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Long-term protective immunity from an influenza virus-like particle vaccine 2 administered with a microneedle patch 3

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Fu-Shi Quan1*, Yeu-Chun Kim2*, Jae-Min Song3, Hye Suk Hwang6, Richard W. Compans4, 5 Mark R. Prausnitz5, Sang-Moo Kang6,+ 6

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1Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea; 8

2Department of Chemical and Biomolecular Engineering; Korea Advanced Institute of Science and 9

Technology (KAIST), Daejeon, Korea; 3Department of Global Medical Science, Sungshin Women's 10

University, Seoul, Korea; 4Department of Microbiology and Immunology and Emory Vaccine Center, 11

Emory University School of Medicine, Atlanta, GA 30322, USA; 5School of Chemical and Biomolecular 12

Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA; 6Center for Inflammation, 13

Immunity & Infection, and Department of Biology, Georgia State University, Atlanta, Georgia 30303, 14

USA 15

16 17 18 Key words: microneedle, VLPs, influenza, long term 19 Running title: Influenza VLP vaccination with microneedles 20 21 22 23 * These authors contributed equally to this work 24 + To whom correspondence should be addressed 25 E-mail:skang24@gsu.edu (S.M.K.) 26 Telephone: 1-404-413-3588 27 Fax: 1-404-413-3580 28 29 30 31 32 33 34 35

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00251-13 CVI Accepts, published online ahead of print on 17 July 2013

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Abstract 36

37

Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown 38

to induce similar or better protection compared to intramuscular immunization. In this study, we 39

examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin 40

vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice 41

in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 42

virus 14 months after a single vaccine dose. Influenza virus-specific total IgG response and 43

hemagglutination inhibition (HAI) titers were maintained at high levels for over one year after 44

microneedle vaccination. Microneedle vaccination also induced substantial levels of lung IgG 45

and IgA antibody responses, and antibody-secreting plasma cells from spleen and bone marrow, 46

as well as conferring effective control of lung viral loads, resulting in complete protection 14 47

months after vaccination. These strong and long-lasting immune responses were enabled in part 48

by stabilization of the vaccine by formulation with trehalose during microneedle patch 49

fabrication. Administration of the stabilized vaccine using microneedles was especially effective 50

at enabling strong recall responses measured four days after lethal virus challenge, including 51

increased HAI and antibody-secreting cells in the spleen and reduced viral titer and 52

inflammatory response in the lung. The results in this study indicate that skin vaccination with 53

VLP vaccine using a microneedle patch provides long-term protection against influenza in mice. 54

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Introduction 59

Influenza is a serious respiratory disease spreading around the world, causing seasonal epidemics 60

and recurrent outbreaks, resulting in more than 220,000 hospitalizations. Approximately 36,000 61

people die in the United States every year (1, 2). The experience with the 2009 H1N1 pandemic 62

demonstrated that conventional vaccination showed a significant delay in controlling the new 63

pandemic spread. Significant shortages and delays happened in the supply of the 2009 pandemic 64

vaccine, due in part to lower growth in egg substrates compared to those observed with seasonal 65

vaccines. New approaches are therefore needed to develop effective influenza vaccine that can 66

be rapidly produced on a large scale with low production cost. 67

68

Virus-like particles (VLPs) are noninfectious, thus require no exceptional biosafety containment 69

and can be manufactured rapidly. They present structurally native, immunologically relevant 70

viral antigens. Influenza VLPs, as a promising vaccine candidate, have been shown to induce 71

high neutralizing antibody titers and strong protective immunity (3-7). Influenza VLP vaccines 72

were shown to be more immunogenic and to provide better protection compared to a commercial 73

split vaccine in ferrets (8) or a soluble protein hemagglutinin vaccine (9), indicating the 74

possibility that influenza VLPs could be a new vaccine platform (10, 11). 75

76

Skin is considered an important peripheral immune organ rich in potent immune-inducing cells, 77

including Langerhans cells (LCs), dermal dendritic cells (DCs), and keratinocytes (12-15). Thus, 78

vaccine delivery via skin has been suggested to be an attractive approach for vaccination, 79

especially using a microneedle patch (16-23). Microneedles are micron-scale needles that can be 80

coated with vaccine for simple, painless and targeted delivery of the vaccine to the skin (24). It 81

