Enhanced Humoral and Cell-Mediated Immune Responses

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Enhanced Humoral and Cell-Mediated Immune Responses Generated by Cationic Polymer-Coated PLA Microspheres with Adsorbed HBsAg Xiaoming Chen,†,‡ Yuying Liu,§ Lianyan Wang,*,† Yuan Liu,†,‡ Weifeng Zhang,†,‡ Bei Fan,∥ Xiaowei Ma,∥ Qipeng Yuan,§ Guanghui Ma,*,† and Zhiguo Su*,† †

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China ‡ Graduated University of Chinese Academy of Sciences, Beijing, 100049, PR China § College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China ∥ Hualan Biological Engineering Inc., Henan 453003, PR China ABSTRACT: Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1β, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses. KEYWORDS: PLA, microsphere, cationic polymer, HBsAg, adsorption, Th1



INTRODUCTION Hepatitis B is a potentially life-threatening disease caused by the hepatitis B virus (HBV). It is estimated that approximately 30% of the world’s population has been infected with HBV at some point in their lives, and more than 350 million people are chronically infected, serving as the viral reservoir. Moreover, about 60 million people worldwide die each year due to infection with HBV.1 Infection with HBV causes acute and chronic liver necroinflammation, which often develops into more serious conditions, such as liver cirrhosis or hepatocellular carcinoma.2 The hepatitis B vaccine, containing hepatitis B surface antigen (HBsAg), has been available since 1982 and is recommended by the World Health Organization for administration to all infants in order to control the spread of HBV. Initially, HBsAg was purified from the plasma of HBsAg carriers. However, the development of genetic engineering has allowed for the safer and easier production of yeast-derived recombinant HBsAg, which has replaced the plasma-derived antigen. Like other recombinant peptide and protein antigens, HBsAg does not elicit a robust immune response and therefore requires an adjuvant to enhance immunogenicity.3 Commercially available aluminum-adsorbed hepatitis B vaccines can © 2014 American Chemical Society

effectively stimulate humoral immunity but are poor inducers of cell-mediated immunity, which plays a key role during intracellular virus clearance.4 In addition, aluminum has the potential to cause severe local and systemic side effects, including sterile abscesses, eosinophilia, and myofascitis; fortunately, most of these serious side effects are relatively rare.5 There are also concerns about the possible role of aluminum in neurodegenerative diseases, such as Alzheimer’s disease.6 Because of these potential adverse effects of aluminum, there is urgent demand for safer and more effective adjuvants suitable for human use, particularly safe and nontoxic adjuvants available to stimulate cellular (Th1) immunity. Microspheres formulated with biodegradable polymers, such as polylactic acid (PLA) and poly(lactide-co-glycolide) (PLGA), are being extensively investigated as vaccine delivery systems due to their controlled release characteristics and biocompatibility.7−9 A great number of studies have confirmed that microsphereReceived: Revised: Accepted: Published: 1772

October 10, 2013 February 20, 2014 April 15, 2014 April 16, 2014 dx.doi.org/10.1021/mp400597z | Mol. Pharmaceutics 2014, 11, 1772−1784

Molecular Pharmaceutics



based vaccine formulations have the capacity to elicit strong cellular and humoral immune responses. For example, particulate vaccine delivery systems can facilitate the uptake of antigens by antigen-presenting cells (APCs), such as dendritic cells (DCs) or macrophages,10 and activation of DCs is enhanced by PLGA or γ-PGA-Phe nanoparticle-pulse due to upregulation of costimulatory molecule expression (CD40, CD80, CD83, CD86, MHC I, and MHC II) and cytokine release.11,12 Additionally, particle-based vaccines may serve as a depot for controlled release of antigen, thereby increasing the availability of antigens to APCs which will enhance not only the level but also the quality of immune responses.13 Particle-based adjuvants may also possess the ability to modulate induced immune responses when used alone or in combination with other immunostimulatory molecules.14 Specifically, particulate vaccines can potentially cross-present exogenous antigen, which is especially important in the generation of CD8+ T cell-mediated cell immune responses against intracellular viral infections.15 However, most microsphere-based vaccine delivery systems are dependent on the encapsulation of protein antigen or DNA into microspheres. Although novel strategies could be employed to improve the stability of the encapsulated antigen, these will inevitably expose antigens to harsh conditions, such as water−oil interfaces, shear forces, heat, etc., leading to protein instability and denaturation during the microencapsulation process.16,17 Antigen loading efficiency is always relatively low due to the washing, centrifugation, or decantation steps during microsphere preparation.18 In addition, the moisture, acidity, temperature, and unphysiological salt concentration of the microenvironment generated during the course of microsphere degradation and protein release may also cause destabilization and aggregation of the encapsulated antigens.19 Typically, antigens adsorbed on the surface of polymer microspheres represent a potential antigen display form to induce a robust immune response.20,21 High antigen loading efficiency can be achieved by surface adsorption, avoiding unfavorable conditions during preparation. Most importantly, adsorbed antigen carriers make the vaccine ready to use, like the aluminum vaccine. Several studies have demonstrated that antigen-adsorbed microspheres are as immunogenic as encapsulated antigens.22,23 Interestingly, compared with neutral microspheres, surface-charged microspheres are more conducive to adsorbing antigen, especially positively charged microspheres, which enhance microsphere interactions with the negatively charged cell membrane and promote antigen uptake by APCs.24,25 Unfortunately, PLA or PLGA microspheres prepared by conventional methods are always negatively charged, which limits surface antigen adsorption. Polycation polymer coating modification is a potential strategy to build cationic microspheres for vaccine delivery.26 Chitosan (CS), polylysine, and polyethylenimine (PEI) are the most widely used cationic polymers for coating. Recent studies have also confirmed the capacity of cationic polymers, like PEI and polylysine, to induce the Th1 cell immune response.27 Numerous studies have focused on the delivery of DNA vaccines using coated cationic microspheres; however, few studies have investigated recombinant subunit antigens in this context. The present investigation was therefore aimed at developing PLA microspheres coated with cationic polymers and having surface-adsorbed HBsAg and evaluating the ability of these microspheres to stimulate humoral and cell-mediated immune responses after immunization in BALB/c mice.

