Skin delivery of trivalent Sabin inactivated poliovirus vaccine using dissolvable microneedle patches induces neutralizing antibodies

Journal of Controlled Release 311–312 (2019) 96–103

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Skin delivery of trivalent Sabin inactivated poliovirus vaccine using dissolvable microneedle patches induces neutralizing antibodies ⁎

Agnese Donadeia, ,1, Heleen Kraanb, Peter C. Soemab, Anne C. Moorea

⁎,1

T

, Olga Ophorstb, Olivia Flynna, Conor O'Mahonyc,

a

School of Pharmacy, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland Intravacc (Institute for Translational Vaccinology), Bilthoven, The Netherlands c Tyndall National Institute, University College Cork, Cork, Ireland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Inactivated polio vaccine Microneedle Skin Immunization Sabin polio vaccine Neutralizing antibody

The cessation of the oral poliovirus vaccine (OPV) and the inclusion of inactivated poliovirus (IPV) into all routine immunization programmes, strengthens the need for new IPV options. Several novel delivery technologies are being assessed that permit simple yet efficacious and potentially dose-sparing administration of IPV. Current disadvantages of conventional liquid IPV include the dependence on cold chain and the need for injection, resulting in high costs, production of hazardous sharps waste and requiring sufficiently trained personnel. In the current study, a dissolvable microneedle (DMN) patch for skin administration that incorporates trivalent inactivated Sabin poliovirus vaccine (sIPV) was developed. Microneedles were physically stable in the ambient environment for at least 30 min and efficiently penetrated skin. Polio-specific IgG antibodies that were able to neutralize the virus were induced in rats upon administration using trivalent sIPV-containing microneedle patches. These sIPV-patch-induced neutralizing antibody responses were comparable to higher vaccine doses delivered intramuscularly for type 1 and type 3 poliovirus serotypes. Moreover, applying the patches to the flank elicited a significantly higher antibody response compared to their administration to the ear. This study progresses the development of a skin patch-based technology that would simplify vaccine administration of Sabin IPV and thereby overcome logistic issues currently constraining poliovirus eradication campaigns.

1. Introduction Since the introduction of the Global Polio Eradication Initiative (GPEI), the incidence of polio has decreased by > 99%. However, polio remains endemic in three countries that have never stopped polio transmission: Afghanistan, Pakistan and Nigeria [1]. Currently, two types of vaccines are in use to prevent paralytic polio and stop poliovirus transmission in the world; inactivated poliovirus vaccine (IPV, based on the Salk strains) and live-attenuated oral poliovirus vaccine (OPV, based on Sabin strains). The attenuated polioviruses contained in OPV confer effective mucosal immunity at the primary site of poliovirus entry, thereby blocking the shedding of viruses through faeces and person-to-person transmission. However, in rare cases, the vaccine virus may genetically mutate and become neurovirulent, which might cause paralysis. On the contrary, the inactivated polioviruses contained in IPV eliminates the possibility of vaccine-derived poliomyelitis and prevents the recirculation of vaccine-derived polioviruses that might

cause new polio outbreaks after vaccination. The current strategy towards polio eradication includes a phased withdrawal of OPV and the inclusion of IPV into all polio immunization programmes [2]. However, the switch from OPV to injected IPV brings challenges. The limited IPV supply and relatively high manufacturing costs encourage accelerated efforts to develop better and more affordable IPV options [3,4]. The significant advantage of ease of oral administration of OPV is lost with injected IPV as the latter requires needles, syringes, biohazardous sharps waste and suitably trained personnel. Furthermore, storage and distribution of polio vaccines requires refrigerated conditions. The costs of this cold chain distribution and logistics chain can add up to 50% to the total cost of fully immunizing a child [5]. Several alternative administration approaches for IPV for its use in routine immunization programmes in low and middle income countries have been evaluated [6]. Microneedles devices have been evaluated for polio vaccination in preclinical research [7–10]. The feasibility of fractional doses of IPV using needle-free injector

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Corresponding authors. E-mail addresses: [email protected] (A. Donadei), [email protected] (H. Kraan). 1 Authors contributed equally. https://doi.org/10.1016/j.jconrel.2019.08.039 Received 29 May 2019; Received in revised form 27 August 2019; Accepted 31 August 2019 Available online 01 September 2019 0168-3659/ © 2019 Elsevier B.V. All rights reserved.

