Conjugation of β-glucan markedly increase the immunogencity of meningococcal group Y polysaccharide conjugate vaccine

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Conjugation of ␤-glucan markedly increase the immunogencity of meningococcal group Y polysaccharide conjugate vaccine Weilin Qiao a,b , Shaoyang Ji b , Yubao Zhao a , Tao Hu b,∗ a b

School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, Hunan Province, China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

a r t i c l e

i n f o

Article history: Received 18 November 2014 Received in revised form 17 February 2015 Accepted 17 February 2015 Available online xxx Keywords: Meningococcal polysaccharide Conjugate vaccine ␤-Glucan Adjuvant

a b s t r a c t Meningococcal disease is a fatal illness of sudden onset caused by Neisseria meningitides. Meningococcal capsular polysaccharide (CPS) is a major virulence factor that generally does not induce immunological memory. Conjugation with a carrier protein can significantly increase the immunogenicity of CPS and induce immunological memory. However, it is highly desired to optimize the CPS-specific immunogenicity of the conjugate vaccine. Although adjuvant has been widely used to improve the immunogenicity of antigens, co-administration and conjugation of adjuvant with the conjugate vaccine has rarely been investigated. As a stimulator of humoral and cellular immunity, ␤-glucan can activate macrophages and trigger intracellular processes to secrete cytokines initiating inflammatory reactions. In the present study, a conjugate vaccine (CPS-TT) was generated by conjugation of tetanus toxoid (TT) with meningococcal group Y CPS. CPS-TT was further conjugated with ␤-glucan to generate CPS-TT-G. Immunization with CPS-TT-G led to an 8.2-fold increase in the CPS-specific IgG titers as compared with CPS-TT. Presumably, conjugation of ␤-glucan ensured the two components to simultaneously reach the antigen presenting cells and stimulate the immune response. In contrast, co-administration of ␤-glucan suppressed the CPSspecific immunogenicity of CPS-TT. Thus, conjugation of ␤-glucan is an effective strategy to markedly improve the CPS-specific immunogenicity of the conjugate vaccine. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Meningococcal disease is a fatal illness of sudden onset caused by Neisseria meningitides [1,2]. As a major meningococcal virulence factor, the bacterial-cell surface capsular polysaccharide (CPS) has been used to control the incidence of meningococcal disease [3,4]. However, plain CPS is a T-cell independent antigen that fails to induce memory T cell responses in infants and young children [5,6]. In order to induce a satisfactory immune response in infants and young children, CPS was conjugated with a carrier protein to generate the conjugate vaccine [7,8]. Conjugation to a carrier protein can significantly improve the immunogenicity of CPS and induces the immunological memory. Accordingly, a tetravalent meningococcal conjugate vaccine (Menactra, Sanofi Pasteur) has been developed by conjugation of diphtheria toxoid with CPS from seragroups A, C, W135 and Y [9]. Another tetravalent meningococcal conjugate vaccine (Menveo, Novartis Vaccines) has been developed by conjugation of CRM197 with CPS from seragroups A, C, W135 and Y [10,11].

∗ Corresponding author. Tel.: +86 10 62555217; fax: +86 10 62551813. E-mail address: [email protected] (T. Hu).

In addition, a monovalent conjugate vaccine (MenAfriVac, Serum Institute of India) has been developed by conjugation of tetanus toxoid with CPS from seragroup A [12]. In the field of glycoconjugate vaccines, however, it is still interesting to understand if specific conjugation parameters and the use of adjuvant can improve the immunogenicity of glycoconjugate vaccines. Recently, CPS-to-protein ratio, carrier protein, conjugation chemistry, and spacer arm between CPS and protein have been investigated to improve the CPS-specific immunogenicity of the conjugate vaccine [13–15]. For example, immunization with the conjugate vaccine using polyethylene glycol (PEG) as the spacer arm led to a 3.0-fold increase in the CPS-specific IgG titers as compared to that without PEG [15]. Adjuvant has been widely used to significantly improve the immunogenicity of microbial antigens [16,17]. However, co-administration and covalent conjugation of adjuvant with the conjugate vaccine have rarely been investigated. ␤-Glucan is a stimulator of humoral and cellular immunity [18,19]. Binding of ␤-glucan to a specific receptor (especially CR3 and Dectin-1) activates macrophages and triggers intracellular processes, leading to activation of other phagocytes and secretion of cytokines that initiates inflammatory reactions (e.g. IL-1, IL-9 and TNF-␣) [20]. Studies on combination of antigen and adjuvant in one

http://dx.doi.org/10.1016/j.vaccine.2015.02.045 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Qiao W, et al. Conjugation of ␤-glucan markedly increase the immunogencity of meningococcal group Y polysaccharide conjugate vaccine. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.02.045