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was also reported that microneedle vaccination induced protective immunity at a lower dose and 82

provides vaccine dose-sparing effects (25). In addition, skin immunization with microneedles 83

coated with influenza VLPs or inactivated viral vaccines in the presence of a stabilizer Trehalose 84

was shown to induce better protection compared to intramuscular immunization (19, 20, 26, 27). 85

However, protective immunity longer than 6 months has only received limited attention after 86

microneedle vaccination (28). 87

88

In this study, we determined the protective efficacy of influenza VLP vaccine delivered to the 89

skin using coated microneedles. Microneedle vaccine effects after over a year of immunization 90

were compared in formulations with or without trehalose as a stabilizer. We found that stabilized 91

microneedle vaccination in the skin provided improved efficacy of protection after 14 months of 92

vaccination. 93

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Materials and Methods 98

Virus and cells 99

Influenza virus, A/PR/8/1934 (H1N1, abbreviated as A/PR/8) was grown in 10-day old 100

embryonated hen’s eggs for two days at 36-37oC. Allantoic fluids were harvested from infected 101

eggs after storing overnight at 4oC and centrifuged to remove cell debris. The virus was purified 102

from allantoic fluids by using a discontinuous sucrose gradient (15%, 30% and 60% layers) and 103

ultracentrifugation (at 28,000 rpm for 60 min). The purified virus was inactivated by mixing the 104

virus with formalin at a final concentration of 1:4000 (v/v). For use in challenge experiments, 105

mouse adapted A/PR/8 was prepared as lung homogenates of infected mice as described (6). 106

Spodoptera frugiperda Sf9 cells were maintained in suspension in serum free SF900II medium 107

(GIBCO-BRL). MDCK cells were grown and maintained in Dulbecco’s modified Eagle’s 108

medium (DMEM). 109

110

Preparation of influenza VLPs and microneedle patches 111

Influenza VLPs containing hemagglutinin (HA) and matrix M1 derived from A/PR/8 112

were prepared as described (6). Briefly, Sf9 insect cells were co-infected with recombinant 113

baculoviruses expressing HA and M1 at a multiplication of infection 2 and 1 respectively. 114

Influenza VLPs released into culture media were purified using (15%, 30% and 60% layers) 115

ultracentrifugation (28,000 rpm, 60 min). The purified VLPs were characterized by western blot 116

and hemagglutination activity analysis (6, 29). The content of HA was approximately 10 % of 117

total proteins of influenza VLPs, which is similar to that found in a previous report (6). 118

Microneedle preparations and coatings were performed as described (27, 30). Briefly, 119

rows of solid metal microneedles were fabricated by cutting needle structures from stainless 120

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sheets (SS304, 75µm thickness, McMaster-Carr, Atlanta, GA) using an infrared laser (Resonetics 121

Maestro, Nashua, NH) and the microneedles measured 700 µm in length and 160 µm in width. In 122

order to coat a vaccine on the surface of microneedles, microneedles were dipped six times at 123

25oC into coating solution using a dip-coating device. The coating solution was composed of 1% 124

(w/v) carboxymethylcellulose (CMC) sodium salt (Carbo-Mer, San Diego, CA), 0.5% (w/v) 125

Lutrol F-68 NF (BASF, Mt.Olive, NJ) with or without 15% (w/v) D-(+)-trehalose dihydrate 126

(Sigma Aldrich, St.Louis, MO). Four μg of influenza VLPs (total proteins) were coated onto a 127

microneedle array with 5 needles in the presence or absence of a trehalose dissacharide stabilizer 128

(15% (w/v), Sigma Aldrich). Although trehalose may slightly improve delivery kinetics due to 129

its solubility characteristics, the main function of trehalose is to stabilize the HA of VLPs (20, 130

25, 26, 30). Microneedles coated with VLP vaccines were used to vaccinate animals. Mock 131

vaccination was carried out using microneedles without VLP vaccine. 132

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Immunization and challenge infection 134

