Virus-Mimetic Polymer Nanoparticles Displaying Hemagglutinin as an Adjuvant-free

Influenza Vaccine

Chaeyeon Leea, Jonghwa Jeonga, Taeheon Leea, Wei Zhangb, Li Xub, Ji Eun Choic, Ji Hyun

Parkc, Jae Kwang Songc, Sinae Jangd, Chi-Yong Eomd, KyuHwan Shime, Seong Soo A.Ane,

Young-Sun Kangf, Minseok Kwakg, Hyeong Jin Jeonh, Jeung Sang Goh, Yung Doug Suhi,

Jun-O Jinb,j* and Hyun-jong Paika,*

aDepartment of Polymer Science and Engineering, Pusan National University, Busan, 46241,

Republic of KoreabShanghai Public Health Clinical Center, Shanghai Medical College, Fudan University,

Shanghai, 201508, ChinacResearch Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology

(KRICT), Daejeon, 34114, Republic of KoreadSeoul Center, Korea Basic Science Institute (KBSI), Seoul, 02481, Republic of KoreaeDepartment of Bionano Technology, Gachon University, Sungnam, 461-701, Republic of

KoreafDepartment of Biomedical Science & Technology (DBST), Colleage of Veterinary Medicine,

Konkuk University, Seoul, 27478, Republic of KoreagDepartment of Chemistry, Pukyong National University, Busan, 48547, Republic of KoreahSchool of Mechanical Engineering, Pusan National University, Busan, 46241, Republic of

KoreaiLaboratory for Advanced Molecular Probing (LAMP), Research Center for Convergence

Nanotechnology, Korea Research Institute of Chemical Technology (KRICT), Daejeon,

34114, Republic of Korea jDepartment of Medical Biotechnology, Yeungnam University, Gyeongsan 38541, South

Korea

Corresponding Authour: Hyun-jong Paik (Tel: +82-51-510-2402, E-mail: hpaik@pusan.ac.kr)

and Jun-O Jin (Tel: +86-21-3799-0333-7338, E-mail: junojin1@gmail.com)

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Abstract: The generation of virus-mimetic nanoparticles has received much attention in

developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method,

encompassing most viral features for their size, hydrophobic domain and antigen display,

would represent a meaningful direction for the vaccine development. In the present study, a

polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface

(H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features.

Moreover, the concentrations of antigen on the particle surface were controlled, and its effect

on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted

H1-specific immune activation and cross-protective activities, which consequently prevented

H1N1 infections in mice.

Keywords: in situ method, protein-polymer nanoparticle, virus-mimicking, antigen-specific

immune activation, influenza vaccine

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1. Introduction

Various types of protein-polymer hybrid structure have been developed as the

potential aids in biomedicine and biotechnology.[1-5] Displaying proteins on polymer

particles or supports with controlled orientation is an ideal direction to introduce the

biological recognition function of protein component.[6-10] The polymer-templated protein

nanoball (PTPNB) system is in situ assembly to manufacture polymer-protein core-shell

nanoparticles.[11-13] PTPNB is based on the specific chelating interaction of hexa-histidine

tag (His6-tag) of protein and nickel(II)-complexed nitrilotriacetic acid (Ni2+-NTA) of the

polymer chain end. In the PTPNB system, the size of the protein nanoballs and the orientation

of proteins on nanoballs can be controlled. Furthermore, a wide range of proteins, including

enzymes, antibodies, and antigens, can be introduced to the PTPNB, providing and sustaining

the corresponding biological functions. Depending on the functions of the designated proteins,

the PTPNB can be designed for diverse bio-applications. As the first application, here we

report the generation of a direction oriented antigen-displaying PTPNB, as a new influenza

vaccine.

Among the conventional vaccine formulations, vaccine subunits of highly purified

antigens would be much safer than the inactivated or live-attenuated pathogens, but they

readily presented less immunogenic to elicit immunity of disease protection in challenging

study.[14] The convergent approaches of vaccine subunits and the synthetic nanoparticle

system improved the vaccine efficiency in terms of 1) the preferred size for dendritic cell

(DC) uptake, 2) the co-encapsulation of adjuvants in particles, 3) the prevention of antigen

degradation, and 4) the multi-valent presentations of antigens.[15-18]

Designing vaccines that mimic pathogens is the current direction of vaccine

development.[19] The system of virus-like particles (VLPs) assembled with viral structural

proteins have been powerful method to prepare virus-mimetic vaccines, but this method

revealed the limitations with the delicate genetical processes of developing specific vaccine.

