>−</sup> mice were immunized once with 0.5 μg of Zika prME alone or 0.5 μg of Zika prME and 11 μg of opt-36γt in 30 μL of water delivered intramuscularly as described above.