Female inbred BALB/c mice (Charles River) aged 6 to 8 weeks were used. Groups of 135

mice (12 mice per group) were immunized with a microneedle array coated with VLP vaccine (4 136

μg total VLP proteins) for delivery to the skin. For microneedle delivery, mice were anesthetized 137

with ketamine (110 mg/kg, Abbott Laboratories) mixed with xylaxine (11 mg/kg, Phoenix 138

Scientific). Hair on the dorsal surface of mice was removed by hair-removing cream (Nair) with 139

a moisturized cotton stick. After cleaning with a soaked cotton ball (70% ethanol) and drying 140

with a hair dryer, an array of vaccine-coated microneedles was inserted into the skin and held for 141

10 min for release of the vaccine antigen from the coated microneedle. 142

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For challenge infections, mice lightly anesthetized with isoflurane were intranasally 143

infected with a lethal dose of A/PR8 virus (10 x LD50) in 50 μl of PBS at 14 months after a 144

single VLP vaccine dose. Mice were observed daily to monitor changes in body weight and to 145

record mortality. We followed an approved Emory IACUC protocol for this study, in which 146

animals losing more than 25% body weight were euthanized. 147

Antibody responses and hemagglutination inhibition titer (HAI) 148

Influenza virus-specific total IgG or IgA antibody responses were determined by enzyme-linked 149

immunosorbent assay (ELISA) plates coated with inactivated A/PR8 viral antigen and by using 150

anti-mouse IgG isotype specific secondary antibodies as described (6). ELISA titers were 151

defined as the reciprocal highest dilution with an OD value that is twice the OD background. For 152

determination of hemagglutination-inhibition (HAI) titers, serum samples were first treated with 153

receptor destroying enzyme (Denka Seiken) by incubation overnight at 37°C, and then incubated 154

30 min at 56°C. Sera were serially diluted, mixed with 4 HA units (HAU) of influenza A/PR8 155

virus, and incubated for 30 min at room temperature prior to adding 0.5% chicken red blood 156

cells. The reciprocal of the highest serum dilution preventing hemagglutination was scored as the 157

HAI titer. 158

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Lung viral titer and lung inflammatory cytokine assays 160

Lung viral titers at day 4 post challenge were determined by counting plaques formed on the 161

MDCK cells as described (6). The whole-lung extracts prepared as homogenates using frosted 162

glass slides were centrifuged at 1,000 rpm for 10 min to collect supernatants. The lung 163

supernatants were frozen and kept at −70°C until used for immunoglobulin and virus titers and 164

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cytokine assays. Inflammatory cytokines IFN-γ, IL-6, TNF-α in lungs and bronchoalveolar 165

lavage fluid (BALF) collected at day 4 post challenge were analyzed by Ready-Set-Go cytokine 166

kit (eBioscience) following the manufacturer’s procedure as previously described (31, 32). 167

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Analysis of antibody secreting cell response in vitro and antibody responses post challenge 169

Virus specific antibody responses were determined from serum, lung, bone marrow and spleen at 170

day 4 post-challenge using ELISA (6, 20, 29). To determine antibody-producing cell responses 171

in vitro, bone marrow and spleen cells were cultured in multiscreen 96-well filtration plates 172

(Millipore) coated with inactivated A/PR/8 viral antigen with RPMI media for 2 and 4 days 173

(5x105 cells per well), and levels of virus specific antibodies secreted into the culture media were 174

determined (6, 20, 29). 175

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Histopathology 177

Mice were challenged with A/PR/8/34 virus after 6 months of immunization with 4 µg of 178

influenza VLPs. For histological analysis of lung tissue, mice were anesthetized with isoflurane 179

and exsanguinated from the abdominal aorta. Lung samples were fixed in 10% neutral buffered 180

formalin for 48hrs, transferred to 70% ethanol, embedded paraffin blocks, sectioned into a 181

thickness of 5 μm and stained with hematoxylin and eosin (H&E) as described (33, 34). 182

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Statistics 184

All parameters were recorded for individuals within all groups. Three independent experiments 185

have been performed and the data shown in the figures are consisted of the average of several 186

independent experiments. Statistical comparisons were carried out using the analysis of variance 187