[20] Thus, the simple and switchable method based on the combination of nanoparticle and

recombinant antigen was proposed and should be regarded as a promising platform

mimicking viruses. With respect to viral features, the general factors of their size, existence of

a hydrophobic domain and the antigens located on the cell membrane must be considered in

the design of such vehical and for the optimal antigenicity. The synthetic methods fulfilling

these factors were reported, showing stronger immune responses than antigen proteins.[21-30]

However, the development of new vaccines with few side effects and sufficient

immunogenicity was still required. The other important factors to induce superior immune

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responses of virus-mimetic structures included the consistent orientation and density of the

antigens on the viral particles.[17, 31] Previous synthetic methods based on non-specific

interactions, however, were not adequate to control the antigen orientation. Thus, a new

platform encompassing most viral features, including direction oriented antigen display,

would represent a meaningful approach for the vaccine development.

The PTPNB system is an one-step process which can mimic influenza virus with

respect to the size, hydrophobic domain, the direction orientated antigen display, and density

of antigen on the surface. Ni2+-NTA on the polymeric core can conjugate with His6-tag

attached to the stem (C-terminus) of hemagglutinin, an important influenza virus antigen. The

head, including the receptor binding pocket of hemagglutinin, can be exposed as in original

influenza structures (Fig. 1a, b).

In the present study, we designed a PTPNB conjugated hemagglutinin1 (H1) (H1-NB)

for preventing influenza viral infection in mice as a first case to demonstrate the high potential

of the PTPNB system, as a new vaccine platform. With respect to the control of antigen

density, we verified the ability to introduce multiple proteins and to control the surface

density of H1 by adjusting the ratio of dummy and antigen proteins. The present study also

compared the immunogenicity of free H1, PTPNB without H1 and H1-NB to determine

whether the factors inducing immune responses were only properties of polymer template,

such as size and hydrophobicity or the synergic effects of antigen display. Finally, to confirm

this PTPNB as a putative influenza vaccine, the immune response without adjuvant and the

cross-activity, which can cope with virus mutations, were estimated in vivo.

2. Materials and methods

2.1. Materials

The gene encoding hemagglutinin1, His6-H1 protein of influenza A virus

(A/Texas/2009/H1N1), was synthesized based on the format available from GenBank

accession No. ACP41934. The recombinant protein purified from 293 cell culture, His6-H1’

from A/Puerto Rico/8/1934 (H1N1), was purchased from Immune Technology Corp (New

York, USA). His6-green fluorescent protein (GFP) was expressed and purified as previously

described.[12] Alexa647 Maleimide was purchased from Thermo Fisher Scientific Solutions

LLC (Massachusetts, USA). Ni2+-NTA-end-functionalized polystyrene (Ni2+-NTA-PS)

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(Mn=5620 g/mol and PDI=1.28) was synthesized by atom transfer radical polymerization

(ATRP) as previously described.[13]

2.2. Methods

2.2.1. H1-NBs preparation with the different H1 concentrations

His6-H1 labeled with Alexa 647 (His6-H1-647) was used for H1-NBs formation to verify the

immobilization and the protein contents on the particle surface. Non-labeled His6-H1 was

introduced for in vitro and in vivo examinations. Ni2+-NTA-PS (0.05 mg, 8.95 x 10-6 mmol)

was dissolved in 0.1 mL N,N-dimethylformamide (DMF). The mixtures of His6-H1 labeled or

not with Alexa 647 and His6- GFP in 5 mL phosphate buffer solution (PBS) (10 mM, pH 7.5)

were prepared depending on the mole ratio of both proteins (1, 5, 10 and 20 mol% of H1).

The total protein concentration was fixed at 1.16 μmol/L. The polymer stock solution was

slowly added to the protein solution by using a syringe pump under rapid stirring at 25°C.

Structure formation and particle sizes were characterized by dynamic light scattering (DLS)

and transmission electron microscopy (TEM). TEM images were obtained on a Hitachi H-

7600 instrument at 80 kV. For microscopic analysis, the TEM sample was prepared by

dipping a TEM grid (carbon coated grid) into the respective solutions. DLS was performed

with a 90 plus Particle Size Analyzer (Brookhaven Instruments Corporation). To immunize

the mice with H1-NBs, H1-NBs were concentrated through centrifugal filtration (10K

MWCO cellulose filter) without the removal of free proteins not conjugated onto polymer

particles for adjusting total protein concentration into 11.6 μmol/L. In each case of 1, 5, 10

and 20 mol% of H1, H1 concentration was 7.6, 38, 76 and 152 μg/mL.

