Demonstration of d-Octaarginine-Linked Polymers as Promising

Jul 27, 2016 - School of Medicine, Miyazaki University, Kihara 5200 Kiyotake-cho, Miyazaki, Miyazaki 889-1692, Japan. ∥ Graduate School of Medical a...
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Demonstration of D‑Octaarginine-Linked Polymers as Promising Adjuvants for Mucosal Vaccination through Influenza Virus Challenge Kohei Miyata,†,‡ Kohta Mohri,† Tomomi Egawa,† Rikito Endo,† Naoki Morimoto,‡ Kyohei Ochiai,‡ Ken-ichiro Hiwatari,‡ Kazufumi Tsubaki,‡ Etsuo Tobita,‡ Tomofumi Uto,§ Masanori Baba,*,∥ and Shinji Sakuma*,† †

Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan Life Science Materials Laboratory, ADEKA Co., 7-2-34 Higashiogu, Arakawa-ku, Tokyo 116-8553, Japan § School of Medicine, Miyazaki University, Kihara 5200 Kiyotake-cho, Miyazaki, Miyazaki 889-1692, Japan ∥ Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan ‡

ABSTRACT: Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-coacrylic acid) bearing D-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and D-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.

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INTRODUCTION

prediction or virus mutation-based changes in antigenic properties of HA proteins. The respiratory tract is a primal site of influenza virus infection. Immunoglobulin A (IgA) is secreted on the mucosa of the respiratory tract when mucosal immunity is acquired through natural infection of influenza viruses. Not only does secreted IgA block mucosal invasion of influenza viruses, but also the antibody is cross-protective against heterologous virus infection.5−8 Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics which result from incorrect prediction of epidemic strains or virus mutation.9−11 FluMist was initially launched as a mucosal vaccine which included cold-adapted influenza viral strains selected on the basis of WHO prediction.12−14 The vaccine-induced mucosal IgA and cell-mediated immunities protect hosts from infection with antigenically matched and distant viral strains. FluMist, however, is not indicated for high-risk groups such as infants

Influenza viruses are categorized into three types (A, B, and C) on the basis of antigenic properties of proteins that are composed of virus particles. There are furthermore a large number of subtypes (H1N1−H16N9) of influenza A viruses with different combinations of hemagglutinin (HA) and neuraminidase (NA) on virus envelopes, and a diversity of genes is also observed in the same subtype.1 Vaccination is the most effective tool for the prophylaxis of influenza viruses-related diseases. HA proteins, which bind to sialic acids on the surface of susceptible cells in the process of infection,2 are targets of vaccine-induced antibodies. The World Health Organization (WHO) annually predicts epidemic strains of two subtypes of influenza A viruses (H1N1 and H3N2) and influenza B viruses. Clinically used parenteral vaccines include HA proteins of the predicted strains and induce serum immunoglobulin G (IgG) which is highly protective against homologous virus infection. However, since the antibody is much less effective against heterologous virus infection,3,4 influenza epidemics often result from incorrect © XXXX American Chemical Society

Received: June 4, 2016 Revised: July 12, 2016

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Therefore, the reduction described above had been out of examination, so that the number of inoculations remained unchanged. Doses of inactivated A/Puerto Rico/8/34 viruses and Doctaarginine-linked PNVA-co-AA and a period of mice maintenance after the final inoculation were listed as experimental conditions that would influence immune response. They were set to 1 μg/mouse, 100 μg/mouse, and 7 days in our previous study.19 The appropriateness of these conditions was first validated. Figure 1 shows the effect of the