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(ANOVA) and Npar one-way Kruskal–Wallis tests of the PC-SAS system. P values of 0.05 or 188

less were considered significant. A P value less than 0.05 was considered to be significant. 189

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Results 191

High levels of long-lasting virus-specific antibodies were achieved after microneedle VLP 192

vaccination 193

Our previous studies demonstrated that trehalose-stabilized microneedle influenza 194

vaccines were effective in inducing higher recall immune responses and enhanced immunity (20, 195

26, 30). In the present study to determine long-term antibody responses after skin delivery of 196

vaccines, groups of mice were immunized in the skin with influenza VLPs using coated 197

microneedles with and without trehalsoe stabilizer. Levels of total IgG antibody titers specific to 198

influenza virus (A/PR/8) were determined in serum samples collected at 1 and 2 weeks, and 1, 2, 199

3, 7, 10, and 13 months after a single vaccination, and at month 14 after challenge (Fig. 1). At an 200

early time point of 1 week, virus-specific IgG antibody levels were detected at very low levels 201

(Fig. 1A). At week 2 post microneedle vaccination, IgG antibodies were detected at increased 202

levels (Fig. 1A, *P<0.05). At month 1 post microneedle vaccination, both trehalose-stabilized 203

microneedle group (VT) and trehalose-free microneedle group (V) showed high levels of virus-204

specific antibodies compared to that at week 2 (Fig. 1A, **P<0.01), and then, maintained high 205

level at month 2, 3, 7, 10 and up to the month 13 after microneedle vaccination (Fig. 1AB). IgG 206

titers in VT group showed significantly higher than that in V group at all time points except W1 207

(P<0.05). Interestingly, the trehalose-stabilized VT group showed a trend of inducing higher 208

levels of IgG2a isotype antibody levels (supplementary Fig. S1). 209

Groups of mice were challenged 14 months after microneedle vaccination. A significant 210

increase in levels of virus-specific antibodies was observed at day 4 post challenge in both 211

stabilized (VT) and unstabilized (V) microneedle groups compared to that prior to challenge (Fig 212

1B, **P<0.01). In particular, we observed a significant increase in virus specific antibody titers 213

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from the trehalose-stabilized microneedle group (VT) compared to that in the trehalose-free 214

microneedle group (V) as well as higher levels of IgG2a isotype antibodies in the VT group at 215

day 4 postchallenge (supplementary Fig. S1). Taken together, stabilized microneedle 216

immunization could induce effective memory B cells that differentiate rapidly into antibody 217

secreting cells upon exposure to viral infection. The results also suggest that microneedle 218

immunizations in the skin with VLP vaccines (VT, V) were effective in inducing virus-specific 219

antibodies for a long period of time. 220

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Stabilized microneedle vaccine contributes significantly in enhancing recall HAI activity 222

compared to trehalose-free microneedle vaccine 223

Titers of hemagglutination-inhibiting activity are used as a criterion in assessing vaccine 224

efficacy. We determined HAI titers in serum samples collected at different time points after 225

microneedle vaccination. At week 2 post microneedle vaccination, increased HAI titer was 226

observed (Fig 2, *P<0.05) compared to week 1 and week 0. At one month post microneedle 227

vaccination, a HAI titer of approximately 80 was observed and maintained up to 13 months (Fig 228

2, *P<0.05)). No significant differences were found between the trehalose stabilized (VT) and 229

unstabilized (V) microneedle influenza VLP vaccines. However, when serum samples were 230

collected at day 4 post challenge, significant increases of HAI titers were detected in both 231

trehalose-stabilized and non-stabilized group (Fig 2, *P<0.05, **P<0.01). Interestingly, 232

trehalose-stabilized group showed much higher increase of HAI titer (Fig 2, *P<0.05) than non-233

stablized group. These results suggest that stabilization of microneedle influenza vaccines using 234

trehalose may be important in inducing a high level of recall HAI activities, which is functional 235

and protective antibody responses to influenza virus. 236

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Stabilized microneedle influenza VLP vaccine confers improved protection 237

To determine and compare the long-term protective efficacy after delivery of influenza 238

