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Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides
Accepted Manuscript Title: Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides Author: Guiqiang Zhang Peiyuan Jia Gong Cheng Siming Jiao Lishi Ren Shaoyang Ji Tao Hu Hongtao Liu Yuguang Du PII: DOI: Reference:
S0144-8617(17)30188-1 http://dx.doi.org/doi:10.1016/j.carbpol.2017.02.058 CARP 12037
To appear in: Received date: Revised date: Accepted date:
11-11-2016 13-2-2017 16-2-2017
Please cite this article as: Zhang, G., Jia, P., Cheng, G., Jiao, S., Ren, L., Ji, S., Hu, T., Liu, H., and Du, Y.,Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides, Carbohydrate Polymers (2017), http://dx.doi.org/10.1016/j.carbpol.2017.02.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlight PCV2-COS conjugates were designed by covalent linkage of PCV2 molecules to COS.
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COS conjugation significantly strengthened the immunogenicity of PCV2 vaccine.
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Immunization of PCV2-COS conjugates showed no pathological signs at
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injection sites.
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*Manuscript Click here to view linked References
Enhanced immune response to inactivated porcine circovirus type 2 (PCV2)
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vaccine by conjugation of chitosan oligosaccharides
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Guiqiang Zhanga,b,1, Peiyuan Jiaa,1, Gong Chenga, Siming Jiaoa, Lishi Rena, Shaoyang
4
Jia, Tao Hua,*, Hongtao Liua,*, Yuguang Dua,*
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1
State Key Laboratory of Biochemical Engineering and Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of
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Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China
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b
University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
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1
Authors contributed equally to this work.
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* To whom correspondence should be addressed:
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E-mail: [email protected]; [email protected]; [email protected]
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Phone: (86) 10-82545070
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Fax: (86) 10-82545070
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Full address: No.1 Bei-er-tiao, Zhong-guan-cun, Haidian, Beijing 100190, P.R. China.
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Abstract This study aimed to investigate the effect of chitosan oligosaccharide (COS)
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conjugation on the immunogenicity of porcine circovirus type-2 (PCV2) vaccine. Two
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conjugates (PCV2-COS-1 and PCV2-COS-2) were designed by covalent conjugation
20
of an inactivated PCV2 vaccine with COS, and administered to C57BL/6 mice three
21
times at two-week intervals. The results indicate that, as compared to PCV2 alone
22
group, the PCV2-COS conjugates remarkably enhanced both humoral and cellular
23
immunity against PCV2 by promoting T lymphocyte proliferation and initiating a
24
mixed Th1/Th2 response, including the elevated production of PCV-2 specific
25
antibodies and up-regulated secretion of inflammatory cytokines. Noticeably, the
26
immunization with PCV2-COS-1 conjugate displayed similar or even better
27
immune-stimulating effects than that by PCV2/ISA206 (a commercialized adjuvant)
28
and showed no infection or pathological signs at injection sites of the mice.
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Presumably, the covalent linkage of PCV2 vaccine to COS might be a viable strategy
30
to increase the efficacy against PCV2-associated diseases.
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Key words: Porcine circovirus type 2 (PCV2); Vaccine; Adjuvant; Chitosan
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oligosaccharide; Conjugation
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1. Introduction Porcine circovirus type 2 (PCV2), an important viral pathogens essentially in all major
37
Buechner-Maxwell, 2009), is the main cause of post-weaning multi-systemic wasting
38
syndrome (PMWS) and other PCV-associated diseases with 5-30% morbidity (Chae,
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2005). PMWS mainly affects 5-12 week old pigs and is characterized by progressive
40
weight loss, dyspnea, jaundice and enlargement of the inguinal lymph nodes (Guo,
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Wang, Qiao, Yang, Yang & Chen, 2015). It was estimated that this disease had led to
42
around £52.6 million loss per year during the epidemic period (Alarcon, Rushton &
43
Wieland, 2013). So far, PCV2 vaccination is one of the most effective methods to
44
control PMWS outbreak. And commercialized PCV2 vaccines include inactivated
45
whole PCV2 virus, subunit of open reading frame 2 and inactivated chimeric PCV1-2
46
(Chae, 2012). Although these PCV2 vaccines have been demonstrated to be effective
47
at reducing clinical signs and improving production parameters in farms with PCV2
48
infection, they fail to completely block the infection and transmission of PCV2
49
(Beach & Meng, 2012). Given the huge economic loss to herd production caused by
50
PCV2, it is vital to raise immune efficacy of PCV2 vaccines to minimize the pathogen
51
exposure.
countries
(Gillespie,
Opriessnig,
Meng,
Pelzer
&
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swine-producing
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To improve the vaccine immunogenicity, several commercialized adjuvants have
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been developed and widely applied to the breeding of livestock, including oil
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emulsion, carbomer and light paraffin oil (Chae, 2012). Regretfully, almost all these
55
adjuvants were found to cause side effects to certain extents, including the injection
56
site lesions, sterile granuloma, ulceration and other systemic responses such as fever,
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nausea and lethargy (Petrovsky, 2015). And some of the above adverse symptoms can
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even be detected in animal products that might bring potential food safety issues. It 3
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seems that new adjuvants are urgently needed to improve vaccine potency without
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compromising the safety. Chitosan oligosaccharides (COS), deriving from degradation and deacetylation
61
of
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tumor, immune-stimulation, anti-inflammation and anti-oxidation (Qiao et al., 2010).
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Moreover, COS was shown to be well biocompatible, biodegradable, non-toxic and
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non-allergenic (Dash, Chiellini, Ottenbrite & Chiellini, 2011; Yeh et al., 2013).
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Recently, it was reported that the administration of physical mixture of COS and
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inactivated vaccine significantly activated humoral immune response of host and be
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beneficial to the inhibition of pathogens (Liu, Zhang, Gao, Zhang, Wu & Zhang,
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2015). However, the physical mixture of antigen and adjuvant may be not the most
70
effective way of drug delivery. Recent studies demonstrated the importance of
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incorporating both antigen and adjuvant into one entity by using delivery system
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(Salman, Irache & Gamazo, 2009; Standley et al., 2007), or using covalent linkage of
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the antigen to an adjuvant for maximal immune-stimulation (Qiao, Ji, Zhao & Hu,
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2015; Slutter, Soema, Ding, Verheul, Hennink & Jiskoot, 2010; Vecchi et al., 2014).
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Similarly, the antigen presenting cells (APCs) taking up both antigen and adjuvant
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were proved to activate T-cells, whereas APCs with only either of the two components
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failed to stimulate T-cell proliferation (Blander & Medzhitov, 2006b; Schlosser et al.,
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2008). Besides, the maturation of dendritic cells was identified to be induced by such
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newly-conjugated vaccines, which increased the antigen capturing and subsequent
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presenting (Slutter, Soema, Ding, Verheul, Hennink & Jiskoot, 2010). From the above,
81
we hypothesize that the covalent conjugation of COS may help to improve the
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specific immunogenicity of PCV2 vaccine.
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chitosan,
has
displayed
versatile
biological
functions
such
as anti-
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In this study, the enhanced immunogenicity of PCV2 by COS conjugation was 4
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investigated for the development of high-efficient and safe PCV2 vaccines. To this
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aim, we covalently linked COS to inactivated PCV2 vaccine, followed by the
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assessment of physicochemical characterization of newly-prepared PCV2-COS
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conjugates. Further, we evaluated the effect of COS conjugation on PCV2-induced
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serum antibody response and cytokine production in mice, as well as the promotion to
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lymphocyte proliferation. Additionally, the adjuvant effect of COS on PCV2 vaccine
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was also compared with that by MONTANIDE™ ISA206, a commercial adjuvant as a
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positive control.
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2. Materials and methods
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2.1. Reagents
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COS was prepared as previously described with the deacetylation degree over 95%
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and average molecular weight below 1 kDa (Supplementary Fig. 1), and no endotoxin
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was detected using the limulus amebocyte lysate test (Zhang, Du, Yu, Mitsutomi &
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Aiba, 1999). The contents of COS mixture were determined by HPLC and the
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percentages of oligosaccharides with polymerization degree 2-7 were 5.9%, 18.1%,
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30.9%, 28.5%, 12.6% and 4.0%, respectively (Supplementary Fig. 2). Inactivated
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PCV2 vaccine was obtained from WINSUN PHARM, Inc. (Guangdong, China).
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Mouse
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(HRP)-conjugated
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3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) were purchased from
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Sigma (MO, USA). Enzyme linked immunosorbent assay (ELISA) kits for detection
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of mouse tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were purchased
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from BioLegend (CA, USA). Platinum ELISAs for detection of mouse interleukin-2
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(IL-2) and IL-5 were purchased from eBioscience (CA, USA).
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monoclonal
antibody
goat
isotyping
anti-mouse
reagents,
IgG
horse
antibody,
radish
peroxidase
2-iminothiolane
and
5
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2.2. Preparation and purification of PCV2-COS conjugates For PCV2-COS conjugate preparation, the inactivated PCV2 vaccine (3 ml, 1.7
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mg/ml) was incubated with 100-fold molar excess of MBS in phosphate buffer saline
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(PBS, 20 mM, pH 7.4) at 4 °C for 3 h. Then, the reaction mixture was centrifuged at
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4,000 g using Amicon membrane with 3 kDa cutoff (Millipore, USA) for five times to
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remove the free MBS.
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COS solution (2 ml, 2.5 mg/ml) was incubated with 5-fold molar excess of IT in
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20 mM PB (pH 7.4) at 4 °C for 3 h, followed by the removal of free IT using
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desalting column with 20 mM PBS (pH 7.4).
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The PCV2-COS conjugates were obtained by incubating maleimide-activated
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PCV2 (2 ml, 2.5 mg/ml) with thiolated COS (2 ml, 2.5 mg/ml). The incubation was in
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20 mM PB (pH 7.4) at 4 °C overnight.