Article

EXPERIMENTAL DETAILS

Materials and Methods. Materials. Polylactic acid (molecular weight [MW], 10 000 Da) was obtained from Shandong Institute of Medical Instrument (Jinan, China). CS (MW, 50 000 Da) and chitosan chloride (CSC; MW, 57 000 Da) were obtained from Xiamen Blue Bay Science & Technology Co., Ltd. (Xiamen, China). PEI (MW, 25 000 Da) was obtained from Sigma (St. Louis, MO, USA), and poly(vinyl alcohol) (PVA-217) was obtained from Kuraray (Japan). Dichloromethane was obtained from Sinopharm Chemical Reagent Beijing Co., Ltd., and recombinant HBsAg, derived from Hansenula polymorpha (320 μg/mL in 6.2 mM PBS buffer), and the aluminum adjuvant (aluminum hydroxide gel) were kindly supplied by Hualan Biological Engineering Incorporation (Henan, China). The Microbicinchoninic acid protein assay (Micro BCA) kit was obtained from Thermo Fisher Scientific Inc. (USA), SPG membranes were purchased from SPG Technology Co. Ltd. (Japan), and Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were ordered from Hyclone (USA). All other reagents were analytical pure. Microparticles Preparation and Characterization. Preparation of PLA and Surface-Coated Microspheres. PLA microspheres were prepared by a method combining SPG premix membrane emulsification and emulsion−solvent evaporation, as described previously,28 with slight modifications. In brief, 10 mL of 4% (w/v) PLA solution in dichloromethane was added to a 1.5% (w/v) PVA aqueous solution to prepare the primary emulsion using low-speed magnetic stirring. The solution was then poured into the premix reservoir. Subsequently, nanodroplets were achieved by extruding the coarse emulsions through the membrane pores under a pressure of 0.75 MPa. The ultimate emulsions were stirred and solidified overnight for the evaporation of volatile organic solvent. Formed PLA microspheres were washed 3 times using ultrapure water and collected by centrifugation (4000g for 5 min) and lyophilization. For the preparation of cationic polymer-coated PLA microspheres, lyophilized PLA microspheres were dispersed into a 1% (w/v) CS solution (acetate buffer, pH 4.5), 1% (w/v) CSC solution (acetate buffer, pH 4.5), or 1% (w/v) PEI solution (ultrapure water) and incubated for 4 h at room temperature in a shaking incubator. Coated cationic PLA microspheres were washed 3 times using ultrapure water to remove the unattached polymers, followed by centrifugation (4000g for 3 min) and lyophilization. Characterization of Microspheres. The surface morphology of PLA microspheres was observed by scanning electron microscopy (SEM) using a JEM-6700F (JEOL, Japan). Samples for SEM were prepared by spreading a suspension of the microspheres on a flat aluminum stub. After allowing the suspension to dry naturally, the stub was coated with gold using a sputter coater. The samples were then randomly scanned and photographed. The average size, surface charge, and polydispersity index (PDI) of PLA microspheres and surfacemodified CS-PLA, CSC-PLA, and PEI-PLA microspheres were measured by dynamic light scattering (DLS) using a computerized inspection system (Malvern UK, Malvern Zetasizer Nano ZS). For the measurement of surface charge, microspheres were dispersed in phosphate-buffered saline (PBS, 6.2 mM, pH 6.5). 1773

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HBsAg Adsorption on Microspheres. 500 μL of HBsAg (80 μg/mL in PBS, pH 6.5) was incubated with 500 μL of the different microsphere suspensions (10 mg/mL in PBS, pH 6.5) overnight at 25 °C in a shaking incubator. After incubation, the solutions were centrifuged, and the antigen concentration in the supernatant was analyzed using a bicinchoninic acid (BCA) protein assay. Antigen adsorption efficiency was calculated as follows:

