Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus

Abstract

This 'proof-of-concept' study aimed to test the microparticulate vaccine delivery system and a transdermal vaccine administration strategy using dissolving microneedles (MN). For this purpose, we formulated poly(lactic-co-glycolic) acid (PLGA) microparticles (MP) encapsulating the inactivated canine coronavirus (iCCoV), as a model antigen, along with adjuvant MP encapsulating Alhydrogel^® and AddaVax. We characterized the vaccine MP for size, surface charge, morphology, and encapsulation efficiency. Further, we evaluated the in vitro immunogenicity, cytotoxicity, and antigen-presentation of vaccine/adjuvant MP in murine dendritic cells (DCs). Additionally, we tested the in vivo immunogenicity of the MP vaccine in mice through MN administration. We evaluated the serum IgG, IgA, IgG1, and IgG2a responses using an enzyme-linked immunosorbent assay. The results indicate that the particulate form of the vaccine is more immunogenic than the antigen suspension in vitro. We found the vaccine/adjuvant MP to be non-cytotoxic to DCs. The expression of antigen-presenting molecules, MHC I/II, and their costimulatory molecules, CD80/40, increased with the addition of the adjuvants. Moreover, the results suggest that the MP vaccine is cross presented by the DCs. In vivo, the adjuvanted MP vaccine induced increased antibody levels in mice following vaccination and will further be assessed for its cell-mediated responses.

Keywords: antibody response; antigen presentation; cytotoxicity; immunogenicity; microneedles; microparticles.

Figure 1

Preparation of vaccine-loaded MN: Vaccine/adjuvant MP were mixed with the polymer gel base, added to MN molds, and centrifuged to form the MNs. A backing layer of 10% HA was added, and the needles were allowed to dry overnight. The prepared MN were then removed and used for in vivo immunization. Image was created using Biorender.com (accessed on 3 August 2022).

Figure 2

Timeline of in vivo immunization study in mice. The mice were vaccinated in three doses at W0, W3, and W5. The sera of the mice were collected and assessed for their antibody levels at W2, W4, W6, and W8. The mice were then sacrificed at W10.

Figure 3

Characterization of MP. A. Scanning electron microscope image of MP at a magnification of 5500× (A) and 7100× (B). The MP were spherical with smooth surfaces.

Figure 4

Nitrite release from DCs following treatment with MP. Cell density was adjusted to 3 × 10⁴ cells/well and then treated with the following groups for 24 h: No Treatment (-ve control), iCCoV suspension (2 μg), iCCoV MP (2 μg), Alhydrogel^® MP (3 μg), AddaVax™ (0.5 μg), iCCoV MP (2 μg) + Alhydrogel^® MP (3 μg), iCCoV MP (2 μg) + AddaVax™ (0.5 μg) MP, iCCoV MP (2 μg) + Alhydrogel^® MP (2 μg) + AddaVax™ (0.5 μg) MP. The nitrite release in the supernatant was assessed using the Griess assay. Data are expressed as mean ± SEM (n = 3), One-way ANOVA test, Post hoc Dunnett’s multiple comparisons test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 5

Percent Viability of DCs pulsed with varying concentrations of iCCoV MP, Alhydrogel^® MP, and AddaVax™ MP as measured using MTT assay. Cell density was adjusted to 1 × 10⁴ cells/well. The cells were exposed to two-fold serial dilutions of the corresponding MP groups ranging from 31.25 ug/mL to 500 ug/mL in cDMEM (100 μL/well) for 24 h. DMSO (50 μL) was used as a -ve control, and cells only were used as a +ve control. Data are expressed as mean ± SEM (n = 3). One-way ANOVA test, Post hoc Dunnett’s multiple comparison test, **** p ≤ 0.0001, ns, non-significant.

Figure 6

Expression of MHC I and CD80 on the surface of murine DCs pulsed with various groups. Cells were plated at a density of 3 × 10⁴ cells/well. The cells were exposed to the following treatment groups for 24 h: No Treatment (-ve control), iCCoV suspension (2 μg), iCCoV MP (2 μg), and iCCoV MP (2 μg) + Alhydrogel^® MP (3 μg) + AddaVax™ (0.5 μg) MP. Data are expressed as Mean ± SEM (n = 3), One-way ANOVA test, Post hoc Dunnett’s multiple comparisons test, ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 7

Expression of MHC II and CD40 on the surface of murine DCs pulsed with various groups. Cells were plated at a density of 3 × 10⁴ cells/well. The cells were exposed to the following treatment groups for 24 h: No Treatment (-ve control), iCCoV suspension (2 μg), iCCoV MP (2 μg), and iCCoV MP (2 μg) + Alhydrogel^® MP (3 μg) + AddaVax™ (0.5 μg) MP. Data are expressed as Mean ± SEM (n = 3), One-way ANOVA test, Post hoc Dunnett’s multiple comparisons test, ns, non-significant, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 8

Characterization of MN. Scanning electron microscope and Digital images of microneedle arrays. Scanning electron microscope image of MN patch at a magnification of 180× (A). Digital image of MN depicting its size when placed on a fingertip (B).

Figure 9

Assessment of serum IgG antibody levels in mice. (Absorbance at 450 nm). Responses obtained are compared to the naïve group (No treatment control) and the iCCoV suspension group. Data are expressed as mean ± SEM, n = 4 mice, Two-way ANOVA, Post hoc Tukey’s multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 10

Assessment of serum IgA antibody levels in mice. (Absorbance at 450 nm). Responses obtained are compared to the naïve group (No treatment control) and the iCCoV suspension group. Data are expressed as mean ± SEM, n = 4 mice, Two-way ANOVA, Post hoc Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Figure 11

Assessment of serum IgG1 and IgG2a antibody levels in mice. Serum IgG1 Levels (A) and serum IgG2a levels (B). (Absorbance at 450 nm). Responses obtained are compared to the naïve group (No treatment control) and the iCCoV suspension group. Data are expressed as mean ± SEM, n = 4 mice, Two-way ANOVA, Post hoc Tukey’s multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.