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The size exclusion chromatograph based on a Superdex 200 column (2.6 cm ×
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70 cm, GE Healthcare, USA) was used to purify the conjugates from reaction
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mixtures. The column was equilibrated and eluted by PBS (20 mM, pH 7.4) at a flow
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rate of 3.0 ml/min. The fractions corresponding to the conjugates were pooled and
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concentrated using Amicon membrane with 3 kDa cutoff at 4 °C.
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2.3. Physicochemical characterization of PCV2-COS conjugates
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2.3.1. Quantitative assay
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Total carbohydrate content of PCV2-COS conjugates was measured using the
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phenol-sulphuric acid colorimetric method as described (Stefanetti, Rondini, Lanzilao,
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Saul, MacLennan & Micoli, 2014). The quantification of unconjugated carbohydrate
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in the conjugates was performed by the ethanol precipitation method (Qiao, Ji, Zhao
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& Hu, 2015). The protein content was measured using micro bicinchoninic acid 6
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method (Solarbio, Beijing, China), and the ratio of carbohydrate to protein (w/w) of
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PCV2-COS conjugates was calculated.
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2.3.2. Circular dichroism (CD) spectroscopy assay The CD measurement of PCV2 and its conjugates was carried out on a Jasco
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J-810 spectropolarimeter (JASCO, Tokyo, Japan) using a cuvette with 0.2 cm
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pathlength (Wang, Hu, Liu, Zhang, Ma & Su, 2011). All samples were at a protein
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concentration of 0.1 mg/ml in PBS (20 mM, pH 7.4), and the PBS solution baseline
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was subtracted from experimental spectra for correction.
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2.3.3. Dynamic light scattering (DLS) analysis
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To measure the molecular radii of PCV2, the mixture of COS and PCV2, and the
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PCV2-COS conjugates, DLS analysis was performed using a Malvern Zetasizer Nano
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ZS instrument (Malvern Instruments Ltd, Worcestershire, UK) at a controlled
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temperature of 25 °C (Hu et al., 2012). The samples were at a protein concentration of
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1.0 mg/ml in PBS (20 mM, pH 7.4). All samples were centrifuged at 12,000 g for 10
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min before analysis.
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2.3.4. Fluorescence measurement
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The intrinsic fluorescence was measured by a Hitachi F-4500 fluorescence
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spectropolarimeter (Hitachi, Tokyo, Japan) with a 1.0 cm pathlength cuvette (Suo, Lu,
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Hu, Ma & Su, 2009). The measurement was carried out at a protein concentration of
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0.1 mg/ml in PBS (20 mM, pH 7.4) at room temperature. The emission spectra
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(300-450 nm) were excited at 280 nm with a slit width of 5.0 nm. The PBS solution
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baseline was subtracted from experimental spectra for correction.
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2.3.5.
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H-NMR assay 7
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Identity and structure of PCV2 and PCV2-COS conjugates were analyzed by
155 156
1
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and PCV2-COS conjugates were dissolved in deuterated water to a final protein
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concentration of 2 mg/ml. The 1H-NMR spectra were obtained on a Bruker NMR
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Spectrometer, Avance DRX 600 MHz, equipped with a 5 mm NMR probe (Bruker,
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Karlsruhe, Germany) at 25 ± 0.1 °C. MestReNova software was used to process the
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spectrum data.
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2.4. Animal immunization
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H-NMR at 600 MHz. Freeze-dried mixture of PCV2 and COS (PCV2/COS), PCV2
Thirty-six C57BL/6 mice aged 4-6 weeks (15-20 g) were randomly divided into
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six groups (n=6) and received 0.1 ml of the following injections at a protein
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concentration of 500 μg/ml by intramuscular administration (i.m.): 1) PBS for the
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PBS group, 2) PCV2 for the PCV2 group, 3) PCV2 plus COS for the PCV2/COS
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group, 4) PCV2-COS conjugate-1 for the PCV2-COS-1 group, 5) PCV2-COS
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conjugate-2 for the PCV2-COS-2 group, 6) PCV2 plus ISA206 for the PCV2/ISA206
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group. All mice were administered on day 0, 14 and 28, and blood samples
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were taken via the tail vein at 0, 7, 14, 21, 28, 35 and 42 days post primary
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immunization (dpi). The mice sera were isolated and stored at -80 °C for further
172
experiments.
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The procedures of animal experiments were approved by the Animal Ethical
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Experimentation Committee of Institute of Process Engineering, Chinese Academy of
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Sciences (Beijing, China) and in accordance with the National Act on Use of
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Experimental Animals (China).
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2.5. Detection of PCV2-specific antibodies
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For detection of PCV2-specific antibodies including IgG, IgG1, IgG2a, IgG2b, 8
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IgG3 and IgM, the serum samples were analyzed by modified ELISA method. Briefly,
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96-well microplates were coated with 5 μg/well PCV2 antigen in carbonate buffer (50
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mM, pH 9.6) overnight at 4 °C. After that, the plates were washed three times with
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PBS containing 0.1% Tween 20 (PBST, 10 mM, pH 7.4) and blocked with 5 % of
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skimmed milk in PBS for 1 h at 37 °C. After three washes with PBST, 100 μl of
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diluted serum samples were added into each well and incubated at 37 °C for 1 h
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followed by three washes. Then, the plates were incubated with 100 μl of
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HRP-GAM-IgG, IgG isotype or IgM antibody at 37 °C for 1 h. After the wash for five
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times, 100 μl of TMB substrate was added and incubated at 37 °C in the darkness for
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30 min, followed by quenching the reaction with 50 μl of H2SO4 (2 M). The optical
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density were read at 450 nm using a Tecan Infinite M200 Pro microplate reader
190
(Grodig, Austria)．
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2.6. Lymphocyte proliferation assay
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At 42 dpi, lymphocytes were separated from the spleen of each mouse using
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mouse lymphocyte separation medium, resuspended at 5×106 cells/ml with
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RPMI-1640 complete medium containing 10% FBS, 100 units/ml penicillin and 100
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µg/ml streptomycin. For lymphocyte proliferation assay, 100 μl of cell suspension
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was seeded into each well with lipopolysaccharides (LPS, 10 μg/ml) or concanavalin
197
A (ConA, 2 μg/ml). After incubation at 37 °C with 5 % CO2 for 48 h, the culture
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supernatant were removed and washed with PBS. Then, 100 µl of MTT (5 mg/ml) in
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complete medium was added and incubated for another 4 h. Followed by the removal
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of MTT, the colored formazan was dissolved in 100 μl of DMSO. The OD values
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were measured at 570 nm using a microplate reader as above.
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2.7. Cytokine assay After the lymphocytes were prepared and stimulated for 48 h as described above,
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the culture supernatant in each well was collected to determine the levels of IL-2, IL-5,
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TNF-α and IFN-γ by using commercial ELISA kits according to the protocols of
206
manufacturers.
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2.8. Histopathological assay
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At 42 dpi, the mice for each group were euthanized and tibialis muscle tissue
209
samples at injection sites were collected and fixed in 4% neutral-buffered formalin
210
solution. The tissue samples were then embedded in paraffin and cut into 4 µm thick
211
slices. After the hematoxylin and eosin (HE) staining, histopathological analysis was
212
performed and the microscopic images were photographed using a Leica DMI3000 B
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microscope (Wetzlar, Germany).
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2.9. Statistical analysis
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Data are presented as means ± SD. Statistical significance was assessed using
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Student’s two-tailed t test in two groups and one-way ANOVA in multiple groups. The
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P value <0.05 or <0.01 was considered statistically significant. All statistical analyses
218
were performed using SPSS 13.0 software (SPSS, IL, USA).
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3. Results
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3.1. Purification and quantitative assay of PCV2-COS conjugates
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To prepare the PCV2-COS conjugates, primary amine present in COS was
222
reacted with 2-iminothiolane in PBS (Fig. 1a). The resulting product was reacted with
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the PCV2 whose amine side chains of lysine residues were modified by maleimide
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group to obtain PCV2-COS conjugates (Fig. 1b-1c). The conjugation reaction was 10
Page 11 of 34
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confirmed by SDS-PAGE analysis, which showed a shift toward the smear bands with
226
higher mass compared to that of free PCV2 (Supplementary Fig. 3). A Superdex 200 column (2.6 cm × 70 cm) was used to purify the PCV2-COS
228
conjugates from reaction mixture. As shown in Fig. 2a, the conjugates were eluted as
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two major peaks and their corresponding fractions were pooled for further analysis.
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After purification, the conjugates and free PCV2 samples were further assayed on an
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analytical Superdex 200 column (1.0 cm × 30 cm). As shown in Fig. 2b, free PCV2
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was eluted as a single and symmetric peak at 13.3 ml. On the other hand, after free
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PCV2 was linked to COS by chemical conjugation, the separated two PCV2-COS
234
conjugates displayed higher molecular weights than that of free PCVs, and both peaks
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shifted from 13.3 ml to 8.1 ml (PCV2-COS-1) and 12.4 ml (PCV2-COS-2),
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respectively (Fig. 2b). Further, the SDS-PAGE analysis on collected fractions
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confirmed that the peaks eluted at 8.1 ml and 12.4 ml were two conjugates with
238
different molecular weights (Supplementary Fig. 3).
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Next, the carbohydrate/protein ratio (w/w) of PCV-COS-1 and PCV-COS-2 was
240
calculated to range from 0.51 to 0.38, respectively, which indicated higher
241
conjugation efficiency in aggregated protein conjugate than the monomeric one. In
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addition, the unconjugated carbohydrate was efficiently removed by purification
243
process, leaving low residual levels (2%-5%).
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Fig. 1. Reaction scheme of PCV2-COS conjugation.
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3.2. Physicochemical characterization of PCV2-COS conjugates
247
3.2.1. CD spectroscopy assay As compared to free PCV2, the far UV spectra of CD spectroscopy of
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PCV2/COS mixture was not changed, whereas both PCV2-COS conjugates displayed
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lower ellipticity values at 210 nm than that of free PCV2, indicating the slightly
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decreased α-helix content in PCV2 by COS conjugation (Fig. 2c).
