The orchestration of cellular and humoral responses is facilitated by divergent intracellular antigen trafficking in nanoparticle-based therapeutic vaccine

Pharmacological Research 65 (2012) 189–197

Contents lists available at SciVerse ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

The orchestration of cellular and humoral responses is facilitated by divergent intracellular antigen trafficking in nanoparticle-based therapeutic vaccine Hua Yue a,b,1 , Wei Wei a,1 , Bei Fan c , Zhanguo Yue a,b , Lianyan Wang a , Guanghui Ma a,∗ , Zhiguo Su a,∗ a b c

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Hualan Biological Engineering Inc., Henan 453003, PR China

a r t i c l e

i n f o

Article history: Received 5 July 2011 Received in revised form 23 September 2011 Accepted 23 September 2011 Keywords: Cellular immune response Humoral immune response Intracellular trafficking Polylactide nanoparticles Therapeutic vaccine

a b s t r a c t Therapeutic vaccination for the treatment of chronic hepatitis B is promising but has so far shown limited clinical efficacy. Herein, we employ polylactide nanoparticles (NPs) as the vaccine adjuvant and systematically explore their effect on activation of specific immunity and the underlying theoretical mechanisms. In vitro studies show that hepatitis B surface antigen (HBsAg) accumulates in antigen-presenting cells (APCs) to a larger content (270%) with the assistant of NP in comparison with the pure-antigen group. Besides the elevated costimulators (CD80/86) and increased major histocompatibility complex (MHC) II molecules, the MHC I molecules are also found upregulated. This result is mostly owing to the divergent antigen trafficking ways of NP–antigen in APCs, especially for the escape of exogenous HBsAg from the lysosomes to the cytosol. Interestingly, the MHC I level is downregulated in alum–antigen group, indicating a possible reason for its inefficiency in priming cellular response. Further in vivo experiments establish that NP–antigen group indeed enhances the CD8+ CTL cytotoxicity and IFN-␥ cytokine secretion. Meanwhile, specific antibody titer is also upregulated, and even surpasses that of the commercialized alum–antigen. All these results strongly support that NP-based antigen promotes an orchestration of cellular and humoral immune response, exhibiting favorable intrinsic properties to be applied in therapeutic vaccines. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Approximately 2 billion people of the world have been infected by hepatitis B virus (HBV), which can cause chronic liver disease and put people at high risk of death from hepatic cirrhosis and liver cancer. Various types of drugs have been tried to deal with HBVrelated chronic liver diseases, but it is more and more evident that eradication of HBV by anti-viral drugs is an unachievable goal [1]. Currently, therapies are turning to aim at reducing HBV replication like using ␣-interferon and lamivudine [2]. However, these treatments can lead to frequent relapse or adverse drug reaction under different treatment terms, which compromise the therapy effect and restrict their utilities [3]. To develop an effective strategy, it is beneficial to understand the immune response with HBV infection. Nearly 0.5% of hepatitis B surface antigen (HBsAg) carriers will clear this antigen yearly,

∗ Corresponding authors at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, PR China. Tel.: +86 10 82627072; fax: +86 10 82627072. E-mail addresses: [email protected] (G. Ma), [email protected] (Z. Su). 1 These authors contributed equally to this work. 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.09.008

and patients who control the virus replication show a multi-specific antiviral cell-mediated immunity and high anti-HBV antibody level [4]. Unfortunately, any case of immune deficiency, such as lack of major histocompatibility complex (MHC) molecules or costimulatory signals, may lead to weak cellular response or HBV immunity tolerance, thus causing chronic liver diseases [5,6]. In this aspect, redirecting the weak response with immunotherapy vaccine is considered a great promising alternative to tackle HBV, as this strategy utilize natural functions of immune system to acquire antigen presentation and further trigger T cell response. To enhance the immune-modulatory capacity of hepatitis B vaccines, several adjuvant-dependent systems have been developed [7,8]. Alum, a generic term for salts of aluminium, is the only approved adjuvant for human use over several decades. Although it can enhance the adjuvanticity by indirectly activate the antigen-presenting cells (APCs), they are deficient at initiating cell-mediated immunity [9,10]. Other experimental adjuvants (e.g. lipopolysaccharide or CpG DNA) have also been widely studied. Albeit effective in animal models, they may cause toxic side effects involving anaphylactic shock or systematic inflammation, which far preclude their clinical application. Since nanoparticles (NPs) can offer preferred attributes for bioapplication, NP-based adjuvants are emerging for the development

