International Journal of Biological Macromolecules 86 (2016) 929–936
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
The enhanced immune response of PCV-2 vaccine using Rehmannia glutinosa polysaccharide liposome as an adjuvant Yee Huang, Zhenguang Liu, Ruonan Bo, Jie Xing, Li Luo, Sisi Zhen, Yale Niu, Yuanliang Hu, Jiaguo Liu, Yi Wu, Deyun Wang ∗ Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China
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Article history: Received 5 January 2016 Received in revised form 27 January 2016 Accepted 1 February 2016 Available online 3 February 2016 Keywords: Rehmannia glutinosa polysaccharide liposome Adjuvant PCV-2
a b s t r a c t Liposomes, one kind of vaccine adjuvants, have been demonstrated as effective adjuvants and vaccine delivery system. Immunization against PCV-2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV-2 infection. Inactivated vaccines represent a complex mixture of viral antigens closely resembling the native virion. In the present study, PCV-2 attenuated antigen was encapsulated within Rehmannia glutinosa polysaccharide liposome, instead of oil adjuvant which is the mainstream adjuvant. Our results showed that RGPL could elicit a strong IgG response and significantly increased the production of Th1 and Th2 associated IgG subtypes and cytokines. R. glutinosa polysaccharide liposome showed excellent particle stability. In vitro, R. glutinosa polysaccharide liposome could also significantly promote phagocytic activity of macrophage and the levels of cytokines it produced. Overall, the results demonstrated that R. glutinosa polysaccharide liposome has the potential to be developed into a more effective and safer vaccine adjuvant. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Vaccine plays an important role in protecting food-producing animals against pathogens, as well as in preventing and controlling infectious diseases. With the development of modern vaccines, a variety of materials have been investigated as vaccine adjuvants. Over the past 20 years there has been increasing interest in the use of nanoparticles as vaccine adjuvants [1,2], as well as carriers for the targeted delivery of antigens to immune competent cells [3]. Formulating protein antigens in nanoparticles has indeed emerged as one of the most promising strategies to augment both humoral and cellular immune responses to vaccine antigens [4–8]. Liposomes, one kind of vaccine adjuvants, have been demonstrated as an effective adjuvant and vaccine delivery system [9]. The adjuvanticity of them can be attributable to their capability of protecting antigen from degradation [10], forming a depot at the site of injection [11], and facilitating Ag uptake [12]. Moreover, liposomes also demonstrate intrinsic immunostimulatory proper-
Abbreviations: RGPL, Rehmannia glutinosa polysaccharide liposome; RGP, Rehmannia glutinosa polysaccharide; PCV-2, porcine circovirus type 2; BL, blank liposome; PBS, phosphate buffered saline. ∗ Corresponding author. Fax: +86 25 84398669. E-mail address: [email protected] (D. Wang). http://dx.doi.org/10.1016/j.ijbiomac.2016.02.003 0141-8130/© 2016 Elsevier B.V. All rights reserved.
