Potential Hepatitis B Vaccine Formulation Prepared by Uniform-Sized

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Potential hepatitis B vaccine formulation prepared by uniform-sized lipid hybrid PLA microparticles with adsorbed hepatitis B surface antigen Qi Liu, Xiaoming Chen, Jilei Jia, Ting Lu, Tingyuan Yang, and Lianyan Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00722 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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Molecular Pharmaceutics

Potential hepatitis B vaccine formulation prepared by uniform-sized lipid hybrid PLA microparticles with adsorbed hepatitis B surface antigen Qi Liu,†,‡,‖ Xiaoming Chen, †,‡,‖ Jilei Jia,† ,‡ Ting Lu,† ,‡ Tingyuan Yang, † Lianyan Wang*,† †

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, Beijing, 100190, PR China. ‡

‖

*

University of Chinese Academy of Sciences, Beijing, 100049, PR China

These authors contributed equally. Corresponding authors.

Tel/fax: 8610-82544931, E-mail addresses: [email protected] (L.Y. Wang)

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract For the purpose of strengthen the immunogenicity of Hepatitis B vaccine, which contains hepatitis B surface antigen (HBsAg), the development of biodegradable poly (lactic acid) (PLA) microparticles (MPs) modified with the cationic surfactant didodecyldimethylammonium bromide (DDAB) was attempted. DDAB-PLA MPs with uniform size about 1 µm were prepared in a simple and mild way. DDAB-PLA MPs with increased surface charge enhanced antigen adsorption capacity compared to plain PLA MPs. After immunization, DDAB-PLA MPs induced the gene expression of inflammatory cytokines and chemokines which facilitated the following immune response. DDAB-PLA MPs augmented the expression of costimulatory molecules along with activation of bone-marrow derived dendritic cells (BMDCs). DDAB-PLA MPs-based vaccine formulations efficiently induced antibody production than aluminum-based vaccine and plain PLA MPs-based formulation in vivo. Moreover, DDAB-PLA MPs were more likely to generate the polarization of the Th1 response indicating the cytotoxic ability against infectious pathogens. In conclusion, DDAB-PLA MPs could be potent vaccine formulation to prime robust cellular and humoral immune responses. Keywords: DDAB-PLA, microparticles, HBsAg, antibody, cellular response


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Introduction Hepatitis B virus (HBV) has been known as one of the most serious and potentially life-threatening pathogens1, 2. Although vaccines against HBV have been available since 1982, it is estimated that more than 350 million chronic HBV carriers are at high risk to developing as hepatic decompensation, cirrhosis, and even liver cancer3. With the development of genetic and cell engineering, the yeast-derived recombinant hepatitis B surface antigen (HBsAg) was put into use instead of the purified plasma-derived antigen from HBsAg carriers. Although recombinant HBsAg-based vaccines being quite safer and much cheaper than the plasma-derived vaccines, in most cases they have poor immunogenicity and require additional adjuvants to generate

potent

and

long-lasting

immunity4.

Commercially

available

aluminum-adsorbed hepatitis B vaccines could elicit antibody responses, which are suitable for pathogens that could be prevented by generation of neutralizing antibodies 5. But alum-based adjuvants surely have limitations which include elevation of IgE antibody, ineffectiveness to every antigen, and only serum antibody responses without adequate cellular immune responses, especially cytotoxic T cell responses6. Therefore, there is an urgent needed for particular safer and nontoxic adjuvants that can effectively stimulate both humoral and cellular immunity against HBV. In recent years, biodegradable synthetic polymer microparticles (MPs) and nanoparticles (NPs) have extensively aroused researchers’ concerns with potential capacity to promote antigen internalization by antigen-presenting cells (APCs) dependent on their size7, 8, serve as an antigen depot9-11, activate and maturate APCs12-14, and thus improve the subsequent immune responses. Poly (lactic acid) (PLA) / poly (D,L-lactic-co-glycolic acid) (PLGA), approved by FDA as pharmaceutical excipients, become one of the most popular materials for MPs/NPs fabrication. It is well known that many physiochemical characteristics could affect the immune

response

induced

by

MPs/NPs

including

particle

size12,

15,

16

,

hydrophobicity17-19, administration route20, 21, antigen release kinetics22-24, and surface charge25. Among these characteristics described above, surface charge is an especially ACS Paragon Plus Environment