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2.3. Microneedles patch physical characterization

devices and hollow microneedle systems have already been demonstrated in several clinical trials [11–14]. However, these systems still require liquid form IPV, which requires the cold chain. An administration system that overcomes the need for IPV reconstitution to a liquid state and injection would make IPV as simple to administer as OPV. If this technology eliminated associated hazardous waste in addition to improved ease of use, then it would have significant complementary benefits to immunization programmes. The use of dissolvable microneedle patches could address all of these issues. Microneedles are micron-scale protrusions designed to penetrate the skin creating conduits for vaccine administration [15–17]. We previously described a dissolvable microneedle (DMN) vaccine patch that demonstrated excellent vaccine stabilizing and immunogenicity properties [18]. A simple production method that eliminates vaccine wastage during incorporation into these patches was developed [19]. This system is based on a patch comprising of vaccine-containing microneedles that, when applied to the skin, pierce into the skin and subsequently dissolve, thereby releasing the vaccine to the body. These dissolvable microneedles are manufactured with the vaccine embedded in a dried formulation, which permits vaccine stabilization. No reconstitution of the solid vaccine form, with needles, syringes or saline, is required. The only waste material is the adhesive backing used to temporarily hold the patch in place on the skin. This vaccine delivery system is aimed to be deployed with minimum ancillary support, training or cost. The aim of the current study was to develop an immunogenic dissolvable microneedle patch containing trivalent inactivated Sabin IPV (sIPV). This proof of concept study combines the advantages of a new generation of IPV, the Sabin IPV technology, with the simplicity of a patch for skin application. This combination could meet important characteristics of the ideal target product profile of an IPV for use in routine immunization programmes [20].

2.3.1. Environmental stability To examine the rate at which microneedles dissolved in an ambient environment after removal from their primary packaging, patches were imaged using a light microscope attached to a photo camera (Olympus Corporation, Tokyo, Japan) at various times after exposure to the ambient environment (18–21 °C) containing at least 50% relative humidity (RH). The microneedle stability is assessed microscopically by capturing the loss of the sharp sides seen at time zero as it takes up water from the environment thereby increasing its moisture content. Microneedles are considered stable over time if there is no change in their physical appearance, i.e., the sharp tip and all the eight sides of the microneedle are maintained. 2.3.2. Skin penetration To evaluate the ability of DMNs to pierce skin, patches were applied with thumb pressure (approximately 10 N) to full thickness ex vivo pig skin. Patches were applied to the skin for 10 min, then removed. Methylene blue (Sigma-Aldrich, St. Louis, MO) was applied for 5 min and subsequently the stratum corneum was removed by 5 tape strips. The skin layer was imaged using a light microscope attached to a photo camera to identify where microneedles had penetrated. The “penetration success rate” is related to the number of microneedles on each patch that penetrate the skin. A 100% success rate indicates that 25 skin penetration events were observed in the skin. 2.4. D-antigen ELISA To determine D-antigen content of DMN formulations, sIPV-containing patches were dissolved in assay buffer, PBS supplemented with 0.005% (v/v) Tween 80 (Merck, Darmstadt, Germany), and tested in ELISA. High binding 96 wells microtiter plates were coated overnight with serotype-specific bovine anti-polio serum and subsequently blocked with 1% (w/v) albumin bovine fraction V (Serva Electrophoresis GmbH, Heidelberg, Germany) for 30 min at 37 °C. After washing with 0.05% (v/v) Tween 80 in tap water using Biotek platewasher, serial dilutions of sIPV samples (dissolved patches or liquid controls) were added and incubated for 2 h at 37 °C. Subsequently, plates were washed and serotype-specific monoclonal antibodies (HYB295-17-02 (type 1), HYB294-06-02 (type 2) from Thermo Fisher Scientific, or 4-8-7 (type 3) (Bilthoven Biologicals, Bilthoven, The Netherlands) were added and incubated for 2 h at 37 °C. After another washing step, HRP-labeled anti-mouse IgG (GE Healthcare, Buckinghamshire, UK) was added, plates were incubated for 1 h at 37 °C, washed, and SureBlue tetramethylbenzidine (TMB) Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. After 10 min, the reaction was stopped with 0.2 M sulfuric acid and absorbance at 450 nm was measured. Assay data were analyzed by four-parameter logistic curve fitting using Gen5 2.0 Data Analysis software (BioTek Instruments) and D-antigen content was calculated relative to the reference standard.

2. Materials and methods 2.1. Inactivated polio vaccine Monovalent Sabin IPV bulk material used in this study was produced at Intravacc as described previously [21]. For the preparation of trivalent sIPV, monovalent bulks of type 1, type 2 and type 3 were mixed and, subsequently concentrated using 10 kDa Amicon© Ultra Centrifugal filters (Merck Millipore, Billerica, MA) to a nominal antigen concentration of 5000, 8000 and 16,000 D-antigen units/mL for Sabin type 1, type 2 and type 3 respectively.

2.2. Dissolvable microneedle (DMN) patch fabrication Pyramidal-shaped silicon microneedles consisting of 5 × 5 array of 600 μm (height) were used as a moulding template for fabrication of microneedle cavities in polydimethylsiloxane (PDMS) moulds, as previously described [22]. Approximately 7 μL of formulation was delivered directly into microneedle pores using a thin silicon capillary (100 μm ID) connected to a syringe pump delivering formulation at a rate of 10 μL/min [18]. The previously described trehalose, polyvinyl alcohol (PVA)-based formulation [18] was supplemented with magnesium chloride hexahydrate, monosodium glutamate and sorbitol (Sigma-Aldrich, St. Louis, MO). Following delivery of the formulation onto moulds, microneedles were dried for 36 h at 23 °C. After transfer onto medical grade adhesive tape (1525 L Poly Med tape, 3 M) arrays were hermetically sealed in anti-moisture bags with desiccant. Each microneedle patch dose was loaded with a target dose of 1.5 single human dose (shd) of trivalent sIPV; equivalent to 15 DU of type 1, 24 DU of type 2 and 48 DU of type 3 [23].