VLP vaccines, groups of mice including microneedle-treated with (VT) or without trehalose (V) 239

were challenged with a lethal dose of A/PR/8 virus (10LD50) 14 months after a single 240

microneedle vaccination. All mice in the mock-vaccinated negative control group rapidly lost 241

body weight and euthanized when body weight loss is over 25%. Mice vaccinated with 242

unstabilized microneedle vaccine exhibited substantial body weight losses up to approximately 243

10%, whereas mice vaccinated with stabilized microneedle vaccine showed no body weight loss 244

(Fig 3A). Both groups of mice showed 100% survival protection (Fig 3B). 245

To better assess the efficacy of vaccines, we determined viral titers in lungs at day 4 post 246

challenge infection. The mock control group showed the highest viral titers of 5 x 105 plaque 247

forming units (pfu). Virus titers were below the limit of detection in the mice vaccinated with 248

stabilized microneedle vaccine (Fig 4A). The unstabilized microneedle vaccination group 249

showed 2 x 103 pfu/ml (Fig 4A, *P<0.05), which is 200-fold less compared to the mock-250

vaccinated control group. To determine whether microneedle vaccination would diminish an 251

inflammatory response due to influenza viral replication in lungs, we measured levels of an 252

inflammatory cytokine, IFN-γ and IL-6, in lung extracts at day 4 post challenge (Fig. 4B). IFN-γ 253

was not detected in lung extracts of mice vaccinated with stabilized microneedle vaccine, while 254

mice immunized with unstabilized microneedle vaccine and the mock-vaccinated control group 255

showed 200 and 500 pg/ml IFN-γ, respectively (Fig. 4B, *P<0.05). IL-6 levels showed a similar 256

patterns of IFN-γ (Fig. 4C). These results indicate that stabilized microneedle influenza VLP 257

vaccination was more effective in inducing protection and in controlling lung viral replication 258

than unstablized microneedle VLP vaccination. 259

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Levels of lung IgG antibodies were detected at higher levels than IgA antibodies (Fig. 260

5AB). In particular, the stabilized microneedle group showed significantly higher levels of virus-261

specific lung IgG and IgA antibodies than those in the unstabilized microneedle group (Fig. 5, *P 262

< 0.01). Thus, these results further indicate that trehalose-mediated stabilization of microneedle 263

influenza VLP vaccines is important for improving long-term protective immunity. 264

265

Microneedle vaccination induces long-lived antibody-secreting cell responses 266

In general, vaccine antigen-specific cells such as memory B cells and plasma cells are 267

present at low levels at the memory state after vaccination. Therefore, we determined recall 268

antibody-secreting cell responses shortly after challenge. Spleen and bone marrow cells were 269

harvested at day 4 post challenge, which was a time point of 14 months after microneedle 270

vaccination. The stabilized microneedle vaccination elicited higher levels of recall antibody-271

secreting cells from spleens after 2 and 4 days of in vitro cultures in the presence of inactivated 272

A/PR/8 viral antigen compared to unstablized microneedle or placebo vaccination (Fig. 6A, P < 273

0.05). Similar levels of antibody secreting cell responses were detected from bone marrow cell 274

cultures in both microneedle vaccine groups (Fig. 6B). These results indicate that microneedle 275

vaccination in the skin induces long-lived cells capable of producing antibodies specific to virus. 276

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Discussion 279

280

Our previous studies showed that skin vaccination with influenza VLP-coated microneedles 281

provided higher short-term efficacy of protection compared to intramuscular immunization. Thus, 282

the capability of inducing long-term protective immunity by vaccination using VLP-coated 283

microneedles would be an important added strength of this approach. In this study, we focused 284

on long-term protective immunity after single microneedle vaccination with influenza VLPs in 285

the presence and absence of a trehalose stabilizer. Results indicate that complete virus clearance 286

was found 14 month after stabilized microneedle vaccination without any body weight loss. 287