important aspect. Positively surface charged MPs could argument antibody release and CD8+ T cell responses compared with MPs which were negatively or neutrally charged26. An approach involving the preparation of MPs with positive surface charge was utilized to enhance DNA vaccine efficacy27-29. MPs were also proved to adsorb antigen and make the vaccine ready to use, promote the interaction with cell membrane (negatively charged) to facilitate antigen phagocytosis by APCs, and attain as immunogenic as encapsulated antigens30-33. The cationic polymers, including chitosan, polylysine, and polyethylenimine, were recently demonstrated to induce potent Th1-polarized response. The results mentioned above strongly proved that MPs with positive surface charge were superior than plain MPs25, 34. Since these cationic polymers might induce cytotoxicity caused by plasma membrane instability and generation of reactive oxygen species (ROS)35, it is evident that there still exists a need for pursuing more biosafe and effective materials to decorate the PLGA/PLA MPs to regulate the adjuvant activity toward stronger immune response. In this study we construct a robust and versatile MP-based vaccine formulation based on PLA MPs hybrid with cationic surfactant, didodecyldimethylammonium bromide (DDAB). DDAB, possessing polar amino groups at one end, could make the surface of MPs positively charged to facilitate antigen adsorption and uptake by APCs; at the same time, containing two non-polar aliphatic chain, could insert into the shell of MPs to fully expose the hydrophobic danger signal of PLA itself19. Furthermore, DDAB, as one of the most commonly used material to prepare cationic liposomes, could regulate immune response and have anti-bacterial and anti-inflammatory functions.36-38 DDAB-based liposome vaccine has now completed phase I clinical trials, which has been proven to be effective and showed great clinical prospective significance in prevention and therapy in tuberculosis39,

40

. The present research is intended to

develop PLA MPs modified with cationic lipid -- DDAB and by surface-adsorption of HBsAg so as to evaluate the efficacy to induce humoral and cellular immune response after administration in vivo.


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Materials and Methods Preparation of DDAB-PLA Particles DDAB-PLA MPs were fabricated by the SPG premix membrane emulsification and emulsion-solvent evaporation method as described previously41. The lipid was added as follows: 0.55% (w/v) DDAB (optimized concentration, data not shown) was mixed with 5.5% (w/v) PLA in dichloromethane solution. Characterization of DDAB-PLA Particles Surface morphology was observed by scanning electron microscopy (SEM) using a JEM-6700F (JEOL, Japan). Dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, UK) was used to detect the size, surface charge, and polydispersity index (PDI) of prepared MPs. For MPs surface charge measurement, phosphate-buffered saline (PBS, 6.2 mM, pH 6.5) was used for dispersion. The coating of DDAB on the surface of PLA MPs were further confirmed using X-ray photoelectron spectroscopy (XPS, AXIS Ultra. Kratos Analytical, UK) with Al Kα radiation (hν= 1486.6 eV). The performance of Axis spectrometers was demonstrated by the guaranteed performance on PET where the full width at half maximum of the component corresponding to the ester group was < 0.68 eV with a sensitivity defined by the maximum of the hydrocarbon peak >12 kcps, providing quantitative chemical state information from the upper 10 nm of the polymers. HBsAg concentration was measured using a bicinchoninic acid (BCA) protein assay , and HBsAg adsorption by MPs was calculated based on previous methods25. Cell viability assays Based on previously established protocol, BMDCs were extracted and cultured42. Colorimetric cell counting kit-8 (CCK-8) was used to measure BMDCs viability. BMDCs (1× 105, 150 µL growth medium per well) were seeded in 96-well plate. Then, DDAB-PLA MPs with various concentrations were prepared by diluting the appropriate amount in growth medium. 50 µL of these particles were added to treat cells. A two- stage incubation was employed, i.e. 24 h incubation firstly, and another 4-h incubation with CCK-8 reagent (10 µL/well). The results were measured using absorbance at 450 nm and analyzed according to the results of OD450 ratio between ACS Paragon Plus Environment


