An additional preclinical study was conducted in guinea pigs using the same H5N1 rHA antigen in doses ranging from 0.001–10
μg. Both IM and ID routes were used in a two-dose prime/boost regimen. Animals received an IS patch containing 10 μg of LT
or a placebo patch at the time of boost. A 10-fold dose sparing was observed using the LT-IS patch for the IM route (data
not shown), and ≥10-fold using the ID route (Figure 2). Taken together, the preclinical data in both animal models show that
the LT-IS patch can dose spare 10– to 100–fold.
Figure 2. Dose sparing in the guinea pig model using rH5 (Anti-A/Vietnam/1203/2004). Animals received two intradermal injections
21 days apart of either 0.001, 0.01, 0.1, 1, or 10 μg rHA and an IS patch containing either 10 μg LT or a placebo with no
LT at the time of second injection. Serum IgG titers were determined by an ELISA method two weeks after the final immunization.
Results of individual animals (5–6 per group, open circles) and the group geomeans (bars) are shown.
Universal Adjuvant Effect for Seasonal Flu Vaccines
We also evaluated the LT-IS patch effect using different manufacturers' influenza vaccines and demonstrated that the adjuvant
effect is not antigen-dependent. Four commercial annual vaccines were evaluated: FluShield, Fluzone, Fluvirin, and InFlexal.
Trivalent influenza vaccine at 4.5 μg HA dose per virus strain was injected ID into mice with and without an LT-IS patch (10
μg LT). The mice were immunized twice, two weeks apart, with serum collection two weeks after the second immunization. The
animals receiving the LT-IS patches had significantly higher anti-influenza IgG titers (as determined by ELISA) than those
receiving flu vaccines with placebo patches (no LT). Only anti-A/New Caledonia serum IgG titers are shown in Figure 3 as representative
of IgG response in all three strains. Figure 3 indicates that influenza vaccines obtained from different manufacturers were
enhanced equivalently with an LT-IS patch and reinforces the idea of a potential universal dose sparing strategy using an
Figure 3. IS patch effect of different seasonal flu vaccines in the mouse. Mice received two intradermal immunizations, 14
days apart, of a commercially available seasonal flu vaccine and IS patches containing either 10 μg LT or a placebo with no
LT. Serum IgG titers were determined by an ELISA method. Results of individual animals (8 per group, open circles) and the
group geomeans (bars) are shown. Groups receiving LT have statistically greater titers than groups receiving placebo patches.