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3.2.2. Fluorescence measurement
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Intrinsic fluorescence was measured to detect the conformational variation in
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PCV2 by COS conjugation, as revealed by any changes of Trp, Tyr and Phe residues
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exposed to the solvent. As shown in Fig. 2d, we failed to detect any changes in the
256
emission fluorescence intensity of PCV2-COS-2 as compared to that of PCV2,
257
whereas a slight decreased fluorescence intensity was observed for PCV2-COS-1
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sample. The changed fluorescence intensity indicated the minor conformational
259
variation in PCV2 upon conjugation with COS. In contrast, the PCV2/COS sample
260
showed a slightly red-shift in the maximum wavelength. This difference may possibly
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be due to the impact by large amount of COS.
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Fig. 2. Separation, purification and physical characterization of PCV2-COS
264
conjugates. (a) The separation of PCV2-COS-1 and PCV2-COS-2 conjugates by size
265
exclusion chromatography analysis. (b) The purification of PCV2-COS conjugates
266
performed on a Superdex 200 column (2.6 cm × 70 cm) by the same analytic method
267
as Fig. 2a. (c-f) Physical characterization of PCV2-COS-1 and PCV2-COS-2
268
conjugates measured by CD spectroscopy (c), fluorescence analysis (d), DLS (e) and 13
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1
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3.2.3. DLS analysis
H-NMR (f).
As measured by DLS, free PCV2 exhibited a molecular radius of 12.79 nm,
272
slightly lower than that of PCV2/COS mixture (14.27 nm). Noticeably, the two
273
conjugates, i.e., PCV2-COS-1 and PCV2-COS-2, separately showed much higher
274
molecular radii of 52.82 nm and 28.91 nm than that of the free PCV2 (Fig. 2e). It can
275
be presumed that the molecular volume of PCV2 might be increased by COS
276
conjugation.
277
3.2.4.
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H-NMR assay
Next, the identity and structure of free PCV2, PCV2/COS mixture and
279
PCV2-COS conjugates were characterized by 1H-NMR spectroscopy. The result
280
showed that COS conjugation had no effect on the integrity and identity of PCV2
281
molecule (Fig. 2f). It can be observed that the peak of deuterated water was at 4.7
282
ppm, and sharp signals obtained from PCV2/COS indicated the unambiguous
283
assignment of proton signals of acetamide groups at C-2 positions in the range of
284
1.8-1.9 ppm. Compared to PCV2/COS,
285
conjugates showed not only the same assignments as described for PCV2/COS, but
286
additional chemical shifts in 3.6 ppm related to methylene and a peak at the 7.3-7.4
287
ppm range related to proton signal of benzene ring.
288
3.3. PCV2-specific serum antibody production
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H-NMR spectra of both PCV2-COS
289
PCV2-specific antibody levels of IgG, IgG isotypes (IgG1, IgG2a, IgG2b and
290
IgG3), IgA and IgM in mouse serum were assessed by ELISA. The result shows that 14
Page 15 of 34
low levels of PCV2-specific IgG and its isotypes antibodies were produced during the
292
whole vaccination period in groups administered by PBS, PCV2 or PCV2/COS
293
mixture (Fig. 3 and Fig. 4a-4d). In contrast, the conjugation of COS with PCV2 led to
294
rapid and significant increase in the titers of IgG or its isotypes (P<0.05 or 0.01, vs
295
PCV2 alone group) by day 14 after the first immunization, which remained at high
296
levels until 42 dpi. It is important to note that the PCV2-COS-1 group even
297
predominated in IgG2a, IgG2b and IgG3 levels (P<0.05 or 0.01) as compared to the
298
group vaccinated by a mixture of PCV2 and ISA206, a commercialized oil emulsion
299
adjuvant.
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As an indicator to analyze the Th1- or Th2-biased immune responses to
301
vaccination, the IgG2a/IgG1 ratio was also obtained from each group after PCV2
302
immunization as presented in Table 1. In comparison with PCV2 alone group, the
303
IgG2a/IgG1 ratio of groups administered with PCV2-COS-1 was significantly
304
increased during the whole immunization period (P<0.05), indicating that COS
305
conjugation increased the Th1-biased immune response initiated by PCV2 vaccine.
306
Moreover, the PCV2-COS-1 group displayed a higher value of IgG2a/IgG1 than that
307
of PCV2/ISA206 group (P<0.05 or 0.01).
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In addition, the PCV2-specific antibody levels of IgA and IgM in mouse serum
309
were also detected. As shown in Fig. 4e-4f, PCV2 alone or PCV2/COS mixture failed
310
to produce high levels of IgA and IgM antibodies after vaccination. However, both
311
antibody levels were strongly induced by COS conjugation after the first
312
immunization (P<0.05, vs PCV2 alone group), which were further enhanced after the
313
second immunization (P<0.01, vs PCV2 alone group) and then slightly decreased
314
during the third immunization period.
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15
Page 16 of 34
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Fig. 3. Effect of COS conjugation on PCV2-specific IgG antibody production in
317
mouse serum. Mice were intramuscularly injected with PBS, PCV2 only, PCV2/COS
318
mixture, PCV2-COS-1, PCV2-COS-2 or PCV2/ISA206 mixture on day 0, 14 and 28.
319
The blood samples from each mouse were collected at day 0, 7, 14, 21, 28, 35 and 42
320
dpi, and the IgG titer was determined by ELISA. Data are represented as means ± SD
321
(n=6) of duplicate wells. *P<0.05 or **P<0.01, compared to PCV2 alone group;
322
#
ed
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an
316
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P<0.05, compared to the group vaccinated with PCV2/ISA206 mixture.
16
Page 17 of 34
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323
Fig. 4. Effect of COS conjugation on PCV2-specific antibody levels of IgG isotypes,
325
IgA and IgM in mouse serum. Mice were intramuscularly injected with PBS, PCV2
326
only, PCV2/COS mixture, PCV2-COS-1, PCV2-COS-2 or PCV2/ISA206 mixture on
327
day 0, 14 and 28. The antibody titers were determined by ELISA after blood samples
328
from each mouse were collected at day 14, 28 and 42 dpi. Data are represented as
329
means ± SD (n=6) of duplicate wells. *P<0.05 or **P<0.01, compared to PCV2 alone
330
group; #P<0.05 or
Ac
324
##
P<0.01, compared to the group vaccinated with PCV2/ISA206 17
Page 18 of 34
331
mixture.
332
Table 1. The effects of COS conjugation on anti-PCV2 IgG2a and anti-PCV2 IgG1
333
ratio (IgG2a/IgG1) in immunized mice Groups
14 dpi
28 dpi
42 dpi
1.05±0.17**##
0.95±0.01**##
0.96±0.10**
PCV2
0.55±0.09##
0.28±0.11##
0.31±0.04##
PCV2/COS
0.41±0.09*
0.24±0.02##
0.28±0.04##
PCV2-COS-1
0.87±0.10**##
1.04±0.05**##
1.02±0.09**#
PCV2-COS-2
0.22±0.03**
0.27±0.08##
PCV2/ISA206
0.28±0.06**
0.53±0.06**
cr
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PBS
us
0.28±0.02## 0.80±0.11**
IgG2a/IgG1 ratio for each mouse serum was calculated and representative results for
335
each experimental group (n=6) were presented as means ± SD. *P<0.05 or **P<0.01,
336
compared to the PCV2 alone group;
337
PCV2/ISA206 group.
338
3.4. Lymphocyte proliferation assay
P<0.05 or
##
P<0.01, compared to the
ed
M
#
an
334
T- and B-lymphocyte proliferation can be separately activated by ConA or LPS.
340
To determine whether the lymphocyte proliferation response to PCV2 vaccination was
341
boosted by COS conjugation, the primary lymphocytes were isolated from mouse
342
spleens at 42 dpi and the proliferation assay was assessed by MTT analysis. As
343
indicated in Fig. 5, the PCV2/COS group showed a slight but no statistically
344
significant increase in T-cell proliferation after ConA (2 μg/ml) stimulation for 48 h
345
compared with PCV2 alone group. By contrast, remarkable T cell proliferation
346
responses were observed in the groups administered with both PCV2-COS conjugates
347
or PCV2/ISA206 (P<0.01). On the other hand, though the PCV2/ISA206 group
348
displayed a considerable increase in B-cell proliferation (P<0.01, vs PCV2 alone
349
group) after LPS (10 μg/ml) stimulation for 48 h, which failed to be promoted in the
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18
Page 19 of 34
groups administered by PCV2/COS or PCV2-COS conjugates.
us
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351
Fig. 5. Effect of COS conjugation on lymphocyte proliferation in PCV2-vaccinated
353
mice. On day 14 after the third immunization, mice were euthanized and splenic
354
lymphocytes were prepared. After treatment with ConA (2 µg/ml) or LPS (10 µg/ml)
355
for 48 h, the lymphocyte proliferation was analyzed by MTT assay. Data are
356
represented as the means ± SD (n=6) of duplicate wells. **P<0.01, compared to
357
PCV2 alone group.
358
3.5. Cytokine assay
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The activation of immune responses was usually characterized by up-regulation
360
of IL-5 (typical of a Th2 response) or IL-2 and IFN-γ (typical of a Th1 response). To
361
monitor the effects of COS conjugation on PCV2-specific cytokine secretion, the
362
primary lymphocytes were isolated from mouse spleens at 42 dpi. Cells from different
363
groups were treated with Con A (2 μg/ml) or LPS (10 μg/ml) for 48 h. After that, the
364
levels of above-mentioned cytokines in culture supernatant were assayed by ELISA.
Ac
359
365
As shown in Fig. 6, the levels of above four cytokines in mice from PCV-COS-1
366
group were statistically higher than those of PCV2 alone group (P<0.05 or 0.01). 19
Page 20 of 34
Especially, the stimulant effects of PCV2-COS-1 on secretion of IL-2, IL-5 and IFN-γ
368
were even stronger than that by PCV2/ISA206 (P<0.05 or 0.01). For PCV-COS-2
369
group, a higher levels of IL-2 and TNF-α were observed (P<0.05 or 0.01, vs PCV2
370
alone group), but not for IL-5 and IFN-γ.
