Hepatitis B vaccination using a dissolvable microneedle patch is immunogenic in mice and rhesus macaques

Abstract Chronic Hepatitis B virus infection remains a major global public health problem, accounting for about 887,000 deaths in 2015. Perinatal and early childhood infections are strongly associated with developing chronic hepatitis B. Adding a birth dose of the hepatitis B vaccine (HepB BD) to routine childhood vaccination can prevent over 85% of these infections. However, HepB BD coverage remains low in many global regions, with shortages of birth attendants trained to vaccinate and limited HepB BD supply at birth. To address the challenges, we developed coated metal microneedle patches (cMNPs) and dissolvable microneedle patches (dMNPs) that deliver adjuvant‐free hepatitis B vaccine to the skin in a simple‐to‐administer manner. The dMNP contains micron‐scale, solid needles encapsulating vaccine antigen and dissolve in the skin, generating no sharps waste. We delivered HepB BD via cMNP to BALB/c mice and via dMNP to both mice and rhesus macaques. Both cMNP and dMNP were immunogenic, generating hepatitis B surface antibody levels similar to human seroprotection. Biomechanical analysis showed that at high forces the microneedles failed mechanically by yielding but microneedles partially blunted by axial compression were still able to penetrate skin. Overall, this study indicates that with further development, dMNPs could offer a method of vaccination to increase HepB BD access and reduce needle waste in developing countries.

of delivering a birth dose of the hepatitis B vaccine (HepB BD) followed by timely receipt of the routine childhood HepB vaccine series, is a critical component of the elimination strategy. While global coverage of the routine HepB series reached 84% in 2015, addition of the HepB BD lags far behind at 39%, providing incomplete coverage. 1 Often the areas with low HepB BD delivery rates are also areas with higher population (including maternal) rates of HBV infection, leaving many infants at risk for early childhood infection with subsequent HBV-associated cirrhosis and liver cancer. The still frequent occurrence of births outside of larger health facilities that have skilled birth attendants trained to administer HepB BD and unreliable vaccine supply and storage at the site of births contribute substantially to the lower rates of HepB BD delivery in these regions. 4 This is particularly true in parts of Africa, where only about 60% of births in 2016 were attended by a skilled health professional who would not always be trained in or have access to timely delivery of the hepatitis B BD. 5,6 Further increasing the demands on training, logistics and cost, current HepB BD and routine HepB delivery require intramuscular (IM) injection, generating the risk for unsafe needle injection (itself a risk factor for HBV infection) and disposal of needle sharps waste.
While particularly relevant to limited resource settings, injection safety and disposal of biohazardous waste from injections, especially needles, remain a global concern, even in economically developed regions.
Microneedle patches (MNPs) have been proposed as an alternate mode of vaccination. These patches consist of micron-scale, solid needles capable of puncturing across the skin's stratum corneum barrier to deliver vaccines into the epidermis and superficial dermis layers of the skin without the need for hypodermic needles. [7][8][9][10] The microneedles can be made of biocompatible, water-soluble materials that encapsulate the vaccine and dissolve in the skin, thereby generating no sharps waste. 11,12 Because they are small, simple-to-administer and often thermostable without refrigeration, MNPs could be administered by minimally trained birth attendants, thereby enabling increased coverage of the hepatitis B BD.
The high density of antigen-presenting cells in the skin make intradermal immunization via MNPs advantageous. For example, prior studies have shown dose sparing, longer-lived immune responses, greater breadth of immunity, isotype class switching and other immunologic advantages and differences when vaccinating in skin using MNPs as opposed to IM or subcutaneous injection. 13,14 While microneedles do not typically insert fully into skin due to skin surface deformation, prior studies indicate that insertion is sufficient to access both epidermis and at least superficial dermis, thereby accessing Langerhans cells and dermal dendritic cells, respectively. 15 In this work, we use the term "intradermal" immunization to refer to stratum corneum puncture accompanied by delivery of vaccine into the epidermis and dermis.
A number of studies have investigated different methods of intradermal hepatitis B vaccine delivery, notably to increase immunogenicity in IM vaccine non-responders. 16,17 These studies have incorporated aluminum adjuvants into the vaccine preparations, which have been shown to cause substantial irritation when administered to the skin. [18][19][20][21] Several studies in animal models have delivered the vaccine intradermally by jet injection, which yielded immune responses comparable to subcutaneous or IM injection. 18,22 MNPs have also been used in animal studies to administer hepatitis B vaccines using microneedles as a pretreatment to permeabilize skin, followed by topical application of a vaccine-containing solution, [23][24][25] by coating metal microneedles with vaccine 26 and by encapsulating vaccine in dissolving MNPs. 27,28 A critical aspect of MNP design is the use of materials and microneedle geometry that enable reliable insertion into skin without mechanical damage to the microneedles. Microneedle design also needs to account for skin deformation during microneedle insertion, which can be accomplished by using microneedles of sufficient length to account for deformation and maximal sharpness to minimize that deformation. 12,29 Insertion of shorter microneedles can be performed at a high velocity that allows skin penetration to occur on a timescale faster than the mechanical relaxation time of the skin. [30][31][32] Failure of microneedles due to the compressive forces present during insertion into skin has been studied experimentally and modeled. [33][34][35][36][37][38] These studies show that microneedles can be designed to insert into skin at forces substantially lower than those associated with microneedle fracture.
In this study, we developed MNPs to administer adjuvant-free hepatitis B surface antigen (HBsAg) vaccine, evaluated their mechanical properties in the context of skin insertion and mechanical failure, and studied their immunogenicity in mice and rhesus macaques.