2.2.2. Verification of the immobilization and the density control of H1 on particle surface

To calculate the conjugation ratio of the proteins, we removed free proteins not conjugated

onto polymer particles via Ni2+-NTA agarose resin filtration, since free proteins can be well

attached to the resin through exposure of the histidine tag. The conjugation ratio of each

protein was calculated by comparing the emission intensity of both His6-H1-647 and GFP

before and after the removal of free proteins. To confirm whether the proportions of His6-H1-

647 and His6-GFP conjugated on particles were controlled, photoluminescence (PL) and super

resolution confocal microscope (SRCM) images of H1-NBs removed free-proteins were

analyzed. In the PL analysis, the normalized fluorescence values of Alexa 647 divided by the

maximum value of GFP fluorescence was calculated. In the SRCM analysis, we obtained the

values of Iparticle/Ibackground (Iparticle=Fluorescence Intensity of Particle Region,

Ibackground=Fluorescence Intensity of Background) by using the ImageJ program. The PL spectra

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were recorded using the Ocean Optics HR4000CG Composite-grating spectrophotometer with

excitation wavelengths at 450 and 645 nm. The nanoparticles were imaged on the Leica TCS

SP8 inverted microscope using a 488-nm laser light and HyD detector (490-530 nm) for GFP

excitation and emission, and a 640 nm laser and HyD detector (645-740 nm) for Alexa647

excitation and emission.

2.2.3. Mice

The C57BL/6 mice (6 weeks old) and BALB/c mice were obtained from Shanghai Public

Health Clinical Center and maintained under pathogen-free conditions. All experiments were

performed under the guidelines of the Institutional Animal Care and Use committee at the

Shanghai Public Health Clinical Center. The protocol was approved by the committee on the

Ethics of Animal Experiments of the Shanghai Public Health Clinical Center (Mouse Protocol

Number: SYXK-2010-0098). The mice were sacrificed by CO2 inhalation euthanasia, and all

efforts were made to minimize suffering.

2.2.4. Dendritic cell (DC) analysis

Spleen DCs were analyzed as described elsewhere.[32, 33] Briefly, the spleens were cut into

small fragments and digested with 2% fetal bovine serum (FBS) containing collagenase for 20

min at room temperature. The digested cells were centrifuged, and the pellet was re-

suspended in 5 mL of Histopaque 1.077 (Sigma-Aldrich). An additional 5 mL of Histopaque

was layered below, and culture medium was layered above the cell suspension, which was

subsequently centrifuged at 1700 g for 10 min. The light density fraction (< 1.077 g/cm3) was

collected and incubated for 20 min with the following FITC-conjugated monoclonal

antibodies (mAbs): anti-CD3 (17A2), anti-Thy1.1 (OX-7), anti-B220 (RA3-6B2), anti-Gr-1

(RB68C5), anti-CD49b (DX5) and anti-TER-119 (TER-119). CD11c+ cells, defined as cDCs,

were further divided into CD8α+CD11c+ and CD8α-CD11c+ cDC lineages. The analysis was

performed on a FACS Aria II (Becton Dickinson).

2.2.5. Immunization of mice

BALB/c mice were immunized i.v. with 100 μL of PBS alone, H1 (7.6 µg) in PBS, a

combination of H1 (7.6 µg) and GFP-NB, 10% H1-NB solution containing 7.6 µg of H1 or

H1 (7.6 µg) mixed with 50 µg Alum in PBS on days 0, 15 and 30. On day 35, some mice

were challenged with PR8 virus and measured survival rate and changes of body weight.

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After immunization, some mice were sacrificed, sera were collected, and splenocytes were

harvested for further analysis.

2.2.6. Influenza viral infection

BALB/c mice were intranasally (i.n.) infected with A/Puerto Rico/8/34 (PR/8) under

isoflurane anesthesia in a 50 µL volume with a 50% Tissue Culture Infectious Dose (TCID50)

of 5 x 103.

2.2.7. ELISPOT assay

Mouse IFN-γ ELISPOTs were performed using pre-coated plates (eBioscience) according to

the manufacturer’s protocol. Briefly, splenocytes were stimulated with 2 μg/mL of the MHC-I

peptide IYSTVASSL, the MHC-II peptide SVSSFERFEIFPK, or a negative control peptide at

37°C for 24 hours. The number of spots was observed by ChampSpot III (Sagecreation,

Beijing, China).

2.2.8. ELISA

The IL-6, IL-12p70, IFN-γ and TNF-α concentrations in the sera were measured in triplicate

using standard ELISA kits (Biolegend).

2.2.9. Tetramer assay

Splenocytes (5 × 106 cells) were cultured with 1 μg/mL of the HA240–248 IYSTVASSL peptide.

After incubation at 37°C for 4 days, the cells were further incubated with recombinant IL-2

for an additional 3 days. The cells were stained with an APC-conjugated H-2Kd-HA240–248

peptide tetramer (MBL) for 20 min, followed by incubation with PE-cy7-conjugated anti-

CD8α antibodies for 20 min. The analysis was performed on a FACS Aria II (Becton

Dickinson).