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2. Materials and methods 2.1. Materials ␤-Glucan from barley (Cat No. G6513), sodium cyanoborohydride, sodium periodate, adipic dihydrazide (ADH), 1-(3dimethylaminopropyl)-3-ethylcarbodimide (EDC), and 3,3 ,5,5 tetramethylbenzidine (TMB) were purchased from Sigma (USA). Horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG Fc antibody (HRP-IgG), IgG1 Fc antibody (HRP-IgG1), IgG2a Fc antibody (HRP-IgG2a) and IgM antibody (HRP-IgM) were ordered from Abcam (USA). Meningococcal group Y capsular polysaccharide and tetanus toxoid with electrophoretic purity were kindly provided by Hualan Biological Engineering, Inc. (China). 2.2. Preparation and purification of CPS-TT Meningococcal seragroup Y CPS (PS, ∼100 kDa) consists of a repeating unit of →6)-␣-d-Glc(1 → 4)-␣-dNeuAc(2 → disaccharide [25]. CPS solution (4 mg/ml, 2 ml) was allowed to react with 8 mg of CNBr (16 ␮l, 50% (w/v) in chloroform) in 0.15 M NaCl at room temperature for 15 min (Scheme 1, Fig. 1). NaOH (0.5 M) was occasionally added to keep the solution at pH 10.5. Then, 2 ml of 12 mg/ml ADH was added. HCl (0.5 M) was added to obtain a final pH value of 8.5. The reaction mixture was incubated at room temperature overnight (Scheme 2, Fig. 1), followed by removal of ADH through extensive dialysis against

CNBr

(1)

CPS -OH

(2)

CPS -OCN + H2N-R-NH2

(3)

CPS-OC-NH-R-NH2 + TT -COOH

CPS -O-CN NH CPS -OC-NH-R-NH2

NH

EDC

NH

O

CPS -OC-NH-R-NH-C- TT (CPS-TT)

NaIO4

(4)

Glu -OH

(5)

CPS-OC-R1-C- TT -NH2 + Glu -CHO

Glu -CHO

TT

CPS-TT-G

TT

CPS-TT 0

20

40

60

80

Time (min)

b CPS-TT-G

CPS-TT

CPS

TT

0

10

20

30

40

Elution time (min) Fig. 2. SEC purification and analysis of CPS-TT and CPS-TT-G. SEC purification (a) was carried out using a Sephacryl S-300 column (2.6 cm × 60 cm) at a flow rate of 3.0 ml/min. The fractions corresponding to CPS-TT and CPS-TT-G were pooled as the arrows indicated. SEC analysis (b) was carried out using a Superose 6 column (1 cm × 30 cm) at a flow rate of 0.5 ml/min.

2 l of 20 mM MES buffer (pH 6.0), using a Slide-A-Lyzer dialysis cassette (Thermo Scientific, MWCO 10 kDa) for 3 h at 4 ◦ C. The dialysis solution was changed with 2 l of 20 mM MES buffer (pH 6.0) with dialysis for another 3 h at 4 ◦ C. The resultant CPS (2 mg/ml, 3 ml) was allowed to react with 1.5 ml TT (4 mg/ml) in the presence of 12 mg EDC in 20 mM MES buffer (pH 6.0) at room temperature overnight (Scheme 3, Fig. 1). The excessive EDC was removed by extensive dialysis against 2 l of 0.15 M NaCl solution, using a Slide-A-Lyzer dialysis cassette (Thermo Scientific, MWCO 10 kDa) for 3 h at 4 ◦ C. The dialysis solution was changed with 2 l of 0.15 M NaCl solution with dialysis for another 3 h at 4 ◦ C. The conjugate (CPS-TT) was purified by size exclusion chromatography (SEC) using a Sephacryl S-300 column (2.6 cm × 70 cm, GE Healthcare, USA). The column was equilibrated and eluted by PBS buffer (pH 7.4) at a flow rate of 3.0 ml/min. The fractions corresponding to CPS-TT were pooled as the arrow indicated in Fig. 2a. The fractions were concentrated using Amicon (Millipore) with 10 kDa cutoff membrane and stored at −80 ◦ C. 2.3. Preparation and purification of CPS-TT-G

