Microneedle patch as a new platform to effectively deliver inactivated polio vaccine and inactivated rotavirus vaccine

Vaccine preparation

CDC-9, a human G1P[8] RV strain, was cultivated in Vero cells and triple-layered particles (TLPs) and double-layer particles (DLPs) were purified from cell supernatants by using CsCl gradient centrifugation³⁸. The ratio of TLPs and DLPs was approximately 9:1 as measured by protein concentration with a Bradford assay. TLPs and DLPs in 50 mM HEPES, 150 mM NaCl, 5 mM CaCl₂ [pH 7.2-7.5] supplemented with 7% D-sorbitol were inactivated at 62 °C for 6 h and protein concentration was determined by Pierce™ Coomassie Bradford protein assay kit (Thermo Fisher Scientific, IL, USA) before being used for the fabrication of dMNP. Mono-bulks containing approximately 900, 450, and 650 DU/ml of IPV types 1, 2, and 3, were donated by GlaxoSmithKline, and were concentrated approximately 150, 100, and 75-fold by volume, respectively, and suspended in 0.375 mM histidine buffer (J.T. Baker^®, PA, USA). Mono-bulk concentration and buffer exchange were performed using Amicon Ultra centrifuge spin filters with 100 kDa MW cutoff at 4 °C, 4000 × g (Millipore Sigma, MA, USA). D-antigen contents were determined by ELISA³⁹.

Dissolving microneedle patch (dMNP) fabrication

Dissolving microneedle patches were fabricated from polydimethylsiloxane (Dow Corning, MI, USA) molds using a two-step casting method (i.e., first an antigen-containing solution followed by a polymer matrix solution).

First-generation dMNP consisted of 112 700-µm-tall microneedles (MNs) with a total MN volume of 1.8 µl. For the fabrication of IRV dMNP to deliver 5 μg IRV, the antigen casting solution had a concentration of 1.13 mg/ml IRV, 5% w/v sucrose (VWR, OH, USA) and 1% w/v sodium carboxymethylcellulose (CMC, Spectrum, CA, USA) in 50 mM HEPES, pH 7.3 (Amresco, OH, USA), 150 mM NaCl and 5 mM CaCl₂ (VWR) buffer. For the fabrication of IPV dMNP to deliver 40, 8, and 32 of IPV types 1, 2, and 3, respectively, the casting solution had a concentration of 12.5, 2.5, and 10.0 DU/μl, 3.75% w/v maltodextrin (Sigma-Aldrich, MO, USA) and 1.25% w/v xylitol (Alfa Aesar, MA, USA) in 0.375 mM histidine buffer. First-generation combination patches were generated from IRV and IPV dMNP manufactured at 20% more than the target dose, which was sectioned in half and assembled on an adhesive backing. Combination patches had a target delivery dose of 5 μg IRV and 40, 8, and 32 DU of IPV types 1, 2, and 3, respectively. To manufacture dMNP at lower doses, solutions were prepared by diluting higher antigen concentration solutions, such as the antigen casting solution used for the full dose combination patch described above, with excipient solution to keep excipient concentration constant while decreasing vaccine dose. Antigen solutions were cast onto molds under vacuum of ~27 inHg. The molds were then dried for an hour (4 °C, 3000 × g).

Polymer matrix solutions, composed of maltodextrin, xylitol, sodium carboxymethylcellulose in histidine buffer for IPV MNs and polyvinyl alcohol (EMD Millipore, MA, USA) and sucrose in HEPES, NaCl, and CaCl₂ buffer for IRV MNs, were deposited to form the base of the MN arrays. The IRV MN arrays were dried at 35 °C overnight; IPV MN arrays were dried refrigerated for 2 days. Once dried, an adhesive backing was adhered to the base of the MN arrays and peeled from the mold. All patches were packaged in foil pouches with a 5-g silica gel desiccant sachet.

To improve manufacturing and delivery efficiency, second-generation dMNP, composed of 163 700-µm-tall MNs with a total MN volume of 2.7 µl, were fabricated similarly with the following differences. For the IRV-containing dMNP, the CMC in the formulation was replaced with methylcellulose (MC) and CMC was removed from the polymer matrix solution of IPV-containing dMNP. Since CMC is unable to be sterilized by filtration, methylcellulose (MC) was evaluated in combination with the other excipients and found to be a suitable replacement for CMC in the formulation to be more compatible with future GMP manufacturing. In addition, the combination IRV-IPV dMNP were fabricated as a single patch and not assembled halves of two separate arrays, as done with the first-generation combination dMNP.