DND-99 (Invitrogen, USA) for 30 min. After washing 3 times with sterile PBS, the cells were fixed with formaldehyde for 1 h and then washed 3 times with PBS. Cell membranes were stained with Alexa Fluor 635 Phalloidin (Invitrogen, USA) at 37 °C for 30 min. Images of the corresponding fluorescent cells were obtained using a Leica TCS SP5 confocal laser scanning microscope (CLSM, Leica). A representative cell was selected at random, and captured images were overlaid to determine localization and colocalization of fluorescent HBsAg within macrophages. Expression of MHC I, MHC II, and CD86 Molecules on Macrophages. Expression of the surface molecules MHC I, MHC II, and CD86 was analyzed by flow cytometry after incubation of macrophages with antigen suspension, microsphere-adsorbed antigen formulations, or aluminum-adsorbed vaccine. After incubation, the cells were washed and incubated with APC mouse anti-MHC class II antibodies, PE mouse antiCD80 antibodies, and FITC-labeled mouse anti-CD86 antibodies (eBioscience, CA) for 30 min followed by washing. Cells were then analyzed using a CyAnTM ADP flow cytometer (Beckman Coulter, USA). Animals and Immunization. Animals. Female BALB/c mice (4−6 weeks old) were purchased from Vital River Laboratories (Beijing, China). The mice were monitored to be pathogen-free, and all animals were treated according to the regulations of Chinese law and the local Ethical Committee. Briefly, throughout the study, animals were housed in climatecontrolled (23 ± 2 °C; relative humidity: 60%) and photoperiod-controlled (12 h light−dark cycles) animal quarters. Mice were fed standard pelleted rodent chow supplemented with grain and had free access to drinking water. The study included 7 groups with 6 animals in each group, as follows: control mice, mice treated with free HBsAg, mice treated with aluminum-adsorbed vaccine, and mice treated with suspensions of PLA, CS-PLA, CSC-PLA, and PEI-PLA microspheres harboring surface-adsorbed antigen. A two-step immunization regimen (“prime and boost”) was applied; each animal was treated with 4 μg of antigen on days 0 and 28 by intraperitoneal (I.P.) injection. Sera and Spleen Cell Culture Supernatant Collection. Blood samples were collected from the retro-orbital plexus of mice on days 7, 15, 28, 35, and 42 after prime immunization. Sera were collected by centrifugation (10,000 rpm for 10 min) and stored at −80 °C until analysis. On day 42 after immunization, mice were sacrificed, and spleens were aseptically removed to prepare cell suspensions by gently grinding the spleen on a fine wire screen. Red blood cells in the suspensions were removed using ACK Lysis Buffer (Beijing CellChip Biotechnology Co., Ltd.), followed by washing and centrifugation. Cell numbers were diluted to 3 × 105 cells/mL in RPMI 1640 medium containing 10% FBS and 1% penicillin−streptomycin. Cell culture supernatants were collected after a 60-h incubation with 5 μg/mL HBsAg. Determination of Serum IgM, IgG, IgG1, and IgG2a. Serum HBsAg-specific IgM (day 7) and IgG antibody levels were determined by indirect ELISA on days 7, 15, 28, 35, and 42 as described previously,29 with minor modifications. Briefly, 96-well microtiter plates (Corning, USA) were coated with 100 μL of HBsAg (5 μg/mL in 50 mM carbonate buffer, pH 9.6). The plates were incubated at 4 °C in moist chambers overnight and then washed 3 times with PBS-T (0.01% v/v Tween-20 in PBS) to remove the unbound antigen. The unbound free sites of each well were further blocked with 200 μL of 2% w/v

AE (%) = (mt − ms)/mt × 100

where AE is the adsorption efficiency, mt is the total amount of antigen added to the system, and ms is the amount of antigen in the supernatant. Isolation and Culture of Mouse Peritoneal Macrophages. Mouse peritoneal macrophages were harvested from 6−8-weekold euthanized BALB/c mice by lavage of their peritoneal cavity with 5 mL of ice-cold PBS (with 3% fetal calf serum (FCS)). The isolated cells were centrifuged at 400g for 5 min and then resuspended in culture medium to a concentration of 5 × 105 cells/mL. Cell suspensions were then added to 24-well plates and cultured in a humidified atmosphere (37 °C, 5% CO2) for cell adhesion. Nonadherent cells were removed by washing with culture medium after 2 h. Prepared macrophages were then applied for antigen uptake and APC activation studies in vitro. Macrophage Viability upon Coincubation with Microspheres. Colorimetry-based CCK-8 assays were used to assess the viability of macrophages upon exposure to PLA microspheres or cationic polymer-coated CS-PLA, CSC-PLA, and PEI-PLA microspheres. In this assay, water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to generate a yellow formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. Cell viability was tested in 96-well plates. Macrophages were plated at a density of 2.5 × 105 cells/ well and incubated with serial dilutions of microspheres (0− 210 μg/mL) for 36 h. After incubation, WST-8 reagent was added into each well, and the absorbance of formazan dye was measured at 450 nm using an Infinite M200 microplate spectrophotometer (Tecan, USA), with a reference absorbance at 600 nm. Viability was calculated according to the manufacturer’s instructions, and each experiment was performed in triplicate. Antigen Uptake by Macrophages. Uptake. FITClabeled HBsAg was used to investigate antigen uptake behaviors by macrophages in vitro using flow cytometry. Macrophages (5 × 105/mL) were seeded into 24-well plates for 12 h at 37 °C in an atmosphere containing 5% CO2 to obtain firmly adhered cells. 20 μL of antigen suspension, microsphere-adsorbed antigen formulations, or aluminum-adsorbed vaccine were added into each well in triplicate, and incubation was continued for an additional 24 h. After incubation, cells were collected by centrifugation, and noningested antigens and microspheres were washed off with cold PBS. Cells were fixed with 3.7% paraformaldehyde, and antigen uptake was assessed by determining the mean fluorescence intensity of HBsAg-positive macrophages. Antigen internalization into macrophages was also observed by confocal microscopy as follows. Petri dishes were precoated with 0.01% poly-L-lysine for 4 h, air-dried, and washed with DMEM 3 times. Then, 4 × 105 cells/mL were seeded into 24-well plates and treated as previously described. After incubation, cells were stained with Lyso Tracker Red 1774

