Development of a single-dose Q fever vaccine with an injectable nanoparticle-loaded hydrogel: effect of sustained co-delivery of antigen and adjuvant

Abstract

Q fever is a zoonotic infectious disease caused by Coxiella burnetii, and there is currently no FDA-approved vaccine for human use. The whole-cell inactivated vaccine Q-VAX, which is only licensed in Australia, has a risk of causing severe adverse reactions, making subunit vaccines a good alternative. However, most subunit antigens are weak immunogens and require two or more immunizations to elicit an adequate level of immunity. We hypothesized that by combining a nanoparticle to co-deliver both a protein antigen and an adjuvant, together with a hydrogel depot for sustained-release kinetics, a single-administration of a nanoparticle-loaded hydrogel vaccine could elicit a strong and durable immune response. We synthesized and characterized a protein nanoparticle (CBU-CpG-E2) that co-delivered the immunodominant protein antigen CBU1910 (CBU) from C. burnetii and the adjuvant CpG1826 (CpG). For sustained release, we examined different mixtures of PLGA-PEG-PLGA (PPP) polymers and identified a PPP solution that was injectable at room temperature, formed a hydrogel at physiological temperature, and continuously released protein for 8 weeks in vivo. Single-dose vaccine formulations were administered to mice, and IgG, IgG1, and IgG2c levels were determined over time. The vaccine combining both the CBU-CpG-E2 nanoparticles and the PPP hydrogel elicited a stronger and more durable humoral immune response than the soluble bolus nanoparticle vaccines (without hydrogel) and the free antigen and free adjuvant-loaded hydrogel vaccines (without nanoparticles), and it yielded a balanced IgG2c/IgG1 response. This study demonstrates the potential advantages of using this modular PPP hydrogel/nanoparticle system to elicit improved immune responses against infectious pathogens.

Keywords: Nanoparticle vaccine; Q fever; co-delivery; hydrogel; sustained delivery.

Graphical abstract

Figure 1.

Overview of the injectable nanoparticle-loaded hydrogel vaccine. The nanoparticle-PPP solution is injected at room temperature and forms a sustained-release depot at physiological temperature.

Figure 2.

Conjugation of CBU1910 antigen and CpG adjuvant onto E2 nanoparticles. (A) Schematic of vaccine nanoparticle synthesis, with components ST-E2 (SpyTag [ST] in yellow, E2 in grey, and cysteines on E2 in black), adjuvant CpG1826 (red), and SC-CBU (SpyCatcher [SC] in blue and CBU in green). CpG1826 and SC-CBU are conjugated on E2 nanoparticle on the interior and exterior surfaces, respectively, to form CBU-CpG-E2. (B) Representative hydrodynamic diameter of E2 (unconjugated) and CBU-CpG-E2. (C) Representative TEM image of the CBU-CpG-E2 nanoparticle.

Figure 3.

Rheological properties of PPP. (A) PLGA-PEG-PLGA (PPP) mixtures comprise PLGA-PEG-PLGA (Mw 1000:1000:1000 Da) LA:GA 1:1 (PPP 1000) and PLGA-PEG-PLGA (Mw 1500:1500:1500 Da) LA:GA 6:1 (PPP 1500), mixed in different ratios (0/100, 25/75, 50/50, 75/25, 100/0; vol/vol), and at total weight concentrations ranging 5–25 wt%. (B) Representative rheological measurement for PPP 1000/PPP 1500 at a 25/75 ratio, and 20 wt%. shown are the T₁, T₂, T_peak, and the corresponding storage modulus, in which T₁ and T₂ are the onset and endset temperatures of storage modulus, T_peak is the temperature where the storage modulus G′ is at the maximum. (C) T_peak of PPP mixtures at different PPP 1000/PPP 1500 ratios and at different concentrations. (D–H) T1 and T2 of PPP solution mixtures at different PPP 1000/PPP 1500 ratios and at different concentrations. (I) Storage modulus (G′) at T_peak of 25/75 PPP mixture at different concentrations. (J) T_peak of 20 wt% PPP mixture (PPP 1000/PPP 1500 = 25/75) loaded with model protein E2 at different concentrations. Data points for panels C–J are average ± S.E.M. values of n ≥ 3 individual samples.

Figure 4.

Characterization of PPP micelles and thermal-triggered self-assembly. (A) Hydrodynamic diameters of micelles or assemblies formed in 0.1 wt % PPP solution at 5 °C, 20 °C, 36 °C, and 50 °C, measured with DLS. TEM images of 1 wt % PPP, at (B) 25 °C and (C) 37 °C.

Figure 5.

The PPP mixture can serve as a depot material to release protein in a sustained profile. (A) In vitro release profile of fluorescently-labeled E2 NP from PPP gel at 37 °C (n = 3; average ± SD). (B) Representative circular dichroism (CD) spectra of ovalbumin (OVA) released in vitro from PPP gel measured on day 4. (C) Representative images of AF750-E2 NP remaining at the injection site in vivo, after injection with formulations that were encapsulated in PPP and as a bolus administration (control). (D) In vivo release profile of fluorescently labeled E2 NP, quantified by IVIS. Each data point is shown as average ± SD (n = 2).

Figure 6.

Antibody responses of nanoparticle-loaded hydrogel vaccine. (A) Table of vaccine formulation of each group and their components. (B) Vaccination and serum collection schedule. (C) Total CBU-specific IgG in serum over time (durability). (D, E) Total CBU1910-specific IgG in serum collected at 8 weeks and 16 weeks after vaccination. Each dot represents a biological replicate (n = 5 mice). (F, G) CBU-specific IgG1 and IgG2c in serum at week 8 and week 16 after vaccination. Each dot represents a biological replicate (n = 5). Data in panels C, D, E, F and G are presented as an average ± SEM of 5 mice per group. Statistical significance was determined by one-way ANOVA followed by a Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7.

Antibody response of nanoparticle-loaded hydrogel vaccine. (A) Table of vaccine formulation of each group and their components. (B) Total CBU-specific IgG in serum over time (durability). (C, D) Total CBU-specific IgG in serum collected at 8 weeks and 16 weeks after vaccination. Each dot represents a biological replicate, n = 5. (E, F) CBU-specific IgG1 and IgG2c in serum at week 8 and week 16 after vaccination. Each dot represents a biological replicate, n = 5. Data in panels B, C, D, E, and F are presented as the average ± SEM of 5 mice per group. Statistical significance was determined by one-way ANOVA followed by a Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.