Glucan particles are a powerful adjuvant for the ... - ACS Publications

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Glucan particles are a powerful adjuvant for the HBsAg, favoring antiviral immunity Edna Soares, Zwier MA Groothuismink, Andre Boonstra, and Olga Borges Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01322 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Molecular Pharmaceutics

338x190mm (300 x 300 DPI)

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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

Glucan particles are a powerful adjuvant for the HBsAg, favoring antiviral immunity Edna Soares§,#; Zwier MA Groothuismink$; André Boonstra$; Olga Borges§,#,*

Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517

§

Coimbra, Portugal #Faculty

of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde

Azinhaga de Santa Comba 3000-548, Coimbra, Portugal $Department

of Gastroenterology and Hepatology, Erasmus MC, University

Medical Center Rotterdam, the Netherlands

KEYWORDS Vaccine adjuvants, β-glucan, chitosan, polymeric particles, hepatitis B antigen

1 ACS Paragon Plus Environment

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ABSTRACT The lack of vaccine adjuvants that are able to induce robust T cell responses fosters the search for more powerful options. Pathogen-like particles are a promising approach. The adjuvant activity of pathogen-like particles is highly influenced by size and surface composition. This study aimed to evaluate the adjuvant potential of two different β-glucan-based particles: blend chitosan/β-glucan particles (ChiGluPs), which are positively charged and have mean size of 1276 nm, and neutral yeast-derived glucan particles (GPs) with a mean size of 3 µm. Additionally, chitosan particles (ChiPs) were used to understand the effect of β-glucan addition (ChiGluPs). Mouse spleen cells responded through the production of either TNF-α or RANTES, following in vitro stimulation with particles containing either β-glucan (ChiGluPs and GPs) or chitosan (ChiGluPs and ChiPs). Human monocytes responded to all particles through TNF-α secretion. Subcutaneous vaccination of mice with the hepatitis B surface antigen (HBsAg) showed increased serum IgG for all particles compared to HBsAg alone (435fold, 4500-fold or 2500-fold increase for either ChiPs, ChiGluPs or GPs). Interestingly, only GPs elicited the secretion of HBsAg-specific Th1, Th2, Th9, Th17, Th22 and Treg related cytokines. This study demonstrates, for the first time, that GPs can have a significant role against the hepatitis B virus, by favoring antiviral immunity. 1. INTRODUCTION Vaccination is the most cost-effective measure to prevent mortality 1. Advancing technology and the desire for safer options led to the development of subunit vaccines with poorly immunogenic antigens that require the addition of immunostimulatory agents 2. Aluminum salts are the most commonly used adjuvants to date by promoting the generation of humoral immune responses

3, 4.

Although prophylactic vaccines are



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highly effective, the lack of robust T cell responses makes their use deceptive for some viral infections or therapeutic interventions. Modern vaccinology still has some challenges to solve, namely the search for better adjuvants to use in therapeutic vaccines against infectious diseases such as hepatitis B, in which restoration of T cell function is considered crucial for a functional cure 5. Therefore, new rationally designed adjuvants are needed and must be used to modulate the desired immune response, through the stimulation of both humoral and cellular adaptive immune responses

4, 6.

Dendritic cell

(DC) activation is a key factor in vaccine design for viral infections as they can orchestrate innate and adaptive immunity to induce both cytotoxic T lymphocyte (CTLs) and helper T (Th) cell differentiation, crucial for HBV-infected hepatocytes destruction. Adjuvant tailoring to reproduce pathogen physicochemical properties represents a promising approach 7. Natural polysaccharides are known to function as pathogenassociated molecular patterns (PAMPs) and can be recognized by pattern recognition receptors (PRRs – e.g. toll-like receptors (TLR) and C-type lectin receptors (CLR)) of the innate immune system allowing a prompt immune response

3, 8.

Interestingly, most

innate immune responses are directed against fungal cell wall polysaccharides, such as chitosan and β-glucan 9. Biocompatibility and reduced side effects are additional advantageous features of those biopolymers

2, 3, 10.

β-glucans are commonly found in

bacteria, fungi and yeast and are characteristically recognized by Dectin-1 receptors (a CLR), which are widely expressed by immune cells, like macrophages and DCs

11.

Tumor necrosis factor (TNF)-α release from macrophages is the hallmark of Dectin-1 activation, and is only induced by particulate β-glucans 8. In fact, a particulate design is also strongly correlated with the generation of a pro-inflammatory environment and recruitment of immune cells

7, 12, 13.

