Intradermal delivery of receptor‐binding domain of SARS‐CoV‐2 spike protein with dissolvable microneedles to induce humoral and cellular responses in mice

Abstract The S1 subunit of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein contains an immunogenic receptor‐binding domain (RBD), which is a promising candidate for the development of a potential vaccine. This study demonstrated that intradermal delivery of an S‐RBD vaccine using a dissolvable microneedle skin patch can induce both significant B‐cell and significant T‐cell responses against S‐RBD. Importantly, the outcomes were comparable to that of conventional bolus injection.

protein are under evaluation. One candidate is the receptor-binding domain (RBD) in the S1 subunit of the S protein that specifically binds to angiotensin-converting enzyme 2 (ACE2) receptor on target cells. 4 For example, Ravichandran et al recently immunized rabbits with different targets of the SARS-CoV-2 S protein and found that immunization with RBD induced high-affinity antibodies. 5 There is also an emerging global phase 3 clinical trial testing the efficacy, safety and immunogenicity of Ad5-nCoV (adenovirus type 5 vector expressing S-RBD). 6 After a viable vaccine is developed and manufactured, the storage, transportion, and administration of the vaccine will be of prime Inovio Pharmaceuticals is delivered through intradermal injection. 7 Both IM and intradermal injections rely on conventional techniques, but they can have serious limitations including the need for trained professionals to accurately inject the formulation safely and the potential for blood-related infections. [8][9][10][11] Therefore, other nonconventional techniques should be considered to minimize repeat doses and the need for trained personnel.
Microneedle (MN)-based intradermal delivery is an emerging delivery method for vaccines. The skin is an immunologically active tissue and contains antigen-presenting dendritic cells, Langerhans cells and other cells that can transfer antigens via lymphatic drainage to initiate antigen-specific adaptive immune responses. 12 Compared with other delivery strategies, MN delivery of vaccines offers advantages such as smaller doses, reduced biohazard waste, and pain-free and fast vaccination. The MNs can be pre-formulated and stably stored for extended periods of time at room temperature (RT), which facilitates vaccine usage in developing countries with limited cold chain.
This addresses the common problems of vaccine storage stability and distribution challenges. For example, Kim et al recently used dissolvable MNs with carboxymethyl cellulose to deliver SARS-CoV-2 S1 subunit vaccines in mice, which induced significantly increased antigen-specific antibodies by 2 weeks. 13 Inspired by these pioneering works, this study investigated the potential use of dissolvable MN-based intradermal delivery of S-RBD proteins as a potential vaccine for COVID-19. The MN device was made from a mixture of S-RBD proteins and low-molecular weight hyaluronic acid (HA) using the micro-molding method. 14-18 HA is a naturally occurring substance in skin with no known side effect and the low molecular weight HA (<50 kDa) quickly dissolves in skin as well. The MN device was effective in penetrating the mouse skin and the resulting immunization elicited significant B cell antibody responses and interferon-gamma (IFN-γ)-based T-cell responses compared to nonimmunized controls. In contrast to conventional subcutaneous injection, MN-based intradermal delivery of S-RBD vaccine is a minimum invasive method that could facilitate rapid control of the COVID-19 pandemic. However, we discovered that this platform is unsuitable for the delivery of mRNA. For example, we showed that luciferase mRNA embedded in the dissolvable MNs did not induce protein expression comparable to that of bolus injection.

| Synthesis of S-RBD protein
The S-RBD protein domain of the spike protein (amino acid residues 306 to 543) was cloned and purified from Escherichia coli as reported.
This was formulated at a ratio of 9:1 in aluminum hydroxide gel (InvivoGen).

| Transfection reagent preparation
InstantFECT liposomes donated by PGR-Solutions (Pittsburgh, Pennsylvania) 19 were prepared by adding the recommended amounts (100-1000 μl) of the reconstitution solution to a dried film. The film was allowed to set for 1 min and then vortexed for 1 min to rehydrate the lipid film into a slightly translucent suspension. and T7 buffer mix (2 μl) was kept at 37 C for 2 h. The mRNA product was purified by lithium chloride precipitation followed by washing in 70% ethanol. The modified IVT mRNA was then 5 0 -capped using the vaccinia-virus-capping system (NEB). The 5 0 -cap-modified IVT mRNA products were stored at −20 C.

