Conjugation with an InulinâChitosan Adjuvant Markedly Improves the

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Conjugation with an inulin-chitosan adjuvant markedly improves the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein Weili Yu, and Tao Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00138 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 11, 2016

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Molecular Pharmaceutics

Conjugation with an inulin-chitosan adjuvant markedly improves the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein

Weili Yu 1, 2, and Tao Hu 1,*

1

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

2

University of Chinese Academy of Sciences, Beijing 100190, China

Running title: An inulin-Cs adjuvant system

* To whom the correspondence should be addressed. Tao Hu, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences No. 1 Bei-Er-Tiao Street, Haidian District, Beijing 100190, China. E-mail: [email protected]. Tel: +86-10-62555217. Fax: +86-10-62551813.

1

ACS Paragon Plus Environment


Table of contents

Inulin Chitosan IFN-γ (pg/ml)

16000

Conjugation

Inulin-Cs-CT

CT Inulin-CT Cs-CT Inulin-Cs-CT

40 30 20 10

Second dose

0 0

CT specific IgG titers

CFP10-TB10.4 (CT)

CT (µg/ml)

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

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8000 4000 0

4000

CT

Cs-CT Inulin-Cs-CT

2000 1000

Second dose

0 50 100 150 200 250 300

Time (h)

Time (h)

Inulin-CT

CT Inulin-CT Cs-CT Inulin-Cs-CT

3000

0

50 100 150 200 250 300

12000

2


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Abstract Protein-based vaccines are of potential to deal with the severe situations posed by Mycobacterium tuberculosis (Mtb). Due to inherently poor immunogenicity of Mtb protein antigens, a potent immunostimulatory adjuvant is needed to enhance the cellular and humoral immune response to Mtb protein antigens. Inulin and chitosan (Cs) are polysaccharide adjuvants that can be used as achieve such an objective. The inulin-Cs conjugate (inulin-Cs) acted as a potent adjuvant through a synergistic interaction of inulin and Cs. CFP10 and TB10.4 are two important virulent protein antigens of Mtb. The CFP10-TB10.4 fusion protein (CT) was constructed and used as the protein antigen. In the present study, an adjuvant delivery system (inulin-Cs-CT) was developed by covalent conjugation of CT with inulin-Cs. Conjugation with inulin-Cs significantly increased the hydrodynamic volume of CT and did not alter the structure of CT. High levels of Th1-type cytokines (IFN-γ, TNF-α and IL-2) and Th2-type cytokine (IL-4) were secreted by provocation of inulin-Cs-CT. Inulin-Cs-CT elicited high CT-specific antibody titers, mostly in the form of IgG1 and IgG2b. Pharmacokinetics revealed that conjugation with inulin-Cs could prolong the serum exposure of CT to the immune system. Pharmacodynamics suggested that conjugation with inulin-Cs led to an efficient production of CT-specific IgG. Thus, conjugation of inulin-Cs can serve as a potent adjuvant delivery system to improve the immunogenicity of the Mtb protein antigens.

Keywords: Mycobacterium tuberculosis, inulin, chitosan, adjuvant delivery system, subunit vaccine

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INTRODUCTION Mycobacterium tuberculosis (Mtb) is a major cause of tuberculosis (TB), one of the most leading public diseases in the world [1]. Vaccination is an effective way to deal with the severe situations posed by TB [2]. Although Mycobacterium bovis BCG vaccine has been used for over ninety years, it can not reliably prevent the prevalence of TB [3]. Vaccines based on Mtb protein antigens have potential to replace or boost BCG vaccine and have received much attention [4]. However, protein antigens alone are typically very poorly immunogenic due to lack of exogenous immune activating components such as nucleic acids, lipids and cell membrane components [5]. Immunostimulatory adjuvants can turn the protein antigens into effective vaccines by increasing their immunogenicity [6]. In general, cell-mediated immunity has been considered to play a predominant role in protection against Mtb infection [7]. The adjuvants (e.g., CAF01, AS01A and IC31) presently used for the protein-based vaccines can elicit a strong cellular immunity against Mtb [8-10]. However, some evidence suggests that humoral immune response is also indispensible for protection against Mtb [11]. Thus, there is an increasing need to achieve a robust cellular and humoral immunity against Mtb. Inulin is a biocompatible and nontoxic polysaccharide (PS) from dahlia tubers [12]. Inulin can promote cellular and humoral immunity and has been used as a vaccine adjuvant against HBV and HIV [13-14]. Chitosan (Cs) is structurally a cationic poly-β-(1→4)-D-glucosamine obtained by N-deacetylation of chitin with biodegradable and nontoxic properties [15]. Cs has been used to enhance the ability of antigens to elicit cellular and humoral immunity [15]. Recently, a potent adjuvant has been developed by conjugation of two different adjuvants in one entity [16]. For example, CpG and lipoteichoic acid were conjugated in one entity and showed a