ties. They can encapsulate one or more ingredients either within the aqueous core or integrated within the lipid bilayer [13,14]. Currently, there are about 12 liposome-based drugs approved for clinical use and more are in various of clinical trials, such as delivery of surface antigens derived from the hepatitis A or influenza virus and Inflexal V [15]. Porcine circovirus type 2 (PCV-2) is considered the causative agent of the post-weaning multisystemic wasting syndrome (PMWS), one of the major disease that has a significant threat to the global swine industry. The control of PCV-2 disease is based on management strategies, control of co-infections, and mainly on vaccination. Immunization against PCV-2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV-2 infection. Over the years, various PCV-2 vaccines, including DNA vaccines [16,17] and subunit vaccines [18], have been described for the control of PCV-2 infections. DNA vaccination is not suitable for clinical application because of the possibility of carcinogenesis [19]. Subunit vaccination is safer but not suitable for farmers in developing countries due to its high cost. Inactivated vaccines were made from PCV-2 infected cell cultures which were lack of infectivity but still maintain good immunogenicity. They are theoretically advantageous because they represent a complex mixture of viral antigens closely resembling the native virion. Without the help of appropriate adjuvants, immune response induced by vaccine would be suboptimal. So it’s of great impor-
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tance to develop new types of vaccine adjuvant. The study of heral medicine has become a hotspot nowadays [20,21]. Our previous study demonstrated that liposome encapsulated with herbal active ingredient, such as gypenosides [22], propolis flavonoids [23] salidroside [24] and glycyrrhetinic acid [25], could serve as favorable adjuvant. In the present study, we investigated the adjuvanticity of liposome encapsulated with Rehmannia glutinosa polysaccharide (RGP). R. glutinosa is a natural medicine which has been used in China for thousands of years. According to the record in Chinese medical classics “Shennong’s Herba”, R. glutinosa is basically thought as a drug for nourishing Yin (Yin means negative, dark, and feminine and Yang, positive, bright, and masculine in traditional Chinese medicine, TCM), and enriching the blood and benefitting the marrow, etc. RGP is one of the major active components of R. glutinosa. Thus far, several studies have proved that RGP could enhance the immunity [26–28]. Our previous study also demonstrated that RGP could significantly promote lymphocyte proliferation, increase the concentration of IFN-␥ and IL-2 secreted by T lymphocyte, and promote DCs stimulation on T lymphocyte proliferation and its ability on antigen presentation [29]. Furthermore, when polysaccharide encapsulated within liposome, the immunological enhancement activity would be further improved [30–32]. This study aim to investigate the adjuvanticity of liposome encapsulated with R. glutinosa polysaccharide (RGPL) in vitro and in vivo. PCV-2 attenuated antigen was encapsulated within RGPL. The effect of it as the adjuvant system for PCV-2 vaccine was explored in C57BL/6J mice. Compared with oil adjuvant, RGPL could lead to an enhanced long-lasting immune response, and showed excellent particle stability. In vitro, the results demonstrated that RGPL could significantly promote phagocytic activity of macrophage.
Inc.) for 20 min to form the uniform liposome. Ultimately, the solution was successively filtered using 0.45 m and 0.22 m millipore membrane. The resulting solution was kept at 4 ◦ C for subsequent use. Inactivated PCV-2 antigen was provided by Jiangsu Academy of Agricultural Sciences. The inactivated antigen was used as vaccine antigen and mixed thoroughly with different adjuvants (Vantigen :Vadjuvant = 1:3) respectively on a vortex mixer until a milky suspension was obtained, and then freezing and thawing repeated three times. RGPL adjuvant contained 2 mg RGP and 20 mg lipid in 0.25 mL of vaccine. RGP adjuvant vaccine contained 2 mg RGP in 0.25 mL of vaccine. Blank liposome adjuvant contained 20 mg lipid in 0.25 mL of vaccine. Oil adjuvant (ISA 206 adjuvant) was also provided by Jiangsu Academy of Agricultural Sciences. All the vaccines contained the same amount of inactivated PCV-2 antigen. Mice of the blank control group (BC) were injected with PBS (0.25 mL/mouse, pH 7.4). Encapsulation efficiency (EE) of RGP in the liposome was measured by phonel-sulfate method [33] and the EE was 72.75 ± 0.32%. The free RGP was separated from encapsulated RGP through Sephadex G50 column. Liposome sizes and PDI value were measured at 25 ◦ C using a Zetasizer (Nano-ZS, Mavern Instruments, UK) at determined time intervals. 2.3. Mouse immunization Groups of mice received 0.25 mL of vaccine formulations. Vaccine formulations were administered subcutaneously in the dorsal skinfold, and each mouse received on day 0, and a booster dose was given to each primed mouse 7 days after the first immunization. 2.4. Serum antibody response