Dectin-1 activation leads to an immune response

characteristic of a Th1/Th17 differentiation 14. On the other side, chitosan is a cationic polymer prepared by chitin deacetylation with excellent bioadhesive properties for controlled drug delivery and significant immunostimulatory properties, including enhanced antigen presentation to DCs and NRLP-3 inflammasome activation 9, 15, 16. The global aim of the present work was to study and compare the immune outcome

of

β-glucan-based

“pathogen-like”

adjuvants,

regarding

different

physicochemical properties. It has been reported that hollow porous Saccharomyces cerevisiae derived glucan particles (GPs) 17 are associated with cell-mediated immunity following repeated subcutaneous vaccination with model antigens 18-20. For that reason, 3 ACS Paragon Plus Environment

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in the present study, we decided to evaluate for the first time the GPs adjuvant effect in a HBsAg-based vaccine. This will be done in comparison to new blend chitosan/βglucan particles (ChiGluPs), recently developed by our group based on a precipitation/reticulation technique

21.

ChiGluPs were demonstrated to be a good

adjuvant for the HBsAg inducing high antibody titers in vivo, but not for HBsAgspecific cytokine production, following two subcutaneous injections. Additionally, when compared to similarly produced plain chitosan particles (ChiPs), ChiGluPs revealed the beneficial β-glucan incorporation for increased antibody titers 21. However, mechanistic studies using ChiGluPs to understand the role of β-glucan are missing and is part of the aim of the present work, in comparison to both the plain β-glucan adjuvant (GPs) and the plain chitosan adjuvant (ChiPs). It is important to note that both the βglucan source for ChiGluPs (curdlan) and the yeast derived β-glucan zymosan (similar to our GPs) are 1,3-β-D-glucans that induce a TNF-α secretion profile in vitro, dependent on Dectin-1 receptor

18, 22.

Proof-of-concept in vivo studies were done to

evaluate the vaccine efficacy with (ChiPs, ChiGluPs and GPs) and without adjuvant (No Adjuvant) in the generation of humoral-mediated (antigen-specific antibodies) and cellmediated (Th cytokines profile) immunity 6. The number of times the vaccine is administrated is known to influence the immune responses elicited, and was chosen based on a study with ovalbumin loaded GPs that showed increased immunogenicity with three consecutive administrations comparing to two or just one dose

20.

Also two

ChiGluPs/HBsAg vaccine doses did not induce cytokine mediated responses in our recent report 21. 2. MATERIALS AND METHODS 2.1. MATERIALS Curdlan (Lot 60201c - Alcaligenes faecalis extracted β-1,3-glucan), the β-glucan was acquired from Megazyme (Ireland). Saccharomyces cerevisiae was kindly donated by AB MAURI (Mauripan, Portugal). Chitosan (ChitoClear™ - 95 % DD and 8 cP viscosity) was purchased from Primex BioChemicals AS (Norway) and purified as previously described 23. CBC Genipin was purchased from Challenge Bioproducts Co., Ltd. (China). The hepatitis B surface antigen (HBsAg - purity > 98 %; subtype adw, VLP with approximate 25 nm) was acquired from Aldevron (USA). Brefeldin A, concavalin A (ConA), 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), heat4 ACS Paragon Plus Environment


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inactivated fetal bovine serum (FBS), lipopolysaccharide (LPS), MEM amino acids and MEM non-essential amino acids, Roswell Park Memorial Institute (RPMI 1640), sodium pyruvate, thiazolyl blue tetrazolium bromide (MTT) reagent, torula yeast type IV (tRNA) were purchased from Sigma Aldrich Corp. (USA). Nuclear Labeling Kit and Image-iT™ were obtained from Life Technologies Corporation (UK). IgG1, IgG2c, IgG3 and IgE horseradish peroxidases (HRP) were purchased from Rockland Immunochemicals Inc. (Limerick, PA, USA.) and IgG HRP from Bethyl Laboratories (USA). Goat anti-mouse HBsAg (ad/ay) HRP was bought from Meridian, Life Science, ®Inc. (USA). Fluorescein isothiocyanate (FITC) was acquired to Santa Cruz Biotechnology

(USA).