NH

NH O

O

CPS -OC-R1-C- TT -NHCH2- Glu (CPS-TT-G)

O

a

Absorbance at 280 nm

delivery system have shown beneficial effects of the cointernalization of an antigen with adjuvant [21]. Thus, covalent conjugation and co-administration of ␤-glucan can possibly improve the CPSspecific immunogenicity of the conjugate vaccine. The present study was aimed to improve the CPS-specific immunogenicity of a meningococcal CPS conjugate vaccine by conjugation with ␤-glucan. Tetanus toxoid (TT) was used as the carrier protein for its wide clinical acceptance. Meningococcal group Y has been recognized as a major cause of invasive meningococcal infections in recent years [22]. A conjugate vaccine (CPS-TT) was generated by conjugation of the capsular CPS with TT, using adipic acid dihydrazide as the spacer arm [23,24]. ␤-Glucan was oxidized with sodium periodate to obtain the aldehyde groups, followed by conjugation with CPS-TT to generate a new conjugate vaccine (CPSTT-G) (Fig. 1). The immunological characteristics of CPS-TT-G were investigated and compared with those of CPS-TT co-administrated with ␤-glucan and CPS-TT.

Absorbance at 280 nm

2

O

R: HN-C(CH2)4C-NH

R1: HN-R-NH

Fig. 1. Schematic representation of CPS-TT and CPS-TT-G.

␤-Glucan (3 mg/ml, 6 ml) was oxidized by 20 mM sodium periodate in 20 mM acetate buffer (pH 5.8) (Scheme 4, 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 2 l of PBS buffer (pH 7.4), using a Slide-A-Lyzer dialysis cassette (Thermo Scientific, MWCO 10 kDa) for 3 h at 4 ◦ C. The dialysis solution was changed with 2 l

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of PBS buffer (pH 7.4) with dialysis for another 3 h at 4 ◦ C. The oxidized ␤-glucan (2 mg/ml, 6 ml) was allowed to react with CPS-TT (2 mg/ml, 6 ml) and 1 ml sodium cyanoborohydride (6 mg/ml) in PBS buffer (pH 7.4) at 4 ◦ C overnight (Scheme 5, Fig. 1). The resultant conjugate (CPS-TT-G) was purified by SEC using a Sephacryl S-300 column (2.6 cm × 70 cm, GE Healthcare, USA). The column was equilibrated and eluted by PBS buffer (pH 7.4) at a flow rate of 3.0 ml/min. The fractions corresponding to CPS-TT-G were pooled as the arrow indicated in Fig. 2a. The fractions were concentrated using Amicon (Millipore) with 10 kDa cutoff membrane and stored at −80 ◦ C.

respectively. The resultant samples were defined as CPS-TT/G1 and CPS-TT/G2, respectively. Thirty-six mice were randomly divided into six groups of six animals each. The groups were the CPS, CPSTT, CPS-TT/Al (CPS-TT and 0.35 mg aluminum adjuvant), CPS-TT-G, CPS-TT/G1, and CPS-TT/G2 groups. The mice were immunized subcutaneously with 0.5 ml of the five samples at a CPS concentration of 10 ␮g/ml on days 0, 7 and 14. Blood samples were taken from the mice on days 7, 14 and 21 after the first injection. The mice sera were isolated and stored at −20 ◦ C.