288

Immune parameters such as IgG response, HAI titer, lung virus titer, lung IgG or IgA response, 289

lung inflammatory cytokine IFN-γ response, cellular response or memory response, body weight 290

change, and survival rate are informative in assessing the influenza vaccine-induced efficacy. All 291

these immune parameters are likely contributing to clearing lung viral loads and protection 292

against lethal influenza challenge. Inflammatory cytokines (IFN-γ, IL-6, TNF-α) in lung 293

homogenates (lungs) and BALF at an early time point of virus challenge probably were induced 294

non-specifically and measured as a result of inflammation due to influenza virus infection (Figs. 295

5 and S2). We have not determined what cells produce inflammatory cytokines although it is 296

assumed that natural killer cells, non-specifically activated CD4 and CD8 T cells would 297

contribute to the excess production of inflammatory cytokines as natural innate immune 298

responses to viral infection. The levels of inflammatory cytokines in lung extracts were highest 299

in unvaccinated naïve mice that were infected with A/PR8 virus (Fig. S2) in contrast to those in 300

the VLP vaccinated and A/PR8 virus infected mice (Vaccine+Virus, Fig. S2). Pheripheral 301

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bronchioles of lung sections after H&E staining showd necrotizing bronchiolitis and alveolar 302

space contained infiltrates of mixed inflammatory cells after A/PR8 virus infection (Fig. S3). 303

There was a correlation between levels of IFN-γ in lung homogenates and infiltrates in H&E 304

staining of lung histology (Fig. S3). 305

In particular, clearance of virus in lungs is considered to be an important and sensitive 306

barometer for evaluating the vaccine efficacy. Different types of microneedle influenza vaccines 307

and efficacies have been demonstrated. Substantial amounts of viral loads with a range of 104 to 308

105 pfu titers were detected in mice that were immunized on the skin using microneedles coated 309

with 0.4 to 2 µg H5 HA VLPs derived from influenza A/Vietnam/1203/04 (21, 35). Also, 102 to 310

103 pfu lung viral titers and weight losses of 5-10% were observed in mice immunized with 311

microneedle vaccines coated with 3 µg of split A/Brisbane/59/2007 vaccine, which are 312

approximately 10 to 50 folds less than the infected naïve control group (36). A low level of lung 313

viral titers (102 pfu titers) was reported in mice after 5 weeks of microneedle vaccination with 314

0.4 µg inactivated A/PR/8/34 H1N1 virus (20). In summary, previous studies demonstrated a 315

certain level of lung virus titers and body weight loss. Also, microneedle vaccine-induced 316

efficacies in mice are different depending on influenza virus strains, vaccine doses, prime-boost 317

immunizations, and timing of post immunization at the time of challenge. In this study, 318

immunization with a high dose (4 µg) of microneedle vaccines induced high levels of IgG 319

antibody responses (before challenge) regardless of trehalose stabilization, which is consistent 320

with an early study of microneedle vaccination without trehalose (23). Dosage effects and higher 321

immunogenicity of microneedle VLP vaccines were previously demonstrated when a low dose of 322

0.4 µg of vaccines was used to immunize mice (20, 25, 26). Nevertheless, we observed that 323

stabilized microneedle vaccination with 4 µg HA VLPs (derived from A/PR/8/34) was able to 324

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provide lung virus clearance and prevent weight loss against lethal challenge infection for a long 325

period of 14 months. 326

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Influenza hemagglutinin (HA) stability is likely to play an important role in providing long-term 328

protective immunity. Our previous studies demonstrated that inclusion of 15% trehalose in the 329

microneedle coating formulation resulted in retaining the HA activity of VLPs over 60% after 1 330

day drying process in contrast to less than 10% HA activity of VLPs from coated microneedles 331

without trehalose (26, 30). HAI titers were maintained at similar levels for 13 months after 332

vaccination between stabilized and non-stabilized microneedle vaccination. Interestingly, HAI 333

titers were found to increase rapidly in the stabilized microneedle vaccine group upon challenge. 334

Differences in host protective immune responses of two microneedle vaccine groups were found 335

to be more evident after challenge. The experimental data for improved protection include no 336

detectable lung viral titers, no loss in body weight, and higher levels of protective host responses 337

such as significant increases in HAI titers and vaccine antigen specific recall antibody secreting 338

cell responses. We have shown that IgG2a isotype antibody and Th1 dominant immune 339

responses were due to increased stability of the HA in the trehalose-stabilized micorneedle 340

vaccination (20). There was no significant difference in bone marrow antibody secreting cell 341

responses between stabilized VT and non-stabilized V groups. However, higher levels of splenic 342