| Concentration of hepatitis B vaccine
Concentrated bulk solution of aluminum adjuvant-free hepatitis B vaccine (HBsAg) was generously provided by the Serum Institute of India (Pune, India). Starting antigen concentration was 2.4 mg/mL. The bulk HBsAg was further concentrated using Vivaspin 20 centrifuge spin filters with a 10 kDa molecular weight cutoff (GE Life Sciences, Pittsburgh, PA) by centrifugation at 700 g for 2 min. HBsAg bulk was concentrated 4-fold (9.5 mg/mL) and 5.4-fold (12.9 mg/mL) for the dissolvable and the metal microneedles, respectively (see below). For our initial mouse study, unmodified bulk HBsAg (2.4 mg/mL) was used instead of the concentrated HBsAg solution. HBsAg concentration was measured via the VITROS Immunodiagnostic System (Ortho Clinical Diagnostics, Raritan, NJ).

| Microneedle patch fabrication 2.2.1 | Coated microneedle patches
In-plane rows of stainless steel microneedle arrays were coated using an in-house dip-coating device to make coated MNPs (cMNPs). The

| Dissolvable microneedle patches
Dissolvable microneedle patches (dMNPs) were fabricated as described previously. 39,40 In this study, the adjuvant-free HBsAg vaccine was mixed into a casting solution at a 60:40 ratio with casting solution containing 25% w/v trehalose and 2.5% w/v CMC in PBS.  Systems, San Jose, CA). While data were collected on a continuous basis, we present the data as a series of discreet points on a forcedisplacement graph to facilitate averaging among multiple replicate measurements.
A yield point was also determined for each force-displacement curve, defined as the first detected change in the force-displacement curve slope, which we consider to be the lowest force at which detectable deformation of the microneedle tip takes place. We call this a yield force rather than a fracture force because microscopic examination of the microneedles indicated a deformation in the tips of the microneedles rather than breaking off of microneedle tips (see "Section 3"). We did not study the mechanical properties of stainless steel cMNPs because previous work has shown the ability of stainless steel cMNPs to reliably insert into skin without deformation or fracture. 41 This method of insertion simulates the intended use of the dMNPs, for example, as used in a recent clinical trial of influenza vaccination. 42 Twenty minutes later, the dMNP was removed and gentian violet dye (HUMCO, Texarkana, TX) was applied to the skin to stain puncture spots on the stratum corneum. The skin was imaged using an Olympus SZX16 microscope (Tokyo, Japan) to determine insertion efficiency.