the wells with DDAB-PLA MPs-treated cells and the control with only cells and medium. Endotoxin Levels. To exclude the impact of endotoxin contamination, the endotoxin level was measured using LAL assay method (Pyrosate 0.25 EU/Ml, Falmouth, MA) so that to ensure the endotoxin in each formulation was lower than 0.05 EU/mg MPs. Determination of Local Inflammation by Quantitative Real-Time PCR Vaccine-associated inflammation at injection site was evaluated by analyzing related cytokine gene expression quantitative real-time PCR. The injection site was excised 3 h after vaccine administration. After extracting total RNA and reverse-transcribe to cDNA, SYBR Green PCR kit and CFX96 RT-PCR system (Bio-Rad) were utilized to amplify. The calculation of gene expression level and primers used were based on our previous methods43. Activation and Maturation of BMDCs in vitro BMDCs with 24 h MP-treatment, were further labeled with flow antibodies against CD 11c, CD40, CD80, CD83 and CD86 (eBioscience, CA).The expression of those co-stimulatory molecules on BMDCs was determined as percentages by flow cytometer (CyAnTM ADP, USA). Animals and immunization scheme Each immunization group has 6 mice (BALB/c, female, 4-6 weeks old) as follows: control, alum, PLA MPs, and DDAB-PLA MPs. Traditional immunization scheme was applied (three-step: prime, boost and booster), and each mouse was intramuscularly injected with 2 µg of antigen on day 0, 14 and 28. Determination of antigen-specific antibodies On day 7, 21 and 35, serum was collected using previous method25. The levels of HBsAg-specific IgM, IgG, IgG1, and IgG2a antibody were determined by ELISA as described previously44. Serum samples were diluted at indicated dilutions: for IgM, 1:400; for IgG, IgG1, and IgG2a, 1:100. HRP-conjugated goat anti-mouse antibodies were diluted as following: for IgM and IgG, 1:6000; for IgG1 and IgG2a, 1:8000. Splenocyte proliferation assay ACS Paragon Plus Environment

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Mice were sacrificed on day 35, and spleens were collected to prepare splenocytes suspensions25. To determine activation of antigen-specific splenocyte, splenocyte proliferation assay and proliferation index was adapted and calculated from previous methods45. The HBsAg concentration for ex vivo stimulating was 5 µg/mL. Evaluation of cytokine levels by ELISA The supernatant of restimulated splenocytes was collected and measured for IL-2, IL-12, IFN-γ, Granzyme B, IL-4, IL-5, IL-6 and IL-10 levels using ELISA kits (eBioscience, CA). Determination of intracellular IFN-γ and CTL-related factors expression For the intracellular IFN-γ and CTL-related factors expression, splenocytes were restimulated with HBsAg (5 µg/mL) for 60 h followed by additional 8-h treatment of brefeldin A.

For intracellular IFN-γ expression, cells were stained with AlexaFluor

488-CD4, PE-CD8, and APC-IFN-γ flow antibodies; for CTL-related factors expression, splenocytes were stained with AlexaFluor488-CD8, eFluor®450-107a, PE-FasL, and APC-perforin flow antibodies, then detected and analyzed by flow cytometry. Statistical Analysis. The data were presented as the mean ± SD of at least three independent experiments. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) and Tukey adjusted t-tests. Statistical significance thresholds were set at *p < 0.05; **p < 0.01; ***p < 0.001.



Results and discussion Characterization of DDAB-PLA MPs PLA MPs were prepared as control formulation and coated the cationic compound lipid, DDAB, on their surface to fabricate DDAB-PLA MPs. From scanning electron micrographs (SEM) analysis, both MPs were spherical and had surface smooth (Figure1.A1-B1). By measuring size and zeta potential, the size of DDAB-PLA MPs was ~1.0 µm, and DDAB-PLA MPs had positive surface charge (~+16 mV) , demonstrating the successful decoration of DDAB (Table 1). For the control formulation, the size of PLA MPs was also about ~1.0 µm, and the surface charge was found to be electronegative (~ -20 mV) (Table 1). Then, compared with PLA MPs, the DDAB coating on PLA MPs was also proved by the peak appearance of Br5p, Br3p, Br3s, and N1s core-level spectrum of XPS at the binding energy of ~175, 185, 260, and 400, eV on the surface of DDAB-PLA MPs, respectively (Figure. 1 A2-B2). The biocompatibility of DDAB-PLA MPs tested on BMDCs demonstrated their excellent biocompatibility (Figure 1C). HBsAg adsorption efficiency was increased following the increasing surface charge of the MPs (Figure 1D), which showed that the main adsorption force should be electrostatic interaction between antigen and MPs. Since the isoelectric point of HBsAg is 4.7 in the buffer (pH~6.5), negatively charged HBsAg had preferable adsorption with positively charged DDAB-PLA MPs. PLA MPs, with relatively low antigen adsorption, might have contributed to the weak interactions between both negatively charged HBsAg and MPs (Figure 1D). The effect of antigen absorption on MPs’ size and surface charge was subtle. The increment of MPs’ size was about 2-8 nm, and surface charge went down to ~+10-15 mV. These data showed that the cationic compound decorated PLA MPs (DDAB-PLA MPs) were suitable as vaccine delivery and adjuvant system.