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bovine serum albumin (BSA) in PBS-T (blocking buffer) for 2 h at 37 °C, and wells were then washed 3 times with PBS-T. For the measurement of IgM, 100 μL serum samples at 1:400 dilutions in diluent was added to the wells in duplicate and incubated for 1 h at 37 °C. Plates were washed 5 times with PBS-T, and 100 μL of horseradish peroxidase (HRP)conjugated goat antimouse IgM (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added at a 1:6000 dilution. After a 1-h incubation, the plates were washed 5 times and developed with 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB, in citrate phosphate buffer, containing H2O2) in the dark for 10−20 min at room temperature. The reaction was stopped by adding 50 μL of 2 M H2SO4 to all wells. The color produced by the reaction was measured at 450 nm using an Infinite M200 microplate spectrophotometer with a reference absorbance at 620 nm. For the measurement of IgG, IgG1, and IgG2a, serial dilutions of each serum sample (100 μL/well) from individual mice were tested in duplicate, starting from a 1:100 dilution in prepared diluent. Other steps were similar to those described above, and HRP-conjugated goat antimouse IgG, IgG1, and IgG2a (1:6000, 1:8000, and 1:8000 dilutions, respectively) were used. Enzyme-linked immunosorbent assay (ELISA) titers were expressed as the maximum dilution ratio whose absorbance was 2.1 times more than that of the control serum (dilution at 1:100). Cytokine Determination and ELISPOT Assay for CytokineProducing T Cells. Levels of the cytokines interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in cell culture supernatants of macrophages and spleen cells were measured using Platinum ELISA Kits (eBioscience) according to the manufacturer’s protocols. For the ELISPOT assay, 96-well polyvinylidene difluoride (PVDF)-backed plates (Mabtech, SE) were presoaked using 75% (v/v) ethanol followed by washing with sterile water. Plates were then coated with antibodies targeting IL-2, IL-4, IL10, and IFN-γ and incubated overnight at 4 °C. After incubation, plates were washed 3 times with PBS and then blocked with RPMI-1640 medium containing 10% FBS. Cells were cultured at a density of 3 × 105 cells/well in 0.1 mL medium for restimulation with HBsAg for 36 h (37 °C, 5% CO2). After incubation, spots on each plate were detected following the manufacturer’s instructions, and results were collected using a ChanmpSpot Elispot II system (Sagecreation, Beijing, China). Statistical Analysis. All experiments were performed in triplicate, and the data were presented as the mean ± SD. Statistical differences between groups were determined by oneway analysis of variance (ANOVA) and Tukey posthoc tests. Pvalues of less than 0.05 were considered statistically significant.

Table 1. Characterization of PLA Microspheres microspheres PLA CS-PLA CSC-PLA PEI-PLA

size (nm) 802.05 823.29 825.01 835.22

± ± ± ±

5.2 3.7 2.5 3.4

PDI

zeta potential (mV)

0.112 0.136 0.201 0.175

−17.087 ± 3.2 +3.998 ± 2.7 +16.182 ± 2.1 +33.534 ± 1.9

microspheres showed gradually increasing positive charges (+3.998 mV, +16.182 mV, and +33.134 mV, respectively) due to the polycation polymer coating of the PLA microsphere surface. To determine whether the positive charge of these cationcoated microspheres may be toxic, we next assessed the viability of macrophages exposed to PLA microspheres and surfacemodified PLA microspheres in vitro. All treatment groups retained more than 95% viability (Figure 1C), suggesting that surface-modified microspheres (up to a concentration of 210 μg/mL) exhibited negligible cytotoxicity in macrophages. The low cytotoxicity of CS-PLA, CSC-PLA, and PEI-PLA microspheres may be related to the reduced amount of polycation polymers adsorbed on the PLA microsphere core, and the biocompatibilities of CS and PEI have been well documented in previous studies.30 These data suggested that our prepared cationic polymer-coated PLA microspheres may be safe to use during vaccine delivery. Characterization of Antigen Adsorption on Microspheres. HBsAg adsorption by aluminum adjuvant and microspheres was investigated by Micro BCA and flow cytometry, as described in Figure 2. Antigen adsorption efficiency was enhanced with the increased positive surface charge of the microspheres. PEI-PLA microspheres, having the highest surface charge, exhibited the highest antigen adsorption capacity, similar to that of aluminum hydroxide (95%), followed by CSC-PLA and CS-PLA microspheres. PLA microspheres only adsorbed less than 5% of the antigen (Figure 2A). A similar trend of antigen adsorption by microspheres was also obtained by flow cytometry using FITC-labeled HBsAg, which showed an enhanced fluorescence intensity of the microspheres with increased microsphere surface charges (Figure 2B and C). These results indicated that the electrostatic attraction between HBsAg and microspheres was the predominant adsorption force. Since the isoelectric point of HBsAg is 4.7, positively charged microspheres is more favorable to adsorption of negatively charged HBsAg in PBS buffer at pH of 6.5. Unmodified PLA microspheres exhibited the lowest antigen adsorption due to the weak interactions between negatively charged antigen and negatively charged microspheres. Our results demonstrated that electrostatic interactions play a key role during microsphere adsorption of HBsAg. Positively charged microspheres with adsorbed antigen may be potential carriers for antigen delivery. Evaluation of HBsAg Internalization and Intracellular Distribution in Vitro. A key requirement for the induction of immune responses is the internalization of antigen into APCs. DCs and macrophages are the primary APCs circulating in the body and function to sense pathogens and antigens. Therefore, we next isolated mouse peritoneal macrophages and investigated antigen uptake in vitro. To evaluate the uptake efficiency of antigen, FITC-labeled HBsAg was adsorbed with aluminum or microspheres. Peritoneal macrophages were incubated with free antigen or antigen−adjuvant formulations for 24 h. After extensive



RESULTS Characterization of PLA, CS-PLA, CSC-PLA, and PEIPLA Microspheres. The sizes and surface charges of PLA, CSPLA, CSC-PLA, and PEI-PLA microspheres were studied as shown in Table 1 and Figure 1. Surface-modified CS-PLA, CSC-PLA, and PEI-PLA microspheres showed size distributions and surface morphologies similar to those of PLA microspheres, suggesting that polycation surface modification had no influence on the size, dispersion, and morphology of microspheres (Figure 1A, B, and D). All microspheres were about 800 nm in size but differed in their surface charges. PLA microspheres showed a relatively negative surface charge (−17.087 mV), while CS-PLA, CSC-PLA, and PEI-PLA 1775

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Figure 1. Characterization of prepared microspheres. (A) Scanning electron micrographs of PLA and surface-modified PLA microspheres. A1, PLA; A2, chitosan-coated PLA microspheres (CS-PLA); A3, chitosan chloride-coated PLA microspheres (CSC-PLA); and A4, polyethylenimine-coated PLA microspheres (PEI−PLA). (B and D) Dynamic light scattering zetasizer analysis of microsphere size distribution and surface charge. (C) CCk-8 assay of macrophage viability after incubation with different concentrations of microspheres for 36 h.