| Microneedle insertion
For this work, we did not study antigen release kinetics of cMNPs and dMNPs. Instead, we relied on previous work from our group showing that 20 min insertion time is enough to achieve complete delivery (c) IM injections of reconstituted cMNP; (d) two dMNPs applied to skin for 10 min per array; (e) two cMNPs applied to skin for 10 min per array. MNPs were reconstituted in saline for injection (Hospira, Lake Forest, IL). The backs of mice receiving MNP vaccination were shaved with electric shears followed by application of a depilatory cream (Nair, Princeton, NJ) one day before vaccination. The mice were anesthetized using isoflurane for the vaccination and blood collection.
All mice received two vaccine doses (by the same method of delivery) separated by 3 weeks. Estimated HBsAg doses administered to the mice were 2.5 μg, 1.1 ± 0.1 μg, 1.9 ± 0.04 μg, 1.2 ± 0.3 μg (2 patches) and 1.1 ± 0.2 μg (2 patches) HBsAg for groups 1-5, respectively. Dosing variability may be related to fabrication variability and/or analysis of concentrations at the limits of the platform measuring range. Because 40 μg of HBsAg was applied onto each dMNP mold during fabrication, only 1.5% of the applied vaccine was administered to the animals in an active form. Based on measurements of HBsAg in patches before and after application to skin, these losses in vaccine activity mostly occurred while making the dMNPs using a non-optimized formulation and fabrication method, although 30% loss was due to incomplete delivery from the dMNPs into the skin. The goal of this study was to conduct an initial assessment of HBsAg immunogenicity in animals and not to develop an optimized dMNP.
Visual evidence of skin punctures by dMNPs was noticeable (to a trained eye) immediately after administration; no bleeding was observed. Mice were re-shaved and depilated prior to the second vaccination using MNPs. Blood was collected at weeks 2, 4, 5, and 8 to measure antibody to HBsAg (anti-HBs) as described in Section 2.6.
After week 8, the mice were sacrificed by isoflurane euthanasia. The protocol for these experiments was approved by the Institutional Animal Care and Use Committee (IACUC) at Georgia Tech.

| Macaque immunogenicity study
The immunogenicity of HBsAg vaccine administered by dMNPs was

| Mechanical characterization of microneedle patches
While stainless steel microneedles have been shown to have sufficient mechanical strength to penetrate into skin, 44,45 dissolving polymer microneedles require more careful optimization to assure mechanical strength. We therefore characterized the mechanical properties of the dMNPs used in this study. In response to pressing dMNPs against a rigid aluminum block at a constant rate, force generally increased with increasing applied displacement, which could be divided into two regions ( Figure 2). Based on data collected from 11 dMNPs, we found that at small displacement (i.e., less than 0.15 ± 0.05 mm; Region I) there was a progressive increase in force that led to an apparent yield point at 1.1 ± 0.6 N of axial force applied to 100 microneedles (i.e., 11 ± 6 mN per microneedle). Above this yield point, force While microneedle tip deformation should not be good for insertion into skin, it may be that some deformation is acceptable. We therefore assessed the ability of microneedles to penetrate into skin after different amounts of tip deformation. Intact microneedles were able to penetrate the skin efficiently, as indicated by an assay that stains sites of microneedle penetration into skin (Figure 3d (Figure 4b). The acceptable degree of insertion efficiency loss will depend on the application, but the important point of these findings is that minor tip blunting may be tolerated and that major tip blunting only occurred at forces higher than those usually associated with successful microneedle insertion into skin. 11,29,46 3.2 | Immunogenicity in mice We next administered adjuvant-free HBsAg using dMNPs and cMNPs to characterize the magnitude of anti-HBs responses ( Figure 5). Two weeks after administration of the first vaccine dose, 38% of mice in the dMNP group had antibody responses that surpassed 10 mIU/mL, which in humans is taken to represent seroprotection against HBV infection. 47 All mice in the cMNP group or the three IM groups receiving bulk HBsAg, HBsAg reconstituted from cMNP or HBsAg reconstituted from dMNP had no detectable antibody titers (i.e., below the detection limit of 2 mIU/mL). The two reconstituted vaccine patch groups were included to determine if anything associated with the MNP fabrication process affected HBsAg immunogenicity (independent of the MNP route of administration to the skin).
One week after the second vaccination (i.e., 4 weeks into the study), all groups had at least 50% of mice with anti-HBs levels ≥10 mIU/mL, except the cMNP group that had only 25% mice with anti-HBs levels >10 mIU/mL ( Figure 5). At the end of the study after 8 weeks (i.e., 5 weeks after the second vaccine dose), all groups had at least 50% mice with anti-HBs levels >10 mIU/mL. Overall, no significant differences between groups was found based on one-way ANOVA analysis (p > .05). Overall, we can conclude that HBsAg vaccine administered by cMNPs and dMNPs was immunogenic in mice.