Th1/Th2/Th9/Th17/Th22/Treg

cytokine

17-Plex

Mouse

ProcartaPlex™ Panel + RANTES (simplex) multiplex immunoassay (Target list: IFN-γ, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF-α, GM-CSF, IL-18, IL-10, IL-17A, IL-22, IL-23, IL-27, IL-9 and RANTES), TNF-α-PE-Cy7 (Mab11) and IL-10PacificBlue (JES3-9D7) antibodies and sandwich ELISA kits specific for human IL-10, IL-12p70, TNF-α and IL-6 from eBioscience (USA). IL-6-FITC (MQ2-13A5) and IL12p40-PE (C11.5) antibodies from BD Pharmingen (BD Biosciences, USA). XVIVO™15 culture medium, L-glutamin, HEPES, penicillin/streptomycin and PYROGENT™ Gel Clot LAL Assays (Lymulus Amebocyte Lysate - 0.125 EU/ml Sensitivity) from Lonza (Belgium). Ultrapure LPS-SM from InvivoGen (USA). R848 from Alexis Biochemicals (USA). Ficoll-Paque™ plus from GE Healthcare (Sweden). Magnetic CD14 MicroBeads from Miltenyi Biotech (Germany). Murine interferon (IFN)-γ standard ABTS ELISA development kit from PeproTech (USA). All other chemicals and reagents used were of analytical grade. 2.2. PARTICLE PRODUCTION Particle production was performed under a laminar flow cabinet, using pyrogenfree water, to avoid LPS contamination. After particle production a Gel Clot LAL Assay with a sensitivity of 0.125 EU/ml was performed to attest contamination or not. Chitosan (ChiPs) and β-glucan/chitosan (ChiGluPs) particles were produced by precipitation followed by genipin reticulation, as recently described in detail by us

21.

Briefly, the acidic chitosan solution is precipitated, under high speed homogenization, in a basic solution containing (ChiGluPs) or not (ChiPs) the β-glucan (Curdlan). Then, the crosslink was performed with genipin in PBS pH 7.4. Glucan particles (GPs) were 5 ACS Paragon Plus Environment

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produced from Saccharomyces cerevisiae, by alkaline/acidic treatments, as firstly described by Soto and Ostroff

17

with minor modifications already reported by our

group 24. For laser scanning confocal microscopy (LSCM) and flow cytometry studies, chitosan was labeled with FITC and used to prepare the particles in a proportion of 25 % of total chitosan used and GPs were labeled with DTAF as previously described 24. For in vivo studies, the HBsAg was adsorbed onto ChiPs and ChiGluPs in water, under mild agitation, 30 min prior vaccination. HBsAg loading into GPs was done using tRNA, as described before

24.

GPs were centrifuged to eliminate residual amounts of

tRNA and suspended in water for use. For HBsAg loading efficiency (LE) assessment, the HBsAg quantification in the resultant supernatant of HBsAg-loaded ChiPs and ChiGluPs and GPs centrifugation, was done by a direct ELISA method, previously described 24 and calculated as follows (Eq. 2): LE (%) =

total HBsAg (µg/mL) - free HBsAg in supernatant (µg/mL) totalHBsAg (µg/mL)

x 100

(Eq. 2)

2.3. SIZE AND ZETA POTENTIAL MEASUREMENTS HBsAg-loaded and unloaded particles mean hydrodynamic diameter was measured by dynamic light scattering (DLS), and zeta potential (ZP) measured by electrophoretic light scattering (ELS) using a DelsaTM Nano C particle analyzer (Beckman Coulter, USA). Size analyses were performed at 25 °C and scattered light collected at a 165° angle. 2.4. HUMAN MONOCYTE STIMULATION STUDIES The capacity of ChiPs, ChiGluPs and GPs to stimulate immune cells was tested using human monocytes. Venous blood was collected from healthy volunteers at the Erasmus MC University Medical Center (Rotterdam) and a written informed consent was obtained from all participants. The peripheral blood mononuclear cells (PBMCs) were isolated by gradient density centrifugation (Ficoll-Paque™ plus). Sequentially monocytes were purified from PBMCs using magnetic CD14 MicroBeads in LS columns for positive selection (purity 95-99 %). Monocytes (3x105) were cultured overnight in 96-well plates in 250 µL of X-VIVO™15 culture medium containing penicillin/streptomycin, L-glutamin and HEPES either unstimulated or stimulated with 6 ACS Paragon Plus Environment