2.5. Immunization study The welfare of the animals was maintained in accordance with the general principles governing the use of animals in experiments of the European Communities (Directive 2010/63/EU) and Dutch legislation (The revised Experiments on Animals Act, 2014). This included approval of the project by the Central Committee for Animal Studies and approval of the study by the Authority for Animal Welfare. Wistar female rats (6–8 weeks old from Charles River, Germany) were anesthetized during immunization and for 30 min afterwards. Immunizations were performed on days 0 and 21. The target dose was 1.5 single human dose of trivalent sIPV, that is 15-24-48 DU of type 1, type 2 and type 3, respectively. Blood samples were taken on days 0 (prior to 97

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immunization), 21 and 42. Antibody titres to all three serotypes were negative in all animals at day 0. Four groups of ten rats per group were included in this study: Group 1 and 2 received sIPV vaccination intramuscularly (IM) injecting 0.1 mL, divided over both hind limbs, of conventional liquid vaccine (positive control, benchmark) or dissolved patches respectively. Two other groups were immunized by microneedle patch applied on the ear or flank respectively. For application to the flank, a section of hair on each side of the animal was removed using an electric shear before application. Patches (2 × 1.44 cm2 in size per dose) were pressed into the ear or hair-free section of the flank and then covered with medical tape. Patches were removed on the following day.

Fig. 1. Sabin IPV-containing dissolvable microneedle patch design. Patches with an area of 1.44 cm2 contain 25 dissolvable microneedles. Each microneedle has an eight-sided pyramidal shape and is 600 μm in height.

2.6. Polio-specific IgG ELISA Enzyme-linked immunosorbent assays (ELISA) were performed to determine polio-specific (total IgG) antibody titres in sera. Polystyrene 96 wells high binding microtiter plates (Greiner Bio-One) were coated overnight at 4 °C with bovine anti-poliovirus serum (Bilthoven Biologicals) in PBS (Gibco). After washing coated plates with 0.05% (v/ v) Tween 80 (Merck) in tap water (using Biotek plate washer), trivalent inactivated polio vaccine diluted in assay buffer, PBS containing 0.5% (w/v) Protifar (Nutricia) and 0.05% (v/v) Tween 80, was incubated and added for 2 h at 37 °C. Subsequently, plates were washed and threefold sera dilutions in assay buffer were added and incubated for another 2 h at 37 °C. After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (Southern Biotech). After 1 h incubation at 37 °C, plates were washed and SureBlue tetramethylbenzidine (TMB) Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories) was added to each well. After 10–15 min, the reaction was stopped with 0.2 M sulfuric acid (Fluka) and absorbance was measured at 450 nm by using a BioTek Synergy Mx plate reader (BioTek Instruments). Endpoint titers were determined by 4-parameter analysis using the Gen5 2.0 Data Analysis software (BioTek Instruments) and defined as the reciprocal of the serum dilution producing a signal identical to that of the negative control samples at the same dilution plus three times the standard deviation.

administration of microneedle patches into skin, which can be easily applied with the thumb pressure during administration [18,22]. Microneedles were tested for their skin penetration characteristics. Full thickness porcine cadaver skin was imaged after patch application to evaluate the ability of DMNs to pierce the skin. Three different batches of patches, 2 patches from each batch, were tested to evaluate the penetration success rate of each patch as reported in Supplemental Fig. 2. Vaccine-containing patches had 100% success rate of microneedle penetration (Fig. 2A). Microscopic imaging of the back layer of a patch after application (Fig. 2B) showed that microneedles had dissolved completely after 10 min of application. As the formulations contain hygroscopic sugars, atmospheric moisture can be absorbed by dissolvable microneedles. The retention of the mechanical strength and the structure of DMN is important to ensure reproducible and effective administration. Increased moisture content will result in decreased strength, as the DMN begin to dissolve. Therefore, we visually assessed the dissolution of microneedles, on removal from primary packaging and placed in ambient environmental conditions over time, as a measurement of the physical stability of the sIPV-containing DMN patch. An internal scoring system was used to evaluate the stability of the needles, by capturing the loss of the sharp sides seen at time zero as it takes up water from the environment increasing its moisture content. Microneedles are considered stable over time if there is no change in their physical appearance, i.e., the sharp tip and all the eight sides of the microneedle are maintained. The microneedles in the sIPV-patch retained sharp tips and all the eight faces even after 30 min of exposure (Fig. 3).