371
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Fig. 6. Effects of COS conjugation on the production of IL-2 (a), IL-5 (b), IFN-γ (c)
373
and TNF-α (d) secreted by spleen lymphocytes from immunized mice. The mice were
374
sacrificed at 42 dpi and the primary spleen lymphocytes were prepared. After cells
375
were treated with Con A (2 μg/ml, for IL-2, IL-5 and IFN-γ assay) or LPS (10 μg/ml,
376
for TNF-α assay) for 48 h, the culture supernatant was collected for cytokine detection
377
by ELISA. Data are represented as the means ± SD (n=6) of duplicate wells. *P<0.05
378
or **P<0.01, compared to PCV2 alone group; #P<0.05 or ##P<0.01, compared to the
379
group vaccinated with PCV2/ISA206 mixture.
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20
Page 21 of 34
380
3.6. Histopathological assay To investigate the possible side effects by COS conjugation or ISA206, the mice
382
from each group were euthanized and the tibialis muscle tissues were collected for
383
histopathological analysis. In this study no lethality or clinical signs were observed
384
for all vaccinated groups during the whole immunization period. However, the
385
remarkable lesions were observed at injection sites of the mice from PCV2/ISA206
386
group after each immunization, whereas no pathological signs were found in other
387
groups. The histopathological examination of tibialis muscle section near injection
388
sites shows that, the mice with PCV2/ISA206 immunization displayed severe
389
infectious symptoms characterized by inflammatory cell infiltration as the arrow
390
indicated in Fig. 7, while no microscopic lesions were observed in the groups
391
administered with PBS, PCV2/COS, PCV2 alone or PCV2-COS conjugates. As the
392
major metabolic and excretory organs responsible for the elimination of drugs and/or
393
their metabolites, the liver and kidney were also collected for histopathological
394
examination, and no microscopic lesions were observed in all groups including the
395
mice administered with PCV2/ISA206 (Supplementary Fig. 4).
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381
396 397
Fig. 7. Histopathological examination of vaccinated mice after the immunization. The 21
Page 22 of 34
mice were euthanized at 42 dpi and the tibialis muscle tissues at near injection sites
399
were collected for pathological examination by HE staining. Solid arrows represented
400
the inflammatory cell infiltration. Magnification: × 200.
401
4. Discussion
ip t
398
PCV-associated disease was first described in the early 1990s and has led to great
403
economic losses worldwide during the past twenty years. To date, vaccination is still
404
adopted as the most effective tool in the prevention of PCV infection. And some
405
adjuvants have been used to enhance the immune efficacy of PCV vaccines. However,
406
the main pitfall for adjuvants is the potential health risk associated with their use as
407
immune stimulating compounds (Singh & O'Hagan, 1999), so do the PCV adjuvants.
408
Thus, a novel and safe adjuvant capable of eliciting both humoral and cellular
409
immune responses is necessary to enhance the efficacy of PCV vaccines. In this study,
410
we for the first time evaluated the adjuvant effect of COS which was conjugated to an
411
inactivated PCV2 vaccine. And the strongly enhanced immune responses of host to
412
PCV2 were observed after the immunization. Noticeably, it showed no infection or
413
pathological signs at injection sites of the mice.
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402
In present study, PCV2 vaccine as a proteinaceous antigen was coupled to COS
415
using the thiol-maleimide reaction. For several decades, such method has been widely
416
applied to bio-conjugation and bio-molecular labeling given its reliability, efficiency,
417
and selectivity (Gindy, Ji, Hoye, Panagiotopoulos & Prud'homme, 2008; Miyadera &
418
Kosower, 1972). Once again, the thiol-maleimide method was proved to be a useful
419
way to covalently link COS and PCV2 vaccine in our experiment. By this method we
420
produced a heterogeneous mixture of aggregated PCV2-COS conjugates with
421
different molecular radii and molecular weights by using size exclusion
422
chromatograph, DLS analysis and 1H-NMR assay. Due to the multiple reaction groups
Ac
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Page 23 of 34
of COS accessible to PCV2 molecules, the structure of PCV2-COS-1 should be a
424
multi-component lattice-type which conjugated several PCV2 molecules, while
425
PCV2-COS-2 may be the COS conjugate with a monomeric PCV2 molecule. In
426
addition, only slight changes were detected in the structure of PCV2 by using CD
427
spectroscopy analysis and intrinsic fluorescence spectroscopy assay, suggesting that
428
such modification method has little effect on the conformation of PCV2. The
429
preservation of protein structure makes this method an interesting strategy to couple
430
those protein-based antigens to an adjuvant carrying primary amine groups.
us
cr
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423
Research data have shown that PCV-specific antibodies are associated with
432
pathogen prevention, as evidenced by the development of PCV-related diseases
433
accompanied by reduced serum antibodies (Carasova et al., 2007; McIntosh, Harding,
434
Ellis & Appleyard, 2006). In this study, though PCV2-specific antibody levels elicited
435
by PCV2-COS-1 and PCV2-COS-2 vaccines were different to some extents, both
436
were significantly higher than that by PCV2/COS mixture and PCV2 vaccine alone,
437
indicating that COS conjugation enhanced the humoral immune response to PCV2
438
vaccine. Moreover, PCV2-COS-1 rapidly induced the antibody production of IgG,
439
IgG isotypes, IgA and IgM in the first and booster immunization, even superior to the
440
commercialized adjuvant ISA206. It was proposed that COS conjugation to PCV2 can
441
activate the immune system in a short time and may have more advantages in the
442
prevention of emergent PCV2 infections. Also, the PCV2-COS-1 group showed an
443
increase in IgG2a/IgG1 ratio compared with PCV2 alone group, implicating that COS
444
conjugation can increase the Th1-biased immune response.
Ac
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an
431
445
Previous studies have demonstrated that saccharide adjuvants can promote T
446
lymphocyte proliferation and augment cytokine production during the immune
447
responses to vaccination (Fan et al., 2015; Fan et al., 2016). In the present study, we 23
Page 24 of 34
found that COS conjugation not only significantly increased T-cell proliferation after
449
ConA stimulation, but markedly produced higher levels of IL-2, IL-5, IFN-γ and
450
TNF-α as compared to PCV2 alone group. Since IL-2 is the central regulators of Th1
451
immune response while IL-5 is proved to enhance Th2 responses, this study displayed
452
a mixed Th1/Th2 responses in groups with PCV2-COS conjugates. CD4+ T cell
453
mediates the killing of organisms responsible for a variety of intracellular infections
454
through the production of IFN-γ and TNF-α (Seder & Hill, 2000). And our results
455
suggest that COS conjugation dramatically increased the production of IFN-γ and
456
TNF-α compared with PCV2 alone group, indicating the strengthening of
457
cell-mediated response to PCV2. It was reported that the sole induction of humoral
458
response might not guarantee a full protection against PCV2 infection, and
459
cell-mediated immune response might contribute together with antibodies to viral
460
clearance and help avoid the progression of PCV-associated diseases (Fort, Sibila,
461
Allepuz, Mateu, Roerink & Segales, 2008; Fort, Sibila, Perez-Martin, Nofrarias,
462
Mateu & Segales, 2009; Martelli et al., 2011). Therefore, the conjugation of COS to
463
PCV2 displayed a great potential in initiating both humoral and cellular responses to
464
PCV2 vaccine, and this may help to provide a better protection against infection than
465
other commercialized PCV2 adjuvants.
Ac
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448
466
In this study, we show that PCV2-COS-1 led to its best immunogenicity.
467
Previous studies demonstrated that the protein aggregation by chemical treatment
468
could increase the uptake of antigen by dendritic cells (Kastenmuller et al., 2011).
469
And the ability of aggregated or particulate antigens to enhance cellular immunity and 24
Page 25 of 34
specifically increase the efficiency of cross-presentation has been proved with a
471
variety of antigens and formulations (Schnorrer et al., 2006; Trombetta & Mellman,
472
2005), which may in part explain the strengthened immunogenicity of PCV2 by COS
473
conjugation. A second mechanism to explain the potency of PCV2-COS conjugates
474
may related to the activation of macrophages and dendritic cells via mannose receptor
475
or Toll-like receptor 4 (TLR4) by COS (Han, Zhao, Yu, Feng & Yu, 2005; Wu & Tsai,
476
2007). Results from other groups showed that the enhancement of cellular immunity
477
is associated with the antigen linkage to ligands targeting TLR2, TLR4, or TLR9
478
(Blander & Medzhitov, 2006a; Khan et al., 2007). In the future, the detailed molecular
479
mechanism of how COS conjugation affects the biological performance of PCV2
480
should be further elucidated by in vitro and in vivo studies.
481
Conclusions
ed
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470
In summary, the conjugation of COS markedly enhanced both humoral and
483
cellular immunity against PCV2 by promoting T lymphocyte proliferation, which in
484
turn skewed towards a mixed Th1/Th2 response, including the elevated production of
485
IgG, IG istoypes, IgA and IgM, and up-regulated secretion of inflammatory cytokines,
486
i.e., IL-2, IL-5, IFN-γ and TNF-α. Moreover, the immunization with PCV2-COS-1
487
conjugate displayed similar or even better immune-stimulating effects on antibody
488
production and cytokine secretion than that by PCV2/ISA206, suggesting that the
489
covalent linkage of PCV2 vaccine to COS might be a potential solution to increase the
490
efficacy against PCV2-associated diseases.
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482
25
Page 26 of 34
491
Acknowledgements We are grateful for the support by National Programs for High Technology
493
Research and Development (863 Programs, 2014AA093604), National Natural
494
Science Fund of China (NO. 31500747 and NO. 31570801), and by Postdoctoral
495
Science Fund of China (NO. 2014M561069).