2.2.10. Ag-specific antibody analysis

Anti-influenza virus antibodies were measured by ELISA by using standard methods and

purified virus as the substrate. Endpoint titers were defined as the highest dilution of serum

that resulted in a signal three times above the background level. Total IgG, IgG1, and IgG2a

levels were detected as previously described.[32] Hemagglutination-inhibiting (HAI) titers to

the PR/8 strain were assayed using trypsin-heat-periodate-inactivated sera.[34]

2.2.11. Statistical analysis

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Results are expressed as the means ± standard error of the mean (SEM). The statistical

significance of the differences between experimental groups was calculated using analysis of

variance with Bonferroni’s post-test or unpaired Student’s t-test. All p-values <0.05 were

considered significant.

3. Results and Discussion

3.1. Preparation of H1-NBs with the H1 density control on the particle surface

(Fig. 1)

The PTPNB system was introduced to prepare virus-like protein-polymer nanoparticle,

incorporating His6-H1 from A/Texas/2009 (H1N1) and His6-GFP. In this study, H1 of

monomeric form was introduced. A schematic illustration of H1-NB was shown in Fig. 1c.

Ni2+-NTA was introduced to the polymer chain end, as a tool to immobilize proteins on

surface of the polymer nanoball. Ni2+-NTA could complex with the His6-tag of recombinant

proteins, and this conjugation indicated good structural properties, such as fast, reversible and

selective interactions.[35, 36] Based on the selectivity, the NTA-Ni2+/His affinity interactions

can lead the controlled immobilization of proteins. PTPNBs were spontaneously formed by

using a one-pot procedure by mixing Ni2+-NTA-PS in DMF and protein in PBS.

In the present study, PTPNBs containing His6-H1 and His6-GFP were prepared using a

mixed protein solution of His6-H1 and His6-GFP. After the addition of the Ni2+-NTA-PS

solution into the mixed protein solution, unstable polymeric aggregates preserving Ni2+-NTA

on the surface were formed by hydrophobic effects, and subsequently, His6-proteins would

coat the polymeric aggregates from the NTA-Ni2+-His interaction. Here, His6-GFP was

introduced, as a fluorescent dummy protein for controlling the surface density of His6-H1 and

the fluorescence analysis of cellular uptake.

To confirm whether both proteins were conjugated together on the particles and to

determine whether the ratios of H1/GFP on the particle surface were controlled, His6-H1-647

was introduced to prepare H1-NBs. Four types of H1-NBs were prepared according to the H1

ratio in contradistinction to GFP at 1, 5, 10 and 20 mol%. Fig. 1d shows the successful

nanoparticle formation of all samples. The similar diameters around 170 nm of H1-NBs were

confirmed by DLS measurement without reference to the H1 content (Fig. 1e, S1a). The

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conjugation ratio of each protein was calculated as over 80% by comparing the fluorescence

intensity before and after the removal of the non-conjugated proteins (Fig. S1b).

To verify that both His6-H1 and His6-GFP coexisted in a particle, SRCM was used to

analyze and observe nanoscale structures. As result, the confirmed H1-NBs exhibited the

green fluorescence of His6-GFP and red fluorescence of His6-H1-647 (Fig. 1f). By comparing

the relative fluorescence intensity of H1 and GFP after the removal of non-conjugated

proteins with PL analysis, we confirmed that the content of H1 immobilized on each particle

was increased depending on the H1 mol% of the reacting protein solution (Fig. S1c). To show

that the proportions of H1 and GFP conjugated on particles were controlled by changing the

protein ratio of the reactant solutions, the IH1/IGFP value calculated from PL emission spectra

and the fluorescence of the particles in confocal images was used (Fig. 1g). The fluorescence

intensity (IH1 and IGFP) was selected from a maximum value in the PL analysis, and the IH1 and

IGFP from the confocal analysis was calculated as Iparticle/Ibackground by using the ImageJ program

(Fig. S1d). In all the cases of PL analysis, before and after removal of the non-conjugated

proteins, the relative red intensities of His6-H1-647 were increased with increasing H1

content. A similar tendency was also observed in the analysis of the confocal microscope

images.

3.2. Release of His6-proteins from particles under excess imidazole or the serum environment

The NTA-Ni2+/His interaction can be dissociated by the addition of excess imidazole.

To verify that the proteins were immobilized on particles through the NTA-Ni2+/His

interaction, H1-NB containing 10 mol% His6-H1 was incubated in 250 mM imidazole for 1 h.