B cells secreting antibodies were detected in the stabilized microneedle vaccination at an early 343

time point after challenge, which was also reflected by enhanced serum total IgG antibodies (Fig. 344

1) and IgG2a isotype antibody levels (Fig. S1). Stabilized microneedle vaccination was highly 345

effective in inducing improved protection. Therefore, maintaining influenza HA antigen stability 346

in vaccines is important in providing long-term protective immunity. 347

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348

The present study implies an important aspect in evaluating the efficacy of experimental 349

vaccines. Immunogenicity itself may not reflect a good correlation with protective efficacy. At 350

the month of 13 after vaccination and at an early time point of challenge, levels of binding 351

antibodies did not show obvious differences between the two groups of microneedle vaccination. 352

Even the HAI titers, a measure of functional antibodies for predicting protection against 353

influenza virus, did not show a significant difference at 13 months after vaccination. However, 354

protective efficacy was significantly improved in the stabilized microneedle group after 355

challenge (Fig. 3). It is likely that coating influenza vaccines onto microneedles in the absence of 356

stabilizer might have resulted in exposing non-neutralizing domains of influenza HA proteins in 357

a denatured conformation. These conformational changes are speculated from a result of 358

lowering hemagglutination activity of coated microneedle vaccines (25, 27). This might explain 359

similar levels of binding antibodies in both stabilized and unstabilized groups. An insignificant 360

difference in qualitative antibody levels was rapidly expanded upon infectious virus challenge as 361

shown by a significant increase in HAI titers. Therefore, developing an appropriate challenge-362

protection animal model should be an essential feature of evaluating the vaccine efficacy. 363

Another important arm of host immunity to pathogens is T cell immune responses. 364

Although we have not investigated T cell immunity in this study, there is a possibility that T cell 365

immune responses induced by stabilized microneedle vaccination might have contributed to 366

improved protection observed. In previous studies, we observed enhanced T cell responses after 367

stabilized microneedle vaccination compared to intramuscular immunization or unstabilized 368

microneedle vaccines (20, 26). Also, intradermal vaccination in the elderly population was 369

reported to be more effective in inducing protective immune responses including CD4 T or CD8 370

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T cell responses (37-42). As a future direction, it is important to study long-term T cell immune 371

responses after microneedle skin vaccination. 372

373

Overall, the literature suggests that microneedle skin vaccination can offer important advantages. 374

Influenza VLP microneedle vaccination generated dose-sparing effects and robust HAI titers (20, 375

26, 43). Microneedle skin delivery of inactivated influenza vaccine induced better control of 376

viral replication and reduced inflammatory responses in the lungs (43). In addition, skin 377

microneedle vaccination provides important logistic advantages. Microneedle skin vaccination 378

can probably be administered by patients themselves, significantly increasing the coverage of 379

vaccination. It can also eliminate or reduce the risk of needle-associated injury and re-use of 380

needles and syringes (43). These advantages from both immunologic and logistic benefits, 381

combined with long-term protective immunity as presented in this study, indicate that 382

microneedle delivery to the skin may offer a strategy for improved influenza vaccination. 383

384

In conclusion, this study demonstrated that highly effective long-term immunity to influenza was 385

induced by skin vaccination using microneedles coated with VLPs. Stabilized microneedle VLP 386

vaccine conferred rapid increases in functional antibody responses and effective viral clearance 387

resulting in improved long-term protection after lethal challenge. Thus, microneedle delivery to 388

the skin using non-replicating influenza VLPs has the potential to become effective vaccines for 389

inducing long-term protective immunity. 390

391

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Acknowledgments 394

This work was supported by a grant from the Kyung Hee University in 2012 (KHU-20120459), 395

and grants from NIH, AI105170 (S.M.K.), AI093772 (S.M.K.), AI087782 (S.M.K.), AI068003 396

(R.W.C) and EB006369 (M.R.P.). This work was carried out at the Emory Vaccine Center and at 397

the Center for Drug Design, Development and Delivery and the Institute for Bioengineering and 398