Figure 2. Antigen adsorption characterization. (A) MicroBCA assay of HBsAg adsorption by microspheres and aluminum adjuvant. HBsAg (40 μg/ mL) was incubated with 5 mg/mL microspheres or aluminum hydroxide for 12 h, followed by centrifugation. Adsorption efficiency was calculated by comparing the amount of antigen in supernatants with total input antigen. The load of HBsAg/particles (μg/mg) was 0.24, 3.2, 8, and 9.2, respectively, for PLA, CS-PLA, CSC-PLA, and PEI−PLA. (B and C) Flow cytometry of antigen adsorption by microspheres. FITC-labeled HBsAg was used, and the antigen adsorption capacity is shown, based on the fluorescence intensity of different microspheres.

aluminum-adsorbed antigen, while surface-modified microsphere adjuvant formulations facilitated phagocytic antigen localization independent of the lysosome, particularly in the case of PEI-PLA microspheres (Figure 4). This would be expected to afford antigens more opportunities to be degraded in the cytoplasm. Additionally, lysosome localization is always accompanied by antigen degradation and presentation via MHC II, while cytoplasmic degradation, mainly in the proteasome, may promote antigen cross-presentation via MHC I, thereby enhancing cellular immune responses.31 In conclusion, these results suggest that the higher positive surface charge of microspheres favored antigen adsorption and

washing, antigen internalization into macrophages was determined by the percentage of CD11b+ fluorescence-positive cells. As shown in Figure 3A, compared with free HBsAg, the addition of adjuvant (aluminum or microspheres) significantly enhanced antigen internalization, especially PEI-PLA microspheres, which showed a 2-fold increase in fluorescence intensity compared to that of aluminum and a 10-fold increase compared to that of free antigen. Next, CLSM was used to visualize the internalization of HBsAg within the cells (Figure 3B−G). Results indicated that most of the phagocytic antigens were intracellularly localized (green fluorescent). Moreover, antigen mostly colocalized with lysosomes (red florescence) in cells exposed to free antigen and 1776

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Figure 3. Antigen uptake by macrophages in vitro. Different vaccine formulations containing FITC-labeled HBsAg were incubated with mouse peritoneal macrophages for 24 h. (A) Quantitative analysis of HBsAg+ macrophages by flow cytometry. Results are shown as the mean fluorescence intensity of HBsAg+ cells. (B−G) Confocal images of HBsAg phagocytic macrophages. Cell membranes were stained red, and green fluorescence represents HBsAg. (B) HBsAg, (C) aluminum-HBsAg, (D) PLA-HBsAg, (E) CS-PLA-HBsAg, (F) CSC-PLA-HBsAg, and (G) PEI-PLA-HBsAg. Results confirmed that the antigen localized within the cells but was not attached to the cell surface. ***p < 0.001.

antigen. The secretion of these cytokines in cells treated with microspheres was mostly related to the microsphere surface charge and antigen adsorption capacity, i.e., the greater the antigen adsorption, the higher is the level of cytokines release. Overall, this result may suggest that positively charged microspheres adsorbed with surface antigen favored the activation of macrophages. Serum Anti-HBsAg Antibody Response after Vaccination in Mice. The systemic immune response was studied by measuring HBsAg-specific serum antibody levels with indirect ELISA. Our results suggested that the association of HBsAg with microspheres induced a strong, rapid humoral antibody response compared with free antigen and aluminumadsorbed vaccine, as measured by changes in IgM and IgG titers, which vary during the different stages of the immune response. On day 7 after primary immunization, administration of microsphere−antigen groups generated higher levels of IgM than free antigen and aluminum vaccine (Figure 6A; PLA and PEI-PLA vs aluminum, P < 0.05). In contrast, there were no significant differences in IgM titers among the 4 microspherebased formulations. On day 7, IgG titers were too low to detect, except for CS-PLA and CSC-PLA microsphere formulations (Figure 6B). On day 15, most of the vaccine formulations had detectable IgG titers, and until day 28, mice immunized with free HBsAg showed low levels of HBsAg-specific antibodies. After booster immunization on day 28, IgG titers in all groups reached plateau levels on day 35. All of the microsphereadsorbed vaccines exhibited relatively high IgG levels comparable with that of the aluminum vaccine on days 28, 35, and 42, indicating that PLA microspheres modified with cationic polymers induced enhanced humoral immune response than that of aluminum. The ratio of IgG2a/IgG1 antibody titers on day 42 was also measured, as shown in Figure 6C. Results indicated that aluminum tended to induce

internalization into APCs, potentially improving cell-mediated immunity. Cytokine Secretion and Surface Molecular Expression in Macrophages Incubated with Different Vaccine Formulations. After internalization by macrophages, antigens are processed and subsequently presented, a process that is accompanied by augmented cell surface molecule expression. MHC I, MHC II, and CD86 are important surface molecules related to macrophage activation and antigen presentation. Therefore, we next investigated the surface expression of these markers on macrophages incubated with free or adjuvantadsorbed HBsAg (Figure 5A−C). Compared to macrophages treated with free antigen or aluminum hydroxide-adsorbed antigen, macrophages treated with microsphere−antigen formulations showed marked increases in MHC I, MHC II, and CD86 expression, especially following exposure to CSCPLA and PEI-PLA microspheres. These results were consistent with a study by Ma et al.,32 who also found that liposomes with higher surface charges exhibit elevated expression of costimulatory molecules (i.e., CD86 and CD83) on the APC surface. Surprisingly, the expression of MHC I and MHC II on macrophages treated with aluminum-HBsAg showed a slight decrease compared with free HBsAg. Ulanova et al.33 demonstrated that the aluminum adjuvant had no effect on the expression of MHC II molecules on human monocytes and that the enhanced immune response of aluminum adjuvant is mostly mediated by the NLRP3 inflammasome.34 Macrophages can also produce inflammatory cytokines during the process of phagocytosis and activation. In this study, the activation of macrophages was further determined by measuring the secretion of IL-1β, IL-6, TNF-α, and IL-12 in the supernatant. As shown in Figure 5D−G, microsphere−antigentreated cells generated significantly high levels of cytokines compared to those treated with aluminum vaccine and free 1777