190

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

of therapeutic vaccines [11,12]. To enable this approach to move quickly to the clinic, the FDA (Food and Drug Administration)approved polylactide (PLA) that has undergone significant clinic evaluations is often selected as the nanoparticle material. There are emerging studies using PLA NPs as antigen delivery system or adjuvants, the results are encouraging and promising to provoke T cell immune response [13–16]. Nevertheless, the applied polymer particles in a wide size distribution would inevitably compromise their promising results in pharmacological or immunological researches. Moreover, most of them failed to give detailed mechanistic explanations for the immunological outcome of NP-based HBsAg. Keeping these in mind, we prepared NP–HBsAg formulation to assess their immunological performances on provoking HBVspecific immune response. Uniform-sized PLA nanoparticles were obtained by a facile method using Shirasu Porous Glass (SPG) membrane emulsification technique [17], ensuring more reliable results. Considering the accumulated reports on the much higher cytotoxicity generated by nanomaterial with a very small size (<100 nm) [18,19], we paid more attention to the asprepared 350 nm nanoparticles. In the research field of therapeutic HBV vaccine, to our best knowledge, there are no documents addressing the application of NPs that prepared by SPG technique. To shed light on the theoretical view of the NP-based adjuvant, we attempted to construct a bridge between the in vitro and in vivo performance of asprepared antigen formulation. It was widely accepted that macrophages or other APCs played a critical role in processing the exogenous antigen and priming specific immune response [20–22]. Since murine macrophage J774A.1 cell line was a widely acceptable APC prototype in antigen presentation related study [23,24], we employed this cell type to examine subsequent immunological performance including antigen uptake, cellular trafficking, and antigen-presenting ability in cell-based experiments. For in vivo validation, CTL activity, anti-virus cytokine level, and anti-HBs antibody titer were detected to testify the feasibility of NP adjuvant in therapeutic HBV vaccine.

2. Materials and methods 2.1. Materials and reagents PLA (10 kDa) was obtained from the Institute of Medical Instrument (China). Polyvinyl alcohol (PVA-217) was from Kuraray (Japan). SPG membrane was provided by SPG Technology Co. Ltd. (Japan). HBsAg (Mw ∼24 kDa) and alum adjuvant (aluminium hydroxide gel) were kindly supplied by Hualan Biological Engineering Incorporation (China). HBsAg-specific epitope S28-39 (IPQSLDSWWTSL, purity ≥95%) was synthesized by Institute of Materia Medica (Chinese Academy of Medical Sciences & Peking Union Medical College). Concanavalin A and mitomycin C were bought from Roche (Germany). MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) was obtained from Merck (Germany). Anti-CD8 microbeads were purchased from Miltenyi Biotec (Germany). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were ordered from Gibco. Rhodamine-phalloidin, Lyso Tracker probes, and 4,6-diamidino-2-phenylindole (DAPI) were from Invitrogen. Nile red and Fluorescein isothiocyanate (FITC) were purchased from Sigma–Aldrich. FITC anti-mouse CD80 Ab and PE anti-mouse CD86 Ab were bought from Biolegend. Allophycocyanin (APC) antimouse MHC Class I Ab and FITC anti-mouse MHC Class II Ab were both from eBioscience. Anti-mouse IFN-␥ ELISpot kit and HBsAbIgG ELISA kit were ordered from R&D System. All other reagents were of analytical grade.

2.2. Mice and cells 6–8 weeks old male BALB/c female mice (H-2d) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products. The mice were monitored to be pathogen-free and maintained under standard conditions according to institutional guidelines. Murine macrophage cell line J774A.1 was from ATCC (American Type Culture Collection), which were cultured in DMEM culture medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 g/mL streptomycin. The P815 (H-2d) mastocytomal cell line was kindly supplied by Chinese Center for Disease Control and Prevention and was maintained in complete culture medium (RPMI 1640 medium supplemented with 10% FBS). All these cells were grown in a humidified incubator at 37 ◦ C, 5% CO2 .

2.3. NP preparation and characterization Uniform-sized nanoparticles were prepared by a facile method combining SPG premix membrane emulsification and emulsion–solvent removal method [25]. The size of nanoparticles could be successfully controlled by selecting SPG membrane with a certain pore size. Briefly, 2 wt% PVA was used as the water phase. PLA (5 wt%) was dissolved in ethyl acetate, which was used as the oil phase. (For preparation of the fluorescent PLA particles, Nile red at a final concentration of 50 ␮g/mL was added to the oil phase.) The coarse emulsions were first prepared by low-speed stator homogenization and then poured into the premix reservoir. Subsequently, nanodroplets were achieved by extruding the coarse emulsions through the membrane pores under a pressure of 1 MPa. The droplets were stirred and consolidated overnight for evaporation of volatile organic solvent (ethyl acetate). Following consolidation, the NPs were collected by centrifugation (8000 × g, 10 min) and then resuspended in deionized water. The washing process repeated six times to remove the residue solvent and PVA emulsifier without addition of any other solvent. Finally, the PLA NPs could be well dispersed in water without any aggregation or precipitation. Prior to characterization, the PLA NPs were lyophilized. The surface morphology of NP was observed by a JEM-6700F (JEOL, Japan) scanning electron microscope (SEM). The size distribution and polydispersity index (PDI) of nanoparticles were measured by dynamic light scattering technique (DLS) using a zeta potential analyzer (Brookhaven Instruments Corporation). In detail, NPs were resuspended in purified water (from a Millipore filtration system) at a concentration of 0.1 mg/mL, and the sample was analyzed by using the “Log Normal” fit from the Brookhaven 9KPSDW software. PDI is a measure of the nonuniformities that exists in the particle size distribution. It is denoted as: PDI = 2 / 2 , where 2 = (D2 * − D*2 )q4 and  = Dq2 . q = 4n/0 sin(/2), where  is the scattering angle, 0 is the wavelength of laser light, and n means the index of refraction of the suspending liquid. The D is translational diffusion coefficient, the principle quantity measured by DLS system. The D* is the average diffusion coefficient, the D*2 is the square value of D*, and the D2 * is the mean square value of the D.