| Immunogenicity in rhesus macaques
Guided by outcomes of vaccination using cMNPs and dMNPs in mice, we next vaccinated rhesus macaques using dMNPs, because rhesus macaques are an animal model more closely related to humans and because dMNPs can have immunological 11 and logistical 48 advantages over cMNPs. The rhesus macaques were vaccinated with a standard aluminum adjuvanted (HBsAg) vaccine or non-adjuvanted bulk HBsAg via IM injection or non-adjuvanted HBsAg by dMNP at two different doses. Baseline blood samples at weeks 1 and 2 before vaccination were negative for anti-HBs.
Immediately after removing the dMNP, there was faint evidence of the site where the microneedles had penetrated the skin (square shape in Figure 6b) and where the adhesive had contacted the skin (round shape in Figure 6b) during patch application. No erythema or other signs of irritation were observed. In 10% of the patches, a speck of blood was seen on the residual dMNP post-administration.
No adverse health effects were noted by the veterinary staff.
Seven weeks after vaccination, at least half of the macaques (i.e., at least 2 out of 4 macaques) in all groups had anti-HBs levels At later times, the adjuvanted IM vaccination group achieved significantly higher anti-HBs titers (two-way ANOVA, p < .05), but the other three groups had titers that were not significantly different from each FIGURE 5 Antibody responses in mice. (a) Percent of BALB/C mice with serum antibody response ≥10 mIU/mL anti-HBs at 2-8 weeks after vaccination using bulk adjuvant-free HBsAg vaccine. Vaccine was administered using dissolvable microneedle patches (dMNP) and coated microneedle patches (cMNP), as well as intramuscular (IM) injections of bulk, adjuvant-free HBsAg vaccine, IM reconstituted dMNPs (IM (dMNP) and IM reconstituted cMNPs (IM (cMNP)). In the latter two groups, dMNPs and cMNPs were dissolved in 270 μL and 300 μL of saline for injection, respectively (i.e., reconstituted) and the eluted vaccine was injected IM. Mice were vaccinated at week 0 and again at week 3. (n = eight mice per group). (b) Median anti-HBs titers. Dotted line indicates the seroprotective level in humans (≥10 mIU/mL anti-HBs). Dashed line indicates the detection limit of the anti-HBs assay (≥2 mIU/mL anti-HBs), that is, all points below that line were below detection limit.

| Mechanical characterization of microneedles
The dMNPs used in this study exhibited deformation at an apparent yield point of 11 ± 6 mN per microneedle. In this work, we consider yield point to be the lowest force at which noticeable deformation of the microneedle tip takes place as shown be a slope change on the force-displacement curve. Compared to failure forces measured in prior studies of microneedles, this yield point is comparable to some and significantly lower than others, although direct comparison is difficult because microneedle mechanical strength depends on microneedle geometry, composition and other factors. 30,33,34,36,39,49 From a practical standpoint, however, microneedles only need to be strong enough to perform their function of penetrating skin, which the microneedles in this study were able to do.
This study addressed, for the first time, the relationship between microneedle tip deformation and insertion into skin. There has been a general expectation that microneedle failure force (i.e., leading to tip deformation) must be significantly larger than the microneedle insertion force for successful skin penetration. While our data largely support that expectation, we found that some level of tip deformation may be acceptable. For the dMNPs used in this study, it was possible to insert microneedles into porcine skin ex vivo after dMNPs experienced forces significantly higher than the observed failure force for failure, which means that this failure force is not the maximum force a microneedle can tolerate before being made unable to insert into skin.
The force required to insert a microneedle into skin depends on microneedle tip sharpness, where smaller forces are needed for sharper tips. 29 If the yield force of a microneedle is less than the force needed to puncture skin, then the microneedle tip can be blunted while being pressed against the skin but before it inserts into the skin.
If this happens, then the blunt-tipped microneedle requires a still greater force to insert into skin. In this event the microneedle may yield even more as the greater force is applied, thereby increasing the skin insertion force further. This could produce a viscous feedback loop, where increased force (above the microneedle yield force) would cause increased tip deformation, which would increase the microneedle insertion force (due to a less sharp tip), leading to increased tip deformation, etc. In this way, understanding forces associated with microneedle tip deformation and microneedle insertion into skin, which were studied here as separate sequential events, might actually be part of an integrated processes during MNP application to skin.