and the elderly, presumably due to the potential jeopardy of live attenuated vaccines. Inactivated vaccines are much safer than live attenuated ones. However, since most antigens in the former vaccines are poorly immunogenic when solely applied to the mucosa, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity. Ichinohe et al. have reported that the synthetic double-stranded RNA poly(I:C) and Ampligen, whose structure is similar to poly(I:C), were potent adjuvants for mucosal vaccination of influenza viruses.15−17 When mice were nasally inoculated twice with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and poly(I:C), they were protected from not only homologous virus infection but also infection of antigenically drifted variants within a subtype of the inoculated strain. A similar result was obtained when antigens and adjuvants were substituted with H5N1 influenza virus vaccines derived from the A/Viet Nam/1194/2004 strain and Ampligen, respectively. Other mice experiments revealed that intranasally secreted IgA induced by clinically used influenza virus HA vaccines in the presence of Ampligen prevented mice from infecting with highly pathogenic avian H5N1 influenza viruses beyond the subtype. We have also been investigating the potential of cellpenetrating peptide-linked polymers as adjuvants for mucosal vaccination.18,19 Antigen-specific IgG and secreted IgA were produced in sera and nasal cavities, respectively, when antigens of influenza viruses were coadministered with poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing D-octaarginine, which is a typical cell-penetrating peptide, in mice. The intranasal IgA induced by inactivated H1N1 A/New Caledonia/20/99 IVR116 influenza viruses in the presence of D-octaarginine-linked polymers cross-reacted to recombinant hemagglutinin (rHA) proteins of the H1N1 A/Puerto Rico/8/ 34, H1N1 A/Solomon Islands/03/2006, H3N2 A/Brisbane/ 10/2007, and H5N1 A/Viet Nam/1194/2004 strains. Our polymer is a potential candidate for an adjuvant that possesses a couple of important properties required for mucosal vaccination: mucosal IgA induction and its cross-reactivity. Here, infection experiments were initially performed to evaluate the protective abilities of acquired humoral immunity against virus challenge. We focused on whether secreted IgA protected immunized mice from infection of inoculated viral strains. The H1N1 A/Puerto Rico/8/34 strain was chosen as an antigen because much information on this strain, which was isolated about 80 years ago, was reported in the literature20,21 and expertise of staff in the Kitasato Institute for Life Sciences enabled us to do infection experiments using mouse-adapted H1N1 A/Puerto Rico/8/34 viruses.

Figure 1. Levels of H1N1 A/Puerto Rico/8/34 (PRI) virus-specific IgG in sera (a) and secreted IgA in nasal wash fluids (b) of mice. Mice were nasally inoculated 4 times with inactivated PRI viruses with (black bar) or without (white bar) D-octaarginine-linked PNVA-co-AA at 7-day intervals. They were maintained for 7 days after the final inoculation. Doses of inactivated viruses and polymers were set to 1 μg and 40−100 μg, respectively, /20 μL of dosing solution/mouse/time. Each bar represents endpoint titers of the antibody (mean ± SD, n = 4).

polymer dose on antibody production. Serum IgG was clearly produced when inactivated virus was solely administered. A little production of intranasally secreted IgA was also observed. Both humoral immune responses were significantly enhanced when D-octaarginine-linked PNVA-co-AA was coadministered with antigens at a dose of 40 μg/mouse. The antibody levels elevated with an increase in the polymer dose. Our previous mice experiments revealed that cytotoxicity did not result from nasal administration of D-octaarginine-linked PNVA-co-AA at a dose of 100 μg/mouse, although the weight-average molecular weight of polymers was larger than that of current ones.18 Since the level of secreted IgA that would protect mice from infection with mouse-adapted A/Puerto Rico/8/34 viruses was unclear, 100 μg/mouse was preferred over 40 μg/mouse as the polymer dose. An insufficient dose of antigens possibly results in unsatisfied immunization profiles. On the other hand, an excessive dose of antigens may cause moderate immune responses even when mucosal adjuvants are free from the dosing solution. As shown in Figure 2, the dose of inactivated viruses was next evaluated. The dose of D-octaarginine-linked PNVA-co-AA was set to 100 μg/mouse. When mice were inoculated with inactivated viruses at a dose of 1 μg/mouse with or without the polymers, antibody induction was observed with good reproducibility, as compared with Figure 1. The immune responses were reduced, irrespective of the antibody type, when the antigen dose was halved (0.5 μg/mouse). Production of secreted IgA was hardly

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RESULTS AND DISCUSSION Design of Immunization Protocols. Our previous studies demonstrated that 4× inoculations with a mixture of antigens of influenza viruses and D-octaarginine-linked PNVA-co-AA were a prerequisite for induction of antigen-specific secreted IgA in nasal cavities, although 2× inoculations were sufficient for induction of the corresponding IgG in sera.18,19 The difference indicated that IgA-mediated topical immunity was less susceptible to antigens when compared with an IgG-mediated systemic one. A reduction in the number of inoculations is needed for the practical use of our technology. However, the primal objective in the present study is to evaluate protective abilities of secreted IgA against the homologous virus challenge. B