| MN fabrication
MN patches were made using a micro-molding method. Briefly, HA (molecular weight: 48 K, 100 mg/ml) was dissolved in deionized water (100 mg/ml). Alexa Fluor-546 rabbit IgG (Z25304, Thermo Fisher) or RBD protein (25 μg) formulated with aluminum hydroxide gel or 5 μg luciferase mRNA formulated with 4 μl InstantFECT liposomes was mixed with the HA solution. Next, 50 μl of the mixture was added to a PDMS negative mold and centrifuged at 4000 rpm for 3 min to ensure all cavities in the mold were filled. After drying overnight at RT, additional HA solution was added to form the backing of the patch. After drying, the patch was peeled off from the mold and preserved in a dry box until use.

| In vivo imaging
For luciferase mRNA delivery, 24 and 48 h after injection, BALB/C mice were anesthetized with ketamine and dopamine (25:1 ratio). Next, 100 μl of luciferin substrate (30 mg/ml, Gold Biotechnology) was injected intraperitoneally into the mice. After 5 min, all mice were viewed under an in vivo imager (IVIS SPECTRUM) to monitor luciferase signals.

| Histological analyses
Paraffin-embedded skin sections were stained with hematoxylin and eosin (HE) and viewed under a BX51 Olympus Light microscope.

| IFN-γ ELISPOT assay
On Day 28, mice were sacrificed and spleen cells were collected for IFN-γ ELISPOT analysis. Briefly, 100 μl of spleen cells were incubated on the IFN-γ ELISPOT plate, which was preactivated for 30 min using 200 μl DMEM media without FBS. Subsequently, 5 μg of the peptide (S-RBD and the positive inducer) were added and cells were incubated for 20 h at 37 C in 5% CO 2 . After the media was discarded, cells were washed in a washing buffer and incubated with the secondary biotinylated antibody for 1 h at RT. Finally, cells were incubated with the biotin substrate, followed by washing and drying to yield visible spots in positive wells.

| Enzyme-linked immunosorbent assay (ELISA)
At Days 14, 21, 28, 67, and 97 from the first vaccination, blood collected from tail vein was centrifuged at 3000 rpm for 30 min. The separated serum was stored at −80 C until use. A 96-well ELISA plate was coated with 10 μg/ml of antigen (S-RBD) in coating medium. Specifically, 100 μl of the solution was added to each well at 4 C overnight. After 12 h, the solution was discarded and the plate was blocked with blocking buffer (5% milk in tris-buffered saline mixed with tween 20 [TBST]) for 3 h at RT. The wells were washed with TBST six times to remove milk precipitates. Next, serum collected from above was serially diluted in milk-TBST solution at a ratio of 1:3, Finally, the reaction was stopped with H 2 SO 4 (50 μl per well) and the absorbance was read at 450 nm. ELISA data were collected on a Varioskan Flash spectral scanning multimode reader (Thermo Scientific).

| Statistical analysis
All results were plotted in Prism 7 (GraphPad Software Inc, San Diego, California). Statistical comparisons between groups for ELISA were determined by unpaired nonparametric t-test (Mann-Whitney test) using Prism 7. Statistical comparisons between groups for ELISPOT were determined by unpaired parametric t-test using Prism 7. For all tests, p < 0.05 was considered statistically significant. To optimize these parameters, we chose Alexa Fluor-546 tagged rabbit IgG as the model protein to prepare the model MNs.
The MN patch was made through micro-molding. 20,21 Firstly, we prepared HA solution with distilled water. There are three types of medical grade HA (i.e., 48, 300, and 1100 K). However, HA with molecular weight larger than 300 K cannot dissolve well in water and forms a super viscous solution even at a concentration of 20 mg/ml. We examined the concentration effect in 48 k HA by preparing 100 and 50 mg/ml solutions, which showed that 100 mg/ml was the highest concentration for a manipulable solution. We used a two-step procedure: (1) the Alexa Fluor-546 tagged rabbit IgG protein was used to fill the MN tips and (2) blank HA solution was used to form the backing (Figure 1(a)). The prepared MN patch (Figure 1(b)) can be seen as a fluorescent tip (Figure 1(c-e)) with nonfluorescent backing.