2.4. Preparation of antigen formulations HBsAg was employed as the vaccine antigen for in vitro incubation and in vivo injection. To prepare the alum–antigen formulation, the commercialized aluminium salts and soluble HBsAg were mixed extensively at an appropriate ratio (alum:antigen = 1 mg:40 ␮g). Similarly, 5 mg particles were added to the soluble HBsAg, and the NP–antigen formulation at a final

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

191

concentration of 1 mg/40 ␮g (NP/antigen) was acquired by a 12 h mixing/adsorbing process.

MHC molecules expressions were acquired using a 3 laser/9 color CyAnTM ADP flow cytometer (Beckman Coulter).

2.5. Cytotoxicity test

2.8. In vitro evaluation of costimulatory molecules

MTT test was carried out to evaluate the cytotoxicity of asprepared PLA NPs. J774A.1 cells at a density of 5000 cell/200 ␮L were cultured and allowed to adhere in each well of 96-well plate. Then, serial dilutions of particles ranging from 0 to 120 ␮g/mL were added to the culture medium. After 48 h incubation, cell samples were treated with MTT for 4 h, which was followed by addition of isopropanol to dissolve the formazan crystals. Infinite M200 microplate spectrophotometer (Tecan) was used to measure the absorbance at 570 nm. Percent viability was normalized to cell viability in the absence of particles. Each experiment was performed in triplicate, and data were represented as means ± standard deviation.

To study the effect of adjuvants on the expression of CD80 and CD86 costimulatory molecules, cells were seeded in 6-well plate (3 × 105 cell/well) and stimulated with different antigen formulations for 24 h. Then cells in each group were rinsed and resuspended with PBS. After that, PE anti-mouse CD86 Ab and FITC anti-mouse CD80 Ab were simultaneously incubated with cells on ice for 30 min. The fluorescent intensity was quantified with CyAn ADP flow cytometer using Summit 4.3 software (Beckman Coulter). For further confirmation, cells were seeded in Petri dish, and corresponding confocal images of the treated cells were obtained by a TCS SP5 CLSM. 2.9. Immunization

2.6. In vitro cellular uptake and intracellular antigen trafficking study In vitro studies were carried out using FITC labeled HBsAg in J774A.1 cells. FITC was covalently linked to HBsAg in a way as described previously [26]. Briefly, FITC (2 mg) was slowly added into 50 mL HBsAg solution (pH 8.0). After a 12 h intensive stirring procedure at 4 ◦ C in dark, the unbound FITC was removed with an ultra-centrifuge tube (molecular weight cutoff 50 kDa, Millipore). To investigate the effects of particles or alum on antigen uptake using flow cytometry technique, 2 mL J774A.1 cells were seeded in 6-well plate at a concentration of 3 × 105 cell/well for 24 h. Then cells were added with 25 ␮L 40 ␮g/mL different antigen formulations (including pure-antigen, conventional alum–antigen or NP–antigen) at a final HBsAg concentration of 1 ␮g/mL in cell medium for 24 h. Subsequently, cells were extensively washed by PBS, detached by TE (Tris–EDTA), resuspended and fixed in 3.7% paraformaldehyde (pH 7.4). These samples were analyzed on a LSR flow cytometer (Becton Dickinson) through FL1 channel, and data were acquired from 15,000 cells per sample. Cells without any treatment were taken as the blank control, and its fluorescence intensity in FL1 was subtracted before the calculation of relative intensity of FITC–antigen. For the confocal laser-scanning microscope (CLSM) images, 5 × 105 /mL cells were seeded in 35 mm diameter Petri dish and allowed to grown for 24 h, and then different antigen formulations were added. After incubation for a desired time, particles adhering to the cell surface were removed by washing with PBS, and the cells were fixed in 3.7% paraformaldehyde for 30 min. In order to give a visualization of the interaction between particles and cells, the cell membrane and nucleus were stained with rhodamine-phalloidin and DAPI, respectively. To track the cellular location of nanoparticles and FITC labeled HBsAg, lysosomes were stained with Lyso Tracker probes. The corresponding fluorescent images were taken by CLSM TCS SP5 (Leica). 2.7. In vitro evaluation of MHC molecules Effect of adjuvant on T cell recognition signals was analyzed in terms of surface MHC levels on J774A.1 cells in vitro. Briefly, cells at a concentration of 3 × 105 cell/well were cultured in 6-well plate for 24 h and then incubated with different antigen formulations for 24 h at 37 ◦ C. After that, cells were washed with PBS, detached by TE and resuspended in PBS. According to the manufacturer’s instruction, samples were incubated with APC anti-mouse MHC Class I Ab and FITC anti-mouse MHC Class II Ab on ice for 30 min, and untreated cells were used as control. Quantification data of