2.7. Neutralizing antibody measurement Neutralizing antibody titres against all three poliovirus types in day 42 serum samples were measured separately by inoculating Vero cells with 100 CCID50 of the wild-type Salk strains (Mahoney, MEF-1 and Saukett). Twofold serial serum dilutions were made, serum/virus mixtures were incubated for three hours at 36 °C and 5% CO2 followed by overnight incubation at 5 °C. Subsequently, Vero cells were added and after 7 days of incubation at 36 °C and 5% CO2, the plates were stained and fixed with crystal violet and results were read macroscopically. Virus neutralizing (VN) titres were expressed as the last serum dilution that has an intact monolayer (no signs of cytopathogenic effect). 2.8. Statistics Data comparison among all immunization groups were done using an ordinary one-way ANOVA followed by a Tukey multiple comparisons test. Probability (p) values of p < .05 were considered statistically significant. Statistics were performed using GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA). 3. Results

Fig. 2. Skin penetration properties of sIPV-containing DMN patches. Full thickness porcine cadaver skin stained with methylene blue to reveal sites of skin puncture after DMN patch insertion. (A) Skin layer stained after the application of a DMN patch. (B) Example of the back layer of a patch imaged after microneedle dissolution into the skin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.1. Development of a stable DMN patch Sabin IPV-containing patches contained 25 microneedles per 1.44 cm2. Each microneedle had distinct 8 sides and measured 600 μm in height (Fig. 1). The presence of sharp tips facilitates the 98

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but different format. Three weeks after the first immunization, detectable serum IgG antibody responses against all three serotypes were detected in groups which received either IM liquid sIPV or IM dissolved patches and in some animals that were vaccinated in the ear and on the flank with patches (Fig. 5A). No significant differences in serum IgG antibodies were observed in animals immunized once with dissolved patches or with liquid vaccine delivered IM for polio type 1 and type 2 (Fig. 5A). For type 3, a significant dose response between the liquid IM and dissolved patch IM was observed. Both DMN patch groups induced IgG responses that were significantly lower compared to the liquid sIPV delivered via the IM route after the primary immunization for all three serotypes. However, no significant difference in anti-type 2 or type 3 IgG antibody titres were detected between the dissolved patches (given IM) and the patches applied to the flank skin, after the prime. As the doses delivered in the prime were similar for the dissolved patches and the skin-administered patches, this indicates that the skin route, particularly on the flank of the animal, is as immunogenic as IM for inducing poliovirus-specific serum IgG for serotypes 2 and 3. Two weeks after the second immunization, polio-specific IgG antibody titres were assessed in serum (Fig. 5B). Administration of patches to the flank induced significantly higher polio-specific IgG responses compared to patches applied to the ear, for all 3 serotypes (Fig. 5B). Interestingly, for type 2 and 3, flank applied DMN patches elicited serum IgG titres that were comparable to those obtained for the IM dissolved patch group, which contained similar vaccine doses. These results suggest that DMN patch-based delivery is equivalent to IM delivery on a dose-per-dose level for type 2 and type 3. The differences in IgG titres between the liquid sIPV and dissolved patches, both given by the same IM route, for types 2 and 3 (Fig. 6B) can be explained by the lower doses present in the dissolved patches compared to the liquid vaccine (Fig. 4); type 2 and type 3 recoveries were lower than the target dose in the patches.

Fig. 3. Environmental stability of sIPV-containing DMN patches. A bench stability study was performed at 19 °C/50% RH environmental conditions. (A) Microneedle image directly after removal from the mould. (B) Microneedle after 30 min of environmental exposure.

3.2. Production of trivalent sIPV DMN patches for immunogenicity analysis As these patches demonstrated enhanced mechanical robustness, bench stability and skin penetration, this design was used to evaluate the immunogenicity of trivalent sIPV-containing patches in a rat study. Each microneedle patch was produced with an intended dose of 0.75 shd of each IPV serotype; animals received two patches to get a target dose of 1.5 shd. DMN patch batches for animal testing were produced as fresh as possible (less than one week before administration) and Dantigen content was determined prior to administration (Fig. 4). Two independently produced batches were prepared, one for prime and one for booster vaccination, and both batches showed comparable D-antigen recoveries (Fig. 4). The mean dose per serotype per batch are outlined in the figure’ capture. Whereas sIPV1 showed full D-antigen recovery during DMN production process, sIPV2 showed 40–50% loss during patch production (Fig. 4). The highest loss in D-antigenicity (60–75%) during DMN production was observed for type 3 and the batch used for booster vaccination showed slightly lower sIPV3 D-antigen content when compared to the batch used for prime immunization (Fig. 4).

