Delivery of small molecule mast cell activators for West Nile Virus vaccination using acetalated dextran microparticles

Abstract

Recently, there has been increasing interest in the activation of mast cells to promote vaccine efficacy. Several mast cell activating (MCA) compounds have been reported such as M7 and Compound 48/80 (C48/80). While these MCAs have been proven to be efficacious vaccine adjuvants, their translatability is limited by batch-to-batch variability, challenging large-scale manufacturing, and poor in vivo stability for the M7 peptide. Due to this, high throughput screening was performed to identify small molecule MCAs. Several potent MCAs were identified via this screening, but the in vivo translatability of the compounds was limited due to their poor aqueous solubility. To enhance the delivery of these MCAs we encapsulated them in acetalated dextran (Ace-DEX) microparticles (MPs). We have previously utilized Ace-DEX MPs for vaccine delivery due to their passive targeting to phagocytic cells, acid sensitivity, and tunable degradation. Four different MCA loaded MPs were combined with West Nile Virus Envelope III protein (EDIII) and their vaccine adjuvant activities were compared in vivo. MPs containing the small molecule MCA ST101036 produced the highest anti-EDIII IgG titers of all the MCAs tested. Further, ST101036 MPs produced higher titers than ST101036 formulated with PEG as a cosolvent which highlights the benefit of Ace-DEX MPs over a conventional formulation technique. Finally, in a mouse model of West Nile Virus infection ST101036 MPs produced similar survival to soluble M7 (80-90%). Overall, these data show that ST101036 MPs produce a robust antibody response against EDIII and survival emphasizing the benefits of using Ace-DEX as a delivery platform for the poorly soluble ST101036.

Keywords: Acetalated dextran; Mast cell activator; Microparticles; Vaccine; West Nile Virus.

Figure 1.

Scanning electron micrographs of (A) blank Ace-DEX MPs and Ace-DEX MPs loaded with (B) ST101036, (C) ST048871, (D) R529877, or (E) ST027688. Scale bars represent 1 μm. Diameter and PDI measured by DLS. Loading is represented in units of mg of MCA per mg of particle.

Figure 2.

Comparison of MCA loaded Ace-DEX MPs in vivo. Mice (n = 5) were vaccinated (SC) on a prime + boost + boost schedule (d0, 14, and 28) with soluble EDIII (15 μg), blank MPs + soluble EDIII (15 μg), alum (1%) + soluble EDIII (15 μg), or MCA loaded (200 nmol) MPs + soluble EDIII (15 μg). EDIII-specific IgG titers from day (A) 21 and (B) 35 were measured via ELISA. Data are shown as the mean ± SD. * = p ≤ 0.05.

Figure 3.

Comparison of PEG400 formulated ST101036 to ST101036 MPs. Mice (n = 3) were vaccinated (SC) on a prime + boost + boost schedule (d0, 21, and 42) with soluble EDIII (15 μg), alone or with Blank MPs, alum (1%), 50% PEG400, or ST101036 loaded (200 nmol) MPs. EDIII-specific IgG titers from day 62 were measured via ELISA. Data are shown as the mean ± standard deviation. * = p ≤ 0.05, ** = p ≤ 0.01.

Figure 4.

MC/9 cells were stimulated for 24 hours with soluble M7, soluble ST101036, ST101036 MPs, and blank MPs. (A) Cell viability was measured via the resazurin assay. (B) IL-6 and (C) TNF-α concentration in the culture supernatant was measured via ELISA. (D) Degranulation activity of the compounds was also measured after a 30-minute stimulation via the release of β-hexosaminidase. Data are shown as the mean ± standard deviation.

Figure 5.

Efficacy of ST101036 MPs in a mouse model of West Nile infection. Mice (n = 10) were vaccinated (SC) on days 0, 14, and 28 with the either soluble EDIII (15 μg) alone or soluble EDIII (15 μg) combined with one of the following adjuvants: blank MPs, alum (1%), soluble M7 (20 nmol), or ST101036 MPs (200 nmol). (A) On day 42 sera was collected and analyzed for EDIII-specific IgG, IgG1, and IgG2a via an ELISA. (B) On day 56 mice were challenged with 0.1 mL of 1.6 x 10⁵ pfu/mL of West Nile Virus by IP injection. Mice that lost over 20% of their initial weight or developed hind limp paralysis were euthanized. Data are shown as the mean ± SD. * = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001.