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maintained for 7 days and more. A similar elevation was observed for secreted IgA during the initial 7 days; however, a further extension of the maintenance period resulted in a significant reduction of the IgA level. Bergquist et al. reported that the IgA activity was not highly maintained for a long time when compared with the IgG activity in humans who were nasally immunized with cholera toxin B subunit.22 It appeared that a similar phenomenon was observed in our case. Seven-day maintenance was one of the best choices as a method which would provide ideal immunity in mice. Ichinohe et al. have been investigating the potential of the synthetic double-stranded RNA poly(I:C) and Ampligen as potent adjuvants for mucosal vaccination.15−17 Since infection experiments in mice using the A/Puerto Rico/8/34 strain have been done in a series of their studies, the capabilities of Doctaarginine-linked PNVA-co-AA for antibody induction were compared with those of poly(I:C). Doses of inactivated A/ Puerto Rico/8/34 viruses and poly(I:C) and inoculation schedules were the same as those described in their reports.15 Since their experiments were carried out on mice ranging in the poly(I:C) dose from 0.1 to 10 μg, the mucosal adjuvant was maximally given to mice in our experiments. We expected that 10 μg/mouse was the toxicity-free upper limit because Ampligen had been developed as the synthetic double-stranded RNA which was safer than poly(I:C). As shown in Figure 4, the

Figure 2. Levels of H1N1 A/Puerto Rico/8/34 (PRI) virus-specific IgG in sera (a) and secreted IgA in nasal wash fluids (b) of mice. Mice were nasally inoculated 4 times with inactivated PRI viruses with (black bar) or without (white bar) D-octaarginine-linked PNVA-co-AA at 7 day intervals. They were maintained for 7 days after the final inoculation. Doses of inactivated viruses and polymers were set to 0.5−1 μg and 100 μg, respectively, /20 μL of dosing solution/mouse/ time. Each bar represents endpoint titers of the antibody (mean ± SD, n = 4).

ever observed in mice immunized with inactivated viruses alone. Coadministration of D-octaarginine-linked PNVA-co-AA significantly enhanced the antibody production. As mentioned before, the IgA level required for protection against homologous virus infection was unclear. Taking the primal objectives in the present study into account, the higher antigen dose (1 μg/mouse) was chosen to avoid unsatisfied immune responses under coadministration of D-octaarginine-linked polymers. Figure 3 shows the effect of the period of mice maintenance after the final inoculation on antibody production. The IgG level was elevated with an increase in the maintenance period and the constant value was observed when mice were

Figure 4. Levels of H1N1 A/Puerto Rico/8/34 (PRI) virus-specific IgG in sera (a) and secreted IgA in nasal wash fluids (b) of mice. Mice were nasally inoculated with inactivated PRI viruses in the presence of either D-octaarginine-linked PNVA-co-AA (black bar) or poly(I:C) (left hashed bar). The number of inoculations was set to either 2 (days 0 and 21), 3 (days 0, 14, and 28), or 4 (days 0, 7, 14, and 21). Mice were maintained for 14 days (2× and 3× inoculations) and 7 days (4× inoculations) after the final inoculation. Doses of inactivated viruses, polymers, and poly(I:C) were set to 1 μg, 100 μg, and 10 μg, respectively, /20 μL of dosing solution/mouse/time. Each bar represents endpoint titers of the antibody (mean ± SD, n = 4).

large variation in antibody levels was observed when mice were inoculated twice with a mixture of inactivated viruses and poly(I:C). However, there was no statistical difference in antibody levels among three inoculation schedules designed for poly(I:C). Antibody levels in mice inoculated with inactivated viruses in the presence of D-octaarginine-linked PNVA-co-AA were significantly higher than those in the presence of poly(I:C), irrespective of the inoculation schedule. Such a high level may not be required for the homologous virus challenge because Ichinohe et al. demonstrated that mice

Figure 3. Levels of H1N1 A/Puerto Rico/8/34 (PRI) virus-specific IgG in sera (a) and secreted IgA in nasal wash fluids (b) of mice. Mice were nasally inoculated 4 times with a mixture of inactivated PRI viruses and D-octaarginine-linked PNVA-co-AA at 7-day intervals. They were maintained for either 3, 7, or 14 days after the final inoculation. Doses of inactivated viruses and polymers were set to 1 μg and 100 μg, respectively, /20 μL of dosing solution/mouse/time. Each bar represents endpoint titers of the antibody (mean ± SD, n = 4). C