3.4. Sabin IPV-containing DMN patches induce virus neutralizing antibodies Virus neutralisation (VN) titres were determined in post-boost sera (Fig. 6). Patches applied to the ear resulted in lower VN titres compared to all of the other groups for the three polio serotypes. Type 2 poliovirus neutralizing titres were significantly higher when liquid vaccine was administered by the IM route compared to all other groups. This may reflect the higher antigen dose in the liquid vaccine. Patch administration to the flank induced neutralizing antibodies that were equivalent to the liquid vaccine as well as the dissolved patch delivered IM for type 1 and type 3, despite a 60–75% lower dose of type 3 being administered in the patches. Therefore, although the anti-type 1 and antitype 3 serum IgG titres were significantly lower when the vaccine was administered using DMN patches to the flank when compared to liquid sIPV given IM, and a lower type 3 dose was administered, the

3.3. Sabin IPV patches induce polio-specific IgG The in vivo study was completed using patches and liquid vaccine with doses described in Fig. 4, to evaluate the immunogenicity of sIPV in patches. Furthermore, two different microneedle patch delivery sites, the ear versus the flank were compared. In one group of animals, patches were dissolved and administered by the IM route to verify that the process of incorporating sIPV into patches did not impact on immunogenicity and to determine if there were any differences between the skin and intramuscular routes, using the same vaccine formulation

Fig. 4. Dose content in trivalent sIPV loaded DMN patches used for the in vivo study. Two different batches were produced for the in vivo study, one for prime immunization and one for booster immunization. The intended dose per patch was 0.75 shd corresponding to 7.5-12-24 DU, for type 1, 2 and 3, respectively, as indicated by the dotted line. Per batch, D-antigen contents of five patches were determined prior to immunization, mean ± std. deviation is following reported for type 1, 2 and 3, respectively: −7.6 ± 0.5, 5.9 ± 0.3 and 9.8 ± 1.3 in prime patches; −7.5 ± 0.8, 7.6 ± 1.0 and 5.6 ± 0.6 in boost patches. Symbols represent individual values of each patch; bars represent mean values (n = 5) and error bars depict standard deviation. 99

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Fig. 5. Administration of sIPV-containing DMN patches induced polio-specific IgG in serum. Female Wistar rats (n = 10 per group) were intramuscularly immunized with 0.1 mL of liquid sIPV vaccine or dissolved patches (2 patches/animal). DMN patches were applied on ears or flanks (2 patches/animal). IgG titres against polio serotypes in sera three weeks after prime (day 21) (A) and in sera three weeks after boost (day 42) (B) are reported. Bars represent mean values and error bars depict 95% confidence interval values. Asterisks indicate significant differences between groups using one-way ANOVA followed by a Tukey multiple comparisons test (* p < .05; ** p < .01, *** p < .001).

4. Discussion

neutralizing component of the humoral response was equivalent in the patches compared to the IM injected sIPV, despite differences in the dose administered in the different groups.

Dissolvable microneedles (DMN) patches are a promising technology for effective polio vaccine immunization campaigns using Sabin IPV, as they have the potential to simplify the vaccination procedure. Stabilizing and storing vaccine out of cold chain should enhance

Fig. 6. Administration of sIPV-containing DMN patches induced poliovirus neutralizing antibodies. Virus neutralizing (VN) antibodies after booster immunization (day 42) in serum samples from animals immunized either via intramuscular (IM) injection of liquid sIPV or dissolved DMN patches, or via application of DMN patches on ears or flanks (2 patches per animal) were evaluated. Bars represent mean values and error bars depict 95% confidence interval values. Asterisks indicate significant differences between groups (* p < .05; ** p < .01, *** p < .001) using one-way ANOVA followed by a Tukey multiple comparisons test (* p < .05; ** p < .01, *** p < .001). 100