Bioscience at Georgia Tech. 399

400

401

402

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Figure legends 404

405

Fig. 1. Influenza A/PR8 specific IgG responses. Groups of mice (n=12) immunized with a 406

single dose of VLPs. Mice (n=12 per group) were immunized with microneedles coated with 4 407

μg of influenza VLPs. At weeks 1, 2 (W 1, W 2), months 1, 2, 3, 7, 10, 13 (M1, M2, M3, M7, 408

M10, M13) after a single dose vaccination, blood samples were collected. At month 14, mice 409

were challenged with a high lethal dose of A/PR8 virus (10 x LD50) and IgG levels were 410

measured four days after challenge (AC). The groups of immunized mice were designated: V: 411

microneedle vaccine without trehalose, VT: microneedle vaccine with trehalose as a stabilizer, 412

Mock: placebo microneedles with coating buffer only. Significant differences were detected 413

between W1 and W2 (*P < 0.05), W2 and M1 (**P < 0.01), M13 and AC (**P < 0.01). Three 414

independent experiments have been performed and the data shown in the figures are consisted of 415

the average of several independent experiments. Data show average ± standard error of the mean 416

from n=6 mice. 417

418

Fig. 2. Hemagglutination inhibition titers. HAI titers were determined at weeks 0, 1, 2 (W0, W 419

1, W 2), months 1, 2, 3, 7, 10, 13 (M1, M2, M3, M7, M10, M13) post-immunization and at day 4 420

post-challenge (AC). Significantly higher HAI titers were found from week 2, higher from 421

month 1 post-vaccination and maintained until month 13 compared to week 1 or week 2 (*P < 422

0.05, **P < 0.01). V: microneedle vaccine without trehalose, VT: microneedle vaccine with 423

trehalose. Data show average ± standard error of the mean from n=6 mice. 424

425

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Fig. 3. Body weight changes and survival. At month 14 after microneedle vaccination, mice 426

were challenged with a lethal dose (A/PR8 virus, 10LD50) and were monitored daily to record 427

body weight changes (A) and Survival (B). V: microneedle vaccine without trehalose, VT: 428

microneedle vaccine with trehalose, Mock: microneedles with coating buffer only. Data show 429

average ± standard error of the mean from n=6 mice out of 12 mice per group. 430

431

Fig 4. Lung virus titers, lung IFN-γ and IL-6 response. Lungs from individual mice in each 432

group were collected on day 4 post-challenge, and lung virus titers (plaque forming units, pfu), 433

lung IFN-γ and IL-6 responses were determined in the lung extracts at day 4 post-challenge. V: 434

microneedle vaccine without trehalose, VT: microneedle vaccine with trehalose, Mock: 435

microneedles with coating buffer only, Naïve: normal mice. No virus or lower virus titers were 436

detected in VT and V group (* P < 0.01). No inflammatory cytokine IFN-γ and IL-6 were 437

determined in VT group compared to V or mock control group (*P < 0.01). Data show average ± 438

standard error of the mean from n=6 mice. 439

440

Fig 5. Lung IgG and IgA responses after lethal challenge. Lung IgG and IgA responses were 441

determined from the lung extracts collected at day 4 post-challenge. Significantly higher IgG and 442

IgA antibody responses were found in VT compared to V (*P < 0.05) or mock challenge control 443

(*P < 0.01). V: microneedle vaccine without trehalose, VT: microneedle vaccine with trehalose, 444

Mock: microneedles with coating buffer only. Data show average ± standard error of the mean 445

from n=6 mice. 446

447

448

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Fig. 6. Antibody secreting cells (ASC) induced by microneedle VLP vaccination. Spleen and 449

bone marrow samples were collected from individual mice in each group at day 4 post-challenge. 450

Significantly higher ASC were found in VT groups in both spleen and bone marrow compared to 451

V and mock control (P<0.05). V: microneedle vaccine without trehalose, VT: microneedle 452

vaccine with trehalose, Mock: microneedles with coating buffer only. Data show average ± 453

standard error of the mean from n=6 mice. 454

455

456

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Fig 1

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