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stronger stimulation of immune cells relative to a lipoteichoic acid-CpG mixture [16]. Thus, an inulin-Cs conjugate was assumed to be a potent immunostimulatory adjuvant by the synergistic interaction of inulin and Cs. It is hypothesized that an effective adjuvant system for protein antigens includes both a delivery system and adjuvants [17]. The delivery system can trigger the adaptive immune system by prolonging the serum exposure of protein antigens and enhancing uptake by antigen-presenting cells (APCs) [18]. Adjuvants can enhance the apparent “danger” of protein antigens by providing the molecular signals needed to stimulate B- and T-cells or to activate APCs [18]. Covalent conjugation of adjuvant with protein antigens in one entity can prolong the serum exposure of protein antigens to the immune system and insure that both the protein and adjuvant reach the APCs simultaneously [19]. TB antigen fusion proteins typically appear to evoke a more efficient immune response than individual protein antigen or a mixture of two antigens [20]. CFP10 and TB10.4 are two important virulent protein antigens of Mtb, which belong to the ESAT6 protein family [21-22]. CFP10 was demonstrated to be strongly recognized by B and T cells of patients, followed by induction of high IFN-γ level. TB10.4 was able to promote a strong immune response and induce significant protection against Mtb. Thus, a CFP10-TB10.4 fusion protein (CT) was genetically constructed, expressed and used as a model protein antigen in the present study. A potent immunostimulatory adjuvant (inulin-Cs) was prepared by conjugation of inulin with Cs. An adjuvant delivery system was developed by covalent conjugation of inulin-Cs with CT. The immunological properties of the conjugate (inulin-Cs-CT) were measured for evaluating the efficacious adjuvant potency of inulin-Cs. A strong synergistic adjuvant interaction of inulin and Cs was observed to promote a potent

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cellular and humoral immunity to CT. The pharmacokinetic and pharmacodynamic properties of inulin-Cs-CT were measured for evaluating the adjuvant delivery potency of inulin-Cs. Thus, an efficacious protein-based vaccine can be developed using the inulin-Cs adjuvant delivery system for protection against Mtb.

EXPERIMENTAL PROCEDURES

Materials. Chitosan (medium molecular weight), inulin from dahlia tubers, 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), 2-iminothiolane (IT), bovine serum albumin (BSA), NaCNBH3 and 3,3’,5,5’-tetramethylbenzidine (TMB) were purchased from Sigma (USA). Methoxyl PEG propionaldehyde with Mw of 5 kDa

(PEG-ald)

was

ordered

from

Jenkem

Biotech

(China).

Horseradish

peroxidase-conjugated goat anti-mouse IgG Fc antibody (HRP-IgG), HRP-IgG1, HRP-IgG2b and HRP-IgG2c were ordered from Abcam (USA). IFN-γ, TNF-α, IL-2 and IL-4 ELISA kits were purchased from eBioscience (USA). All other reagents were of analytical grade. Construction of CFP10-TB10.4 plasmid. The gene encoding CFP10-TB10.4 contained a linker (G4S)3 and was synthesized according to the nucleotide sequences of CFP10 gene and Mtb10.4 gene of the Mtb H37Rv strain. The target DNA was amplified by PCR, using the synthesized gene as the template. Oligonucleotides (5’-CGCGGATCCATGGCTGAAATGAAAACCG-3’)

and

(5’-ACCCAAGCTTTTAACCACCCCATTTAGCAG-3’) were used as the sense and antisense primers, respectively. The PCR product was cloned into the pET28a vector. The product was excised with BamH and Hind III, followed by transformation into the E. coli host TOP10. The plasmid construct was isolated and re-transformed into E. coli host BL21 as an expression strain. 6