In the TEM images, well-defined core-shell particles were generally observed before

imidazole treatment (Fig. S2a). After imidazole treatment, most particles were collapsed into

macro structures, and the diameters of the scant remaining particles were decreased with loss

of the outer layer (Fig. S2b). The destruction of H1-NB under excess imidazole indicates that

the proteins were conjugated with polymer particles by a specific NTA-Ni2+/His interaction.

However, the conjugation stability of His6-H1 to NB under in vivo conditions is

important. Since the NTA-Ni2+/His interaction is reversible, His6-H1 may or may not

disassociate from H1-NB prior to internalization into DCs. The stability of the protein-

polymer conjugation under the serum 50% environment was measured in a time course by

SRCM (Fig. S2c, d). The degree of His6-protein binding to NB was only slightly changed

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after incubation for 24 h in the serum environment. Thus, the PTPNB system is suitable to

induce immune responses because DCs may uptake these particles within 1 h.

3.3. The effect of H1 density on particles and particle sizes on delivery to DCs

H1-NBs containing non-labeled His6-H1 were prepared for in vitro and in vivo

experiments. The similar diameters around 130 nm of H1-NBs were confirmed by DLS

measurement without reference to the H1 content (Fig. S3). Before evaluation of

immunological activity by H1-NBs, we examined toxicity of H1-NB containing 10 mol% H1

and found that the high concentration (20 ug/mL) of H1-NB did not show cytotoxic effect in

NIH-3T3 cells (Fig. S4). Next, we examined the effect of H1 density of particle surface on

phagocytosis in bone marrow-derived dendritic cells (BMDCs). BMDCs were treated with 1,

5, 10 and 20 mol% H1-conjugated PTPNBs for 1 h. The degree of H1-NB uptake into

BMDCs increased depending on the H1 content, which was saturated after 10 mol% (Fig. S5).

Next, since BMDCs could efficiently uptake H1-NB, spleen DCs were further assessed for the

potential phagocytosis of H1-NB in vivo. C57BL/6 mice were intravenously (i.v.) injected

with 1, 5, 10 and 20 mol% H1-conjugated NBs. Consistent with BMDCs, the uptake

percentage of H1-NB by spleen DCs was saturated at 10 mol% conjugation of H1 (Fig. S6).

Mouse in vivo DCs containes two subsets, CD8α+ DCs and CD8α- DCs.[37, 38] These

DC subsets show different function in antigen presentation and T cell activation. The CD8α+

DCs are specialized to present cytosolic Ag in MHC class I to CD8 T cells, which induced

cytotoxic T lymphocyte (CTL) activation. On the other hand, CD8α- DCs present extracellular

Ag on MHC class II to CD4 T cells.[37-39] Since, the DC subsets promote different immune

responses, we next examined the phagocytosis of H1-NB by spleen DC subsets and found that

both CD8α+ andCD8α- DCs were effectively phagocytosed H1-NBs in a dose-dependent

manner based on H1 percentage (Fig. S7). These results suggested that the H1-NB system

could efficiently deliver H1 Ag to DCs in vivo and in vitro. Interestingly, CD8α+ DCs showed

the potent phagocytosis of H1-NB compared to CD8α- DCs. These differences in the

phagocytic activities of spleen DC subsets may be due to the different expression levels of

pattern recognition receptor (PRR) on DC subsets.[40] In addition, CD8α+ DCs have generally

more potent immunity against viral infection than CD8α- DCs.[41]

We also evaluated size effect in the phagocytosis of H1-NBs by spleen DCs. In

PTPNB system, the particle sizes can be controlled from 80 nm to 250 nm by adjusting the

polymer amount.[13] Three kinds of H1-NBs containing 10 mol% H1 were prepared with

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variation of the polymer amount and their sizes were controlled to 80 nm, 130 nm and 240 nm

(Fig. S8). We examined the phagocytosis of H1-NBs in the spleen DCs and found that H1-

NBs of differenct sizes were efficiently phagocytosed by spleen DCs. Although the

percentage of H1-NB phagocytosis indicating different sizes was similar, the H1-NB of 130

nm were more efficiantly phagocytosed by spleen DCs then other sizes (Fig. S9). Taken

togeher, H1-NB containing 10 mol% H1 and 130 nm of particle size was subjected to further

tests. Henceforth, H1-NB represent H1-NB of 130 nm containing 10 mol% H1 .

(Fig. 2)

Since H1-NBs were phagocytosed by spleen DCs in the mouse in vivo, we further

examined whether other immune cells in spleen also phagocytosed the H1-NB. At one hour

after i.v. injection, macrophages in the spleen also phagocytosed the H1-NB efficiently (Fig.

2a and S10a). In contrast to DCs and macrophages, other immune cells in the spleen,

including natural killer (NK) cells, T cells, B cells and neutrophils, did not phagocytose the

H1-NBs (Fig. S10b).