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potential loss of D-antigen content after patch manufacturing. It can be observed that the post-prime IgG responses to types 1 and 2 were similar between the dissolved patches and the liquid vaccine, both delivered IM, demonstrating that induction of type 2 responses by the IM route are not significantly affected when only 60% of the vaccine is injected. In contrast, induction of IgG responses to type 3 were highly dose-dependent; a significantly lower response was induced when 10 DU compared to 43 DU were administered by the IM route. Furthermore, on a dose-for-dose basis the DMN patch could induce equivalent IgG responses as the IM route in naïve animals, for types 2 and 3, but not for type 1. indicating that immunization with IPV patches to the flank can induce systemic immune responses similar to IM administration for sIPV2 and sIPV3 (Fig. 5A). Neutralizing antibody titres were assessed three weeks after the second immunization (Fig. 6). No effect of route on type 1 poliovirus neutralizing capacity was observed, however administering the vaccine with a patch to the ear induced a lower and more variable neutralizing response. Induction of type 2 neutralizing antibodies by patches also looks promising as equivalent type 2 VN titres were induced when equivalent doses of vaccine were administered by patch or by IM injection of dissolved patches. Only a small number of other studies have examined either the dose-dependency of Sabin IPV-induced neutralizing antibodies using the IM route, or the use of microneedle systems with Salk IPV. Induction of VN titres after a single immunization was demonstrated to be highly dose dependent for types 1 and 3, but less so for type 2 [29]. In contrast, we did not observe a dose-dependent induction of type 1 virus neutralisation, however, these VN titres were analyzed after a second immunization. In a microneedle-based rat study, dose sparing was suggested when 1 DU of monovalent sIPV2, coated onto silicon microneedles induced equivalent anti-type 2 virus neutralisation to an IM injection of 8 DU of sIPV2 [7]. It is difficult to directly compare this with our study, as monovalent IPV can demonstrate increased immunogenicity in rats compared to trivalent vaccine [30] and a spring-loaded applicator that induces strong inflammation was used [31], thereby providing a suggested adjuvating effect. In contrast, a trivalent, unadjuvanted vaccine was used in the current study. Induction of neutralizing antibodies by a trivalent IPV vaccine in a dissolvable microneedle patch in rhesus macaques demonstrates the potential clinical translation of a patch-based (Salk) IPV immunization system [8]. Similar to our study, confounding issues arose with respect to determining actual D-antigen content, in that case, for IPV3 [8]. These studies highlight the requirement for accurate, precise and reproducible D-antigen assays, either ELISA [32] or surface plasmon resonance [33] that should be used in these microneedle studies. At this point, we do not know why administration of sIPV1 in patches induced equivalent NAb but lower IgG. We speculate that the result suggests that different populations of B cells are induced when sIPV1 is administered by patch or IM. This might reflect different cytokine and co-stimulatory environments in germinal centres induced by patch or IM coupled with differing activation and differentiation requirements by B cells that recognise and respond to type 1 compared to types 2 and 3. We hypothesise that intramuscular injection of a bolus of liquid induces strong inflammatory responses in the lymph node. On the contrary, the dissolution of multiple microneedles in the skin may not evoke such an inflammatory response or it results in an altered phenotype or kinetics of the response compared to IM. We previously demonstrated that this difference in inflammation occurred when silicon microneedles were used instead of intradermal immunizations [15,16] however this finding may or may not translate to the delivery of sIPV by a DMN patch. Differences in the inflammatory milieu in the lymph node could affect the affinity, clonality and diversity of the antigen-specific antibody repertoire. The inflammatory environment after IM injection could lead to the development of higher numbers of plasma blasts and plasma cells that secrete high levels of antigen-recognising but non-neutralizing IgG; resulting in a smaller proportion of this total response possessing neutralizing function. In contrast, the patch may

vaccine distribution logistics and contribute to faster vaccine access and coverage at health centres and in door-to-door campaigns; essential components to responding to a public health emergency and to reaching children who have missed immunization [24]. This is important to reach complete polio eradication. The goal of the present work was to develop a dissolvable microneedle patch delivery system that retained or enhanced the immunogenicity of trivalent Sabin polio vaccine. Specifically, we developed an immunologically potent trivalent sIPV-patch, despite lower doses of sIPV being incorporated in the DMN patch, which retained critical physical parameters of the patch delivery system with respect to microneedle skin penetration and environmental stability. This provides an initial proof-of-concept of Sabin IPV immunogenicity when delivered by dissolvable microneedle patches, at lower doses compared to IM for some serotypes. However additional development of this patch needs to be performed to optimize the incorporation of the vaccine into this system. The microneedle design incorporated sIPV in a dried format and permitted dissolution of microneedles in the skin after an efficient skin piercing. In particular, we obtained microneedles that retained sharp tips during their pressure into the skin, making holes below the stratum corneum. Furthermore, the mechanical stability was assured for a time window of at least 30 min from the packaging removal until the skin application. To our knowledge, no previous work involving IPV loadedDMNs has investigated the microneedles' physical stability over time, when exposed to environmental humidity conditions. This is an important aspect, as a time window of operability will reduce variability in immune responses due to unwanted dissolution of microneedles after removal from primary packaging; thereby increasing confidence in this vaccination practice. The vaccine in patches was immunologically active, eliciting comparable neutralizing immune responses with similar or higher vaccine doses delivered intramuscularly. Retaining the antigenic activity of the sIPV after drying the microneedles is an important parameter. It is known that IPV, in particular type 3, are prone to degradation and associated antigenic loss in terms of D-antigenicity when exposed to challenging conditions [25–27]. The recovery of type 1 sIPV in the patches was 100%, indicating that the microneedle fabrication process did not affect type 1 antigenicity. However, sIPV type 2 and 3 recoveries were lower. The loss of antigenicity is likely due to the sensitivity of type 2 and more so type 3, to drying process at temperatures higher than refrigerated conditions. In addition, the residual moisture content of the dried formulations might also affect D-antigenicity. However, other studies examining excipients to incorporate IPV into microneedles suggest that there is a poor correlation between moisture content and loss of IPV activity [28]. Recent studies indicate that maltodextrin, D-sorbitol, glutathione and sulfobutyl ether-β-cyclodextrin (SBE-βCD) can stabilize all three (Salk) IPV serotypes in microneedles dried in desiccant at 5 °C overnight [28] or dried for 17–19 min on polymer discs at an unknown temperature [27]. However, to date, there is no published data in the microneedle field to demonstrate the immunogenicity of IPV formulated with these stabilizing excipient combinations in polio-naïve animals or the mechanical and delivery properties of microneedles produced with IPV in these formulations. Thus, it is unknown if the inclusion of these reported excipients will impact on the mechanical strength of IPV microneedles, the bioavailability of vaccine, the stability of the microneedles when removed from primary packaging or on the vaccine's immunogenicity. Finally, it is presently unknown how stabilizing Sabin vaccines in DMN patches may differ from Salk vaccines. There is, therefore, further work required within the field to identify optimum formulation(s) capable of providing long-term stability to all 3 IPV serotypes of Sabin and Salk vaccines, while also providing the required physical parameters for maximum strength, delivery and ambient stability. The immunogenicity of the sIPV microneedle patches was tested in rats. A control group of dissolved microneedle patches, which were administered via the intramuscular route, was included to adjust for 101

