Sustained delivery of CpG oligodeoxynucleotide by acetalated dextran microparticles augments effector response to Computationally Optimized Broadly Reactive Antigen (COBRA) influenza hemagglutinin

Abstract

A subunit or protein-based influenza vaccine can be a safer alternative to live attenuated vaccine (Flumist) and require fewer boosts than an inactivated vaccine (e.g. Fluzone). However, to form an effective subunit vaccine, an adjuvant is often needed. In this work we used electrospray to encapsulate the hydrophilic adjuvant CpG into microparticles made from the hydrophobic biodegradable polymer acetalated dextran. To understand the rate of particle degradation on CpG release, polymer that was slow (21 h at phagosomal pH 5) and fast (0.25 h at pH 5) degrading was used to encapsulate the adjuvant. The slow-degrading particles exhibited the greatest degree of innate immune stimulation of antigen-presenting cells in vitro. In mice, the broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) Y2 influenza hemagglutinin (HA) antigen was used with CpG particles, soluble CpG, or MF-59 like adjuvant Addavax. Particles and soluble CpG elicited similar induction of anti-HA antibodies and protection against lethal influenza challenge, but the sustained release particles elicited the highest levels antibody effector functions. These results demonstrate a suitable method for encapsulation of CpG oligonucleotide in a hydrophobic particle matrix, and suggest that sustained release of CpG from Ace-DEX microparticles could potentially be used to induce potent antibody effector functions.

Keywords: Intramuscular injection; Nanoparticle vaccine; Seasonal influenza vaccine; Tunable degradation; Universal influenza vaccine.

Figure 1.

Representative scanning electron micrographs of electrosprayed 20 CAC (A) and 60 CAC (B) Ace-DEX MPs encapsulating CpG. Release kinetics of CpG MPs in neutral or acidic conditions over 48 hours (C) or 35 days (D) Data is presented as average ± standard deviation.

Figure 2.

(A,C) TNF-α and (B,D) IL-6 production by (A,B) DC2.4 and (C,D) C57BL/6 bone-marrow derived dendritic cells (BMDCs) after culturing with CpG presented as soluble or encapsulated in Ace-DEX MPs for 48 hours. Cytokines were measured in cell supernatant by ELISA. Statistical significance between the values at the highest CpG treatment concentrations was evaluated using a one-way ANOVA with Tukey’s correction for multiple comparisons and is indicated with * P-value <0.05, *** P-value<0.001, and **** P-value <0.0001. Data is presented as average ± standard deviation.

Figure 3.

C57BL/6 mice were vaccinated intramuscularly on day 0 and 21 with PBS or Y2 COBRA HA (HA) with indicated adjuvant. Antibody endpoint titers were evaluated in day 28 serum for (B) total IgG, (C) IgG2C, and (D) IgG1 titers. Statistical significance is indicated with * P-value <0.05, ** P-value <0.005, *** P-value<0.001, and **** P-value <0.0001. Data is presented as average ± standard deviation.

Figure 4.

C57BL/6 mice (n=10) were vaccinated intramuscularly on day 0 and 21 with PBS or Y2 COBRA HA (HA) with indicated adjuvant and ADCP (A) and ADCD (B) were measured with day 28 sera. Data is presented as average ± standard deviation. Statistical significance is indicated with * P-value <0.05 between 15x dilutions.

Figure 5.

C57BL/6 mice (n=10) were vaccinated intramuscularly on day 0 and 21 with PBS or Y2 COBRA HA (HA) with indicated adjuvant and then challenged on day 35 with 600k pfu H1N1 A/California/2009 intranasally. (A) Body weight after challenge where a 20% loss of weight results in sacrifice of the infected mouse. Data is presented as average ± standard deviation. (B) Survival after challenge. Statistical comparison was performed with log-rank (Mantel-Cox) test with the Bonferroni correction for multiple comparisons.