3.4. DC activation through H1-NB treatment

H1-NB was examined to determine whether it could induce the activation of DCs.

Twenty-four hours after H1-NB treatment, the frequency of spleen DCs substantially

increased, whereas GFP, H1 and GFP-NB treatment did not induce changes (Fig. S11).

Moreover, H1-NB-treated spleen DCs expressed high levels of C-C chemokine receptor type

7 (CCR7), the marker for the migration and activation of peripheral tissue DCs, compared to

other control treatments (Fig. 2b). In addition, the expression levels of co-stimulatory

molecules and MHC class I and II in spleen DCs and its subsets were significantly increased

by treatment with H1-NBs, whereas other treatments did not induce the up-regulation of those

molecules (Fig. 2c and S12). The up-regulation of co-stimulatory and MHC molecules in

splenic DCs in vivo was dependent on the injection amount of H1-NBs (Fig. S13). Moreover,

the mRNA levels of IL-6, IL-12 and TNF-α in the spleen were markedly increased by H1-NB

treatment compared to those in other control treatment groups (Fig. S14). Consistent with the

mRNA expression levels, H1-NB treatment led to marked increases in the percentage of IL-

6-, IL-12- and TNF-α-producing spleen DCs, whereas those treated with PBS or H1 alone did

not increase (Fig. 2d). Hence, these data indicated that treatment with H1-NBs induced the

activation of spleen DCs in the mouse in vivo.

3.5. Protection of mouse from influenza viral infection by immunization with H1-NBs

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(Fig. 3)

Since H1-NBs could deliver H1 Ag to spleen DCs and induce the activation of these

cells, H1-NB was assessed for the potential induction of H1-specific immune responses in the

mouse in vivo. In this stage, a combination system of H1 and GFP-NB that H1 Ag was just

mixed instead of immobilization on NB was additionally tested as control group to ensure the

importance of virus-mimetic structure for immune regulation. To minimize H1

immobilization on NB, we mixed His6-H1 and ready-made GFP-NB just before injection to

mice. BALB/c mice were immunized (i.v.) with H1 alone, a combination of H1 and GFP-NB

or H1-NBs on days 0, 15 and 30. The mice also immunzed i.v. with a combination of 7.6 μg

H1 and 50 μg Alum as a positive control. On day 35, the splenocytes were harvested and re-

stimulated with H1 Ag peptide in vitro for 4 days. The IFN-γ ELISPOT assay showed that

H1-NB immunized splenocytes promoted significantly compared to immunizations with H1

alone and a combination of H1 and GFP-NB through higher numbers of spots, which

indicated IFN-γ production in response to MHC class I (IYSTVASSL)- or MHC class II

(SVSSFERFEIFRK)-restricted peptides. In addition, the induction of H1-specific IFN-γ

production levels were almost similar with those induced by positive control (Fig. 3a). In the

splenocytes from H1-NB-immunized mice, CD4 and CD8 T cells effectively produced IFN-γ

in response to IYSTVASSL or SVSSFERFEIFRK (Fig. S15). In addition, T-bet, the critical

transcription factor for heper T 1 (Th1) and cytotoxic T 1 (Tc1) cells, and IFN-γ mRNA levels

were also significantly increased in the reponses to H1 in H1-NB immunized spleenocytes

compared to PBS or H1 alone immunized splenocytes (Fig. S16). Furthermore, H1-NB

immunization led to marked increases in the proportions of CD44+, a marker of

effector/memory T cells, in the spleen CD4 and CD8 T cells, whereas immunizations with

PBS or H1 alone did not induce these effects (Fig. S17). Thus, these data suggested that H1-

NB immunization promoted H1-specific immune responses.

For effective protection against viral infection, the activation of Ag-specific CTLs is

required. Since H1-NB could induce activation CD8α+ DCs, we next evaluated the capacity of

H1-NB in the promotion of CTL-mediated cytotoxity against H1 Ag. Mice immunized with

H1-NBs showed significantly higher specific killing of MHC class I (IYSTVASSL)-restricted

peptide-coated splenocytes compared to that in other control immunized mice (Fig. 3b).

Moreover, H1-NB immunization promoted substantial increases in the HA-2Kd/HA240-248

tetramer-positive CD8 T cells compared to that after treatment with PBS or H1 alone,

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indicating the activation of CD8 T cells in response to MHC class I-restricted peptides (Fig.

3c). Collectively, these data suggested that the immunization of H1-NB promoted H1-specific

CTL activation in mice in vivo.