Mice were randomly assigned to different groups, and were injected subcutaneously (s.c.) with pure-antigen, alum–antigen, or NP–antigen formulation (each containing 4 ␮g HBsAg) for 14 d separately. All animals were bled through retro-orbital plexus at a desired time. 2.10. Interferon-gamma (IFN-) enzyme-linked immunospot (ELISpot) assay Spleen cells were collected from mice after 14 d immunization, and CD8+ T cells were isolated by positive selection using anti-CD8 Ab-conjugated Microbeads through magnetic-activated cell sorting (MACS). Flow cytometric analysis (FACS) showed that the sorted cells were more than 90% pure. A typical ELISpot assay was used to quantify the number of IFN-␥-secreting CD8+ T cells in response to CTL epitope S28-39 of HBV antigen [27]. For experimental wells, 100 ␮L cell suspension (2 × 106 cell/mL) was incubated with 100 ␮L epitope S28-39 (10 ␮g/mL) for 20 h. The wells with 100 ␮L concanavalin A (10 ␮g/mL) and 100 ␮L cell suspension were used as the positive control, whereas 200 ␮L complete RPMI medium was added to the negative control wells. Following 20 h incubation at a standard procedure, cells producing IFN-␥ were expressed as spot-forming cells (SFC), which were counted with an ImmunoSpot Analyzer (Cellular Technology Ltd.). 2.11. HBsAg-specific cytotoxicity assay A 4 h chromium (51 Cr) release assay that directly measuring cytolytic activity as radioactivity released from killed target cells [28] was applied after a few modifications. To generate stimulated cells, a mixture of splenocytes (5 × 106 cell/mL) were sorted from the unprimed mice, stimulated with mitomycin C for 2 h (at a final concentration of 25 ␮g/mL) and then cultured with HBsAg-specific CTL epitope S28-39 (at a final concentration of 20 ␮g/mL) for 4 h in complete cell culture medium. To generate effector cells, CD8+ T cells from the immunized mice were isolated and cocultured with the stimulated cells (at a ratio of 10:1) for 6 d. As targets, P815 cells were stimulated with CTL epitope and labeled with Na2 51 CrO4 (37 MBq, 1 mCi/mL at 37 ◦ C for 1 h). Afterwards, 100 ␮L 51 Cr P815/S28-39 targeted cells (105 cell/mL) were plated into a 96-well plate, and 100 ␮L effector cells (106 cell/mL) were added at a certain effector to target (E:T) ratio. 100 ␮L 20% Triton X-100 was added to the maximum release well, while 100 ␮L completed medium was added to the spontaneous well. After 4 h incubation, supernatants were harvested and assayed by counting on a scintillation counter (TopCount). Cytotoxicity was defined as: (test

192

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

Fig. 1. Scanning electron microscope image (A) and size distribution profile (B) showing the uniform-sized polylactide nanoparticles (PLA NPs). NPs were prepared by a facile method combining premix membrane emulsification and emulsion–solvent removal method. The polydispersity index (PDI) of NPs was 0.025.

release − spontaneous release)/(maximum release − spontaneous release) × 100%. 2.12. Anti-HBs antibody titer assay To examine the anti-HBs antibody titer level, the tested mice groups (n = 8) were injected s.c. with antigen formulations for 14 d and received a boost immunization for another 14 d at the end of the second week. Mice without antigen immunization were used as the negative control group. Immunized mice were bled at a desired interval and the anti-HBs antibody titer in the serum was detected with an HBsAb-IgG ELISA kit. According to the manufacturer’s instruction, color changes were finally determined at 450 nm using an Infinite M200 microplate spectrophotometer (Tecan), and the calculation of the geometrical mean titers (GMT) of IgG was carried out. 2.13. Statistical analysis Except for the above antibody assay, the other experiments were all performed in triplicate, and data were represented as means ± standard deviation (SD). Statistical differences between groups in the in vitro/in vivo studies were performed by a one-way analysis of variance (ANOVA) and a Tukey post hoc test. P < 0.05 was considered as the level of significance. 3. Results and discussion 3.1. PLA NP characterization Uniform size has been considered as an essential property of NP for the evaluation of their immunological performance [29,30]. However, it is hard to control the particle size of polymerbased material, and PLA NPs prepared by conventional methods often have a broad size distribution. In the pharmacological or immunological studies, using these particles may inevitably lead to ambiguous conclusion or discount the efficacy, which largely delayed the NP-related research progress. To overcome this hurdle, SPG membrane emulsification technique that has been welldeveloped in our lab was applied. By choosing specific pressure and membrane pore size, the required particle size could be acquired. As shown in Fig. 1, PLA NPs with a narrow size distribution (PDI = 0.025) around 350 nm were successfully obtained by using a SPG membrane with the mean pore size of 1.4 ␮m. These uniformsized NP assured the reliability and reproducibility of our findings and promoted us to systematically investigate their subsequent performances.