2. Materials and methods 2.1. Animals The same quantity of female and male C57BL/6J mice (4week-old, weighing between 18 and 22 g), were purchased from Comparative Medicine Centre of Yangzhou University and acclimatized for 7 days prior to use. The experimental protocol involving animal subjects was approved by the University Ethics Committee for the humane care and use of experimental animals, and each mouse was used once and treated in accordance with the National Institutes of Health guide lines for the care and use of laboratory animals. The temperature was maintained at 23–25 ◦ C under the 12/12 light/dark cycle with ∼60% relative humidity. The animals were fed with mouse pellets and distilled water ad libitum. 2.2. Liposome and vaccine formation RGP (purchased from Shanxi Ciyuan Biotechnology Co., Ltd.) liposomes were prepared as described previously [30]. Briefly, appropriate amounts of soybean phospholipid, cholesterol and Tween-80 (40:5:4, mass ratio) were added into a solution with chloroform (5 mL) and methanol (5 mL). After evaporation the remaining lipid film was dissolved by ethyl ether to obtain a liposomal dispersion. RGP solution was injected into the lipid solution. The resulting mixture was homogenized using an ultrasonic cleaner in ice water to form a stabile W/O emulsion, then it was evaporated to form a colloid using a vacuum and appropriate amounts of PBS (pH 7.4) was added to hydrate for an additional 15 min. The resulting mixture was then homogenized using an ultrasonic cell disintegrator (JY92-II DN, Xinzhi Bio-technology and Science
For serum antibody responses, the mice of all groups (6 mice per group) were bled 1 week after the first injection just before the booster and once again 1–5 weeks post-booster. The serum samples were inactivated at 56 ◦ C for 30 min before adding to 96-Well ELISA Plates (BD Biosciences). Their sera were assayed for antigen specific total IgG and IgG subclasses by ELISA. The plates coated with anti-PCV-2 antibody were provided by Jiangsu Academy of Agricultural Sciences. Plates were washed three times and PBS containing 10% BSA was added (150 L/well). The buffer was then discarded and serum diluents (1:400) added to the wells. Plates were incubated for 30 min at 37 ◦ C. After five times of washing, ZyMaxTM goat-anti-mouse IgG (H + L) HRP conjugate (Zymed) was added to each well and incubated at 37 ◦ C for 30 min. Plates were then washed again and then HRP substrate containing 1 L/mL hydrogen peroxide 30% was added. Plates were incubated at room temperature for 10 min in the dark. The reaction was stopped by addition of Stop Reagent (Sigma). The absorbance was finally measured at a wavelength of 630 nm. For specific IgG subtypes determination in the mouse sera, the homologous antigen coated wells reacted with diluted mouse sera (1:200) were appropriately filled with goat-anti-mouse IgG1, IgG2a, IgG2b and IgG3 followed by HRP-labeled rabbit anti-goat immunoglobulins (Sigma, Israel) and ABTS, HRP chromogenic substrate, respectively. The OD at 450 nm of the content in each well was measured against the blank. 2.5. Cytokine study Serum samples were obtained as described above and stored at –70 ◦ C until use. The presence of IL-4, IFN-␥, IL-12, IL-17 and TNF-␣ in the serum samples were determined using the mouse IL-4, IFN-
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Fig. 1. Stability profiles of RGPL. (A) Changes of particle size when stored at 4 ◦ C for 30 days. (Images are representative of three independent experiments.) (B) Changes of PDI value when stored at 4 ◦ C for 30 days. (Images are representative of three independent experiments.)
RGP, blank liposome (BL) and LPS respectively, and serum-free RPMI1640 medium was added as blank control. After incubation for 48 h, macrophages were harvested for phagocytosis activity analysis using Vybrant Phagocytosis Assay Kit (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. The cells were suspended in Hanks balanced salt solution (HBSS) and incubated with Fluorescein-labeled Escherichia coli K-12 Bioparticles at room temperature for 2 h in dark. After the phagocytotic reaction, the cells were washed twice with PBS to remove excessive E. coli particles. The cell viability was assessed by trypan blue staining, and the phagocytic activity was assessed via flow cytometry where positive florescence indicated the phagocytosing cells.
␥, IL-12, IL-17 and TNF-␣ ELISA kits (BD Biosciences) in accordance with manufacturer’s instructions.