| Immune response to adjuvant-free HBsAg vaccine delivery using microneedle patches
The main goal of this study was to assess the immunogenicity of dMNP containing HBsAg vaccine, specifically in the absence of aluminum adjuvant, present in all commercially available hepatitis B vaccine preparations. We showed in two animal models-mice and rhesus macaques-that dMNP delivery of this preparation can elicit anti-HBs responses that exceeded a threshold of ≥10 mIU/mL, levels considered seroprotective in humans. 47 More specifically, anti-HBs response following dMNP delivery of adjuvant-free, bulk vaccine was similar to IM delivery of the adjuvant-free bulk vaccine in both mice and rhesus macaques. IM delivery of the standard aluminum adjuvanted vaccine produced higher antibody titers. These studies therefore show that dMNPs delivering adjuvant-free HBsAg vaccine can induce humoral immune responses comparable to IM vaccination, which provides a proof-of-principle that dMNPs may be further developed for HBsAg vaccination. Additional studies are needed.
In the dMNP-high group in the rhesus macaque study, two of the four macaques were non-responsive to vaccination and the other two animals had anti-HBs titers ≥10 mIU/mL. It remains unclear why this occurred, as the dMNP-high patches were shown to be antigenic by FIGURE 6 Representative image of dissolvable microneedle patch (dMNP) application to the skin of a rhesus macaque. dMNPs were applied to shaved back skin and left on for 20 min. Images show skin with (a) dMNP in place and (b) immediately after dMNP removal the VITROS immunoassay. These inconsistent data may be related to dMNP-high patch function and/or variability within the small sample size of outbred animals used in this study.
In this study, we evaluated immune responses in adult animals, but were motivated to carry out this study to evaluate possible vaccination using dMNPs in infants, including immediately after birth.
Because infants have weaker immune systems than adults, 50 additional studies will be needed to assess HBsAg vaccination by dMNP in infants, as was done in a recent study of measles and rubella vaccination by dMNP in infant rhesus macaques. 40

| CONCLUSION
There is a significant need for increased hepatitis B vaccination, especially in infants needing HepB BD in order to prevent perinatal HBV transmission. Hepatitis B vaccine administration using a dMNP could facilitate vaccination, especially in developing countries where trained healthcare personnel are in limited supply and many births are performed without the assistance of a birth attendant trained in IM delivery of the vaccine. dMNPs offer simple-to-administer vaccination that generates no sharps waste, which should facilitate hepatitis B vaccination with minimally trained personnel.
In this study, we developed dMNPs that administer the adjuvantfree bulk stock of a licensed monovalent HBsAg vaccine used for HepB BD immunization in newborns and found that they generated robust anti-HBs responses in most BALB/c mouse and rhesus macaque animal models in this study. Biomechanical analysis of the dMNPs showed that the microneedles yielded under axial compression. The resulting microneedle tip deformation impeded insertion into skin, but many microneedles could still be inserted into skin even after significant tip deformation. Overall, we conclude that with further development dMNPs may offer a simple-to-administer method for hepatitis B vaccination that could be used to give birth doses in the absence of trained healthcare professionals.

ACKNOWLEDGMENTS
We thank Donna Bondy (Georgia Tech) for administrative support,