3.2. Cellular uptake profiles of HBsAg Prior to the in vitro/in vivo evaluation, we performed MTT assay to delineate whether the asprepared particles cause any cytotoxicity. PLA NPs were exposed to J774A.1 cells, and no evident reduction of cell viability was found (Fig. S1), suggesting that the particles were nontoxic at present dosage range (up to 120 ␮g/mL). Moreover, a safe dosage of NPs could be easily fixed to assure good cell viability in the following experiments. A key attribute defining the potent adjuvanticity of nanoparticles is their ability to be taken up avidly by APCs [12]. To study the effect on cellular uptake of HBsAg, different antigen formulations were incubated with the professional APCs (J774A.1 macrophages) for 24 h. FACS analysis (Fig. 2A) showed that the fluorescent intensity of FITC–HBsAg in NP–antigen group was nearly 3-fold (∼270%) higher compared with that in pure-antigen group at present antigen dose (1 ␮g/mL). However, when we employed the commercialized alum–antigen formulations, only a slight increase was observed (109% compared with pure-antigen group). CLSM images (Fig. 2B–D) further provided visions that HBsAg accumulated in the cells to a very large extent for the NP–antigen group, comparing with a weak improvement for the alum–antigen group. It has been demonstrated that the enhanced uptake in APCs was a possible stimulation mechanism of adjuvant [31]. In this aspect, such an elevated antigen uptake would be a splendid property for a potent adjuvant. To clarify the role of NP on the improved antigen uptake, we further traced the intracellular location of exogenous HBsAg and particles. As CLSM images exhibited (Fig. 2E), the distribution of fluorescent Ag was highly consistent with the cellular location of NP in APCs, indicating an inseparate relationship between them. This is direct evidence that once the internalization was triggered by NP, the cellular entrance of soluble HBsAg could be simultaneously facilitated. This result thus gave a comprehensive understanding about the reason why antigen was highly internalized in NP-based antigen group. Having revealed the cellular uptake profile, we next asked the subsequent immunological performances of antigenloaded APCs.

3.3. In vitro evaluation of MHC molecules on APCs T-cell activation is dependent on interaction of several molecules between T cells and APCs. Recognition of the epitope presented by MHC molecules is the first step for T cell activation, as MHC I or MHC II molecules present proteins for T cells that perform cellular or humoral response respectively. Herein, we compared the MHC molecule levels of the experimented groups

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

Fig. 2. Effect of different adjuvants on cellular uptake of FITC–HBsAg (green) in antigen presenting cells (APCs). Flow cytometric analysis (FACS) (A) and confocal laser-scanning microscope (CLSM) images showing the cellular internalization of pure-antigen (B), alum–antigen (C), and NP–antigen (D). Confocal images (E) showing the intracellular location of Nile red stained NP and FITC–HBsAg in APCs. The actin segments bound with rhodamine-phalloidin appear red in color, and the nucleus dyed with DAPI is blue. The white spots represent the co-locolization information of FITC–antigen and NP. Scale bars: 5 ␮m. Each experiment was performed in triplicate, and FACS data were represented as means ± SD. Asterisk symbols indicate differences between the tested groups. **P < 0.01 (ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

and found that NP–antigen was potential to induce a harmonious response. As shown in Fig. 3B, both NP–antigen and commercialized alum–antigen apparently upregulated the expression of MHC II molecules, manifesting a similar in vitro outcome in this aspect. Since exogenous antigens are generally loaded onto MHC II molecules for CD4+ T cells, this parallel upregulation means that the NP-based formulation may not differ from the effect of commercialized alum–antigen on CD4+ T mediated humoral immunity. By testing the MHC I molecules, the expression level was also found to be improved on the exogenous antigen treated cells in the presence of NPs (Fig. 3A), in comparison with a visible expression decrease found on the alum–antigen treated cells. Since only MHC I presented peptides are recognized by CD8+ T cells, this result indicated that NP-based formulation was promising to induce CD8+ T mediated cellular response and HBV specific cytotoxicity. In contrast, it also suggested a possible reason for the inefficiency of alum adjuvant in priming cellular response present founding.

193

Fig. 3. FACS indicating the expression of MHC I (A) and MHC II (B) molecules in J774A.1 cells after stimulation with different antigen formulations. Each experiment was performed in triplicate, and FACS data were represented as means ± SD. Asterisk symbols indicate differences between the tested groups. **P < 0.01 (ANOVA).

3.4. Intracellular trafficking of HBsAg in APCs Usually, MHC I molecules presented endogenous peptides derived from cytosolic proteins, and the elevated molecule expression induced by exogenous NP–antigen possibly indicated a “cross-presentation” process. In this process, foreign antigens are presented via somewhat specific trafficking, to generate MHC I complexes. Due to its potent cellular immunity induced by exogenous antigen, it has attracted booming interests in immunological therapies, but details remain unclear [21,32]. To gain a deep insight of aforementioned phenomenon, we directed our interest to explore the intracellular fates of different antigen formulations. As lysosomes were correlated to the main degradative pathway of exogenous antigen [33], we tagged these organelles and chased the cellular localization of FITC–HBsAg. CLSM images displayed that HBsAg was highly co-localized with lysosomes in the pure-antigen treated cells (Fig. S2A), and this situation was not altered when cells were exposed with alum–antigen (Fig. S2B). Distinguishingly, divergent trafficking pathways were identified in the NP–antigen treated cells (Fig. 4). Besides the common fate of FITC–HBsAg which was trapped in lysosome (indicated by white spots), some antigen were found gain access to the cytosol (green spots in the overlay image). To verify the role of NPs in this process, we investigated the intracellular fate of particles (Fig. S3). As expected, the NPs alone were capable to escape from the lysosomes, showing their responsibility for the

194

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

Fig. 4. Intracellular trafficking of internalized NP–antigen in J774A.1 cells and detailed overlay analysis. (A) CLSM images exhibiting the divergent cellular locations of NP–antigens. Scale bars: 5 ␮m. (B) Co-localization analysis showing the detailed overlay information: (I) empty lysosome, (II) escaped antigen, and (III) co-localized antigen. Data of other experimental groups can be seen in Fig. S2.

escape of soluble antigen. Diverse trafficking fate of NP–antigen indicated that the exogenous NP–antigen was not only processed via direct presentation but also presented via MHC I mediated pathway by mimicking the cytosolic antigen. Thus, the particularity of NP–antigen in cross-presentation was confirmed, and its influence in subsequent cellular response was awaited.