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Table 1 Characterization of PLA and DDAB-PLA microparticles Microparticles

Diameter (DLS, nm)

PDI

Zeta potential (mV)

PLA

1012.05 ± 5.6

0.121

-19.62 ± 0.59

DDAB-PLA

1024.02 ± 3.4

0.139

+16.48 ± 1.25

Figure 1. Characterization of PLA and DDAB-PLA MPs: A-B) SEM and XPS spectra of PLA MPs and DDAB-PLA MPs; C) the effect of DDAB-PLA MPs on BMDC viability using CCK-8 assays; D) HBsAg adsorption by alum and MPs.



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DDAB-PLA MP-Enhanced Local Inflammation Response around Injection Site after Immunization After immunization with MP-based vaccine, the local inflammation around the injection sites would be initiated. The expression of typical pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, CCL2 and CXCL 1, was assessed at 3 h after intramuscular immunization in the hind legs of mice. The result was normalized using the basic level of non-immunized mice. From Figure 2 A-B, compared to PLA MPs and alum, injection of DDAB-PLA MPs induced significantly higher expression of pro-inflammatory cytokines. Chemokines played a pivotal role in inducing

immune

cells

recruitment46.

The

expression

of

monocyte

and

granulocyte-attracting chemokines (CCL 2 and CXCL 1, respectively) was significantly elevated by DDAB-PLA MPs (Figure 2 C-D). The endotoxin level assays excluded the mechanism involved by PAMP (pathogen-associated molecular patterns). Cationic particles are known to be more inflammatory than negative or neutral particles47, 48. Moreover, DDAB, used in microparticles formulation, has been tested for eliciting inflammatory local response and cell-mediated immune response49, 50

. The surface charge and intrinsic adjuvanticity endowed by DDAB could make

DDAB-PLA MPs having significant effect on the induction of cytokine and chemokine. Taken together, these results indicated that DDAB-PLA MPs could trigger the early inflammatory response to prime the resultant immune response.


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Figure 2. Gene expression profiles of typical pro-inflammatory cytokine and chemokine around injection site after immunization.

DDAB-PLA MPs-Primed Activation of BMDCs after Stimulating In Vitro In addition to local inflammation response indicating the early innate immune response, MPs would activate APCs by upregulating co-stimulatory markers to induce antigen specific immune responses16. BMDCs were incubated with MPs- or alum-based vaccine for 12 h. The expression of costimulatory markers was measured. Highly expressed CD80 (Figure 3B) and CD86 (Figure 3D), and moderately expressed CD40 (Figure 3A) were significantly upregulated in the presence of DDAB-PLA MPs compared to PLA MPs and alum. Similarly, the expression of lowly expressed CD83 was slightly increased (Figure 3C). Collectively, these results suggested enhanced effect on DC activation by DDAB-PLA MPs.



Figure 3. Expression of the costimulatory markers CD40, CD80, CD83, and CD86 on BMDCs stimulated with various vaccine adjuvants.

DDAB-PLA MP-Induction of HBsAg -Specific Antibodies in Mice Having confirmed DC activation, we next sought to detect the efficacy of HBsAg-specific antibodies production driven by various vaccine adjuvants. IgM, as the earliest antibody generated responding to antigen, would play regulating function on the IgG production51. Both MPs augmented IgM production compared to alum indicating the early protection (Figure 4C; p

Potential Hepatitis B Vaccine Formulation Prepared by Uniform-Sized

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