Antigen casting solutions for second-generation dMNP were prepared at lower vaccine concentrations while keeping the ratio of vaccine to excipient constant. To fabricate full dose IRV dMNP, casting solutions had a concentration of 0.625 mg/ml IRV, 2% w/v sucrose (VWR, OH, USA) and 0.4% w/v sodium methylcellulose. IPV casting solutions had a concentration of 5.8, 1.1, and 5.2 DU/μl of types 1, 2, and 3, respectively, 1.5% w/v maltodextrin (Sigma-Aldrich, MO, USA) and 0.5 % w/v xylitol. Full-dose combination patches were prepared to deliver 5 μg IRV and 40, 8, and 32 DU of IPV types 1, 2, and 3, respectively. Solutions for manufacturing the quarter dose dMNP contained lower vaccine doses while keeping the excipient concentrations constant. After deposition of the polymer matrix solution, all MN arrays were dried at 35 °C overnight.

Stability studies

First-generation IRV-dMNP to deliver 5 µg of IRV were fabricated and stored at 5, 25 or 40 °C for up to 24 months, whereas IPV-dMNP to deliver 20, 4, and 16 DU (half the commercial dose) of IPV types 1, 2, and 3, respectively were fabricated and stored at 5 or 25 °C for up to 12 weeks. IPV-dMNP stability at 40 °C was not evaluated. The dMNP were tested for IRV or IPV potency by ELISA using a rotavirus VP7-specific monoclonal antibody or monoclonal antibodies to poliovirus types 1, 2, and 3^25,40. All dMNP were monitored for appearance (e.g., specified number of MNs, free of debris, appropriate shape, and color) throughout the stability studies.

Animal studies

The immunogenicity of standalone IRV or IPV or combined IRV-IPV dMNP was evaluated in two experiments using four weeks old female Wistar rats (Charles River Laboratories, MA, USA). Animals were divided into 10 groups of 6 rats each to test the first-generation dMNP (Table 1). Rats were anesthetized using 1-2% isoflurane for the vaccination and blood collection. The backs of the rats were shaved with electric shears, followed by the application of a depilatory cream (Nair, NJ, USA) one day before MN patch application. As baseline assessment, pre-vaccination blood was taken from the submandibular vein on the same day of hair removal. Rats were vaccinated with first-generation dMNP to deliver 5 (full dose), 2.5 (half), 1.25 (quarter), 0.625 (eighth) and 0.3125 (sixteenth) µg of IRV for dose range study, or full or half doses of IPV alone or combined IRV-IPV. In a follow-up experiment, rats in 3 groups of 10 each were vaccinated with first- or second-generation full dose IRV-IPV dMNP or second-generation quarter dose IRV-IPV dMNP IRV-IPV (Table 1). Patches were applied with thumb pressure for 1 min on the backs of the rats. After 15 min, the patches were removed. IRV patches were reconstituted in 1 ml of HBSS and IPV patches were reconstituted in 2 ml of blocking buffer to determine the residual antigen amount and dose delivered by RV VP7 ELISA or PV ELISA. Control rats received placebo dMNP in the same manner.

All groups received three doses of vaccine separated by 3 weeks. After week 9 (63 days), the rats were euthanized with isoflurane (3–5%). Blood samples were collected at baseline and three weeks after each vaccination. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the CDC and conducted in accordance with the ethical guideline for animal experiments and safety guidelines. Vaccine dosage refers to the established human dose for commercial IPV or 5 µg as full dose for IRV.

ELISA for antigen measurements

For RV antigen measurements in IRV dMNP, we developed a RV-specific ELISA using a VP7-specific monoclonal antibody (mAb)⁴¹. In brief, 96-well plates were coated with rabbit anti-RV (Wa) polyclonal antibody overnight at 4 °C. The plates were washed, blocked with Superblock™ T20 (TBS) blocking buffer (Thermo Fisher Scientific) followed by incubation with a serially diluted (two-fold) solution which is reconstituted from IRV dMNP for 1 h at 37 °C. After washing, plates were added with biotin-conjugated anti-RV VP7 mAb and incubated for 1 h at 37 °C. Plates were then washed and incubated with diluted (1:10,000) Pierce™ Streptavidin Poly-HRP (Thermo Fisher Scientific) for 1 h at 37 °C and BioFX^® TMB One Component HRP Microwell Substrate (Surmodics Inc., MN, USA). The reaction was stopped by 1 N HCl. Plates were read with an EIA reader (Dynex Technologies, VA, USA) at dual wavelength of 450 nm and 630mn. The amount of protein in a sample was determined from a curve of purified rotavirus standard of known concentration.