Next, H1-NB was assessed for the potentially enhanced production of H1-specific

antibodies in mice in vivo. On day 35, the immunization of H1-NB led to significantly higher

levels of H1-specific total IgG, IgG1 and IgG2a in the serum compared to that in mice after

immunization with H1 alone (Fig. S18). The neutralizing antibody titers were increased from

the second boosting immunization of H1-NB, which were significantly higher than those in

mice immunized with PBS or H1 alone (Fig. 3d). In addition, end-point titers revealed the

continuous increase of Ag-specific total IgG in sera from mice immunized with H1-NB,

whereas those immunized PBS or H1 alone did not show this increase (Fig. 3e). Thus, these

data suggested that the immunization of H1-NB enhanced the production of H1-specific

antibodies in mice in vivo.

(Fig. 4)

Since H1-NB immunization promoted humoral and cellular immunity against H1 Ag

in mice, we next evaluated the vaccine efficacy of H1-NB. PTPNB conjugated with A/Puerto

Rico/8/34 (PR/8) H1’, referred to as H1’-NB, was synthesized (Fig. S19). BALB/c mice were

immunized with PBS, H1’ alone or H1’-NB on days 0, 15 and 30 and subsequently

challenged with 5 × LD50 of PR/8 (H1N1) on day 35 of immunization and monitored to their

survival rate and changes in their body weights. The mice immunized with H1’-NB were

completely protected from PR/8 viral infection, whereas those immunized with PBS or H1’

alone died within 7 days after post infection (Fig. 4a, b). Moreover, twelve weeks after H1’-

NB immunization, the mice almost completely rejected the infection of PR/8 compared to

those immunized with H1’ alone (Fig. 4c, d).

Next, the cross-activities against different type of H1N1 were assessed by the

immunization of H1-NB, since H1-NB could induce CTL activation. In the present study, NB

was conjugated with different H1 Ag from A/Texas/2009 (H1N1). On day 35 of

immunization, the mice immunized were challenged with PR/8 and monitored to their

survival rate and changes in their body weights. As shown in Fig. 3e, f, NB conjugated with

different H1 type with virus also completely prevented the lethal effect of the PR/8 infection.

Taken together, these data suggested that immunization with H1-NB prevented influenza

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infections and promoted long-term memory immune responses. Moreover, H1-NB induced

cross activity against different types of H1N1 influenza viruses.

4. Conclusion

The present study demonstrated a simple platform approach for the preparation of Ag-

displaying polymer particles as new influenza and other vaccines. Based on the structural

control of the PTPNB method, virus-mimetic H1-NBs were designed at sizes similar to those

of viruses[42], optimizing the local concentrations of H1 and exposure to the receptor binding

domain through specific interactions between the Ni2+-NTA of polymer chain end and multi-

His tag located in fusion peptide of H1 Ag for the induced immune response. In the present

study, H1 Ag-conjugated PTPNBs displayed protective immunity against H1N1 infection

without the addition of adjuvant. As described in the scheme of the present study (Fig. S20),

DCs showed effective phagocytic activities against H1-NB without adjuvant and ligand. This

effect may be due to the conjugation of nanoparticle to full-length H1 with controlled

orientation, exposing the H1 head group on the surface and displaying a size similar to that of

influenza virus. Previous studies have shown that mouse and human DCs were infected with

influenza virus and promoted immune responses against this virus. Therefore, both similar

size and Ag exposure on the surface may contribute to the phagocytosis of H1-NB by

DCs[43-45]. Moreover, spleen DCs cross- or directly presented the H1 Ag to CD4 and CD8 T

cells and promoted H1-specific Th1 and CTL activation and enhanced production of

neutralizing antibodies. Finally, H1-NB vaccination protected mice from H1N1 infection and

showed cross-activities against different types of H1N1 infections, which may due to the CTL

activation.

The safety studies of administration of polystyrene (PS) beads revealed little or no lethal

consequence in rats or mice.[46, 47] H1-NB indicated low cytotoxicity and any negative

effect was not observed in the course of overall estimation for immune responses. For the

delivery of H1-NB, even though we did not encounter any complication from the coagulation,

allergenic responses or pulmonary embolism in limited experiments with mouse, we cannot

rule out the potential rise of additional side effects from the PTPNB, which should be

investigated separately.  

In conclusion, the current results provided evidence for the potential synthetic adjuvant-

free influenza vaccine H1-NB. Notably, free-H1 and nanoball without displaying H1 could

not induce immune responses compared with H1-NB, suggesting that the materials alone were

not an immune regulators. The factors inducing the immune response of H1-NBs were the

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synergistic effects of mimicking viruses, indicating the high potential of the PTPNB system as

a platform for developing a virus-mimetic vaccine. Especially a wide range of recombinant

antigens can be introduced to PTPNB system with very few restrictions. Therefore this

method can be applied for various virus-mimetic vaccines with direction oriented antigen

display. Thus further investigations will continue to upgrade the PTPNB system for human

uses, including high bio-compatability as well as multiple Ags system for universal vaccines.