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Expression and refolding of CT. The expression strain was inoculated in the LB medium containing 100 µg/ml kanamycin. The culture was incubated with vigorous aeration at 37 °C until the density was up to ~0.8 of A600. The culture was incubated with 0.05 mM isopropylthiogalactoside (IPTG) at 37 °C for 4 h. The cell pellets were resuspended and disrupted with sonication in an ice bath. The inclusion bodies (IBs) of the CFP10-Mtb10.4 fusion protein (CT) were isolated and washed two times with 50 mM Tris-HCl buffer containing 1 M NaCl and 2 M urea (pH 8.0). Then, the IBs were solubilized by 50 mM Tris-HCl buffer containing 1 mM EDTA, 15 mM DTT and 8 M urea (pH 8.0). The solution was diluted gradually with the refolding buffer (0.1 M Tris-HCl, 0.18 mM EDTA, 4 mM L-arginine, 0.9 mM GSH, 0.18 mM GSSG and 2 M urea, pH 8.0) and gently stirred at 4 oC for overnight. Purification of CT. CT was purified from the refolding solution by anion exchange chromatography, using a Q Sepharose HP column (2.6 cm×15 cm, GE Healthcare, USA). The column was equilibrated and eluted with 20 mM Tris-HCl buffer (pH 8.0, buffer A), followed by gradient elution with 0-0.5 mM NaCl in buffer A. The fractions containing CT were pooled and further purified by a Superdex 200 column (2.6 cm×60 cm, GE Healthcare, USA). The column was equilibrated and eluted with 20 mM phosphate buffer containing 0.15 M NaCl (pH 7.4). The purified CT was analyzed by SDS-PAGE, using 12% gradient Tris-glycine polyacrylamide gel. The molecular weight of CT was determined by MALDI-TOF. Preparation of inulin-CT. Inulin (4 mg/ml, 6 ml) was oxidized by 20 mM NaIO4 in 20 mM acetate buffer (pH 5.8) (Fig. 1). The reaction was kept in dark condition for 20 min at room temperature and was stopped by addition of excessive ethylene glycol, followed by extensive dialysis against 20 mM phosphate buffer (pH 7.4). The inulin-CT conjugate (inulin-CT) was obtained by incubation of CT (2 mg) with the

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oxidized inulin (2 mg) and NaCNBH3 (1 mg) in 20 mM phosphate buffer (pH 7.4) at 4 oC overnight (Fig. 1). Preparation of Cs-CT. Cs (2.5 mg/ml, 4 ml) in 20 mM citric acid monohydrate (pH 3.0) was incubated with 4 mg PEG5K-ald and 4 mg NaCNBH3 at room temperature overnight (Fig. 1). The excessive reagents were removed by ultrafiltration through a 50 kDa cutoff membrane (Millipore, USA). PEG-Cs was thiolated by 2-iminothiolane (IT, 2 mg) at room temperature for 3 h (Fig. 1), followed by extensive dialysis with 20 mM phosphate buffer (pH 7.4). Similarly, the Cs-CT conjugate (Cs-CT) was obtained by incubation of the thiolated PEG-Cs (10 mg) with the MBS-modified CT (2 mg) in 20 mM phosphate buffer (pH 7.4) at 4 oC overnight. (1) Inulin-OH

NaIO4

Inulin-CHO

(2) Inulin-CHO + CT-NH2

(3) R-CHO + Cs-NH2

O

O

CT-NHCH2-inulin

Cs-NHCH2-R

O

(R1: inulin; R2: PEG)

O

O

CT-NHC(CH2)4-N

O

O

O

(6) CT-NHC(CH2)4-N

(Inulin-CT)

HS-Cs-NHCH2-R

N-O-C(CH2)4-N O

O

NaCNBH3

Thiolation

(4) H2N-Cs-NHCH2-R

(5) CT-NH2 +

NaCNBH3

O + HS-Cs-R

O

CT-NHC(CH2)4-N

O

-S-Cs-R

O (Cs-CT and inulin-Cs-CT)

Figure 1 Schematic representation of inulin-CT, Cs-CT and inulin-Cs-CT.