3.5. In vitro evaluation of costimulators on APCs Aside from the MHC molecules, CD80/CD86 are the second signal known as costimulator for T cell activation and survival [34]. Both FACS analysis (Fig. 5A) and CLSM analysis (Fig. 5B) illuminated that NP–HBsAg formulation increased the molecule expression of

Fig. 5. Effect of different adjuvants on the expression of costimulatory molecules CD80 (A) and CD86 (B) in J774A.1. The corresponding CLSM images (C) are displayed below. Scale bars: 5 ␮m. Each experiment was performed in triplicate, and values were represented as means ± SD. Asterisk symbols indicate differences between the tested groups. **P < 0.01 (ANOVA).

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

195

CD80 and CD86. Along with the elevation of the aforementioned activation signals (MHC I and MHC II molecules), costimulator upregulation would make NP–antigen more powerful in provoking efficient T cell immunity. It was clear that the presentation of self-antigen by APCs without adequate costimulation always lead to tolerance of T cells [35]. Distinct from the results in NP–antigen group, the expressions of CD80 and CD86 molecules were found downregulated by alum–HBsAg, suggesting another reason for the inefficiency of alum adjuvant in priming cellular response.

3.6. In vivo evaluation of IFN- secretion and CTL cytotoxicity From the in vitro observations, we concluded in assurance that NP–antigen could efficiently enhance the antigen internalization, and improve the expression of surface molecules for T cell activation in APCs. These results offered us a big hope and promoted us to test the efficacy of NP adjuvant in vivo. Subsequently, different antigen formulations were administrated to mice, and their performance on HBsAg specific immune response was compared. Cellular immunity is critical for vaccines capable of inducing resistance to intracellular virus. This is often measured by determining if CD8 T cells produce the inflammatory cytokine IFN-␥ after exposure to an antigenic stimulus [36]. In present work, CD8+ T cells were isolated from the spleen of immunized mice, and ELISpot assay was carried out to evaluate this cytokine event. As expected, NP–HBsAg increased the IFN-␥ production in contrast with the other antigen formulations (Fig. 6A). Moreover, statistical analysis showed difference between the pure-antigen and NP–antigen treated group (P < 0.05). It has also been reported that higher level of IFN-␥ produced by antigen-specific T cells was associated with lower HBV DNA level [37]. Accordingly, superior cytokine secretion raised by NP–antigen might lead to a direct inhibition of virus replication and benefit the treatment of chronic hepatitis B (CHB) infection. Once CD8+ T cells become activated, they are recognized as Tc, which release the cytotoxin perforin and granulysin to form pores in targeted cell membrane and cause the lysis of infected cells. Herein, CD8+ T cells were isolated, and HBsAg-specific cytotoxic activity against the 51 Cr labeled P815 cell-targets was detected. We observed that NP–antigen improved the HBsAg specific lysis against the P815/S28-39 target cells (Fig. 6B), whereas lower CTL activity was determined in the alum–antigen group. This result recalled the expression level of T cell activation molecules as well as our speculated reasons for the alum’s inefficiency in cellular response. On the other side, our proposal was hence to be confirmed that the immunological approaches towards enhancing the MHC I mediated antigen presentation was practicable to generate higher CTL activity. In terms of the hepatitis B vaccine, therapeutic vaccine and preventive vaccine is quite different. A few studies have attempted to employ polymer particles as vaccine adjuvant to induce strong immune response. However, most of the antigen formulations preferred to play a preventive role, rather than fulfill the treatment demand to clear the virus in infected individuals. This is most likely due to the ordinary process and outcome primed by exogenous antigen. In present work, we introduced the PLA NP–antigen formulation to compensate or “wake up” the flaw immune response through divergent antigen presentation. In addition to the efficient virus suppression performed by IFN-␥, the improved HBsAg specific cytotoxicity would make greater effort to meet the therapeutic need. Noteworthy, this NP–HBsAg formulation was obtained just via a gentle and simple mix process compared with that for the encapsulated system, which not only ensured the antigen activity to a large extent but also made it easier to scale up in the future development. Meanwhile, using the asprepared nanoparticles in

Fig. 6. In vivo evaluation of different antigen formulations on HBsAg specific IFN-␥ secretion (A) and specific lysis activity (B). Mice were immunized with pure-HBsAg, alum–HBsAg or PLA NP–HBsAg, separately. CTL activity data were shown as HBsAgspecific cytotoxic activity against the P815/S28-39 targets by subtracting the HBsAgnonspecific cytotoxic activity against P815 cells. One representative experiment out of three is shown, and the values are means ± SD (n = 6). Asterisk symbols indicate differences between the tested groups. *P < 0.05 and **P < 0.01 (ANOVA).

