Development and Evaluation of Five-in-One Vaccine Microneedle Array Patch for Diphtheria, Tetanus, Pertussis, Hepatitis B, and Haemophilus influenzae Type b: Immunological Efficacy and Long-Term Stability

Abstract

Background and objectives: The development of a five-in-one vaccine microneedle patch (five-in-one MN patch) aims to address challenges in administering vaccines against Diphtheria (DT), Tetanus (TT), Pertussis (wP), Hepatitis B (HBsAg), and Haemophilus influenzae type b (Hib). Combining multiple vaccines into a single patch offers a novel solution to improve vaccine accessibility, stability, and delivery efficiency, particularly in resource-limited settings. Methods: The five-in-one MN patch consists of four distinct microneedle arrays: DT and TT vaccines are coated together on one array, while wP, HepB, and Hib vaccines are coated separately on individual arrays. The patch was tested for long-term stability (12 months at 25 °C) and evaluated for immunogenicity in mice and minipigs. Antibody titers were measured using ELISA to compare immune responses between microneedle-based delivery and traditional intramuscular (IM) injection. Results: The five-in-one MN patch demonstrated stable antigenicity for up to 12 months at room temperature. In animal studies, the patch induced antibody titers comparable to traditional IM injections for all vaccines. Notably, immunogenic responses to Pertussis and Haemophilus influenzae type b vaccines via microneedles were reported for the first time. The patch facilitated the simultaneous yet independent delivery of vaccines, preserving their immunogenicity without interference. Conclusions: The five-in-one MN patch represents a significant advancement in vaccine delivery by enabling stable, minimally invasive, and efficient immunization. Its innovative design addresses the critical limitations of combination vaccines and has the potential to enhance vaccine accessibility in low- and middle-income countries. Future studies will focus on optimizing patch application techniques and evaluating broader clinical applicability.

Keywords: combination vaccine; immunological efficacy; microneedle array; microneedle patch; thermal stability.

Figure 1

Timeline for vaccination and blood collection in mice: the timeline includes the use of either microneedle patches or traditional injection for vaccinations.

Figure 2

Schedule of vaccination and blood collection in minipigs: the timeline includes the use of either microneedle patches or traditional injection for vaccinations.

Figure 3

Concept of 5-in-1 MN patch assembled with four microneedle arrays and four layers: a microneedle patch that integrates combination vaccines into a single application, consisting of four coated microneedle (C-MN) arrays, each coated with different vaccines. (a) The C-MN housing consists of four layers: (1) upper adhesive layer, (2) array-holder layer, (3) lower adhesive layer, and (4) mechanical support layer. (b,c) Fabrication of the patch with four C-MN arrays (front and back). Scale bar represents 1 cm.

Figure 4

(a) Stained porcine tissue after 5-in-1 MN patch insertion showing that all microneedle arrays (four C-MN arrays) were successfully inserted into the porcine skin. (b) Enlarged image of treated skin showing successful penetration of all microneedles, forming blue dots. Scale bar represents 1 cm.

Figure 5

(a) Optical micrographs of vaccine C-MNs showing the overall structure and individual microneedle. (b) SEM image of vaccine C-MNs providing detailed visualization of microneedle tips. (c) Optical microscope images and doses of different vaccine C-MNs (vaccine additives and doses are specified). Scale bar represents 200 μm. The dashed line represents the cross-section of the coated solid formulation.

Figure 6

Long-term storage stability of each vaccine microneedle array for 12 months. (a) DT vaccine C-MN stability. (b) TT vaccine C-MN stability. (c) HBsAg vaccine C-MN stability. (d) wP vaccine C-MN stability. (e) Hib vaccine C-MN stability. Circles in the figure represent the antigenicity of the stock solution stored at 4 °C over time.

Figure 6

Long-term storage stability of each vaccine microneedle array for 12 months. (a) DT vaccine C-MN stability. (b) TT vaccine C-MN stability. (c) HBsAg vaccine C-MN stability. (d) wP vaccine C-MN stability. (e) Hib vaccine C-MN stability. Circles in the figure represent the antigenicity of the stock solution stored at 4 °C over time.

Figure 7

Comparison of DT- and TT-specific IgG levels induced by single-antigen vaccine (IM, MN) and combination vaccine (MN) and the p values p1, p2, p3, and p4. Statistical significance between groups was determined by a t test (p < 0.05), and there was no statistically significant difference (p2 < 0.5).

Figure 8

Comparison of antibody titers of 5-in-1 MN patch and pentavalent vaccine IM injection when mice were inoculated with a fifth of a human dose (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (a) Anti-DT IgG, (b) anti-TT IgG, (c) anti-HBsAg IgG, (d) anti-Pertussis IgG, (e) anti-Hib-PRP IgG.

Figure 9

Comparison of antibody titers of 5-in-1 MN patch and pentavalent vaccine IM injection when minipigs were inoculated with human dose (* p < 0.1, ** p < 0.01). (a) Anti-DT IgG, (b) anti-TT IgG, (c) anti-HBsAg IgG, (d) anti-pertussis IgG, (e) anti-Hib-PRP IgG. Colors: blue (prime), red (1st boosting), green (2nd boosting).