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Figure 4. Antigen distribution within macrophages. After incubation with different vaccine formulations, macrophages were stained with LysoTracker Red DND-99 to label lysosomes and were fixed. A representative cell after treatment with dye was selected at random, and a series of optical sections (z-sections) were taken in dual filter mode. Images captured in bright-field, DND, FITC, and trinal modes were overlaid to determine localization and colocalization of fluorescent antigens.

Cytokine-Secreting T-Cell Response. The ELISPOT assay was used to determine the number of spleen T cells secreting IL-2, IFN-γ, IL-4, and IL-10 upon restimulation with HBsAg in vitro. As shown in Figure 8, mice immunized with CS-PLA-HBsAg, CSC-PLA-HBsAg, and PEI-PLA-HBsAg generated 2-fold or more increases in the number of IL-2 ELISPOTs than the aluminum vaccine, consistent with the previous results. Since IL-2 is considered a typical Th1-type cytokine, our results indicated that positively charged CS-PLA, CSC-PLA, and PEI-PLA microspheres could generate enhanced Th1-type immune responses. Similar to the IL-2 ELISPOTs data, immunization with the CSC-PLA and PEIPLA microsphere−antigen formulations generated a significantly greater number of IFN-γ SPOTs than immunization with the aluminum vaccine, indicating an enhanced cell-mediated immune response since the IFN-γ ELISPOT assay has been used as a surrogate assay to quantify functional CD8+ T-cell responses against pathogens.35 In contrast, the aluminumadsorbed vaccine mostly induced IL-4- and IL-10-secreting T cells (Figure 8C,D) compared with microsphere-based formulations. This result also confirmed that positively charged PLA microspheres could generate elevated cellular immune responses, while the aluminum adjuvant mainly induced humoral-dominated immune responses.

predominantly IgG1, while microsphere adjuvants induced a mixture of IgG2a-IgG1 antibody responses, with a predominance of IgG2a. Together, these results suggested that cationic polymer-coated microspheres with antigen adsorbed on their surfaces induced a rapid, efficient, antigen-specific antibody response compared with that of free antigen and aluminumadsorbed vaccine. Cytokine Release ex Vivo. Studies were also carried out to evaluate the cytokine secretion patterns in cultured spleen cell supernatants of immunized animals. Consistent with the previous data, microsphere-based vaccine formulations, especially CSC- and PEI-coated microspheres, induced an increase in the release of Th1-type cytokines (IL-2, IL-12, and IFN-γ) as compared to that of the aluminum vaccine (Figure 7C−E). The expression of these cytokines in spleen cells from mice immunized with the microsphere formulations was related to the microsphere surface charge and HBsAg adsorption capacity, suggesting that the relatively high surface charge and antigen adsorption promoted Th1 cytokine release. For the secretion of Th2-type cytokine (IL-4 and IL-10), aluminum-adsorbed vaccine induced higher levels of IL-4 and IL-10 compared with those of PLA and CS-PLA. There was no significant difference in Th2 cytokine release among mice immunized with aluminum, CSC-PLA, and PEI-PLA, which may be related to their analogous HBsAg adsorption capacity. This result indicated that positively charged microspheres with high antigen adsorption capacities could induce the release of both Th1 and Th2 cytokines. 1778

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Figure 5. Surface molecule expression (A−C) and cytokine release (D−G) after treatment of macrophages with different vaccine formulations. MHC I, MHC II, and CD86 were detected by flow cytometry with specific fluorescently labeled antibodies. Cytokine release was measured using ELISA kits. Values are the mean ± SD of data from triplicate experiments. *p < 0.05; **p < 0.01; and ***p < 0.001.



DISCUSSION In this study, we present the first systematic in vitro and in vivo analysis of the immune responses elicited by PLA microspheres coated with the cationic polymers CS, CSC, and PEI and having surface adsorbed HBsAg. Our data demonstrated that modified positively charged PLA microspheres were potent delivery systems for recombinant subunit protein vaccines, which could induce significantly enhanced humoral and cellmediated immune responses after I.P. immunization. Importantly, the commercially available aluminum-based prophylactic hepatitis B vaccine induces an antibody response that is consistent with our results as described above. The application of cationic polymer-coated PLA microspheres not only generated an increase in the HBsAg-specific antibody response but also enhanced the release of multiple cytokines, indicating that a variety of specific and polyclonal T-cell responses which play a pivotal role during virus clearance of acutely infected individuals.36 Thus, our data strongly suggested that positively charged PLA microspheres with cationic polymer surface modification were potential vaccine delivery systems for recombinant antigens. PLA- and PLGA-based polymer microspheres and nanoparticles have been extensively studied as drug and vaccine delivery systems due to their excellent properties of degradability, biocompatibility, and controlled release of encapsulated molecules. However, the lack of functional groups