For IPV, D-antigens were measured by ELISA using polio type-specific mAbs for both capture and detection as previously described²⁵. Antibodies were labeled with horseradish peroxidase (HRP) using a Lightning Link Conjugation kit (HRP, 100 µg reaction kit; Novus Biologicals) to be used for antigen detection. Capture antibody solutions were prepared by adding IPV types 1, 2, or 3 (Thermo Fisher Scientific) specific antibodies to 0.05 M carbonate-bicarbonate buffer, pH 9.6. IPV type 1 and type 3 antibodies were diluted at 1:1000, and IPV type 2 antibodies were diluted at 1:500. Capture solution was added to Immulon 2HB high-binding 96-well plates (NUNC, NY, USA) and plates were incubated between > 16 h at 5 °C. Coated plates were washed with wash buffer (1x PBS; Corning, VA, USA) with 0.05% Tween 20 (Sigma-Aldrich) and incubated with blocking/dilution buffer (1x PBS with 0.5% gelatin (BD, MD, USA) and 0.25% Tween 20) for 1 h at 37 °C. After washing, antigen was added and incubated for 1 h at 37 °C. Plates were washed and detection solution, prepared by diluting the HRP-conjugated mAbs 1:1,000 in dilution buffer, was added. Plates were incubated for 1 h at 37 °C and washed. SureBlue Reserve TMB Microwell Peroxidase Substrate (1-Component) (KPL, MD, USA) was added and the reaction was allowed to proceed for approximately 15 min before the addition of TMB BlueSTOP Solution (KPL). Plates were evaluated on a SpectraMax^® Plus 384 microplate spectrophotometer (Molecular Devices, CA, USA) at a wavelength of 620 nm²⁵.

Quantification of IgG in rats by ELISA

Rotavirus-specific IgG in animal sera was measured using a modified enzyme immunoassay²⁴. In brief, 96-well plates were coated with rabbit hyperimmune serum to RV Wa overnight at 4 °C. The plates were washed, blocked with 5% skim milk in PBS, and then incubated with supernatants of rhesus RV (RRV) (~10⁶ FFU/ml) for 1 h at 37 °C. Serial diluted (four-fold) rat serum samples were added and incubated for 1 h at 37 °C. After washing, plates were incubated with biotin-conjugated goat anti-rat IgG (Sigma-Aldrich) and Extravidin (Sigma-Aldrich) for 1 h each. TMB (Sigma-Aldrich) substrate was added for development, and the reaction was stopped with 1 N HCl. Optical density (OD₄₅₀) was determined with an ELISA reader (Dynex Technologies). The antibody titer in serum specimen was defined as the reciprocal of the highest dilution that gave a mean OD greater than 3 standard deviations above the mean OD of the negative-serum wells.

Microneutralization assay for RV and PV

RV-specific neutralizing activity (NA) was measured with a microneutralization assay against a homotypic RV strain, Wa (G1P[8])⁴² or a heterotypic RV strain, CDC-6 (G9P[6]) (only Post dose 3). Each strain was individually tested to optimize the amount of virus for use (700 FFU for Wa and 1,500 FFU for CDC-6 per well). Neutralizing titer was defined as the reciprocal of the highest dilution that gave a greater than 60% reduction in the absorbance (OD₄₅₀) value compared to that in virus-only control wells.

Serum samples were tested using a standard microneutralization assay for antibodies to poliovirus types 1, 2, and 3 according to established protocols at the Global Polio Specialized Laboratory, CDC³⁹. Briefly, diluted serum samples were incubated with polioviruses types 1, 2, and 3 at 35 °C for 3 h prior to addition to HEp-2(C) cells. After incubation for 5 days at 35 °C, cells were stained with crystal violet and cell viability was measured by OD₅₉₅. Titers were determined using the Spearman–Karber method. Seropositivity was defined as antibody titers greater than or equal to 1:8.

Statistics

All immunogenicity results were analyzed by Prism software version 7 (GraphPad, CA, USA). Comparisons among individual samples were done using an unpaired t test. Comparisons among multiple groups were done using a two-way ANOVA. p < 0.05 was considered significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.