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lower D-antigen doses in these patches, equivalent neutralizing responses can be induced when these patches are administered to skin on the flank of animals, particularly for IPV3. This study demonstrates the potential of producing trivalent Sabin IPV loaded patches that can deliver the vaccine into the skin. Further formulation developments are required to minimize D-antigen degradation during production and storage and to maximize immunogenicity of these DMN patches. If these findings clinically translate, then this DMN patch approach would improve the accessibility and uptake of polio vaccine and contribute to global eradication of polio virus.

induce fewer (than IM) effector plasma blasts and plasma cells and/or a different lymph node environment may enhance the induction of higher affinity antibodies that are highly effective at neutralizing. This may result in an overall lower magnitude of the total IgG response but with a proportion of highly efficacious neutralizing antibodies. Higher affinity antibodies, generated by increased somatic hypermutation, have been demonstrated [34–36] Only future studies examining the repertoire, diversity and function of the neutralizing response to poliovirus serotype 1 would further address this point. Alternatively, differences in IgG and NAb responses to sIPV1 could be purely due to the differences what is being measured in an ELISA, compared to a bioassay of virus neutralisation. Finally, it is not unexpected that this skew to a higher proportion of NAb to total IgG may occur with type 1 sIPV. It is known that responses to the different serotypes of IPV differ in dose sensitivity [29] this supports a concept that B cell responses to the different serotypes are different. Differences in both the polio-specific IgG and virus neutralisation responses due to administration to the ear or the flank was unexpected. This highlights the importance of a correct selection of the application site for patches, at least in rodent models. A possible reason of the differences in immune responses for the ear or flank patch application sites may be due to a thinner corneum layer in the flank compared to the ear and/or increased blood flow in the flank [37]. In a clinical trial, no statistical differences were observed when influenza vaccine coated on silicon microneedles was administered, using a spring-loaded applicator, to the upper arm or volar forearm [38]. Different sites have been investigated for IPV intradermal delivery in humans [14]; no clear difference was identified. However, it is difficult to predict how any of these findings would translate to IPV in a dissolvable microneedle patch in humans and administration to different skin sites will likely need to be tested in early clinical trials. IPV is currently given by injection rather than oral drops as for OPV. IPV administration is therefore dependent on the use of needles and syringes as well as the employment of trained personnel and cumbersome logistics of biohazard waste disposal. There is therefore a need to develop alternative vaccine delivery technologies in order to simplify and to reduce costs related to vaccination campaigns [2]. DMN patches permit a simple vaccine administration. Furthermore, no sharp or biohazard waste is associated with this system, thereby eliminating logistics associated with injection practices. Health-care practitioners' and parents' opinions of the suitability for the use of microneedles patches for paediatrics vaccination are overall positive [39,40]. However, the key role of endorsement by healthcare professionals for full acceptance of this new technology has been highlighted [41]. Dissolvable microneedle patches therefore represent an important alternative strategy for polio vaccination that retains the simplicity of OPV administration. This would have a positive impact on campaigns in low income countries, where minimally trained personnel can conduct house-to-house vaccine distribution [2]. Finally, the skin route, specifically intradermal administration of IPV, in some cases using hollow microneedles instead of hypodermic needles [14], can demonstrate good dose-sparing capacity [11,42]. From our pre-clinical results, the level of dose-sparing that could be achieved could be serotype-dependent. Vaccine dose-sparing with DMN patches will need to be demonstrated in large animal models and in the clinic.

