Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity

Abstract

Vaccination is an essential public health measure for infectious disease prevention. The exposure of the immune system to vaccine formulations with the appropriate kinetics is critical for inducing protective immunity. In this work, faceted microneedle arrays were designed and fabricated utilizing a three-dimensional (3D)-printing technique called continuous liquid interface production (CLIP). The faceted microneedle design resulted in increased surface area as compared with the smooth square pyramidal design, ultimately leading to enhanced surface coating of model vaccine components (ovalbumin and CpG). Utilizing fluorescent tags and live-animal imaging, we evaluated in vivo cargo retention and bioavailability in mice as a function of route of delivery. Compared with subcutaneous bolus injection of the soluble components, microneedle transdermal delivery not only resulted in enhanced cargo retention in the skin but also improved immune cell activation in the draining lymph nodes. Furthermore, the microneedle vaccine induced a potent humoral immune response, with higher total IgG (Immunoglobulin G) and a more balanced IgG1/IgG2a repertoire and achieved dose sparing. Furthermore, it elicited T cell responses as characterized by functional cytotoxic CD8⁺ T cells and CD4⁺ T cells secreting Th1 (T helper type 1)-cytokines. Taken together, CLIP 3D-printed microneedles coated with vaccine components provide a useful platform for a noninvasive, self-applicable vaccination.

Keywords: 3D printing; continuous liquid interface production; microneedles; transdermal delivery; vaccine.

Fig. 1.

CLIP-printed MNs for vaccine formulation. (A) Design and environmental scanning electron microscope (ESEM) images. (Top) Square pyramidal MN. (Bottom) Faceted MN. (B) Ovalbumin coating (n = 19). Data are presented as mean ± SD of individual samples, statistical analysis by unpaired Student’s t tests. ****P < 0.0001. (C) Cargo co-coating scheme. A matching coating mask was used to simultaneously load two cargos onto two sections of needle array. (C) Ovalbumin coating (n = 19). Data are presented as mean ± SD of individual samples, statistical analysis by unpaired Student’s t tests. ****P < 0.0001. (D) Photograph of an OVA–Texas Red and CpG-FITC co-coated MN patch. (E) Fluorescence image of the MN patch in D.

Fig. 2.

Transdermal cargo delivery by sectionally coated MN patches. Mice were either treated with OVA + CpG on MN patches or through SC delivery of soluble cargos. (A) Cargo delivery efficiency was calculated for sectionally coated MN patches after 2 min, 2, or 24 h application time. Cargo retention at the delivery site was evaluated at the indicated time points following cargo delivery via IVIS live-animal imaging to track (B) CpG or (C) OVA at the delivery site over time. (D) CpG fluorescence quantified from images in B. (E) OVA fluorescence quantified from C. Data are presented as mean ± SD of samples from individual animals (n = 4 for each group). Statistical analysis was done by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Cargo drainage to lymph nodes (LNs) and activation of LN cells. The draining inguinal LNs from mice (the same animals as in Fig. 2) were harvested and imaged for cargo fluorescence by IVIS. (A) CpG fluorescence in dLNs and (B) OVA fluorescence in dLNs. Dissociated LN cells were then counted and further stained with antibodies for cell markers followed by flow cytometry analysis. (C) Cellularity of dLNs. (D) Number of dendritic cells in dLNs. (E) Expression of costimulatory molecule CD80 on dendritic cells in dLNs. Data are presented as mean ± SD of samples from individual animals (n = 4 for all groups). Statistical analysis by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Fig. 4.

MN vaccine induces potent humoral immune responses. Mice were either untreated (n = 6) or immunized with blank MN (n = 3), OVA (16.7 µg) + CpG (2.5 µg) –coated MN patches (n = 6), OVA (16.7 µg) + CpG (2.5 µg) subcutaneously injected (n = 9) or intradermally injected (n = 6), or SC injection of 500 µg Alum + 16.7 µg OVA (n = 6). All groups received two immunizations of the same doses of antigen and adjuvant on day 0 and 23. Serum samples were collected on day 21, 30, 49, 119, 147, and 196 and analyzed by ELISA. (A) Total IgG on day 30. (B) IgG1 on Day 30. (C) IgG2a on day 30. (D) Percentage of B cells in GCs in the dLNs on day 46 post–prime immunization by flow cytometry analysis. For the dose-sparing study, mice were immunized (n = 5 per group) on days 0 and 21 with MN patches loaded with various amounts of OVA and CpG or the equivalent of soluble OVA and CpG injected by ID, SC, or intramuscular (IM) routes. OVA-specific IgG titers were evaluated on day 28 (E). (F) Kinetics of IgG. All data are presented as mean ± SD of samples of individual animals. For A–E, data were analyzed by one-way ANOVA. In E, the data were analyzed by one-way ANOVA within each administration route (shown as *) or across the groups among the various routes with equivalent dosages (shown as #, all against MN). In F, data were compared by two-way ANOVA. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001.

Fig. 5.

T cell activation by MN vaccine. Spleens from the immunized mice (same as in Fig. 4 A–D) were analyzed for (A) IFN-γ–producing CD8 T cells by ELISpot and (B and C) Cytokine production in CD4 T cells by ELISA. Data are presented as mean ± SD of samples from individual animals, with statistical analysis by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.