(61a) Controlled Release of NKT Cell Agonist and Non-Replicating Pathogen for Single-Dose Vaccination

Introduction. Many vaccines require multiple doses scheduled months or years apart in order to impart successful immunization [1]. This is not compatible with the immediate vaccination timelines of potential biological warfare or worldwide pandemics. Plus, is not feasible for people living in poverty-stricken areas. To address the need for single-dose vaccines, this work examines two avenues: (1) use of an adjuvant, alpha-Galactosylceramide (alpha-GalCer), which can stimulate activity of natural killer T (NKT) cells. NKT cells are an unusual lymphocyte subset that can be therapeutically activated with superagonists to potently boost vaccine-specific CD8 cytotoxic T lymphocytes (CTLs) with long-lasting memory and the production of antibodies; and (2) use of polymeric microparticles for controlled release of adjuvant and antigen for primer and booster doses.

Methods. In vivo studies in mice were performed where mice were injected with PK8 (influenza) and alpha-GalCer separately and combined to determine the effectiveness of alpha-GalCer as an adjuvant. Intraperitoneal and intramuscular injection methods were used and compared. Blood samples were collected every 15 days following vaccination and analyzed for virus specific antibodies by ELISA. PLGA microparticles were synthesized via a double emulsion method [2]. Microparticles were synthesized with encapsulated fluorescein (as an adjuvant model) and gold nanoparticles (as a vaccine model). Microparticles were then analyzed for size using microscopy and ImageJ sizing analysis, loading via indirect method, and release. To analyze release, samples were suspended in PBS and aliquots were assayed using a fluorometer or spectrometer at specific time points. Parameters of the double emulsion were varied and analyzed for their effect on size, loading, and release. Then, microparticles with encapsulated H1N1 vaccine and alpha-GalCer were synthesized and analyzed. H1N1 release was measured via ELISA and alpha-GalCer release was measured using LC-MS/MS as in reference [3]. Microparticles were also imaged using scanning electron microscopy at different timepoints during release to access particle breakdown.

Results. Alpha-GalCer was shown to enhance IgG1 levels when co-administered with PR8, indicating that alpha-GalCer stimulates Th2 immune responses (see Figure 1).

Average loading and sizes can be seen in Table 1. For fluorescein loaded microparticles, 65:35 PLGA resulted in higher loading than 50:50 PLGA. Lower Pluronic F68 in the W2 phase resulted in higher loading (0.25%). 65:35 PLGA resulted in larger microparticles whereas amount of Pluronic in the W2 phase did not significantly affect particle sizes.

The 50:50 PLGA released all encapsulated fluorescein after approximately 130 days. Amount of pluronic F68 used in the formulation did not significantly affect the release profile, which makes sense since it also did not have a significant effect on microparticle size. The 65:35 PLGA has only been releasing for 83 days and has not yet released all of its encapsulated payload.

For microparticles loaded with gold nanoparticles, the amount of gold nanoparticles loaded in the W1 phase was shown to not affect loading significantly. 0.5% pluronic F68 in the W2 phase resulted in the highest loading. Release for gold nanoparticles was not shown in the 60 days it was measured, indicating that the particles will not release any gold nanoparticles until they are fully degraded.

Alpha-GalCer release was shown to have an initial “burst” release followed by minimal diffusional release for 80 days (see Figure 2).

Vaccine release was shown to also have an initial “burst” release followed by minimal diffusional release for 35 days as shown in Figure 3. The plots were not significantly different based on the concentration of vaccine in the W1 phase (0.25-1 mg/ml). Release is still being measured and quantified.

SEM images shown in Figure 4 before release and after 65 days showed that pores begin to form in the microparticles.

Conclusions and future work. Alpha-GalCer was shown to stimulate IgG1 immune responses in vivo when co-administered with PR8. Microparticles were shown to load and release all molecules when using 0.25% pluronic F68 as a stabilizer in the double emulsion. Microparticles were approximately 1-5 μm in size. Release profiles following an initial release of therapeutic followed by minimal passive release. All of the fluorescein was released within 130 days. Release of other molecules, vaccine and alpha-GalCer is still ongoing. SEM images showed that pores begin to form in the microparticles after 65 days of release. After initial release profiles are acquired, the microparticle synthesis will be optimized using different PLGA ratios or drug loading to achieve desired release profiles of burst release, followed by another burst release after 1 month. Optimized microparticle formulations will be tested in vivo.

References:

[1] S. Lu, “Heterologous prime-boost vaccination,” Curr. Opin. Immunol., vol. 21, no. 3, pp. 346–351, 2009, doi: 10.1016/j.coi.2009.05.016.

[2] N. Y. Martinez, P. F. Andrade, N. Durán, and S. Cavalitto, “Development of double emulsion nanoparticles for the encapsulation of bovine serum albumin,” Colloids Surfaces B Biointerfaces, vol. 158, no. 1, pp. 190–196, 2017, doi: 10.1016/j.colsurfb.2017.06.033.

[3] K. Kaneko, A. McDowell, Y. Ishii, and S. Hook, “Selective quantitation of the incorporation of the immunomodulator α-galactosylceramide in liposomes using LC-MS/MS,” Int. J. Mass Spectrom., vol. 392, pp. 96–101, 2015, doi: 10.1016/j.ijms.2015.09.016.