Author contributions AD, HK, OO, OF, CO, PS and AM were involved in the conception and design of the study. AD and HK acquired the data. AD, HK, OO, OF, CO, PS and AM analyzed and interpreted the results. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors had full access to the data and approved the manuscript before it was submitted by the corresponding author. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jconrel.2019.08.039. References [1] Global Polio Eradication Initiative (GPEI), Inactivated Poliovirus Vaccine, http:// polioeradication.org/polio-today/polio-prevention/the-vaccines/ipv/. [2] H. Okayasu, R.W. Sutter, H.S. Jafari, M. Takane, R.B. Aylward, Affordable inactivated poliovirus vaccine: strategies and progress, J. Infect. Dis. 210 (Suppl. 1) (2014) S459–S464, https://doi.org/10.1093/infdis/jiu128. [3] J. Rubin, A. Ottosen, A. Ghazieh, J. Fournier-Caruana, A.K. Ntow, A.R. Gonzalez, Managing the planned cessation of a global supply market: lessons learned from the global cessation of the trivalent oral poliovirus vaccine market, J. Infect. Dis. 216 (2017) S40–S45, https://doi.org/10.1093/infdis/jiw571. [4] UNICEF Supply Division, Inactivated Polio Vaccine: Supply Update, https://www. unicef.org/supply/files/Inactivated_Polio_Vaccine_Supply_Update.pdf, (2018). [5] G. Gandhi, P. Lydon, S. Cornejo, L. Brenzel, S. Wrobel, H. Chang, Projections of costs, financing, and additional resource requirements for low- and lower middleincome country immunization programs over the decade, 2011-2020, Vaccine 31 (Suppl. 2) (2013) B137–B148, https://doi.org/10.1016/j.vaccine.2013.01.036. [6] D. Zehrung, J. Hickling, R. Jones, N. Nundy, Improving the Affordability of Inactivated Poliovirus Vaccines (IPV) for Use in Low- and Middle-Income Countries: An Economic Analysis of Strategies to Reduce the Cost of Routine IPV Immunization, PATH, 2010. [7] D.A. Muller, F.E. Pearson, G.J. Fernando, C. Agyei-Yeboah, N.S. Owens, S.R. Corrie, M.L. Crichton, J.C. Wei, W.C. Weldon, M.S. Oberste, P.R. Young, M.A. Kendall, Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses, Sci. Rep. 6 (2016) 22094, , https://doi.org/10.1038/srep22094. [8] C. Edens, N.C. Dybdahl-Sissoko, W.C. Weldon, M.S. Oberste, M.R. Prausnitz, Inactivated polio vaccination using a microneedle patch is immunogenic in the rhesus macaque, Vaccine 33 (2015) 4683–4690, https://doi.org/10.1016/j.vaccine. 2015.01.089. [9] P. Schipper, K. van der Maaden, S. Romeijn, C. Oomens, G. Kersten, W. Jiskoot, J. Bouwstra, Determination of depth-dependent intradermal immunogenicity of adjuvanted inactivated polio vaccine delivered by microinjections via hollow microneedles, Pharm. Res. 33 (2016) 2269–2279, https://doi.org/10.1007/s11095016-1965-6. [10] P. Schipper, K. van der Maaden, S. Romeijn, C. Oomens, G. Kersten, W. Jiskoot, J. Bouwstra, Repeated fractional intradermal dosing of an inactivated polio vaccine by a single hollow microneedle leads to superior immune responses, J. Control. Release 242 (2016) 141–147, https://doi.org/10.1016/j.jconrel.2016.07.055. [11] S. Resik, A. Tejeda, R.W. Sutter, M. Diaz, L. Sarmiento, N. Alemani, G. Garcia, M. Fonseca, L.H. Hung, A.L. Kahn, A. Burton, J.M. Landaverde, R.B. Aylward, Priming after a fractional dose of inactivated poliovirus vaccine, N. Engl. J. Med. 368 (2013) 416–424, https://doi.org/10.1056/NEJMoa1202541. [12] A. Anand, K. Zaman, C.F. Estivariz, M. Yunus, H.E. Gary, W.C. Weldon, T.I. Bari, M. Steven Oberste, S.G. Wassilak, S.P. Luby, J.D. Heffelfinger, M.A. Pallansch, Early priming with inactivated poliovirus vaccine (IPV) and intradermal fractional dose IPV administered by a microneedle device: a randomized controlled trial, Vaccine 33 (2015) 6816–6822, https://doi.org/10.1016/j.vaccine.2015.09.039. [13] E. Clarke, Y. Saidu, J.U. Adetifa, I. Adigweme, M.B. Hydara, A.O. Bashorun, N. Moneke-Anyanwoke, A. Umesi, E. Roberts, P.M. Cham, M.E. Okoye, K.E. Brown, M. Niedrig, P.R. Chowdhury, R. Clemens, A.S. Bandyopadhyay, J. Mueller,

5. Conclusions This study presents the first assessment of dissolving microneedle patches for Sabin inactivated polio vaccine as an advance over traditional delivery methods. sIPV-containing DMN patches were manufactured which were able to efficiently penetrate the skin and had good environmental stability. Despite loss of D-antigenicity after patch formulation for serotypes 2 and 3, DMN patches were shown to be immunogenic in rats and induce protective neutralizing antibodies. However, it can be observed from the present study that despite the 102

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