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Figure 5. Results of homologous infection experiments. Mice were nasally inoculated 4 times with PBS (Group A), PBS with inactivated H1N1 A/ Puerto Rico/8/34 (PRI) viruses (Group B), or PBS with inactivated PRI viruses and D-octaarginine-linked PNVA-co-AA (Group C) at 7-day intervals. Doses of inactivated viruses and polymers were set to 1 μg and 100 μg, respectively, /20 μL of dosing solution/mouse/time. Seven days after the final inoculation, 4 mice in each group were sacrificed for antibody titration, and the remaining 12 mice in each group were nasally infected with mouse-adapted PRI viruses at a dose of 2 × 106 PFU/20 μL of dosing solution/mouse. Three days after virus infection, 5 mice in each group were sacrificed for virus titration. Body weights and survival of the residual 7 mice in each group were monitored for 14 days after virus infection. (a) Endpoint titers of PRI virus-specific IgG in sera (mean ± SD, n = 4). (b) Endpoint titers of PRI virus-specific secreted IgA in nasal wash fluids (mean ± SD, n = 4). (c) Virus titers in bronchoalveolar lavage fluids (mean ± SD, n = 4 (group B), n = 5 (groups A and C), lower limit of quantification: 101 PFU/mL). (d) Change in body weights after virus infection (mean ± SD, n = 7). (e) Survival rates after virus infection (n = 7).

5 mice for virus titration, and 7 mice for a monitor of body weights and survival. The number of mice assigned to each evaluation were decided on the basis of the literature.15−17,23 Mouse-adapted A/Puerto Rico/8/34 viruses, which Kitasato Institute for Life Sciences possesses, was used in our infection experiment. The dose was set to 2 × 106 PFU/mouse which corresponded to 6 × LD50, because Nagai et al. had confirmed that mice infected with the virus at this dose died within 1−2 week.24,25 As shown in Figure 5ab, antibody induction was observed with good reproducibility. Due to an excess amount of inactivated viruses and/or the excess number of inoculations, the A/Puerto Rico/8/34 virus-specific IgG level elevated considerably when antigens were solely administered (group B); however, there was a small production of corresponding secreted IgA. Both humoral immune responses were significantly enhanced when D-octaarginine-linked PNVA-co-AA was coadministered with antigens (group C). When mice were free from antigens and mucosal adjuvants (group A), rapid proliferation of A/Puerto Rico/8/34 viruses was observed in the respiratory tract. Three days after virus infection, virus titers in bronchoalveolar lavage fluids (PFU/ mL) reached a magnitude of 106 on average (Figure 5c). Body weights decreased and all mice died within 10 days after virus infection (Figure 5de), presumably due to lethal pneumonia. An increase in virus titers was also observed for mice inoculated with inactivated viruses alone (Group B, 4 mice were sacrificed

immunized with inactivated A/Puerto Rico/8/34 viruses in the presence of poly(I:C) completely survived during a monitoring period (10 days) when they were infected with mouse-adapted A/Puerto Rico/8/34 viruses at a dose of 1 × 103 PFU/mouse which corresponded to 40 × LD50.15 It is possible that doses of antigens and mucosal adjuvants and the number of inoculations are reduced. However, heterologous virus challenge on the basis of IgA-possessing cross-reactivity is planned after the homologous one is successfully terminated in the present study. Since mice with high reactivity of secreted IgA to the inoculated H1N1 strain acquired clear cross-reactivity to other H1N1 strains,19 a high IgA level would be advantageous to heterologous virus challenge. There is still room for improvement in immunization protocols; however, we concluded that protective abilities of secreted IgA against homologous virus infection would be evaluated under immunization protocols designed here (inoculation schedule: 4 times with 7-day intervals, a period of mice maintenance after the final inoculation: 7 days, the dose of inactivated A/Puerto Rico/8/ 34 viruses: 1 μg/mouse, the dose of D-octaarginine-linked PNVA-co-AA: 100 μg/mouse). Infection Experiment. Mice were divided into 3 groups, and those in groups A, B, and C were inoculated with antigens/ mucosal adjuvants-free PBS, inactivated A/Puerto Rico/8/34 viruses, and a mixture of the inactivated viruses and Doctaarginine-linked PNVA-co-AA, respectively, before virus infection. Each group comprised 4 mice for antibody titration, D