a narrow size distribution facilitated more confident evaluations, especially in pharmacological and immunological studies. 3.7. In vivo evaluation of anti-HBs titer assay Antibody, which derives from activated B cells, is used to identify and neutralize the foreign antigens (viruses or bacteria) free in serum. Apart from the T cell-mediated immunity, the humoral immunity mediated by antibody is the other crucial arm of the immune system. To examine the anti-HBs antibody level, mice were given a first vaccination for 14 d and a boost immunization for another 14 d, and then the geometrical mean concentration of serum antibody was calculated. As shown in Table 1, both NP and alum successfully facilitated the specific antibody production after the initial vaccination. Although the titer level of NP–antigen group was not very satisfactory at the first dose, it dramatically increased up to 193.85 mIU/mL after a boost immunization, which exceeded that of the alum–antigen group (156.39 mIU/mL). Moreover, the NP–antigen vaccination was found associated with the highest anti-HBs seroconversion rate (a ratio of 7/8), pointing a favorable outcome that was strongly needed in the treatment of CHB. As B cell stimulation process was related to the CD4+ T activation, data obtained here displayed a consistent effect of NP–antigen on the improvement of MHC II and CD80/CD86 expression. It was not surprising that the antibody titer was elevated by alum–antigen, because alum had been reported to be an effective

196

H. Yue et al. / Pharmacological Research 65 (2012) 189–197

Table 1 Serum anti-HBs antibody titer of mice after immunization with different antigen formulations. Groups

Pure-antigen Alum–antigen NP–antigen

14 d

28 d

Antibody titer (mIU/mL)

Positive conversion rate

Antibody titer (mIU/mL)

Positive conversion rate

2.99 7.85 7.66

0/8 3/8 1/8

6.73 156.39 193.85

2/8 6/8 7/8

Fig. 7. A scheme depicting our strategy of how to restore the HBV specific immunity by employing PLA nanoparticle-based vaccine. With the assistant of NP, exogenous HBsAg could escape from the lysosomes and provide recognition signals for CTL activation.

adjuvant in preventive HBV vaccine. However, its capacity in eliciting specific antibody was found lower than that for the NP-based formulation, thus informing a superior property of NP–antigen to clear the HBV from serum and reduce the risk of hepatic decompensation. Referring to the importance of coordinated cellular and humoral antiviral response in HBV control [38], we again confirmed the feasibility as well as our theoretical explorations of employing NP in therapeutic HBV vaccine. 4. Conclusions and perspective We demonstrated that PLA NP–HBsAg possessed tremendous potential to restore the HBV specific immunity owing to its advantages in activating diverse function of APCs (Fig. 7). Besides the facile and highly safe method offered to prepare antigen formulation, a clear in vitro proof of how the NPs facilitated the immunological performance of antigen was provided. In detail, NP–antigen was capable of fulfilling the demands for both cellular and humoral response, which could fall into the following issues. (1) The entrance of antigen into the APCs was benefited significantly, once the cellular uptake process was triggered by NPs. (2) Apart from the elevation of MHC II and costimulators, the CD8 T recognition signals was also upregulated via divergent cellular trafficking of exogenous antigen, showing a strikingly attractive capability in priming CTL response. (3) Based on the prominent properties mentioned above, the in vivo study further revealed that NP–antigen indeed elicited stronger HBsAg specific CTL cytotoxicity as well as higher antibody titer. Although considerable theoretical and practical accomplishments have been made, how to improve the versatile function of NP–antigen remains intriguing for CHB infection treatment.

Fortunately, rapid developments of polymer nanoparticles represent us great opportunities for future research. Novel chemical or bioconjugation approaches for NP modification will, in principle, contribute to higher antigen uptake and stronger HBV specific immunity. Inspired by the performance of NP on crosspresentation, we may also develop well-designed particles and assess their effects on “lysosomal break”, and further on efficient MHC I related antigen processing. In addition, evaluation of the vaccine therapy effect and any potential toxicity should be carried out in extensive HBV animal models, which undoubtedly help us to gain more insight into the immunological performance and promote their application into clinic. Acknowledgments This work was supported by grants from the 973 Program (2009CB724705), the National Nature Science Foundation of China (20536050, 50703043), and the Chinese Academy of Sciences (KJCX2-YW-M02). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phrs.2011.09.008. References [1] Fung SK, Lok AS. Update on viral hepatitis in 2004. Curr Opin Gastroenterol 2005;21:300–7. [2] Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B: 2000—summary of a workshop. Gastroenterology 2001;120:1828–53.

