Multiplexed electrospray enables high throughput production of cGAMP microparticles to serve as an adjuvant for a broadly acting influenza vaccine

Abstract

Subunit vaccines employing designer antigens such as Computationally Optimized Broadly Reactive Antigen (COBRA) hemagglutinin (HA) hold the potential to direct the immune response toward more effective and broadly-neutralizing targets on the Influenza virus. However, subunit vaccines generally require coadministration with an adjuvant to elicit a robust immune response. One such adjuvant is the stimulator of interferon genes (STING) agonist cyclic dinucleotide 3'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). We have shown that encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles through electrospray results in significantly greater biological activity. Electrospray is a continuous manufacturing process which achieves excellent encapsulation efficiency. However, the throughput of electrospray with a single spray head is limited. Here we report the development of a multiplexed electrospray apparatus with an order of magnitude greater throughput than a single-head apparatus. Physicochemical characterization and evaluation of adjuvant activity in vitro and in vivo indicated that microparticles produced with the higher throughput process are equally suited for use as a potent vaccine adjuvant to induce a balanced immune response to COBRA HA antigens.

Keywords: COBRA; Flu; Microparticle; STING; Vaccine adjuvant; Vaccine manufacturing.

Figure 1:

(A) Simplified schematic of single-head electrospray apparatus. (B) Photograph of 10-syringe pump fitted with tubing to the stainless steel needle array, with alligator clip attached to high voltage power supply. (C) Simplified schematic of multiplex electrospray apparatus. (D) Photograph of dried microparticles deposited in characteristic pattern on the stainless steel collection substrate.

Figure 2:

Scanning electron micrographs (SEM) of Ace-DEX particles produced by (A) single-head or (B) multiplex electrospray.

Figure 3:

Bone marrow derived dendritic cells (BMDCs) from C57BL/6 mice were treated with 1 μg/mL soluble cGAMP, or an equivalent dose of cGAMP delivered in Ace-DEX microparticles produced via single-head (SH) or multiplex (MH) electrospray, Lipofectamine 2000 transfection reagent, or 1 μg/mL LPS. Supernatants were collected 24 and 48 h later and assayed for secreted IFN-β by ELISA. n=3 wells per treatment group assayed by three technical replicates. Bars represent the mean ± standard deviation. Statistical comparisons were performed via unpaired t test.

Figure 4:

(A) Timeline of immunizations and bleeds for in vivo evaluation of adjuvants in COBRA vaccine. Serum was collected on the indicated days and assayed for anti Y2 COBRA endpoint titers of total IgG (B), IgG2C (C), and IgG1 (D). Bars represent mean titers ± standard deviation. Statistical comparisons were performed between samples taken on indicated days by a one-way ANOVA using Tukey’s test for multiple comparisons. * = P≤0.05, **=P≤0.01, ***=P≤0.001,****=P≤0.0001.