on the surface of PLA microspheres has limited their potential for the surface attachment of bioactive molecules, such as DNA, ligands,37 or vaccines.38 Various attempts have been made to physically modify the surface of PLA or PLGA microspheres using surfactants or polymers. Among these, polycation coating to create microspheres with a positive surface charge has been considered a potential modification method.39,40 The cell membrane takes on a negative charge, allowing for efficient interaction with positively charged microspheres, thereby facilitating phagocytosis. Cationic microspheres have been widely investigated as gene delivery systems due to the electrostatic interactions between positively charged microparticles and negatively charged DNA.21,41,42 When developing a vaccine formulation, safety is one of the most important aspects to be considered. As with other cationic polymers, the relatively strong positive charge of CS and PEI may cause cytotoxicity. However, the amount of cationic polymers can be substantially decreased by using chitosancoated PLA nanoparticles because only a small amount of CS is expected to coat the surface of a particle. This is particularly true due to the extensive washing we performed after the coating process, removing the unattached or low-affinity polymers from the microsphere surface. The zeta potentials of our prepared CS-PLA and PEI-PLA microspheres were +3.9 and +33.1 mV, respectively, while others have reported values of about +55 and +48 mV;43 this may indicate that a relatively 1779

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Figure 6. Serum antibody measurement using indirect ELISA. (A) Serum anti-HBsAg IgM levels in mice immunized with different formulations on day 7 after primary immunization. The absorbance value of each serum sample was measured. (B) Serum anti-HBsAg IgG levels in mice immunized with different formulations on days 7, 15, 28, 35, and 42. (C) The ratio of serum anti-HBsAg IgG1 and IgG2a levels in mice immunized with different formulations on day 42. Values are the mean ± SD of data from 6 animals. **p < 0.01; and ***p < 0.001.

Figure 7. Release of IL-2, IL-4, IL-10, IL-12, and IFN-γ cytokines by spleen cells from animals immunized with different formulations after incubation in the presence of HBsAg for 60 h in vitro. Results are shown as the mean ± SD (n = 8). Data are from an experiment with n = 6 mice per group. *p < 0.05; **p < 0.01; and ***p < 0.001.

more positive the charge, the higher is the HBsAg adsorption capacity, as confirmed by quantitative micro-BCA assay and flow cytometry measurements. PLA microspheres only adsorbed less than 5% of the available HBsAg. PEI-PLA microspheres, having the highest positive charge, could adsorb more than 90% of the available HBsAg, similar to the aluminum adjuvant. Since increasing or decreasing the ionic strength of PBS had no effect on the antigen adsorption efficiency (data

small amount of polymers were adhered on the microsphere surface in our study. The low cytotoxicity of these coated microspheres was confirmed by a CCK-8 assay, also supporting the above hypothesis. PLA microspheres modified with different cationic polymers (CS-PLA, CSC-PLA, and PEI-PLA) showed different positive charges. The antigen adsorption capacity of these microspheres was correlated with the microsphere surface charge in that the 1780

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Figure 8. ELISPOT assay for the detection of cytokine (IL-2, IL-4, IL-10, and IFN-γ) release from spleen T cells from immunized mice after incubation in the presence of HBsAg. The results are expressed as the mean ± SD spot-forming cells (SFCs) per 3 × 105 spleen cells, n = 6 mice per group. *p < 0.05; **p < 0.01; and ***p < 0.001.

not show), the electronic interactions between coated microspheres and HBsAg likely governed the adsorption process. Antigen phagocytosis by APCs is crucial for generating potent immune responses. Macrophages and DCs are the most important professional APCs in the body. Additionally, microsphere delivery systems are known to enhance antigen uptake by APCs,44 and the higher positive surface charge further increases antigen internalization into macrophages due to higher antigen loading efficiencies and stronger interactions between microspheres and macrophages. Our data showed that microsphere adjuvants significantly enhanced antigen uptake by macrophages, especially PEI-PLA microspheres. Moreover, CLSM imaging confirmed that these antigens were localized within cells rather than adhered on the cell surface. Internalization by APCs would promote antigen processing and presentation. APCs upregulate costimulatory molecules, such as CD80, CD86, MHC I, and MHC II, and, accompanied by antigen presentation, subsequently induce T-cell activation.45 Our results showed that the expression levels of MHC I and CD86 in macrophages treated with CSC-PLA and PEI-PLA microspheres were significantly higher than those in macrophages treated with the aluminum vaccine. MHC I is involved in cross-presentation of exogenous antigen peptide, while CD86 provides costimulatory signals necessary for T-cell activation and survival. Our data suggested that CSC-PLA and PEI-PLA microspheres could potentially promote HBsAg presentation via the MHC I pathway and enhanced CD8+ T cell-mediated immune responses. Antigen distribution within macrophages further confirmed this tendency. After uptake, some of the antigen was localized independent of the lysosome, facilitating antigen processing by the proteasome and crosspresentation via the MHC I pathway. Previous studies have demonstrated that enhanced phagocytosis of antigen-containing microspheres would lead to overloading of phagocytic vesicles, allowing some of the antigen to access the cytoplasm, where it could associate with MHC class I molecules.46