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S0144-8617(17)30188-1 http://dx.doi.org/doi:10.1016/j.carbpol.2017.02.058 CARP 12037
To appear in: Received date: Revised date: Accepted date:
11-11-2016 13-2-2017 16-2-2017
Please cite this article as: Zhang, G., Jia, P., Cheng, G., Jiao, S., Ren, L., Ji, S., Hu, T., Liu, H., and Du, Y.,Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides, Carbohydrate Polymers (2017), http://dx.doi.org/10.1016/j.carbpol.2017.02.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlight PCV2-COS conjugates were designed by covalent linkage of PCV2 molecules to COS.
ip t
COS conjugation significantly strengthened the immunogenicity of PCV2 vaccine.
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Immunization of PCV2-COS conjugates showed no pathological signs at
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injection sites.
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Page 1 of 34
*Manuscript Click here to view linked References
Enhanced immune response to inactivated porcine circovirus type 2 (PCV2)
2
vaccine by conjugation of chitosan oligosaccharides
3
Guiqiang Zhanga,b,1, Peiyuan Jiaa,1, Gong Chenga, Siming Jiaoa, Lishi Rena, Shaoyang
4
Jia, Tao Hua,*, Hongtao Liua,*, Yuguang Dua,*
a
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5
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1
State Key Laboratory of Biochemical Engineering and Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of
7
Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China
an
us
6
b
University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
9
1
Authors contributed equally to this work.
M
8
* To whom correspondence should be addressed:
11
E-mail: [email protected]; [email protected]; [email protected]
12
Phone: (86) 10-82545070
13
Fax: (86) 10-82545070
14
Full address: No.1 Bei-er-tiao, Zhong-guan-cun, Haidian, Beijing 100190, P.R. China.
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1
Page 2 of 34
16
Abstract This study aimed to investigate the effect of chitosan oligosaccharide (COS)
18
conjugation on the immunogenicity of porcine circovirus type-2 (PCV2) vaccine. Two
19
conjugates (PCV2-COS-1 and PCV2-COS-2) were designed by covalent conjugation
20
of an inactivated PCV2 vaccine with COS, and administered to C57BL/6 mice three
21
times at two-week intervals. The results indicate that, as compared to PCV2 alone
22
group, the PCV2-COS conjugates remarkably enhanced both humoral and cellular
23
immunity against PCV2 by promoting T lymphocyte proliferation and initiating a
24
mixed Th1/Th2 response, including the elevated production of PCV-2 specific
25
antibodies and up-regulated secretion of inflammatory cytokines. Noticeably, the
26
immunization with PCV2-COS-1 conjugate displayed similar or even better
27
immune-stimulating effects than that by PCV2/ISA206 (a commercialized adjuvant)
28
and showed no infection or pathological signs at injection sites of the mice.
29
Presumably, the covalent linkage of PCV2 vaccine to COS might be a viable strategy
30
to increase the efficacy against PCV2-associated diseases.
31
Key words: Porcine circovirus type 2 (PCV2); Vaccine; Adjuvant; Chitosan
32
oligosaccharide; Conjugation
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34 35
1. Introduction Porcine circovirus type 2 (PCV2), an important viral pathogens essentially in all major
37
Buechner-Maxwell, 2009), is the main cause of post-weaning multi-systemic wasting
38
syndrome (PMWS) and other PCV-associated diseases with 5-30% morbidity (Chae,
39
2005). PMWS mainly affects 5-12 week old pigs and is characterized by progressive
40
weight loss, dyspnea, jaundice and enlargement of the inguinal lymph nodes (Guo,
41
Wang, Qiao, Yang, Yang & Chen, 2015). It was estimated that this disease had led to
42
around £52.6 million loss per year during the epidemic period (Alarcon, Rushton &
43
Wieland, 2013). So far, PCV2 vaccination is one of the most effective methods to
44
control PMWS outbreak. And commercialized PCV2 vaccines include inactivated
45
whole PCV2 virus, subunit of open reading frame 2 and inactivated chimeric PCV1-2
46
(Chae, 2012). Although these PCV2 vaccines have been demonstrated to be effective
47
at reducing clinical signs and improving production parameters in farms with PCV2
48
infection, they fail to completely block the infection and transmission of PCV2
49
(Beach & Meng, 2012). Given the huge economic loss to herd production caused by
50
PCV2, it is vital to raise immune efficacy of PCV2 vaccines to minimize the pathogen
51
exposure.
countries
(Gillespie,
Opriessnig,
Meng,
Pelzer
&
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Ac
52
swine-producing
ip t
36
To improve the vaccine immunogenicity, several commercialized adjuvants have
53
been developed and widely applied to the breeding of livestock, including oil
54
emulsion, carbomer and light paraffin oil (Chae, 2012). Regretfully, almost all these
55
adjuvants were found to cause side effects to certain extents, including the injection
56
site lesions, sterile granuloma, ulceration and other systemic responses such as fever,
57
nausea and lethargy (Petrovsky, 2015). And some of the above adverse symptoms can
58
even be detected in animal products that might bring potential food safety issues. It 3
Page 4 of 34
59
seems that new adjuvants are urgently needed to improve vaccine potency without
60
compromising the safety. Chitosan oligosaccharides (COS), deriving from degradation and deacetylation
61
of
63
tumor, immune-stimulation, anti-inflammation and anti-oxidation (Qiao et al., 2010).
64
Moreover, COS was shown to be well biocompatible, biodegradable, non-toxic and
65
non-allergenic (Dash, Chiellini, Ottenbrite & Chiellini, 2011; Yeh et al., 2013).
66
Recently, it was reported that the administration of physical mixture of COS and
67
inactivated vaccine significantly activated humoral immune response of host and be
68
beneficial to the inhibition of pathogens (Liu, Zhang, Gao, Zhang, Wu & Zhang,
69
2015). However, the physical mixture of antigen and adjuvant may be not the most
70
effective way of drug delivery. Recent studies demonstrated the importance of
71
incorporating both antigen and adjuvant into one entity by using delivery system
72
(Salman, Irache & Gamazo, 2009; Standley et al., 2007), or using covalent linkage of
73
the antigen to an adjuvant for maximal immune-stimulation (Qiao, Ji, Zhao & Hu,
74
2015; Slutter, Soema, Ding, Verheul, Hennink & Jiskoot, 2010; Vecchi et al., 2014).
75
Similarly, the antigen presenting cells (APCs) taking up both antigen and adjuvant
76
were proved to activate T-cells, whereas APCs with only either of the two components
77
failed to stimulate T-cell proliferation (Blander & Medzhitov, 2006b; Schlosser et al.,
78
2008). Besides, the maturation of dendritic cells was identified to be induced by such
79
newly-conjugated vaccines, which increased the antigen capturing and subsequent
80
presenting (Slutter, Soema, Ding, Verheul, Hennink & Jiskoot, 2010). From the above,
81
we hypothesize that the covalent conjugation of COS may help to improve the
82
specific immunogenicity of PCV2 vaccine.
83
chitosan,
has
displayed
versatile
biological
functions
such
as anti-
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In this study, the enhanced immunogenicity of PCV2 by COS conjugation was 4
Page 5 of 34
investigated for the development of high-efficient and safe PCV2 vaccines. To this
85
aim, we covalently linked COS to inactivated PCV2 vaccine, followed by the
86
assessment of physicochemical characterization of newly-prepared PCV2-COS
87
conjugates. Further, we evaluated the effect of COS conjugation on PCV2-induced
88
serum antibody response and cytokine production in mice, as well as the promotion to
89
lymphocyte proliferation. Additionally, the adjuvant effect of COS on PCV2 vaccine
90
was also compared with that by MONTANIDE™ ISA206, a commercial adjuvant as a
91
positive control.
92
2. Materials and methods
93
2.1. Reagents
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COS was prepared as previously described with the deacetylation degree over 95%
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and average molecular weight below 1 kDa (Supplementary Fig. 1), and no endotoxin
96
was detected using the limulus amebocyte lysate test (Zhang, Du, Yu, Mitsutomi &
97
Aiba, 1999). The contents of COS mixture were determined by HPLC and the
98
percentages of oligosaccharides with polymerization degree 2-7 were 5.9%, 18.1%,
99
30.9%, 28.5%, 12.6% and 4.0%, respectively (Supplementary Fig. 2). Inactivated
100
PCV2 vaccine was obtained from WINSUN PHARM, Inc. (Guangdong, China).
101
Mouse
102
(HRP)-conjugated
103
3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) were purchased from
104
Sigma (MO, USA). Enzyme linked immunosorbent assay (ELISA) kits for detection
105
of mouse tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were purchased
106
from BioLegend (CA, USA). Platinum ELISAs for detection of mouse interleukin-2
107
(IL-2) and IL-5 were purchased from eBioscience (CA, USA).
ce pt
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Ac
monoclonal
antibody
goat
isotyping
anti-mouse
reagents,
IgG
horse
antibody,
radish
peroxidase
2-iminothiolane
and
5
Page 6 of 34
108
2.2. Preparation and purification of PCV2-COS conjugates For PCV2-COS conjugate preparation, the inactivated PCV2 vaccine (3 ml, 1.7
110
mg/ml) was incubated with 100-fold molar excess of MBS in phosphate buffer saline
111
(PBS, 20 mM, pH 7.4) at 4 °C for 3 h. Then, the reaction mixture was centrifuged at
112
4,000 g using Amicon membrane with 3 kDa cutoff (Millipore, USA) for five times to
113
remove the free MBS.
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COS solution (2 ml, 2.5 mg/ml) was incubated with 5-fold molar excess of IT in
115
20 mM PB (pH 7.4) at 4 °C for 3 h, followed by the removal of free IT using
116
desalting column with 20 mM PBS (pH 7.4).
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The PCV2-COS conjugates were obtained by incubating maleimide-activated
118
PCV2 (2 ml, 2.5 mg/ml) with thiolated COS (2 ml, 2.5 mg/ml). The incubation was in
119
20 mM PB (pH 7.4) at 4 °C overnight.
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The size exclusion chromatograph based on a Superdex 200 column (2.6 cm ×
121
70 cm, GE Healthcare, USA) was used to purify the conjugates from reaction
122
mixtures. The column was equilibrated and eluted by PBS (20 mM, pH 7.4) at a flow
123
rate of 3.0 ml/min. The fractions corresponding to the conjugates were pooled and
124
concentrated using Amicon membrane with 3 kDa cutoff at 4 °C.