| Intradermal delivery of S-RBD protein using MNs for immunization
Using the optimized protocol above, we made the S-RBD MN patches containing 25 μg S-RBD protein per patch (1 × 1 cm 2 with 100 MN tips). The dosage of protein was determined in our previous study. 22 Control patches were also made without S-RBD protein.
These HA patches dissolved instantly when they were placed in solution. Both S-RBD and control patches were administered by a thumb press onto the shaved skin of BALB/c mice at Days 0, 3, and 7 (n = 5 per group) (Figure 2(a)). Sufficient force is confirmed when the back part of the MN patch has fully attached to the skin after pressing. As shown in Figure 2 Figure 4(a,b) shows significant

IFN-γ was released by T cells by Day 29 in the S.C. S-RBD and S-RBD
MN groups compared to vehicle and noninduced controls. The high levels of IFN-γ producing T cells may produce strong antiviral protection against SARS-CoV-2. We also noticed that the S.C. S-RBD group had levels of IFN-γ producing T cells comparable to the S-RBD MN group (Figure 4(b)).  S-RBD immunization group, S-RBD microneedle (MN) immunization group, and vehicle control group. ELISA absorbance measurements at 450 nm were normalized to standard cutoff values. Student's unpaired nonparametric t-test (Mann-Whitney) was used with multiple t-test adjustment. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns refers to "not (b) Bar chart shows mean IFN-γ ELISPOT counts for all four groups. Student's unpaired parametric t-test was used with Welch's correction. Data were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns refers to "not significant" injected luciferase mRNA successfully expressed luciferase enzymes after 24 h and the luciferase activities were significantly decreased at 48 h ( Figure 5(b,c)). Unfortunately, MN delivery of luciferase mRNA did not produce significant luciferase compared with the average radiance scores of nonchallenged control mice at 24 and 48 h. However, we cannot rule out degradation of mRNA during the mold fabrication process at RT, which will require further investigation. There is emerging evidence for the use of MN-based delivery of vaccines for COVID-19. 23 Kim et al demonstrated the delivery of recombinant coronavirus vaccines (SARS-CoV-2-S1 and SARS-CoV-2-S1fRS09 subunit) using carboxymethyl cellulose-based MN delivery platforms, which were able to induce high titers of SARS-CoV-2-S1 specific antibodies as detected by ELISA. Our team explored the use of an HA-based MN delivery platform to deliver S-RBD (SARS-CoV-2-S1) in mice. We obtained similar significant antibody responses, for a longer time up to 97 days after administration. Different from their study, we performed additional ELISPOT analysis that showed signifi- Significance of mean average radiance [p/s/cm 2 /sr] between groups was determined by unpaired parametric t-test, p < 0.05 differences from the vehicle-control MN group. Several factors may limit the efficiency of the MN delivery system for S-RBD, such as possible loss of antigen activity during the MN formulation. The HA MN platform was unable to deliver mRNA, as shown through the delivery of luciferase mRNA, which did not produce significant expression of the target protein in vivo.

| MN delivery of mRNA
Finally, considering the clinical translation of this technology, there are a few other key issues to solve in the next stage. Regulatory bodies tend to check for sterility for any medical device like this MN vaccine.
Considering the antigen and HA properties, this device cannot be sterilized using steam, ionizing radiation (gamma and electron-beam radiation), and gas (ethylene oxide, formaldehyde) post the fabrication. One possible solution is to fabricate the MN vaccines in a germ-free production laboratory site. Of course, this idea has to be supported by the regulatory bodies. In addition, it is also important to achieve a costeffective fabrication of this device in the large scale. Method in this article involves the use of PDMS molding, centrifugation, drying, and peeling process. This needs well-trained professionals and multiple equipment, which are expensive. This issue might be solved with 3D printing in the future. Finally, S-RBD MN in this study was applied into the skin through thumb press, which is not ideal for clinical practice. An applicator that ensures consistent and accurate deployment of MNs is needed.
We hope to address the above limitations in our future study to optimize the HA MN delivery as a non-invasive system for vaccines, particularly for COVID-19.