2.4. Analytical methods

The 96-well plates (Corning, USA) were coated with ␤-glucan modified BSA (␤-glucan-BSA) and CPS-BSA conjugate for CPSspecific and ␤-glucan-specific antibody measurement at 4 ◦ C overnight, respectively. ␤-Glucan-BSA and CPS-BSA conjugate were prepared essentially as Huang et al. [15]. ␤-Glucan-BSA (100 ␮l) at a ␤-glucan concentration of 5 ␮g/ml and CPS-BSA conjugate (100 ␮l) at a CPS concentration of 5 ␮g/ml in 50 mM NaHCO3 (pH 9.6) were used for each well, respectively. The plates were also coated with TT for TT-specific antibody measurement. TT (100 ␮l) at a concentration of 5 ␮g/ml in 50 mM NaHCO3 (pH 9.6) was used for each well. Then, the plates were blocked with 200 ␮l of 4% (w/v) skimmed milk in PBS buffer (PBS-milk, pH 7.4) at 37 ◦ C for 1 h and washed with PBS buffer (pH 7.4) for 3 times. The diluted sera (100 ␮l) were incubated in the wells for 1 h at 37 ◦ C, followed by washing with PBS buffer (pH 7.4) containing 0.1% Tween 20 (PBS-Tween) for 3 times. The plates were incubated with 100 ␮l of the diluted HRP-antibodies at 37 ◦ C for 1 h and then washed with PBS-Tween for three times. Then, 100 ␮l of the substrate solution containing 0.015% (w/v) of TMB was added and incubated at 37 ◦ C for 30 min, followed by quenching the reaction with 25 ␮l of 2 M H2 SO4 . The resultant solution was determined spectrometrically at 450 nm.

2.4.1. Quantitative assay of CPS-TT-G Total CPS content of CPS-TT and CPS-TT-G was measured by the resorcinol method [26]. The method for quantitative test of the unconjugated CPS in the conjugates was developed based on the ethanol precipitation [27]. The recovery of CPS was calculated as Huang et al. [15]. The degree of linked ADH was measured by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, using hydrazide as the standard [28]. TT content was measured by the bicinchoninic acid method, using bovine serum albumin (BSA) as the standard. The oxidation extent of the oxidized ␤-glucan was measured by a formaldehyde assay using the Purpald reagent [29]. 2.4.2. Dynamic light scattering The molecular radii of the two conjugates (CPS-TT and CPS-TTG) were determined by dynamic light scattering, using a Wyatt DynaPro Titan TC instrument (Santa Barbara, CA, USA) at 25 ◦ C. BSA was used as the standard to calibrate the instrument. The samples were at a protein concentration of 1 mg/ml in PBS buffer (pH 7.4). The samples were centrifuged at 12,000 × g for 10 min prior to the analysis. 2.4.3. Size-exclusion chromatography SEC analysis of the two conjugates was carried out using an analytical Superose 6 column (1 cm × 30 cm, GE Healthcare, USA). The column was equilibrated and eluted at room temperature with PBS buffer (pH 7.4) at a flow rate of 0.5 ml/min.

2.6. ELISA assay

2.7. Specificity of antibody The specificity of CPS-specific IgG was measured using competitive ELISA [15]. 2.8. Avidity of antibody

2.4.4. NMR spectroscopy The two conjugates were characterized by 1 H NMR at 600 MHz. The freeze-dried samples were dissolved in deuterated water to a final CPS concentration of 5 mg/ml. The 1 H NMR spectra of the samples were obtained on Bruker NMR Spectrometer Avance DRX 600 MHz, equipped with a 5 mm NMR probe (Bruker) at 25 ± 0.1 ◦ C. MestReNova software was used to process the spectra data.

The avidity of CPS-specific IgG antibodies was measured by ammonium thiocyanate elution method [30]. The IgG avidity index (AI) was expressed as ammonium thiocyanate concentration that needed to decrease the absorbance by 50%. 2.9. Statistical analysis

2.4.5. Fourier transform infrared spectoscopy The Fourier transform infrared spectoscopy (FT-IR) of the two conjugates were recorded on an FT-IR 660 Plus spectrometer (Jasco, Japan) in the region of 4000–400 cm−1 . The samples were prepared on KBr discs.

CPS-specific and TT-specific antibody titers of the conjugates were analyzed using GraphPadPrism 5 software (GraphPad Software, San Diego, CA, USA). The values of P < 0.05 (*) and P < 0.01 (**) were considered statistically and highly statistically significant between the experimental groups, respectively.