H. Yue et al. / Pharmacological Research 65 (2012) 189–197 [3] Boni C, Bertoletti A, Penna A, Cavalli A, Pilli M, Urbani S, et al. Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B. J Clin Invest 1998;102:968–75. [4] Rehermann B. Immune responses in hepatitis B virus infection. Semin Liver Dis 2003;23:21–38. [5] Chisari FV, Cytotoxic. T cells and viral hepatitis. J Clin Invest 1997;99:1472–7. [6] Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269–80. [7] Kanchan V, Panda AK. Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response. Biomaterials 2007;28:5344–57. [8] Reddy ST, Swartz MA, Hubbell JA. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol 2006;27:573–9. [9] Kool M, Soullie T, van Nimwegen M, Willart MAM, Muskens F, Jung S, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 2008;205:869–82. [10] Brewer JM. (How) do aluminium adjuvants work? Immunol Lett 2006;102:10–5. [11] Yue H, Wei W, Yue Z, Lv P, Wang L, Ma G, et al. Particle size affects the cellular response in macrophages. Eur J Pharm Sci 2010;41:650–7. [12] Xiang SD, Scalzo-Inguanti K, Minigo G, Park A, Hardy CL, Plebanski M. Promising particle-based vaccines in cancer therapy. Expert Rev Vaccines 2008;7:1103–19. [13] Ahsan F, Thomas C, Rawat A, Hope-Weeks L, Aerosolized PLA. PLGA nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis B vaccine. Mol Pharm 2011;8:405–15. [14] Ionescu RM, Przysiecki CT, Liang XP, Garsky VM, Fan JA, Wang B, et al. Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier. J Pharm Sci-US 2006;95:70–9. [15] Saini V, Jain V, Sudheesh MS, Jaganathan KS, Murthy PK, Kohli DV. Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen. Int J Pharm 2011;408:50–7. [16] Chong CS. Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery. J Control Release 2005;102:85–99. [17] Wei W, Wang LY, Yuan L, Wei Q, Yang XD, Su ZG, et al. Preparation and application of novel microspheres possessing autofluorescent properties. Adv Funct Mater 2007;17:3153–8. [18] Pan Y, Leifert A, Ruau D, Neuss S, Bornemann J, Schmid G, et al. Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small 2009;5:2067–76. [19] Pan Y, Neuss S, Leifert A, Fischler M, Wen F, Simon U, et al. Size-dependent cytotoxicity of gold nanoparticles. Small 2007;3:1941–9. [20] Munic V, Banjanac M, Kostrun S, Nujic K, Bosnar M, Marjanovic N, et al. Intensity of macrolide anti-inflammatory activity in J774A.1 cells positively correlates with cellular accumulation and phospholipidosis. Pharmacol Res 2011;64:298–307.

197

[21] Alatery A, Siddiqui S, Chan M, Kus A, Petrof EO, Basta S. Cross, but not direct, presentation of cell-associated virus antigens by spleen macrophages is influenced by their differentiation state. Immunol Cell Biol 2009;88:3–12. [22] Ramirez MC, Sigal LJ. Macrophages and dendritic cells use the cytosolic pathway to rapidly cross-present antigen from live, vaccinia-infected cells. J Immunol 2002;169:6733–42. [23] Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, et al. Phagosomes are competent organelles for antigen cross-presentation. Nature 2003;425:402–6. [24] Morita H, He F, Fuse T, Ouwehand AC, Hashimoto H, Hosoda M, et al. Cytokine production by the murine macrophage cell line J774.1 after exposure to lactobacilli. Biosci Biotechnol Biochem 2002;66:1963–6. [25] Wei Q, Wei W, Lai B, Wang LY, Wang YX, Su ZG, et al. Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification. Int J Pharm 2008;359:294–7. [26] Wei Q, Wei W, Tian R, Wang LY, Su ZG, Ma GH. Preparation of uniform-sized PELA microspheres with high encapsulation efficiency of antigen by premix membrane emulsification. J Colloid Interface Sci 2008;323:267–73. [27] Smith JG, Liu X, Kaufhold RM, Clair J, Caulfield MJ. Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella–zoster virus. Clin Diagn Lab Immunol 2001;8:871–9. [28] Riedl P, Bertoletti A, Lopes R, Lemonnier F, Reimann J, Schirmbeck R. Distinct, cross-reactive epitope specificities of CD8 T cell responses are induced by natural hepatitis B surface antigen variants of different hepatitis B virus genotypes. J Immunol 2006;176:4003–11. [29] Jiang W, Kim BY, Rutka JT, Chan WC. Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol 2008;3:145–50. [30] Dobrovolskaia MA, McNeil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol 2007;2:469–78. [31] HogenEsch H. Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 2002;20(Suppl. 3):S34–9. [32] Wan Y, Wu Y, Zhou J, Zou L, Liang Y, Zhao J, et al. Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments. Eur J Immunol 2005;35:2041–50. [33] Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 2009;10:623–35. [34] Collins M, Ling V, Carreno BM. The B7 family of immune-regulatory ligands. Genome Biol 2005;6:223. [35] Feldmann M, Steinman L. Design of effective immunotherapy for human autoimmunity. Nature 2005;435:612–9. [36] Reddy ST, Van Der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O’Neil CP, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol 2007;25:1159–64. [37] Ren F, Hino K, Yamaguchi Y, Funatsuki K, Hayashi A, Ishiko H, et al. Cytokinedependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection. J Med Virol 2003;71:376–84. [38] Jung MC, Hartmann B, Gerlach JT, Diepolder H, Gruber R, Schraut W, et al. Virusspecific lymphokine production differs quantitatively but not qualitatively in acute and chronic hepatitis B infection. Virology 1999;261:165–72.