The composition of this adjuvant mimics bacterial DNA and so acts to stimulate the immune system through the TLR9 pathway [20], [21], [22] and [23]. The CpG ODN, is being used in at least one registered FDA monitored clinical trial, but has not yet been approved by the FDA for use in conjunction with a specific vaccine [21]. We found that the presence
of CpG inside the spheres had a significant positive effect on the immune response (Fig. 2a, P = 0.0002). In addition, although previously published findings [24] and [25] showed increased CTL responses when MPLA was placed in the microsphere, we observed strong CTL responses only when MPLA was included in the carrier solution to rehydrate the microspheres for injection ( Fig. 2b, P = 0.0002). We believe MPLA in the carrier solution acts to stimulate the tissue macrophages in the area where transformation to dendritic cells takes place, selleckchem after which phagocytosis and antigen presentation occur. We found that presence of epitope inside the sphere was also critical. In particular, free epitope, even when combined with CpG and MPLA but without the presence of spheres produced essentially no immune response compared to the formulation using the PLGA loaded microspheres for the OVA ( Fig. 2c, P = 0.0015) and for the
VSV epitope ( Fig. 2d, P = 0.0002). We evaluated the dose response to inoculation with 11 μM microspheres loaded with 1%, 10% and 100% of maximum epitope for the OVA and VSV epitopes. The OVA epitope Histone Methyltransferase inhibitor dose response showed a plateau beginning at the lowest level with no statistically significant difference between the 1% and 100% loaded levels ( Fig. 3a, P = 0.25), whereas the VSV epitope showed a statistically significant increase in immune response with increasing loaded concentration at the loading levels tested ( Fig. 3b, P < 0.0001). Also, the difference in immune responses to OVA and VSV both at 1% loading were not statistically significant (P = 0.45), whereas
the difference in responses to OVA and VSV both at 100% were statistically significant (P = 0.0013). We next evaluated the immune response exhibited from two epitopes delivered simultaneously by putting the two epitopes in the same microsphere, with a concentration of OVA and VSV both below at 1% of maximum concentration. We used these concentrations because, as just mentioned, they produced immune responses of similar strength with single-epitope loadings. We administered these spheres in a total amount equal to the amount used previously, with CpG in the spheres and MPLA in the carrier solution. The immune response to OVA in the presence of VSV was not significantly different from the response to OVA in the sphere by itself ( Fig. 4a, P = 0.15), whereas the immune response to VSV in the presence of OVA was slightly greater than the response to VSV in the sphere by itself ( Fig. 4b, P = 0.045).