2.5. Immunization

3. Results

BALB/c female mice of weight 15–20 g were supplied by Animal Center 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). Aliquots of CPS-TT (2 ml) at a CPS concentration of 15 ␮g/ml were mixed with 30 ␮g ␤-glucan (1 ml) and 90 ␮g ␤-glucan (1 ml),

3.1. SEC analysis and quantitative assay CPS-TT and CPS-TT-G were purified by a Sephacryl S-300 column (2.6 cm × 60 cm) (Fig. 2a) and analyzed by an analytical Superose 6 column (1 cm × 30 cm) (Fig. 2b). As shown in Fig. 2a, CPS-TT and CPS-TT-G were both eluted as a major peak and their corresponding fractions were pooled as the arrows indicated. As shown in Fig. 2b, TT was eluted as a single and symmetric peak at 30.3 min. CPS-TT was also eluted as a single and symmetric peak at 15.4 min. The

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Table 1 CPS-specific and TT-specific antibody titers.

a Intensity

CPS-TT-G

CPS-specific IgG1 CPS-specific IgG2 CPS-specific IgG3 CPS-specific IgG3 CPS-specific IgG13 CPS-specific IgG2a3 CPS-specific IgM1 CPS-specific IgM3 CPS-specific IgG/IgM1 CPS-specific IgG/IgM3 TT-specific IgG3 TT-specific IgG13 TT-specific IgG2a3 ␤-Glucan-specific IgG3 ␤-Glucan-specific IgM3

CPS-TT

CPS 8

6

4

2

0

Chemical shift (ppm)

CPS

CPS-TT

CPS-TT-G

20 ± 20 40 ± 40 80 ± 40 – 40 ± 40 0 20 ± 20 40 ± 30 – – – – – – –

100 ± 40 666 ± 200 1066 ± 400 3600 ± 4004 1280 ± 400 150 ± 100 150 ± 50 700 ± 80 0.70 1.25 1400 ± 400 800 ± 250 110 ± 35 – –

150 ± 60 3200 ± 400 8800 ± 800 – 6400 ± 1000 1000 ± 200 120 ± 50 550 ± 80 1.6 16.0 5600 ± 800 4600 ± 700 420 ± 70 1600 ± 400 400 ± 160

1

Sera were obtained on day 7. Sera were obtained on day 14. 3 Sera were obtained on day 21. 4 CPS-TT was mixed with 0.35 mg aluminum adjuvant. CPS-specific and TT-specific antibody titers elicited by CPS-TT and CPS-TT-G were measured using ELISA. 2

b

CPS-TT-G

Glc residues and the H3eq/ax resonances of NeuNAc residues in the range of 3.3–4.2 ppm. It can be observed that the peak of deuterated water was at 4.7 ppm. 1 H NMR spectra of CPS-TT and CPS-TT-G also show the same assignments described for CPS. There were new signals of low intensities in the range 0.5–1.8 ppm. These signals corresponded to the aliphatic amino acids residues of TT.

Intensity

CPS-TT

CPS

3.4. FT-IR 4000 3500 3000 2500 2000 1500 1000

500

-1

Wave number (cm ) Fig. 3. Structural analysis of CPS-TT and CPS-TT-G. 1 H NMR spectra (a) were recorded on a Bruker Avance spectrometer at 600 MHz. FT-IR spectra (b) were recorded on an FT-IR 660 Plus spectrometer from 4000 to 400 cm−1 .

shift of the peak at lower retention time indicates conjugate formation, resulting in a product at higher molecular weight than the protein alone. The elution position of the activated CPS (15.9 min) was slightly right-shifted as compared with that of CPS-TT. In contrast, CPS-TT-G showed a single elution peak at 15.2 min that was slightly left-shifted as compared CPS-TT. This suggested that conjugation of ␤-glucan can slightly increase the hydrodynamic volume of CPS-TT. The ratio of the free CPS to the total CPS in CPS-TT was 5.4% and the recovery of CPS was 87.2%. The degree of linked ADH in CPS-TT was 0.089 ␮mol hydrazide group per mg CPS. The oxidation extent of the oxidized ␤-glucan was 0.155 ␮mol aldehyde group per mg ␤-glucan. 3.2. Dynamic light scattering As measured by dynamic light scattering, TT exhibited a molecular radius of 5.8 nm. CPS-TT showed a molecular radius of 10.7 nm that was higher than TT. Moreover, CPS-TT-G showed a molecular radius of 11.6 nm that was higher than CPS-TT. This confirmed the formation of the two conjugates. 3.3. NMR spectroscopy CPS-TT and CPS-TT-G were characterized by 1 H NMR spectroscopy. As shown in Fig. 3a, the sharp signals obtained for CPS indicated the unambiguous assignment of the anomeric protons of