125
2.3. Physicochemical characterization of PCV2-COS conjugates
126
2.3.1. Quantitative assay
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Total carbohydrate content of PCV2-COS conjugates was measured using the
128
phenol-sulphuric acid colorimetric method as described (Stefanetti, Rondini, Lanzilao,
129
Saul, MacLennan & Micoli, 2014). The quantification of unconjugated carbohydrate
130
in the conjugates was performed by the ethanol precipitation method (Qiao, Ji, Zhao
131
& Hu, 2015). The protein content was measured using micro bicinchoninic acid 6
Page 7 of 34
132
method (Solarbio, Beijing, China), and the ratio of carbohydrate to protein (w/w) of
133
PCV2-COS conjugates was calculated.
134
2.3.2. Circular dichroism (CD) spectroscopy assay The CD measurement of PCV2 and its conjugates was carried out on a Jasco
136
J-810 spectropolarimeter (JASCO, Tokyo, Japan) using a cuvette with 0.2 cm
137
pathlength (Wang, Hu, Liu, Zhang, Ma & Su, 2011). All samples were at a protein
138
concentration of 0.1 mg/ml in PBS (20 mM, pH 7.4), and the PBS solution baseline
139
was subtracted from experimental spectra for correction.
140
2.3.3. Dynamic light scattering (DLS) analysis
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To measure the molecular radii of PCV2, the mixture of COS and PCV2, and the
142
PCV2-COS conjugates, DLS analysis was performed using a Malvern Zetasizer Nano
143
ZS instrument (Malvern Instruments Ltd, Worcestershire, UK) at a controlled
144
temperature of 25 °C (Hu et al., 2012). The samples were at a protein concentration of
145
1.0 mg/ml in PBS (20 mM, pH 7.4). All samples were centrifuged at 12,000 g for 10
146
min before analysis.
147
2.3.4. Fluorescence measurement
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The intrinsic fluorescence was measured by a Hitachi F-4500 fluorescence
149
spectropolarimeter (Hitachi, Tokyo, Japan) with a 1.0 cm pathlength cuvette (Suo, Lu,
150
Hu, Ma & Su, 2009). The measurement was carried out at a protein concentration of
151
0.1 mg/ml in PBS (20 mM, pH 7.4) at room temperature. The emission spectra
152
(300-450 nm) were excited at 280 nm with a slit width of 5.0 nm. The PBS solution
153
baseline was subtracted from experimental spectra for correction.
154
2.3.5.
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H-NMR assay 7
Page 8 of 34
Identity and structure of PCV2 and PCV2-COS conjugates were analyzed by
155 156
1
157
and PCV2-COS conjugates were dissolved in deuterated water to a final protein
158
concentration of 2 mg/ml. The 1H-NMR spectra were obtained on a Bruker NMR
159
Spectrometer, Avance DRX 600 MHz, equipped with a 5 mm NMR probe (Bruker,
160
Karlsruhe, Germany) at 25 ± 0.1 °C. MestReNova software was used to process the
161
spectrum data.
162
2.4. Animal immunization
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H-NMR at 600 MHz. Freeze-dried mixture of PCV2 and COS (PCV2/COS), PCV2
Thirty-six C57BL/6 mice aged 4-6 weeks (15-20 g) were randomly divided into
164
six groups (n=6) and received 0.1 ml of the following injections at a protein
165
concentration of 500 μg/ml by intramuscular administration (i.m.): 1) PBS for the
166
PBS group, 2) PCV2 for the PCV2 group, 3) PCV2 plus COS for the PCV2/COS
167
group, 4) PCV2-COS conjugate-1 for the PCV2-COS-1 group, 5) PCV2-COS
168
conjugate-2 for the PCV2-COS-2 group, 6) PCV2 plus ISA206 for the PCV2/ISA206
169
group. All mice were administered on day 0, 14 and 28, and blood samples
170
were taken via the tail vein at 0, 7, 14, 21, 28, 35 and 42 days post primary
171
immunization (dpi). The mice sera were isolated and stored at -80 °C for further
172
experiments.
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The procedures of animal experiments were approved by the Animal Ethical
174
Experimentation Committee of Institute of Process Engineering, Chinese Academy of
175
Sciences (Beijing, China) and in accordance with the National Act on Use of
176
Experimental Animals (China).
177
2.5. Detection of PCV2-specific antibodies
178
For detection of PCV2-specific antibodies including IgG, IgG1, IgG2a, IgG2b, 8
Page 9 of 34
IgG3 and IgM, the serum samples were analyzed by modified ELISA method. Briefly,
180
96-well microplates were coated with 5 μg/well PCV2 antigen in carbonate buffer (50
181
mM, pH 9.6) overnight at 4 °C. After that, the plates were washed three times with
182
PBS containing 0.1% Tween 20 (PBST, 10 mM, pH 7.4) and blocked with 5 % of
183
skimmed milk in PBS for 1 h at 37 °C. After three washes with PBST, 100 μl of
184
diluted serum samples were added into each well and incubated at 37 °C for 1 h
185
followed by three washes. Then, the plates were incubated with 100 μl of
186
HRP-GAM-IgG, IgG isotype or IgM antibody at 37 °C for 1 h. After the wash for five
187
times, 100 μl of TMB substrate was added and incubated at 37 °C in the darkness for
188
30 min, followed by quenching the reaction with 50 μl of H2SO4 (2 M). The optical
189
density were read at 450 nm using a Tecan Infinite M200 Pro microplate reader
190
(Grodig, Austria)．
191
2.6. Lymphocyte proliferation assay
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At 42 dpi, lymphocytes were separated from the spleen of each mouse using
193
mouse lymphocyte separation medium, resuspended at 5×106 cells/ml with
194
RPMI-1640 complete medium containing 10% FBS, 100 units/ml penicillin and 100
195
µg/ml streptomycin. For lymphocyte proliferation assay, 100 μl of cell suspension
196
was seeded into each well with lipopolysaccharides (LPS, 10 μg/ml) or concanavalin
197
A (ConA, 2 μg/ml). After incubation at 37 °C with 5 % CO2 for 48 h, the culture
198
supernatant were removed and washed with PBS. Then, 100 µl of MTT (5 mg/ml) in
199
complete medium was added and incubated for another 4 h. Followed by the removal
200
of MTT, the colored formazan was dissolved in 100 μl of DMSO. The OD values
201
were measured at 570 nm using a microplate reader as above.
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9
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2.7. Cytokine assay After the lymphocytes were prepared and stimulated for 48 h as described above,
204
the culture supernatant in each well was collected to determine the levels of IL-2, IL-5,
205
TNF-α and IFN-γ by using commercial ELISA kits according to the protocols of
206
manufacturers.
207
2.8. Histopathological assay
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At 42 dpi, the mice for each group were euthanized and tibialis muscle tissue
209
samples at injection sites were collected and fixed in 4% neutral-buffered formalin
210
solution. The tissue samples were then embedded in paraffin and cut into 4 µm thick
211
slices. After the hematoxylin and eosin (HE) staining, histopathological analysis was
212
performed and the microscopic images were photographed using a Leica DMI3000 B
213
microscope (Wetzlar, Germany).
214
2.9. Statistical analysis
ed
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Data are presented as means ± SD. Statistical significance was assessed using
216
Student’s two-tailed t test in two groups and one-way ANOVA in multiple groups. The
217
P value <0.05 or <0.01 was considered statistically significant. All statistical analyses
218
were performed using SPSS 13.0 software (SPSS, IL, USA).
219
3. Results
220
3.1. Purification and quantitative assay of PCV2-COS conjugates
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215
221
To prepare the PCV2-COS conjugates, primary amine present in COS was
222
reacted with 2-iminothiolane in PBS (Fig. 1a). The resulting product was reacted with
223
the PCV2 whose amine side chains of lysine residues were modified by maleimide
224
group to obtain PCV2-COS conjugates (Fig. 1b-1c). The conjugation reaction was 10
Page 11 of 34
225
confirmed by SDS-PAGE analysis, which showed a shift toward the smear bands with
226
higher mass compared to that of free PCV2 (Supplementary Fig. 3). A Superdex 200 column (2.6 cm × 70 cm) was used to purify the PCV2-COS
228
conjugates from reaction mixture. As shown in Fig. 2a, the conjugates were eluted as
229
two major peaks and their corresponding fractions were pooled for further analysis.
230
After purification, the conjugates and free PCV2 samples were further assayed on an
231
analytical Superdex 200 column (1.0 cm × 30 cm). As shown in Fig. 2b, free PCV2
232
was eluted as a single and symmetric peak at 13.3 ml. On the other hand, after free
233
PCV2 was linked to COS by chemical conjugation, the separated two PCV2-COS
234
conjugates displayed higher molecular weights than that of free PCVs, and both peaks
235
shifted from 13.3 ml to 8.1 ml (PCV2-COS-1) and 12.4 ml (PCV2-COS-2),
236
respectively (Fig. 2b). Further, the SDS-PAGE analysis on collected fractions
237
confirmed that the peaks eluted at 8.1 ml and 12.4 ml were two conjugates with
238
different molecular weights (Supplementary Fig. 3).
ed
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227
Next, the carbohydrate/protein ratio (w/w) of PCV-COS-1 and PCV-COS-2 was
240
calculated to range from 0.51 to 0.38, respectively, which indicated higher
241
conjugation efficiency in aggregated protein conjugate than the monomeric one. In
242
addition, the unconjugated carbohydrate was efficiently removed by purification
243
process, leaving low residual levels (2%-5%).
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244 11
Page 12 of 34
245
Fig. 1. Reaction scheme of PCV2-COS conjugation.