In this study, we observed relatively high expression of MHC II in all groups; this may indicate that these macrophages activated their antigen-presenting function.47 Macrophages secrete certain pro-inflammatory cytokines after activation in order to recruit more APCs and effector cells, generating a potent immune response to clear the infections pathogens.48 We found that incubation of antigen-adsorbed microspheres with macrophages induced significantly higher levels of IL-1β, IL-6, TNF-α, and IL-12 than that induced by the aluminum vaccine. The expression of these cytokines increased with the positive surface charge and antigen adsorption capacity of the microspheres, and treatment with cationic microspheres induced high levels of IL-12 expression, especially for CSCPLA and PEI-PLA microspheres. Since IL-12 is one of the cytokines involved in the differentiation of naive T cells into Th1 cells,49 these data also indicated the potential of cationic polymer-coated PLA microspheres to augment the Th1 immune response. IgM is by far the largest antibody in the circulatory system and accounts for 5%−10% of the total serum immunoglobulin. It is the first immunoglobulin to be generated in response to initial exposure to antigen. IgM is also the first antibody to be synthesized by neonates. Because of its high valency, pentameric IgM is more efficient than other immunoglobulin in binding antigens with many repeating epitopes, such as viral microspheres.50 In our study, all of the microsphere-immunized groups generated comparable levels of IgM and IgG compared with aluminum vaccine-immunized mice, suggesting the potential of using PLA microspheres coated with cationic polymers as antigen delivery carriers. CS-PLA microspheres induced the highest IgG but relatively low MHC II molecule expression, which may relate to the proper antigen adsorption capacity (neither too low nor too high) that facilitates antigen uptake and presentation. Although previous studies have confirmed that HBsAg-containing PLGA microspheres could induce efficient humoral immune responses,51 cationic micro1781

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our modified microspheres in phagosomes could lower phagosomal acidification, while the hydrophobic PLA core engulfed in tightly fitting phagosomes inhibiting phagosomal maturation, both of which may permit phagocytic antigens to access the cytoplasm and promote cross-presentation via the MHC I pathway, thereby augmenting the Th1 immune response. In conclusion, the present study revealed that surface modification of PLA microspheres with cationic polymers could be a useful strategy for effective antigen adsorption, enhanced antigen uptake by APCs, and augmented humoral and cellular immune responses. Specifically, HBsAg formulated with cationic PLA microspheres could facilitate antigen localization within the cytoplasm and present via the MHC-I pathway enhancing cell-mediated immune response. These results strongly suggested that positively charged PLA microspheres with cationic polymer modification were potential vaccine delivery systems for recombinant antigens. Further investigations are needed to uncover the specific mechanisms of these processes in the future.

spheres having HBsAg adhered on their surfaces may allow for enhanced availability of antigen to APCs immediately after intracellular uptake of microspheres, helping to induce a powerful immune response.52 HBV is an intracellular infectious virus whose clearance depends on vigorous and efficient multifunctional T-cell responses as follows:53,54 (1) CD4+ T cells serve as master regulators of the adaptive immune response to HBV; (2) CD8+ T cells are the key cellular effectors mediating HBV clearance; (3) CD4+ T cells are required to mount a CD8+ T-cell response to HBV; and (4) viral clearance during HBV infection is associated with the entry of CD8+ T cells into the liver, the production of IFN-γ, and the induction of inflammation. Effector T-cell responses can be observed by the ELISPOT assay and the release of cytokines, such as IFN-γ, IL-2, IL-12, IL-4, and IL-10.55 IFN-γ and IL-2 are Th1-type cytokines, reflecting the cellular immune response, while IL-4 and IL-10 belong to Th2-type cytokines, mediating the humoral immune response. Our results indicated that CSC-PLA and PEI-PLA microspheres induced an increase in the number of T cells releasing IFN-γ and IL-2, while the aluminum vaccine augmented the number of IL-4- and IL-10-producing T cells. Moreover, immunization with CSC-PLA and PEI-PLA microspheres also induced high levels of IL-12, which would promote the polarization of naive T cells into Th1 cells. Together, these results indicated that cationic polymer-coated PLA microspheres with antigen adsorbed on the surface could induce equilibrated Th1/Th2 responses, as shown by higher levels of antigen-specific antibodies and by increased release of multifunctional cytokines. The mechanism through which antigen adsorption by polycation-coated PLA microspheres generate enhanced immune responses, especially Th1 cell responses, is currently unclear. We believe that efficient delivery of the adsorbed antigen to APCs and their localization independent of lysosome within the cells may be important contributing factors and that polycations themselves may stimulate activation of DCs, directing the presentation of antigens and generating T cell help at the same time.26,56 Manmohan et al.21 have demonstrated that efficient delivery of cationic microspheres with adsorbed DNA to APCs induced potent cytotoxic T lymphocyte responses at a low dose, while coadministration of DNA with microspheres (i.e., not adsorbed) does not induce a similar effect. This is consistent with our results demonstrating that antigens formulated with negatively charged PLA microspheres showed lower immunogenicity than polycation polymer-coated decorated PLA microspheres, which induced a potent Th1/Th2 immune response. Chen et al.27 confirmed that cationic polymers, such as PEI, polylysine, and cationic dextran, could increase the percentage of CD4+ T cells in the spleen and induce high expression of IFN-γ and TNF-α, suggesting that cationic polymers strongly stimulated Th1 responses through TLR-4-mediated IL-12 secretion. Finally, the enhanced Th1 immune response generated by cationic microspheres may be attributable to the properties of the hydrophobic core-polyamine-coated cationic shell. Phagocytosed material ends up in phagosomes, where it is often subjected to several “fusion and fission” processes between phagosomes and lysosomes in order to exchange membrane constituents and vesicular contents.57,58 Through this process, the phagosome matures and acquires enzymes and membranebound proton pumps that are typically associated with lysosomes, leading to significant acidification of the matured phagosome.59 Protonation of the polyamine polymer shell of



AUTHOR INFORMATION

Corresponding Authors

*(L.Y.W.) Tel/Fax: 8610-82544931. E-mail: wanglianyan@ home.ipe.ac.cn. *(G.H.M.) Tel/Fax: 8610-82627072. E-mail: [email protected]. ac.cn. *(Z.G.S.) Tel/Fax: 8610-62561817. E-mail: [email protected]. cn. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by the 973 Program (Grant No. 2013CB531500), Special Fund for Agroscientific Research in the Public Interest (Grant No. 201303046), and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-EW-R-19).



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