The two conjugates were analyzed by the FT-IR. As shown in Fig. 3b, the FT-IR spectrum of CPS was characterized by the following absorption bands: ␦ (O H) stretch at 3300 cm−1 , ␦ (O H) wagging at 1020 cm−1 , and ␦ (C H) stretch at 2950–2930 cm−1 . CPS-TT also showed the signals at 3300 cm−1 for ␦ (O H) stretch and 1020 cm−1 for ␦ (O H) wagging. However, these signals were lower than those of CPS, due to that the O H moieties of CPS were activated by BrCN. In addition, a characteristic signal at 1580 cm−1 appeared in case of CPS-TT, which was attributed to ␦ (C O) stretch from the hydrazide. As compared with CPS-TT, the signals at 3300 cm−1 for ␦ (O H) stretch, 1020 cm−1 for ␦ (O H) wagging and 2950–2930 cm−1 for ␦ (C H) stretch increased in case of CPS-TT-G. This indicated the covalent conjugation of ␤-glucan with CPS-TT by formation of CH2 NH moieties (Scheme 5, Fig. 1). 3.5. CPS-specific and TT-specific antibodies CPS-specific and TT-specific antibody titers in mice sera were measured using ELISA assay. As shown in Table 1, free CPS induced IgG titers by a first dose can be undetectable. A second dose and a third one cannot boost the immune response to CPS. This suggested that CPS cannot induce immunological memory in the mice. Similarly, CPS-TT elicited low IgG titers at a first dose. However, CPS-TT can induce a primary CPS-specific immune response that was strongly boosted by a second dose (P < 0.05) and then further boosted by a third dose (P < 0.05). This revealed that CPS-TT can induce immunological memory in the mice. As compared with the CPS group at a third dose, the CPS-TT group showed a 13.3fold increase in the CPS-specific IgG titers (P < 0.01). Conjugation with ␤-glucan led to an 8.2-fold increase in the CPS-specific IgG titers of CPS-TT (P < 0.05). Co-administration of aluminum adjuvant led to a 3.4-fold increase in the CPS-specific IgG titers of CPS-TT. This suggested that conjugation with ␤-glucan showed a higher ability than aluminum adjuvant to increase the CPS-specific

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1500

CPS-specific IgG1 titers

a 1500

1000

500

CPS-specific IgG2a titers

0

CPS-TT

CPS-TT/G1

c 200

100

CPS-TT

CPS-TT/G1

CPS-TT/G2

b 1000

500

0

CPS-TT/G2

TT-specific IgG titers

CPS-specific IgG titers

2000

0

5

1500

CPS-TT

CPS-TT/G1

CPS-TT/G2

CPS-TT/G1

CPS-TT/G2

d

1000

500

0

CPS-TT

Fig. 4. CPS-specific and TT-specific antibody titers elicited by CPS-TT co-administrated with ␤-glucan. The measurements of CPS-specific IgG (a), IgG1 (b), Ig2a (c) and TT-specific IgG (d) were carried out using ELISA. The sera after a third injection were used for analysis. Bar represented mean ± S.D. from 6 mice per group. CPS-TT/G1 and CPS-TT/G2 were defined as the mixture of CPS-TT and ␤-glucan solution at the mass ratios of 1:1 and 1:3, respectively.

IgG titers of CPS-TT (P < 0.05). CPS-TT and CPS-TT-G can elicit clear predominant Th2-type IgG1 titers. The Th1-type IgG2a titers elicited by CPS-TT were lower than those by CPS-TT-G (P < 0.05). The IgG2a/IgG1 ratios of the two conjugates did not show significant difference. The sera after a third injection were used for antibody analysis. As shown in Table 1, the CPS-specific IgM titers elicited by CPS were significantly lower than those by CPS-TT (P < 0.05) and comparable to those by CPS-TT-G. This indicated that conjugation of TT can significantly improve the CPS-specific IgM titers of CPS-TT. In addition, the IgG/IgM ratio of CPS-TT at the third injection was higher than that at the first injection. Interestingly, the IgG/IgM ratio of CPS-TT-G at the third injection was much higher than that of CPS-TT. This suggested that conjugation to ␤-glucan shifted more response from IgM to IgG, which was typical for a conjugate vaccine. CPS-TT can also induce the apparent TT-specific antibody titers. The sera after a third injection were used for antibody analysis. As compared with CPS-TT, the CPS-TT-G group showed 4.0-, 5.8- and 3.8-fold increases in the TT-specific IgG, IgG1 and IgG2a titers, respectively. Thus, conjugation of ␤-glucan can improve the CPS-specific and TT-specific immunogenicity of CPS-TT. However, conjugation of ␤-glucan with CPS-TT could elicit ␤-glucan-specific IgG and IgM titers (Table 1). The background glucan-specific IgG and IgM antibody titers were undetectable in the mice sera. 3.6. Immunogenicity of CPS-TT co-administrated with ˇ-glucan The sera after a third injection were used for analysis. As shown in Fig. 4a and b, CPS-TT/G1 showed apparent CPS-specific IgG and IgG1 titers that were lower than CPS-TT and higher than CPSTT/G2. Similarly, TT-specific IgG titers elicited by CPS-TT/G1 were