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Bioconjugate Chemistry for virus titration because one mouse was lost by anesthesiacaused death on the day of virus infection), although its degree was much less than that of mice in group A. It appeared that the IgA level was too low to protect mice from virus infection, but the IgG level was so high that virus proliferation in mice bodies was suppressed. This hypothesis may be supported by changes in body weight (Figure 5d). As expected from the large variation, the body weight of one mouse decreased extremely (individual data not shown). A 35% reduction in body weight was recorded 10 days after virus infection; however, the weight recovery was observed after the day. No death of mice was observed while survival was monitored (Figure 5e). Serious consequences were circumvented probably because there was a sufficient amount of A/Puerto Rico/8/34 virus-specific IgG. On the other hand, when mice were nasally inoculated with a mixture of inactivated viruses and D-octaarginine-linked PNVAco-AA (group C), viruses were not detected at all in the bronchoalveolar lavage fluids (Figure 5c). It was likely that the A/Puerto Rico/8/34 virus-specific IgA secreted on the mucosa of the respiratory tract perfectly prevented mice from being infected with homologous viruses in their invasion sites. All mice survived without loss of body weight (Figure 5d,e). When immunization protocols were designed, the number of inoculations was not included in the examination. However, current data strongly indicate that homologous virus infection will be protected even if the number of inoculations are reduced. Serum IgG is highly effective for the same viral strains used for immunization. However, cross-reactivity of antibodies is a prerequisite for protection against heterologous virus infection. We finally evaluated cross-reactivity of intranasally secreted IgA for which the sufficient amount was induced (group C). The same test was performed for serum IgG. As shown in Figure 6b, IgA cross-reacted to rHA proteins of not only antigenically drifted variants within a subtype of the inoculated viruses (H1N1) but also viruses categorized into different subtypes (H3N2 and H5N1). On the other hand, IgG reacted only to rHA proteins of homologous viruses used for immunization (Figure 6a), as expected from past reports.26 The present study demonstrates that D-octaarginine-linked PNVA-co-AA will be a promising adjuvant for mucosal vaccination that induces mucosal IgA which perfectly protects against homologous influenza virus infection. The next plan is the heterologous virus challenge. We expect that this challenge will be successfully completed because secreted IgA possesses a broad spectrum of heterosubtype immunity against influenza A virus infection in mice (Figure 6b). The successive study will be discussed in future reports.

Figure 6. Cross-reactivity of serum IgG (a) and intranasally secreted IgA (b) induced by inactivated H1N1 A/Puerto Rico/8/34 (PRI) influenza viruses in the presence of D-octaarginine-linked PNVA-coAA. Mice were nasally inoculated 4 times with a mixture of inactivated PRI viruses and polymers at 7 day intervals. They were maintained for 7 days after the final inoculation. Doses of inactivated viruses and polymers were set to 1 μg and 100 μg, respectively, /20 μL of dosing solution/mouse/time. Reactivity of antibodies to the respective recombinant hemagglutinin (rHA) proteins was measured. Crossreactivity is expressed as the percentage of reactivity to rHA proteins of H1N1 A/New Caledonia/20/99 strain (reverse-stippled bar, NCL), H1N1 A/California/07/2009 strain (rhombic bar, CA), H3N2 A/ Brisbane/10/2007 strain (horizontal lined bar, BNE), or H5N1 A/ Viet Nam/1194/2004 strain (perpendicular lined bar, VNM) to that to rHA proteins of PRI strain (stippled bar) (mean ± SD, n = 4).

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EXPERIMENTAL PROCEDURES Materials. PNVA-co-AA (sodium salts, NVA units/AA units: 70/30, weight-average molecular weight: 350 kDa, production code: GE-160−105) was obtained from Showa Denko Co. (Tokyo, Japan). Octaarginine (D-configuration) with amidated terminal carboxyl groups was obtained from Kokusan Chemical Co. Ltd. (Tokyo, Japan). Poly(I:C) was purchased from Wako Pure Chemical Industries Co. Ltd. (Osaka, Japan). Other chemicals were commercial products of analytical or reagent grade and were used without further purification. Whole particles of the H1N1 A/Puerto Rico/8/34 strain inactivated by β-propiolactone were obtained from Advanced Biotechnologies, Inc. (Columbia, MD). rHA proteins of the H1N1 A/Puerto Rico/8/34 strain and the H5N1 A/Viet Nam/ 1194/2004 strain were purchased from Sino Biological, Inc. (Beijing, China), while the proteins of other H1N1 strains (A/ New Caledonia/20/99 and A/California/07/2009) and one H3N2 strain (A/Brisbane/10/2007) were obtained from eEnzyme LLC (Montgomery Village, MD). Synthesis and Characterization of D-OctaarginineLinked PNVA-co-AA. D-Octaarginine-linked polymers were prepared in the same manner as previously described.18 Briefly, PNVA-co-AA was reacted with N-hydroxysuccinimide under activation of the carboxyl groups of the desalted free form by N,N′-dicyclohexylcarbodiimide. D -Octaarginine was next grafted onto the polymer backbone via amide bonds through

