Our data in the present study showed that protein antigens conjugated onto the surface of nanoparticles prepared with lecithin and GMS induced quick, strong and long-lasting antibody immune responses when subcutaneously injected into mice. Depending on the antigens used, the adjuvanticity of the nanoparticles was stronger than that of the Alum and was comparable to that of the IFA. Moreover, the antibody responses developed more quickly when the antigen was adjuvanted using the nanoparticles than using Alum. The potent adjuvanticity of the nanoparticles was likely due to the nanoparticles' ability to enhance the uptake of the antigens by APCs, to move the antigens into local draining LNs, and to activate APCs.
BSA (~66 KDa) is a serum protein with normal immunogenicity. At the beginning, we used it as an antigen to confirm the conjugation of the protein antigens onto the surface of the nanoparticles and to evaluate the adjuvanticity of the nanoparticles. Our data in clearly showed that successful conjugation of the BSA onto the nanoparticles and that unconjugated BSA can be separated from the nanoparticles by GPC. Importantly, the BSA conjugated onto the nanoparticles generated anti-BSA antibody titers 5-6.5-fold higher than those induced when the BSA was adjuvanted using the Alum (, IgG and IgM). In fact, data in showed that the nanoparticles have similar adjuvanticity as the IFA, a well known strong adjuvant [26
]. The nanoparticles appeared to have adjuvant properties that favor humoral immunity, although less biased than the Alum. If a more balanced antibody response or an increased cell-mediated immune response is needed, one may have the option of including a co-adjuvant such as the low toxicity LPS [27
] or the CpG oligos into the nanoparticle formulation.
In order to evaluate the functionality of the immune response induced by antigens delivered using the nanoparticles, we used the B. anthracis
PA protein. The toxicity of the B. anthracis
toxin is mainly from the lethal factor and the edema factor. PA is needed for the natural entrance of the LF and EF into the host cells [28
]. Thus, antibodies that can neutralize the PA in serum are expected to block the transport of LF and EF into the cytosol and prevent the course of B. anthracis
infection. Similar to the BSA-NPs, the PA-NPs were able to induce a strong anti-PA IgG response, and increasing the dose of the PA tended to increase the resultant anti-PA IgG titer (). Slightly different from the BSA-NPs, at the dose of 5 μg/mouse, the PA-NPs and the PA admixed with Alum induced comparable anti-PA IgG titers (). This may be due to that unlike the BSA, the PA protein is strongly immunogenic in inducing anti-PA antibody responses [29
] and extremely foreign to the mice used in this study. In our splenocyte proliferation assay, we found a proliferation index of 2.5. The relatively small proliferation index may be due to reasons that the mice were dosed with only 1 μg of the PA protein and that the index was derived from the whole splenocytes, not the purified T cells. Shown in are the kinetics of the anti-PA IgG responses induced by PA conjugated onto the nanoparticles. Interestingly, mice immunized with the PA-NPs developed detectable anti-PA IgG following a single immunization (). However, mice immunized with the PA admixed with Alum failed to respond to the priming, and anti-PA IgG was detectable in those mice only after a boost immunization (). The ability of an anthrax vaccine to induce an immune response immediately after the first immunization is likely critical in the case of post-exposure combination therapy of anthrax with a vaccine and an antibiotic. Finally, the anti-PA antibodies induced by PA-NPs were found to be functional. Serum from mice immunized with the PA-NPs was able to neutralize anthrax lethal toxin and protect mouse macrophages in culture (). Moreover, mice immunized with the PA-NPs also survived a lethal dose of anthrax lethal toxin challenge ().
The ability of the nanoparticles to enhance the immune responses induced by antigens conjugated on their surface was likely due to their ability to enhance the trafficking of the antigens into local draining LNs, to improve the uptake of antigens by APCs, and to activate the APCs. For example, our data in clearly showed that nanoparticles facilitated the uptake of the BSA antigen conjugated on their surface, likely because the DCs were more effectively taking up particulates than the BSA protein in solution. Data from previous studies showed that particles with a diameter of 500 nm or below were optimal for uptake by APCs such as DCs and macrophages [6
]. Our BSA-NPs was around 200 nm, which is within the optimal size range for the uptake by APCs. Moreover, our data in showed that close to 4% of the cells in local draining popliteal LNs became FITC+
at 24 h after the BSA-NPs labeled with FITC were injected into the footpad of the hind legs, suggesting that the FITC-labeled nanoparticles were present in ~4% of the LN cells. It is unclear to what extent those particles moved into the LNs by direct draining or by the trafficking of the DCs. However, only about 15% of the FITC+
cells in the popliteal LNs were CD11c+
, suggesting that the direct draining of the nanoparticles into the local draining LNs may have been extensive. Nevertheless, Manolova et al. (2008)
reported that particles of 20-200 nm drained freely to LNs [8
]. Finally, our data indicated that the nanoparticles also activated APCs in the draining LNs because an increased percent of the cells in popliteal LNs became CD11c+
after the subcutaneous injection of the nanoparticles ().
Finally, our nanoparticles were engineered with lecithin and GMS as the matrix materials and Tween 20 as an emulsifying agent. Lecithin is a complex mixture of phosphatides consisting chiefly of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and other substances such as triglycerides and fatty acids. It is GRAS (generally regarded as safe) listed and accepted in the FDA Inactive Ingredients Guide for parenterals (e.g., 0.3-2.3% for intramuscular injection)[30
]. Tween 20 is a polyoxyethylene derivative of sorbitan monolaurate. It is also GRAS listed and included in the FDA Inactive Ingredients Guide for parenterals[30
]. GMS is used in a variety of food, pharmaceutical, and cosmetic applications and is also GRAS listed. Its LD50
in mouse was reported to be 0.2 g/kg by the intraperitoneal route[30
]. Therefore, although more experiments will have to be carried out in the future to define the safety profile of the nanoparticles, we consider this nanoparticle-based antigen delivery system relatively safe.