246
3.2. Physicochemical characterization of PCV2-COS conjugates
247
3.2.1. CD spectroscopy assay As compared to free PCV2, the far UV spectra of CD spectroscopy of
249
PCV2/COS mixture was not changed, whereas both PCV2-COS conjugates displayed
250
lower ellipticity values at 210 nm than that of free PCV2, indicating the slightly
251
decreased α-helix content in PCV2 by COS conjugation (Fig. 2c).
252
3.2.2. Fluorescence measurement
an
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Intrinsic fluorescence was measured to detect the conformational variation in
254
PCV2 by COS conjugation, as revealed by any changes of Trp, Tyr and Phe residues
255
exposed to the solvent. As shown in Fig. 2d, we failed to detect any changes in the
256
emission fluorescence intensity of PCV2-COS-2 as compared to that of PCV2,
257
whereas a slight decreased fluorescence intensity was observed for PCV2-COS-1
258
sample. The changed fluorescence intensity indicated the minor conformational
259
variation in PCV2 upon conjugation with COS. In contrast, the PCV2/COS sample
260
showed a slightly red-shift in the maximum wavelength. This difference may possibly
261
be due to the impact by large amount of COS.
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Page 13 of 34
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262 263
Fig. 2. Separation, purification and physical characterization of PCV2-COS
264
conjugates. (a) The separation of PCV2-COS-1 and PCV2-COS-2 conjugates by size
265
exclusion chromatography analysis. (b) The purification of PCV2-COS conjugates
266
performed on a Superdex 200 column (2.6 cm × 70 cm) by the same analytic method
267
as Fig. 2a. (c-f) Physical characterization of PCV2-COS-1 and PCV2-COS-2
268
conjugates measured by CD spectroscopy (c), fluorescence analysis (d), DLS (e) and 13
Page 14 of 34
269
1
270
3.2.3. DLS analysis
H-NMR (f).
As measured by DLS, free PCV2 exhibited a molecular radius of 12.79 nm,
272
slightly lower than that of PCV2/COS mixture (14.27 nm). Noticeably, the two
273
conjugates, i.e., PCV2-COS-1 and PCV2-COS-2, separately showed much higher
274
molecular radii of 52.82 nm and 28.91 nm than that of the free PCV2 (Fig. 2e). It can
275
be presumed that the molecular volume of PCV2 might be increased by COS
276
conjugation.
277
3.2.4.
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271
H-NMR assay
Next, the identity and structure of free PCV2, PCV2/COS mixture and
279
PCV2-COS conjugates were characterized by 1H-NMR spectroscopy. The result
280
showed that COS conjugation had no effect on the integrity and identity of PCV2
281
molecule (Fig. 2f). It can be observed that the peak of deuterated water was at 4.7
282
ppm, and sharp signals obtained from PCV2/COS indicated the unambiguous
283
assignment of proton signals of acetamide groups at C-2 positions in the range of
284
1.8-1.9 ppm. Compared to PCV2/COS,
285
conjugates showed not only the same assignments as described for PCV2/COS, but
286
additional chemical shifts in 3.6 ppm related to methylene and a peak at the 7.3-7.4
287
ppm range related to proton signal of benzene ring.
288
3.3. PCV2-specific serum antibody production
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1
H-NMR spectra of both PCV2-COS
289
PCV2-specific antibody levels of IgG, IgG isotypes (IgG1, IgG2a, IgG2b and
290
IgG3), IgA and IgM in mouse serum were assessed by ELISA. The result shows that 14
Page 15 of 34
low levels of PCV2-specific IgG and its isotypes antibodies were produced during the
292
whole vaccination period in groups administered by PBS, PCV2 or PCV2/COS
293
mixture (Fig. 3 and Fig. 4a-4d). In contrast, the conjugation of COS with PCV2 led to
294
rapid and significant increase in the titers of IgG or its isotypes (P<0.05 or 0.01, vs
295
PCV2 alone group) by day 14 after the first immunization, which remained at high
296
levels until 42 dpi. It is important to note that the PCV2-COS-1 group even
297
predominated in IgG2a, IgG2b and IgG3 levels (P<0.05 or 0.01) as compared to the
298
group vaccinated by a mixture of PCV2 and ISA206, a commercialized oil emulsion
299
adjuvant.
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291
As an indicator to analyze the Th1- or Th2-biased immune responses to
301
vaccination, the IgG2a/IgG1 ratio was also obtained from each group after PCV2
302
immunization as presented in Table 1. In comparison with PCV2 alone group, the
303
IgG2a/IgG1 ratio of groups administered with PCV2-COS-1 was significantly
304
increased during the whole immunization period (P<0.05), indicating that COS
305
conjugation increased the Th1-biased immune response initiated by PCV2 vaccine.
306
Moreover, the PCV2-COS-1 group displayed a higher value of IgG2a/IgG1 than that
307
of PCV2/ISA206 group (P<0.05 or 0.01).
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In addition, the PCV2-specific antibody levels of IgA and IgM in mouse serum
309
were also detected. As shown in Fig. 4e-4f, PCV2 alone or PCV2/COS mixture failed
310
to produce high levels of IgA and IgM antibodies after vaccination. However, both
311
antibody levels were strongly induced by COS conjugation after the first
312
immunization (P<0.05, vs PCV2 alone group), which were further enhanced after the
313
second immunization (P<0.01, vs PCV2 alone group) and then slightly decreased
314
during the third immunization period.
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Page 16 of 34
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Fig. 3. Effect of COS conjugation on PCV2-specific IgG antibody production in
317
mouse serum. Mice were intramuscularly injected with PBS, PCV2 only, PCV2/COS
318
mixture, PCV2-COS-1, PCV2-COS-2 or PCV2/ISA206 mixture on day 0, 14 and 28.
319
The blood samples from each mouse were collected at day 0, 7, 14, 21, 28, 35 and 42
320
dpi, and the IgG titer was determined by ELISA. Data are represented as means ± SD
321
(n=6) of duplicate wells. *P<0.05 or **P<0.01, compared to PCV2 alone group;
322
#
ed
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P<0.05, compared to the group vaccinated with PCV2/ISA206 mixture.
16
Page 17 of 34
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Fig. 4. Effect of COS conjugation on PCV2-specific antibody levels of IgG isotypes,
325
IgA and IgM in mouse serum. Mice were intramuscularly injected with PBS, PCV2
326
only, PCV2/COS mixture, PCV2-COS-1, PCV2-COS-2 or PCV2/ISA206 mixture on
327
day 0, 14 and 28. The antibody titers were determined by ELISA after blood samples
328
from each mouse were collected at day 14, 28 and 42 dpi. Data are represented as
329
means ± SD (n=6) of duplicate wells. *P<0.05 or **P<0.01, compared to PCV2 alone
330
group; #P<0.05 or
Ac
324
##
P<0.01, compared to the group vaccinated with PCV2/ISA206 17
Page 18 of 34
331
mixture.
332
Table 1. The effects of COS conjugation on anti-PCV2 IgG2a and anti-PCV2 IgG1
333
ratio (IgG2a/IgG1) in immunized mice Groups
14 dpi
28 dpi
42 dpi
1.05±0.17**##
0.95±0.01**##
0.96±0.10**
PCV2
0.55±0.09##
0.28±0.11##
0.31±0.04##
PCV2/COS
0.41±0.09*
0.24±0.02##
0.28±0.04##
PCV2-COS-1
0.87±0.10**##
1.04±0.05**##
1.02±0.09**#
PCV2-COS-2
0.22±0.03**
0.27±0.08##
PCV2/ISA206
0.28±0.06**
0.53±0.06**
cr
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PBS
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0.28±0.02## 0.80±0.11**
IgG2a/IgG1 ratio for each mouse serum was calculated and representative results for
335
each experimental group (n=6) were presented as means ± SD. *P<0.05 or **P<0.01,
336
compared to the PCV2 alone group;
337
PCV2/ISA206 group.
338
3.4. Lymphocyte proliferation assay
P<0.05 or
##
P<0.01, compared to the
ed
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#
an
334
T- and B-lymphocyte proliferation can be separately activated by ConA or LPS.
340
To determine whether the lymphocyte proliferation response to PCV2 vaccination was
341
boosted by COS conjugation, the primary lymphocytes were isolated from mouse
342
spleens at 42 dpi and the proliferation assay was assessed by MTT analysis. As
343
indicated in Fig. 5, the PCV2/COS group showed a slight but no statistically
344
significant increase in T-cell proliferation after ConA (2 μg/ml) stimulation for 48 h
345
compared with PCV2 alone group. By contrast, remarkable T cell proliferation
346
responses were observed in the groups administered with both PCV2-COS conjugates
347
or PCV2/ISA206 (P<0.01). On the other hand, though the PCV2/ISA206 group
348
displayed a considerable increase in B-cell proliferation (P<0.01, vs PCV2 alone
349
group) after LPS (10 μg/ml) stimulation for 48 h, which failed to be promoted in the
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Page 19 of 34
groups administered by PCV2/COS or PCV2-COS conjugates.
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351
Fig. 5. Effect of COS conjugation on lymphocyte proliferation in PCV2-vaccinated
353
mice. On day 14 after the third immunization, mice were euthanized and splenic
354
lymphocytes were prepared. After treatment with ConA (2 µg/ml) or LPS (10 µg/ml)
355
for 48 h, the lymphocyte proliferation was analyzed by MTT assay. Data are
356
represented as the means ± SD (n=6) of duplicate wells. **P<0.01, compared to
357
PCV2 alone group.
358
3.5. Cytokine assay
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The activation of immune responses was usually characterized by up-regulation
360
of IL-5 (typical of a Th2 response) or IL-2 and IFN-γ (typical of a Th1 response). To
361
monitor the effects of COS conjugation on PCV2-specific cytokine secretion, the
362
primary lymphocytes were isolated from mouse spleens at 42 dpi. Cells from different
363
groups were treated with Con A (2 μg/ml) or LPS (10 μg/ml) for 48 h. After that, the
364
levels of above-mentioned cytokines in culture supernatant were assayed by ELISA.