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CONCLUSIONS Infection experiments were performed to evaluate the potential of D-octaarginine-linked PNVA-co-AA as an adjuvant for mucosal vaccination. Antigen-specific IgG and secreted IgA were induced in sera and on the nasal mucosa, respectively, through 4× nasal inoculation in mice with a mixture of inactivated H1N1 A/Puerto Rico/8/34 viruses and the polymers. When immunized mice were inoculated with mouse-adapted A/Puerto Rico/8/34 viruses, secreted IgA perfectly protected them from homologous virus infection. On the other hand, mice inoculated nasally with inactivated A/ Puerto Rico/8/34 viruses alone were infected with the viruses. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination that enabled to protect hosts from homologous influenza virus infection. E

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Bioconjugate Chemistry substitution of the N-oxysuccinimide groups of PNVA-co-AA N-hydroxysuccinimide ester with the terminal amino groups of the peptides. The resulting D-octaarginine-linked PNVA-co-AA was purified in water and final concentration was adjusted to 10 mg/mL. The degree of D-octaarginine grafted onto PNVA-coAA was 15% and 74% as the percentage of monomer units of acrylic acid (AA) grafting the peptides to the total number of monomer units and the weight percentage of the peptides, respectively. The weight-average molecular weight of the polymer was calculated to be 1160 kDa. D-Octaarginine-linked PNVA-co-AA used in this study had been characterized thoroughly in our previous study.19 Animal Studies. Immunization of Mice with Antigens. Animal experiments were approved by the Ethical Review Committee of Setsunan University. BALB/c mice (female, 7 weeks, ca. 20 g, not fasted, n = 4) were used in each experiment. Inactivated H1N1 A/Puerto Rico/8/34 viruses were suspended in PBS with or without D-octaarginine-linked PNVA-co-AA to prepare dosing solution. The polymer was substituted with poly(I:C) when the positive control was needed. Animal experiments were performed in a manner similar to that previously described.18,19 Mice were nasally inoculated with each dosing solution under isoflurane anesthesia. Doses of inactivated viruses, D-octaarginine-linked polymers, and poly(I:C) were set to 0.5−1 μg, 40−100 μg, and 10 μg, respectively, /20 μL of dosing solution/mouse. The inoculation was performed on days 0 and 21 (2 times), days 0, 14, and 28 (3 times), or days 0, 7, 14, and 21 (4 times). After mice were maintained during 3, 7, or 14 days after the final inoculation, sera and nasal wash fluids were collected. The samples were stored at 4 °C and antibody titration mentioned below was immediately performed. Infection Experiment. Animal experiments were approved by the Institutional Animal Care and Use Committee of Kitasato University, and performed in accordance with Guide for the Care and Use of Laboratory Animals in the Kitasato University and the National Research Council Guide for the Care and Use of Laboratory Animals in Japan. Forty-eight BALB/c mice (female, 7 weeks, ca. 20 g, not fasted) were divided into 3 groups (n = 16, each). Mice were nasally inoculated 4 times with PBS (group A), PBS containing inactivated A/Puerto Rico/8/34 viruses (group B), or PBS containing the inactivated viruses and D-octaarginine-linked PNVA-co-AA (group C) at 7 day intervals. Doses of inactivated viruses and polymers were set to 1 μg and 100 μg, respectively, /20 μL of dosing solution/mouse. Seven days after the final inoculation, 4 mice in each group were sacrificed, and sera and nasal wash fluids were collected to estimate antibody levels. On the same day, the remaining 12 mice in each group were anesthetized with sodium pentobarbital and nasally infected with 2 × 106 PFU of mouse-adapted A/Puerto Rico/8/34 viruses suspended in 20 μL of PBS containing 0.1 w/v% bovine serum albumin (BSA). Three days after virus infection, 5 mice in each group were sacrificed to collect bronchoalveolar lavage fluids as mentioned below. Body weights and survival of the residual 7 mice were monitored for 14 days after virus infection. Antibody Titration. Titers of Antigen-Specific Antibodies. Titers of A/Puerto Rico/8/34 virus-specific antibodies were measured in the same enzyme-linked immunosorbent assay (ELISA) as previously described,18,19 except that whole particles of inactivated viruses were directly used as antigens with which assay plates were coated. Endpoint titers of antibodies were determined from the x-axis intercept of the