lower than CPS-TT and higher than CPS-TT/G2 (Fig. 4d). Thus, coadministration of ␤-glucan could suppress the CPS-specific and TT-specific immunogenicity of CPS-TT. However, the CPS-specific IgG2a titers elicited by CPS-TT/G1 were higher than those by CPSTT and lower than those by CPS-TT/G2 (Fig. 4c). This was due to that co-administration of ␤-glucan could increase the cellular immunity of CPS-TT as reflected by the increased Th1-type IgG2a titers. 3.7. Competitive-inhibition ELISA study The ability of the coated CPS-BSA conjugate to bind the CPS-specific IgG in the mice sera was evaluated by a competitiveinhibition ELISA. Free CPS was added to inhibit the binding of CPS-specific IgG with the coated CPS-BSA conjugate. As shown in Fig. 5a, this ability decreased as a function of free CPS amount. The coated CPS-BSA conjugate showed high ability to bind CPS-specific IgG and this ability disappeared at a CPS amount of 15 ␮g. This indicated the presence of high CPS-specific IgG in the mice sera. 3.8. Antibody avidity measurement The antibody avidity is an index to evaluate the bonding degree of antigen and antibody, which can reflect the induction of immunological memory [31–33]. As shown in Fig. 5b, the avidity index (AI) of CPS-specific IgG was 1.10 mol/l for CPS. In contrast, the AI of CPS-specific IgG was 1.72 mol/l for CPS-TT. This indicated that the avidity of CPS-specific IgG can be increased by conjugation of TT. The AI of CPS-specific IgG for CPS-TT-G (1.95 mol/l) was slightly higher than that of CPS-TT. This revealed that conjugation of ␤-glucan can slightly improve the immunological memory of CPS-TT.

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Absorbance at 450 nm

1.2

a

CPS-TT CPS-TT-G

0.9

0.6

0.3

0.0 0

0.015 0.15

1.5

15

150

1500 15000

CPS (ng)

Author contributions

2.5

b

Ms. Weilin Qiao prepared the two polysaccharide conjugate vaccines and measured the immunogenicity of the vaccines. Dr. Shaoyang Ji characterized the physiochemical properties of the conjugate vaccines. Dr. Yubao Zhao designed the experiments of this study. Dr. Tao Hu designed the experiments of this study and prepared the manuscript.

2.0

1.5

Avidity

both CPS-TT and ␤-glucan reach the APCs (e.g. macrophages) at the same time. Thus, the competition effect of ␤-glucan with CPS-TT for the B-cell receptor may be significantly decreased. Moreover, ␤-glucan can activate APCs to produce more signal molecules (e.g. cytokines) to recruit other immune system cells and amplify the specific immune response by elevating circulating antibody titers and numbers of plaque-forming cells. These facts are expected to markedly improve the CPS-specific immunogenicity of the conjugate vaccine. In summary, conjugation of ␤-glucan could increase the CPS-specific and TT-specific immunogenicity of the conjugate vaccine (CPS-TT). In contrast, the CPS-specific and TT-specific immunogenicity of CPS-TT was suppressed by co-administration of ␤-glucan. Thus, our study was of general significance to develop a potent polysaccharide conjugate vaccine for prevention of bacteria infection.

1.0 Conflict of interest statement

0.5

0.0

None. Acknowledgements

CPS

CPS-TT

CPS-TT-G

Fig. 5. Competitive-inhibition ELISA analysis (a) and avidity measurement (b) of CPS-specific IgG. Free CPS was added to inhibit the binding of CPS-specific IgG with the coated CPS-BSA conjugate. Bar represented mean ± S.D. from 6 mice per group.