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365
As shown in Fig. 6, the levels of above four cytokines in mice from PCV-COS-1
366
group were statistically higher than those of PCV2 alone group (P<0.05 or 0.01). 19
Page 20 of 34
Especially, the stimulant effects of PCV2-COS-1 on secretion of IL-2, IL-5 and IFN-γ
368
were even stronger than that by PCV2/ISA206 (P<0.05 or 0.01). For PCV-COS-2
369
group, a higher levels of IL-2 and TNF-α were observed (P<0.05 or 0.01, vs PCV2
370
alone group), but not for IL-5 and IFN-γ.
371
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Fig. 6. Effects of COS conjugation on the production of IL-2 (a), IL-5 (b), IFN-γ (c)
373
and TNF-α (d) secreted by spleen lymphocytes from immunized mice. The mice were
374
sacrificed at 42 dpi and the primary spleen lymphocytes were prepared. After cells
375
were treated with Con A (2 μg/ml, for IL-2, IL-5 and IFN-γ assay) or LPS (10 μg/ml,
376
for TNF-α assay) for 48 h, the culture supernatant was collected for cytokine detection
377
by ELISA. Data are represented as the means ± SD (n=6) of duplicate wells. *P<0.05
378
or **P<0.01, compared to PCV2 alone group; #P<0.05 or ##P<0.01, compared to the
379
group vaccinated with PCV2/ISA206 mixture.
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Page 21 of 34
380
3.6. Histopathological assay To investigate the possible side effects by COS conjugation or ISA206, the mice
382
from each group were euthanized and the tibialis muscle tissues were collected for
383
histopathological analysis. In this study no lethality or clinical signs were observed
384
for all vaccinated groups during the whole immunization period. However, the
385
remarkable lesions were observed at injection sites of the mice from PCV2/ISA206
386
group after each immunization, whereas no pathological signs were found in other
387
groups. The histopathological examination of tibialis muscle section near injection
388
sites shows that, the mice with PCV2/ISA206 immunization displayed severe
389
infectious symptoms characterized by inflammatory cell infiltration as the arrow
390
indicated in Fig. 7, while no microscopic lesions were observed in the groups
391
administered with PBS, PCV2/COS, PCV2 alone or PCV2-COS conjugates. As the
392
major metabolic and excretory organs responsible for the elimination of drugs and/or
393
their metabolites, the liver and kidney were also collected for histopathological
394
examination, and no microscopic lesions were observed in all groups including the
395
mice administered with PCV2/ISA206 (Supplementary Fig. 4).
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396 397
Fig. 7. Histopathological examination of vaccinated mice after the immunization. The 21
Page 22 of 34
mice were euthanized at 42 dpi and the tibialis muscle tissues at near injection sites
399
were collected for pathological examination by HE staining. Solid arrows represented
400
the inflammatory cell infiltration. Magnification: × 200.
401
4. Discussion
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PCV-associated disease was first described in the early 1990s and has led to great
403
economic losses worldwide during the past twenty years. To date, vaccination is still
404
adopted as the most effective tool in the prevention of PCV infection. And some
405
adjuvants have been used to enhance the immune efficacy of PCV vaccines. However,
406
the main pitfall for adjuvants is the potential health risk associated with their use as
407
immune stimulating compounds (Singh & O'Hagan, 1999), so do the PCV adjuvants.
408
Thus, a novel and safe adjuvant capable of eliciting both humoral and cellular
409
immune responses is necessary to enhance the efficacy of PCV vaccines. In this study,
410
we for the first time evaluated the adjuvant effect of COS which was conjugated to an
411
inactivated PCV2 vaccine. And the strongly enhanced immune responses of host to
412
PCV2 were observed after the immunization. Noticeably, it showed no infection or
413
pathological signs at injection sites of the mice.
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In present study, PCV2 vaccine as a proteinaceous antigen was coupled to COS
415
using the thiol-maleimide reaction. For several decades, such method has been widely
416
applied to bio-conjugation and bio-molecular labeling given its reliability, efficiency,
417
and selectivity (Gindy, Ji, Hoye, Panagiotopoulos & Prud'homme, 2008; Miyadera &
418
Kosower, 1972). Once again, the thiol-maleimide method was proved to be a useful
419
way to covalently link COS and PCV2 vaccine in our experiment. By this method we
420
produced a heterogeneous mixture of aggregated PCV2-COS conjugates with
421
different molecular radii and molecular weights by using size exclusion
422
chromatograph, DLS analysis and 1H-NMR assay. Due to the multiple reaction groups
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Page 23 of 34
of COS accessible to PCV2 molecules, the structure of PCV2-COS-1 should be a
424
multi-component lattice-type which conjugated several PCV2 molecules, while
425
PCV2-COS-2 may be the COS conjugate with a monomeric PCV2 molecule. In
426
addition, only slight changes were detected in the structure of PCV2 by using CD
427
spectroscopy analysis and intrinsic fluorescence spectroscopy assay, suggesting that
428
such modification method has little effect on the conformation of PCV2. The
429
preservation of protein structure makes this method an interesting strategy to couple
430
those protein-based antigens to an adjuvant carrying primary amine groups.
us
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423
Research data have shown that PCV-specific antibodies are associated with
432
pathogen prevention, as evidenced by the development of PCV-related diseases
433
accompanied by reduced serum antibodies (Carasova et al., 2007; McIntosh, Harding,
434
Ellis & Appleyard, 2006). In this study, though PCV2-specific antibody levels elicited
435
by PCV2-COS-1 and PCV2-COS-2 vaccines were different to some extents, both
436
were significantly higher than that by PCV2/COS mixture and PCV2 vaccine alone,
437
indicating that COS conjugation enhanced the humoral immune response to PCV2
438
vaccine. Moreover, PCV2-COS-1 rapidly induced the antibody production of IgG,
439
IgG isotypes, IgA and IgM in the first and booster immunization, even superior to the
440
commercialized adjuvant ISA206. It was proposed that COS conjugation to PCV2 can
441
activate the immune system in a short time and may have more advantages in the
442
prevention of emergent PCV2 infections. Also, the PCV2-COS-1 group showed an
443
increase in IgG2a/IgG1 ratio compared with PCV2 alone group, implicating that COS
444
conjugation can increase the Th1-biased immune response.
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445
Previous studies have demonstrated that saccharide adjuvants can promote T
446
lymphocyte proliferation and augment cytokine production during the immune
447
responses to vaccination (Fan et al., 2015; Fan et al., 2016). In the present study, we 23
Page 24 of 34
found that COS conjugation not only significantly increased T-cell proliferation after
449
ConA stimulation, but markedly produced higher levels of IL-2, IL-5, IFN-γ and
450
TNF-α as compared to PCV2 alone group. Since IL-2 is the central regulators of Th1
451
immune response while IL-5 is proved to enhance Th2 responses, this study displayed
452
a mixed Th1/Th2 responses in groups with PCV2-COS conjugates. CD4+ T cell
453
mediates the killing of organisms responsible for a variety of intracellular infections
454
through the production of IFN-γ and TNF-α (Seder & Hill, 2000). And our results
455
suggest that COS conjugation dramatically increased the production of IFN-γ and
456
TNF-α compared with PCV2 alone group, indicating the strengthening of
457
cell-mediated response to PCV2. It was reported that the sole induction of humoral
458
response might not guarantee a full protection against PCV2 infection, and
459
cell-mediated immune response might contribute together with antibodies to viral
460
clearance and help avoid the progression of PCV-associated diseases (Fort, Sibila,
461
Allepuz, Mateu, Roerink & Segales, 2008; Fort, Sibila, Perez-Martin, Nofrarias,
462
Mateu & Segales, 2009; Martelli et al., 2011). Therefore, the conjugation of COS to
463
PCV2 displayed a great potential in initiating both humoral and cellular responses to
464
PCV2 vaccine, and this may help to provide a better protection against infection than
465
other commercialized PCV2 adjuvants.
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448
466
In this study, we show that PCV2-COS-1 led to its best immunogenicity.
467
Previous studies demonstrated that the protein aggregation by chemical treatment
468
could increase the uptake of antigen by dendritic cells (Kastenmuller et al., 2011).
469
And the ability of aggregated or particulate antigens to enhance cellular immunity and 24
Page 25 of 34
specifically increase the efficiency of cross-presentation has been proved with a
471
variety of antigens and formulations (Schnorrer et al., 2006; Trombetta & Mellman,
472
2005), which may in part explain the strengthened immunogenicity of PCV2 by COS
473
conjugation. A second mechanism to explain the potency of PCV2-COS conjugates
474
may related to the activation of macrophages and dendritic cells via mannose receptor
475
or Toll-like receptor 4 (TLR4) by COS (Han, Zhao, Yu, Feng & Yu, 2005; Wu & Tsai,
476
2007). Results from other groups showed that the enhancement of cellular immunity
477
is associated with the antigen linkage to ligands targeting TLR2, TLR4, or TLR9
478
(Blander & Medzhitov, 2006a; Khan et al., 2007). In the future, the detailed molecular
479
mechanism of how COS conjugation affects the biological performance of PCV2
480
should be further elucidated by in vitro and in vivo studies.
481
Conclusions
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470
In summary, the conjugation of COS markedly enhanced both humoral and
483
cellular immunity against PCV2 by promoting T lymphocyte proliferation, which in
484
turn skewed towards a mixed Th1/Th2 response, including the elevated production of
485
IgG, IG istoypes, IgA and IgM, and up-regulated secretion of inflammatory cytokines,
486
i.e., IL-2, IL-5, IFN-γ and TNF-α. Moreover, the immunization with PCV2-COS-1
487
conjugate displayed similar or even better immune-stimulating effects on antibody
488
production and cytokine secretion than that by PCV2/ISA206, suggesting that the
489
covalent linkage of PCV2 vaccine to COS might be a potential solution to increase the
490
efficacy against PCV2-associated diseases.
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25
Page 26 of 34
491
Acknowledgements We are grateful for the support by National Programs for High Technology
493
Research and Development (863 Programs, 2014AA093604), National Natural
494
Science Fund of China (NO. 31500747 and NO. 31570801), and by Postdoctoral
495
Science Fund of China (NO. 2014M561069).
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