Preparation of inulin-Cs-CT. Cs (2.5 mg/ml, 4 ml) in 20 mM citric acid monohydrate (pH 3.0) was incubated with 30 mg oxidized inulin and 10 mg 8


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NaCNBH3 at room temperature for overnight (Fig. 1), followed by extensive dialysis The soluble conjugate (inulin-Cs) was incubated with 3 mg IT at room temperature for 3 h (Fig. 1), followed by extensive dialysis. CT was incubated with 50-fold molar excess of MBS in 20 mM phosphate buffer (pH 7.4), followed by extensive dialysis. Finally, inulin-Cs-CT was obtained by incubation of inulin-Cs (8 mg) with CT (2 mg) in 20 mM phosphate buffer (pH 7.4) at 4 oC for overnight (Fig. 1). Purification of the CT derivatives. Inulin-CT, Cs-CT and inulin-Cs-CT were purified from the reaction mixture by anion exchange chromatography, using a Q Sepharose HP column (1.6 cm×2.5 cm, GE Healthcare, USA). The column was equilibrated and eluted with 20 mM phosphate buffer (pH 7.0, Buffer A), followed by gradient elution with 0-1.0 M NaCl in buffer A for 30 min at a flow fate of 2.5 ml/min. The effluent was detected at 280 nm. The fractions corresponding to inulin-CT, Cs-CT and inulin-Cs-CT were pooled, respectively. Size exclusion chromatography. Size exclusion chromatography analysis of the CT-based vaccines was carried out on an analytical Superdex 200 column (1 cm × 30 cm, GE Healthcare, USA) at room temperature. The column was equilibrated and eluted by 20 mM phosphate buffer containing 0.15 M NaCl (pH 7.4) at a flow rate of 0.5 ml/min. The effluent was detected at 280 nm. Dynamic light scattering. The molecular radii of the CT derivatives were measured by dynamic light scattering (DLS) on a DynaPro Titan TC instrument (Santa Barbara, CA, USA) at 25 oC. The samples were all at a protein concentration of 1.0 mg/ml in 20 mM phosphate buffer (pH 7.4). The samples were centrifuged at 12,000 g for 10 min prior to analysis. NMR spectroscopy. Inulin, Cs, PEG-Cs and inulin-Cs were analyzed by 1H NMR spectroscopy at 600 MHz. In brief, the samples were dissolved in D2O at a final

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concentration of 5 mg/ml, except that Cs was in dissolved in 2% (v/v) DCl. A Bruker NMR Spectrometer Avance DRX 600 MHz was used to record the 1H NMR spectra. MestReNova software was used for the data acquisition and processing. Intrinsic fluorescence. Intrinsic fluorescence spectra of the samples were recorded on a Hitachi F-4500 Fluorescence spectropolarimeter (Hitachi, Japan) at room temperature. A cuvette with 1.0 cm path length was used. The emission fluorescence intensities of the samples were measured from 300 nm to 400 nm with excitation at 280 nm. Both of excitation and emission slit widths were 2.5 nm. All the samples were at a protein concentration of 0.15 mg/ml in 20 mM phosphate buffer (pH 7.4). Quantitative assay. Protein level was measured by the bicinchoninic acid method. Inulin level in inulin-CT was measured by anthrone reaction. Inulin level in inulin-Cs was determined by measuring the difference in the amino level of Cs before and after conjugation with inulin, using the 2,2-dihydroxyindane-1,3-dione method. Cs level in Cs-CT and inulin-Cs level in inulin-Cs-CT were determined by measuring the difference in the maleimide content of CT before and after conjugation with Cs or inulin-Cs. The maleimide level of CT was measured using Amplite colorimetric maleimide quantitation kit (AAT Bioquest, Inc., USA). Animals. Female C57BL/6 mice of weight 18-22 g and adult male Sprague-Dawley rats of weight 220-250 g were supplied by Department of Laboratory Animal Science of Peking University Health Science Center (Beijing, China). All procedures of the animal experiments were approved by the Animal Ethical Experimentation Committee of Institute of Process Engineering, Chinese Academy of Sciences (Beijing, China), according to the requirements of the National Act on the Use of Experimental Animals (China).