4. Discussion In the present study, a meningococcal CPS conjugate vaccine (CPS-TT) was prepared by conjugation of CPS with TT. CPS-TT was then conjugated or co-administrated with ␤-glucan. The immunological characteristics of the resultant samples were investigated and compared with each other. TT possesses T-helper cell epitopes and can activate helper T cells for the CPS-specific B cells through presenting the TT-derived, Class II MHC-bound peptides. CPS-specific B cells can thus act as antigen presenting cells (APCs) for TT and lead to B cell differentiation towards memory or plasma cells [34]. It has been suggested that only an APC that has taken up the antigen and the adjuvant in significant amounts is able to activate T-cells. In contrast, an APC that has only taken up either of the two components does not stimulate T-cell proliferation [21]. When ␤-glucan and CPS-TT were simply co-administrated, ␤-glucan can reach the APC much faster than CPS-TT and occupy the binding sites of B-cell receptors. Although ␤-glucan was a stimulator of humoral and cellular immunity [19], it derived CPS-specific B cells of necessary resources such as T-cell helper by occupying the binding sites of B-cell receptors and reduced the CPS-specific antibody production. Recently, combination of the antigen and the adjuvant in one entity has been considered as a good strategy for future vaccine development [19]. For example, nanoparticles containing both the antigen and the adjuvant like flagellin and CpG have been shown to be very immunogenic in mice [35,36]. Similarly, covalent conjugation of CPS-TT and ␤-glucan in one delivery system can ensure

This study was financially supported by Beijing Natural Science Foundation (7142104), the National Natural Science Foundation of China (grant nos. 20906095, 11405199 and 81402861), and the Science and Technology Service Network Initiative Project of Chinese Academy of Sciences (grant nos. KFJ-EW-STS-027 and KFJ-EW-STS098). References [1] Chang Q, Tzeng YL, Stephens DS. Meningococcal disease: changes in epidemiology and prevention. Clin Epidemiol 2012;4:237–45. [2] Read RC. Neisseria meningitidis; clones, carriage, and disease. Clin Microbiol Infect 2014;20:391–5. [3] Vipond C, Care R, Feavers IM. History of meningococcal vaccines and their serological correlates of protection. Vaccine 2011;30(Suppl. 2):B10–7. [4] Shao PL, Chang LY, Hsieh SM, Chang SC, Pan SC, Lu CY, et al. Safety and immunogenicity of a tetravalent polysaccharide vaccine against meningococcal disease. J Formos Med Assoc 2009;108:539–47. [5] Chang Q, Tzeng YL, Stephens DS. Meningococcal disease: changes in epidemiology and prevention. Clin Epidemiol 2012;4:237–45. [6] Keiser PB, Broderick M. Meningococcal polysaccharide vaccine failure in a patient with C7 deficiency and a decreased anti-capsular antibody response. Hum Vaccine Immunother 2012;8:582–6. [7] Dbaibo G, Van der Wielen M, Reda M, Medlej F, Tabet C, Boutriau D, et al. The tetravalent meningococcal serogroups A, C, W-135, and Y tetanus toxoid conjugate vaccine is immunogenic with a clinically acceptable safety profile in subjects previously vaccinated with a tetravalent polysaccharide vaccine. Int J Infect Dis 2012;16:e608–15. [8] Papaevangelou V, Spyridis N. MenACWY-TT vaccine for active immunization against invasive meningococcal disease. Expert Rev Vaccines 2012;11:523–7. [9] Bilukha O, Messonnier N, Fischer M. Use of meningococcal vaccines in the United States. Pediatr Infect Dis J 2007;26:371–6. [10] Broker M, Dull PM, Rappuoli R, Costantino P. Chemistry of a new investigational quadrivalent meningococcal conjugate vaccine that is immunogenic at all ages. Vaccine 2009;27:5574–80. [11] Cooper B, DeTora L, Stoddard J. Menveo® ): a novel quadrivalent meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135 and Y. Expert Rev Vaccines 2011;10:21–33. [12] Frasch CE, Preziosi MP, LaForce FM. Development of a group A meningococcal conjugate vaccine, MenAfriVac(TM). Hum Vaccin Immunother 2012;8:715–24.

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Please cite this article in press as: Qiao W, et al. Conjugation of ␤-glucan markedly increase the immunogencity of meningococcal group Y polysaccharide conjugate vaccine. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.02.045