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the developed adjuvants (ChiPs, ChiGluPs, GPs - 200 µg/mL) or positive controls (TLR4 - Ultrapure LPS-SM - 2 ng/mL; TLR7/8 – R848 – 1 µg/mL). For intracellular cytokine staining, brefeldin-A (10 µg/mL) was added 2 h after the onset of stimulation and was present until the end. After a total of 18 h of culture, samples were fixed with 2 % formaldehyde, permeabilized with 0.5 % saponin and stained with antibodies against TNF-α-PE-Cy7, IL-6-FITC, IL-12p40-PE and IL-10-PacificBlue. Cytokine-producing monocytes were assessed by flow cytometry (FACS Canto II, BD) and analysed using FlowJo version 10.1 (Tree Star, Inc., USA). For cytokine quantification, after 18 h of culture as described above, supernatants were harvested, and cytokine production determined by a sandwich ELISA kits specific for IL-10, IL-12p70, and TNF-α and IL6. The detection limit for IL-10, IL-6 and TNF-α was 78 pg/mL and for IL-12p70 was 23 pg/mL. 2.5. ANIMAL STUDIES 7/8-week old female C57BL/6 mice were acquired from Charles River Laboratories (Spain), kept in the animal house facility with 12 h light/dark cycle and ad libitum food and water. Animal studies were approved (ORBEA_50_2013/27092013) and carried out in accordance with institutional ethical guidelines and with National (Dec. No. 113/2013) and International (2010/63/EU Directive) legislation. 2.5.1. IN VITRO MOUSE SPLEEN CELL STIMULATION STUDIES ChiPs, ChiGluPs and GPs capacity to stimulate immune cells was achieved using a primary culture of C57BL/6 mouse splenocytes, collected and maintained in RPMI 1640 as previously described by us 25. 50 µL of 1x107 cells/mL cell suspension were plated in a 96-well plate and 150 µL of cell culture medium alone or enriched with ChiPs, ChiGluPs or GPs were added to a final particle concentration of 200 µg/mL. 48 h later, the supernatants were collected and cytokine secretion quantified using a Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex™ Panel + RANTES (simplex) multiplex immunoassay analysed using a Luminex®100™ system. The remaining cells were re-suspended in cell culture medium e proceed to an MTT viability assay with 4 h incubation. The viability of control cells (only culture medium) was established as 100 % and the relative cell viability calculated as follows (Eq. 1): Cell viability (%) =

OD sample (540 nm) - OD sample (630 nm) OD control (540 nm) - OD control (630 nm)


x 100 (Eq. 1)

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2.5.2. MOUSE SPLEEN CELL UPTAKE STUDIES For the uptake experiment, 500 µL of 0.25x107 cells/mL spleen cell suspension were seeded on 48-well for flow cytometry (BD FACSCalibur, BD Biosciences, USA) or 24-well with poly-L-lysine coverslips for LSCM (Zeiss LSM 510 META, Carl Zeiss, Germany) analysis. Then, RPMI either with or without FITC-labeled ChiPs or ChiGluPs and DTAF-labeled GPs was added to a final 100 µg/mL particle concentration. 4 or 8 h later, cells from two wells were pooled, centrifuged for 10 min at 218 x g and suspended in ice-cold PBS for further cytometry analysis. Propidium iodide (PI - 1 µL at 50 µg/mL) was used for cell death assessment and trypan blue (TB – 0.4 % in PBS) was used to quench extracellular fluorescence. A population of 100000 cells was analyzed per sample and the results processed using CellQuestModfit LT software and presented as the frequency of fluorescent cells (%). For LSCM analysis (4 h incubation), cells were fixed for 15 min with 4 % formaldehyde at 37 ºC and the nucleus labeled using Image-iT™ (Hoechst 33342) according to manufacturer’s instructions. After labeling, cells were washed with PBS and the coverslips mounted in microscope slides with DAKO mounting medium. 2.5.3. MOUSE VACCINATION STUDIES Vaccination groups and schedule are listed in table 1. All vaccine doses were suspended in water and administered subcutaneously. 2.5.3.1.

SERUM IMMUNOGLOBULINS

Blood samples were collected by submandibular venipuncture 14 days after each vaccine dose. The determination of serum IgG, IgG2c, IgG1, IgG3 and IgE was performed by ELISA, as previously described26. Serum titers were presented as the endpoint titer (antilog of the last log 2 dilution for which the optical density was at least two-fold higher than naive mouse samples equally diluted). The log 2 endpoint titers were used to normalize the data and equalize variability and then statistically analyzed. 2.5.3.2.

SPLEEN CELL RESTIMULATION WITH HBsAg

For HBsAg restimulation assay, spleens of vaccinated mice were treated as explained in section 2.6.1. HBsAg, LPS and ConA (positive controls) were incubated 8 ACS Paragon Plus Environment


with the cells at a final concentration of 5 µg/mL, 1 µg/mL or 6.25 µg/mL, respectively. Cells were incubated with or without the stimuli for 48 h and then supernatants were collected and analyzed using the same multiplex immunoassay as described above. For the positive controls, a pool of splenocyte supernatants of all mice from each group was used for the multiplex assay. 2.6. STATISTICAL ANALYSIS Results were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism v 5.03 (GraphPad Software Inc., USA). Comparisons between two samples were done using Mann-Whitney U test and multiple comparisons by ANOVA, followed by Tukey’s post-test. A value of p

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