Acknowledgements: We thank the Shanghai Public Health Clinical Center animal facility for

maintaining the animals in this study. This work was supported by Mid-career Researcher

Program (2013R1A2A2A01068818) through the National Research Foundation (NRF) grant

funded by the Korean government (MSIP) and by a grant of the Korea Health Technology

R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by

the Ministry of Health & Welfare, Republic of Korea (HI 16C0973). J.O.J. was also supported

by National Science and Technology Major Project,

Ministry of Science and Technology of China (2017ZX09304027).

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Figure Lists

Fig. 1. Artificial polymeric vaccine displaying H1 antigen on their surface, H1-NB. (a, b) The

overall strategy for designing virus-mimetic structures. (a) Schematic illustration of

recombinant His6-H1. His6-tag was attached to c-terminal located in stem end. (b) The

similarity in aspects to size and orientation between influenza viruses and H1-NB. (c)

Schematic illustration of the process of H1-NB formation. (d, e) Well-defined structure of H1-

NBs regardless of H1 content. (d) TEM images indicating uniform spherical shape about all

samples; %values on images mean H1 mol%; scale bar = 100 nm. (e) Hydrodynamic volumes

and distributions of H1-NBs measured by DLS; H1 content means H1 mol% in total proteins.

(f, g) The successful control of H1 and GFP ratio on particles by adjusting protein ratio of

reactant solutions and co-existence of H1 and GFP in a particle, (f) the images of H1-NBs

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containing H1 and GFP in a single particle obtained through SRCM. (g) IH1/IGFP value drawn

from PL emission spectra and fluorescence of particles in confocal images.

Fig. 2. H1-NB promoted activation of spleen DCs in vivo. (a) C57BL/6 mice were injected

i.v. with PBS, GFP-NB containing 0 µg of H1 or H1-NB containing 7.6 µg of H1 for 1 hour.

Phagocytosis of H1-NB by DC and macrophage in the spleen was measured. (b-d) The mice

were treated with indicated reagents and the spleen were harvested 24 h after injection. The

activation markers of DCs were measured on a flow cytometry. (b) Expression levels of

CCR7 in the lineage–CD11c+ DCs are shown. (c) Expression levels of co-stimulatory

molecules and MHC class I and II in the spleen DCs. (d) Levels of intracellular IL-6, IL-12

and TNF-α production in the spleen DCs. All data are representative of the average of 6

independent samples (2 mice per experiment, total 3 independent experiments). **p < 0.01

versus other control groups.

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Fig. 3. Immunization with H1-NB promoted H1-specific immune responses. BALB/c mice

were immunized with PBS, H1, H1-NB and a combination of H1 and Alum on day 0, 15, 30.

On day 35 of immunization, spleen was harvested and splenocytes were stimulated with

MHC-I peptide IYSTVASSL or MHC-II peptide SVSSFERFEIFPK for 4 days. (a) MHC-I

peptide (left panel) and MHC-II (right panel) specific IFN-γ production was measured by

ELISPOT analysis. (b) The immunized mice were transferred IYSTVASSL-coated

splenocyte. Four hours after the transfer, spleen was harvested and IYSTVASSL-specific lysis

was measured. (c) Mean positive cells of H-2Kd/HA240–248 tetramer in CD8 T cells were

shown. (d) The serum HAI titer to PR/8 virus was assayed at 3 days after first, second and

third immunization, with a detection limit of 1:40 serum dilution. (e) Levels of Ag-specific

total IgG were measured by ELISA. All data are representative of the average of 6

independent samples (3 mice per experiment, total 2 independent experiments). *p < 0.05,

**p < 0.01.

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Fig. 4. Immunization of H1-NB protected H1N1 infection in the mouse. H1-NB were

synthesized by using H1 of A/Puerto Rico/8/34 (PR/8), which called H1’-NB. BALB/c mice

were immunized with PBS, H1’(7.6 µg) or H1’-NB containing 7.6 µg of H1‘ on day 0, 15, 30.

On day 35 of immunization, The mice were challenged with PR8.(a) Mortality rate is shown

(n = 5/group). (b) The changes of body weight were shown (c) Survival rate of PR8-infected

BABL/c mice at 12 weeks after the last immunization (n = 5/group). (d) The body weight was

measured during viral infection. (e) Survival rate of BALB/c mice after immunization with

H1 from A/Texas/2009 (H1N1) by PR8 infection (n = 5/group). (f) Body weight changes

were shown during PR8 infection. All data are the average of 5 independent samples.

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