dilution curve. Each value of endpoint titers is presented as the mean ± standard deviation (SD). The endpoint titers increase as antibody levels elevate. The titer is calculated to be 4.3 when there is no antibody production, because sera and nasal wash fluids are minimally diluted 20-fold before antibody titration. Cross-Reactivity of Antibodies. Reactivity of antibodies to rHA proteins of the H1N1 A/Puerto Rico/8/34 strain was measured using the previously described ELISA method.19 The same measurement was performed for viral strains which were not used for immunization (H1N1 A/New Caledonia/20/99, H1N1 A/California/07/2009, H3N2 A/Brisbane/10/2007, and H5N1 A/Viet Nam/1194/2004). Cross-reactivity was expressed as a percentage of the heterosubtypic reactivity to the homosubtypic reactivity. The percentage increases as crossreactivity becomes high. Virus Titration. Collection of Bronchoalveolar Lavage Fluids. Virus titration was performed under previously reported procedures.24,25 Under isoflurane anesthesia, trachea-bearing lungs were removed from mice exsanguinated through cut of axillary artery and vein. After the surface of the lung was washed with 0.1 w/v% BSA-containing PBS, 2 mL of BSA-containing PBS was poured into trachea twice. Collected fluids were centrifuged at 1200 g for 20 min at 4 °C and the supernatant was used as bronchoalveolar lavage fluids. The samples were stored at −80 °C until virus titration was performed. Virus Titration. Mardin-Darby canine kidney (MDCK) cells were seeded at a density of 7.5 × 104 cells/mL in each well of a 12-well plate and then incubated at 37 °C in humidified air containing 5% CO2 for 4 days. A 10-fold dilution series of bronchoalveolar lavage fluids were separately prepared. After the culture medium was replaced with a fresh one, 0.1 mL aliquots of samples (bronchoalveolar lavage fluids) were added into each well and MDCK cells were incubated at 37 °C for 1 h with tilting every 15 min. The culture medium was then covered with 1.5 mL of minimum essential medium containing 1 w/v% agarose and 2.5 μg/mL trypsin. After incubation at 37 °C for 2 days in humidified air containing 5% CO2, 1 mL of PBS containing 4 w/v% formaldehyde was added into each well and the plate was kept for 1 h at room temperature. After removal of agarose gel, 1 mL of PBS containing 4 w/v% formaldehyde and 1 w/v% crystal violet was added into each well and the plate was kept for 15 min at room temperature. The well was washed with water and then dried. The resulting plaques, which correspond to virus activity (virus titers), were counted (the number increases with an increase in the activities). Statistics. Statistical significance was assessed with the unpaired Student’s t test, and p values of 0.05 or less were considered to be statistically significant.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81-99275-5930. Fax: +81-99-275-5932. *E-mail: [email protected]. Phone: +81-72-8663124. Fax: +81-72-807-6048. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported in part by Adaptable and Seamless Technology Transfer Program through Target-driven F

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Bioconjugate Chemistry

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R&D (A-STEP) Seeds Validation (Research No.: AS2531303Q) from Japan Science and Technology Agency (JST). Infection experiments were performed in Kitasato Institute for Life Sciences, Kitasato University, under contract with the institute. The authors thank Dr. Takayuki Nagai, Mr. Osamu Hiraku, and Mr. Ken Sasaki in Kitasato Institute for Life Sciences, Kitasato University for their technical supports.

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ABBREVIATIONS PNVA-co-AA, poly(N-vinylacetamide-co-acrylic acid); IgA, immunoglobulin A; IgG, immunoglobulin G; HA, hemagglutinin; rHA, recombinant hemagglutinin; NA, neuraminidase

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.6b00283 Bioconjugate Chem. XXXX, XXX, XXX−XXX