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Vaccination. Thirty mice were randomly divided into five groups of six animals each. The groups were the CT, CT/Al (CT containing 0.35 mg aluminum adjuvant), inulin-CT, Cs-CT and inulin-Cs-CT groups, which were defined according to the samples immunized. Mice were immunized subcutaneously with 100 µl of the five samples at 100 µg/ml CT on days 0, 14 and 28. Blood samples were taken from the mice on days 14, 28 and 49. Mice were sacrificed to harvest the spleens on day 49. The mice sera were isolated and stored at -80 °C until use. Antibody determination. CT-specific IgG, IgG1, IgG2b and IgG2c in the mice sera were measured as described previously [23]. Briefly, 96-well ELISA plates (Corning, USA) were coated with CT at 4 °C overnight. CT solution (100 µl, 10 µg/ml) in 50 mM NaHCO3 (pH 9.6) was used for each well. The plates were blocked with 200 µl of 4% (w/v) skim milk in PBS buffer (PBS-milk, pH 7.4) at 37 °C for 1 h, followed by washing with PBS buffer (pH 7.4) for three times. Then, 100 µl of mice sera with an appropriate series of two-fold dilutions was incubated in the wells for 1 h at 37 °C. The plates were washed with PBS buffer (pH 7.4) containing 0.1% Tween 20 (PBST) for 3 times. The plates were incubated with 100 µl diluted HRP-IgG, HRP-IgG1, HRP-IgG2b and HRP-IgG2c at 37 °C for 1 h, respectively. Then, the plates were washed with PBST for 3 times, followed by adding 100 µl of the substrate solution containing 0.015% (w/v) of TMB and incubation at 37 °C for 30 min. The reaction was stopped by adding 25 µl of 2 M H2SO4 to each well. Each well was added with TMB as chromogenic and measured at 450 nm. Antibody titers were defined as the dilution number yielding an OD value of 0.2. The inulin-BSA conjugate was prepared essentially as inulin-CT. The Cs-BSA conjugate was prepared essentially as Cs-CT. The 96-well ELISA plates were coated with inulin-BSA and Cs-BSA at 4 °C overnight, respectively. Inulin- and Cs-specific

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IgG titers in the mice sera were measured as determination of CT-specific antibodies. Splenic cytokine release. The spleen cells were re-suspended in complete medium. The medium was composed of RPMI (Roswell Park Memorial Institute)-1640 supplemented with 0.05 mM 2-mercaptoethanol, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% (w/v) fetal bovine serum (FBS). The cells were plated in duplicate in 24-well plates at 2×106 cells per well, followed by stimulation with CT (10 µg/ml) in 5% (v/v) CO2 at 37 oC for 60 h. The TNF-α, IFN-γ, IL-2 and IL-4 levels in the culture supernatants were measured by the corresponding mouse cytokine ELISA kits. Pharmacokinetics. Twenty-four Sprague-Dawley rats were randomly allocated into four groups of six rats in each group. The four groups were the CT, inulin-CT, Cs-CT and inulin-Cs-CT groups, respectively. The rats were injected twice subcutaneously at 1.0 mg CT/kg body weight. The boost immunization was carried out at 168 h after the primary one. Blood samples (0.1-0.2 ml) were drawn from the rats for serum preparation at the selected time points. The CT concentrations in sera were measured by ELISA. Briefly, 96-well plate was coated with anti-CT polyclonal antibody (PAb) from rabbit immunized with CT diluted in 50 mM NaHCO3 (pH 9.6) overnight. After incubation of the sera at 37 °C for 2 h, the wells were washed five times with PBST, followed by addition of HRP-conjugated anti-CT PAb and incubation at 37 °C for 2 h. The wells were added with TMB substrate after washing with PBST and incubated at 37 °C for 30 min. The optical density was measured at 450 nm for CT determination in sera. Native CT was served as standard. Pharmacokinetic parameters, including half-life (T1/2), plasma peak concentration (Cmax), area under the curve (AUC), peak retention time (Tmax) and clearance over bioavailability (Cl/F) were analyzed by a non-compartmental model using the

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PKsolver 2.0 software (China Pharmaceutical University, Nanjing, China). Results were expressed as mean values ± SD. Pharmacodynamics. The CT-specific IgG titers of serum samples from different time intervals were assayed by ELISA for evaluation of the in vivo pharmacodynamic characteristics of the samples. Statistical analysis. The GraphPadPrism 5 program was used to analyze the results (GraphPad Software, San Diego, USA). The values of P

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