2.7.2. In vitro cytokine production from primary cultured peritoneal macrophages The peritoneal macrophages from C57BL/6J were harvested as above. The cell suspension was plated on 96-well culture plates. After stimulation with RGPL, RGP, BL and LPS respectively, and serum-free RPMI1640 medium for 48 h, the supernatants were collected. The levels of murine IL-6, IFN-␥, IL-12, IL-1 and TNF␣ production in cell culture supernatant were analyzed using ELISA kits (R&D, USA), respectively according to the manufacturer’s instructions.
2.6. Spleen histological analysis
2.8. Statistical analysis
Spleens collected at necropsy were immediately fixed in 4% formaldehyde solution for 24 h, dehydrated in alcohol series and processed for embedding in paraffin wax for subsequent sectioning at 5 mm. Hematoxylin and eosin (H&E)-stained histology slides were subsequently analyzed by a certified histopathologist in a blinded manner using a Coolscope digital light microscope (Nikon Co., Tokyo, Japan).
Data are expressed as mean ± standard errors (S.E.). Duncan and LSD’s multiple range test were used to determine the difference among groups. P-values of less than 0.05 were considered to be statistically significant.
Fig. 2. RGPL encapsulated with PCV-2 induced a long-lasting and strong PCV2-specific antibody response. Bars shown are mean ± SE (n = 6), and differences between groups are determined using one-way ANOVA analysis. * P < 0.05, ** P < 0.001.
3. Results 3.1. Stability of RGPL
2.7. Peritoneal macrophages 2.7.1. Phagocytic activity assay Mice peritoneal macrophages were obtained following the method of Kuan et al. [34], with some modifications. Peritoneal macrophages were obtained from C57BL/6J mice 4 days after intraperitoneal injection of 1 mL 3% thioglycollate medium (Sigma–Aldrich St. Louis, MO) by lavage of the peritoneal cavity with PBS. The cells were washed and suspended in RPMI1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Collected macrophages were plated on 6-well culture plates at a density of 2.0 × 106 cells/ml and cultured for 4–6 h at 37 ◦ C with 5% CO2 humidified air. The non-adherent cells were subsequently washed off by PBS, and the remaining adherent monolayer cells were further cultured and stimulated with RGPL,
Particle size and PDI value were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK). Liposomes either co-encapsulated with RGP and antigen(RGPL–PCV), or encapsulated with RGP alone (RGPL) or antigen alone (BL–PCV) were all included for comparison. A few important observations could be made from Fig. 1A and B. The particle size of RGPL was decreased after being co-encapsulated with PCV-2. The changes of BL were the same. As for PDI value, RGPL–PCV was lower than RGPL, which indicated that homogeneity of RGPL–PCV was improved compared that of RGPL. During the 30 days, the particle size of RGPL was increased by 94.6 nm, from 170.8 nm to 265.4 nm. Whereas that of RGPL–PCV only increased by 51.8 nm, from 138.7 nm to 190.5 nm. Change of RGPL–PCV is nearly half of that of RGPL. The similar results were obtained from
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Fig. 3. The effect of RGPL on PCV-2-induced immune responses in mice. The production of IgG subtypes (IgG1, IgG2a, IgG2b and IgG3) and cytokines (IFN-␥, IL-4, TNF-␣ and IL-17) in serum was measured using ELISA. Bars shown are mean ± SE (n = 6), and differences between groups are determined using one-way ANOVA analysis. * P < 0.05, ** P < 0.001. * Compared with BC group, # compared with oil group.
the changes of BL–PCV and BL. The results may indicate that adding the antigen contributed to the stability of RGPL. 3.2. RGPL enhances the antibody response to vaccine antigens Mice were injected subcutaneously with different adjuvants coencapsulated with PCV-2 inactivated antigen. The PCV-2-specific antibody responses (IgG and its subtypes IgG1, IgG2a, IgG2b, IgG3) were measured at weekly intervals from 0 to 5 weeks after two s.c. vaccinations. Levels of PCV-2-specific IgG after two injections were significantly increased for the mice in RGPL group (RGPL adjuvant with PCV-2), which was significantly higher than that of RGP group (RGP adjuvant with PCV-2), Oil group (206 adjuvant with PCV-2) and blank control (BC) group (PBS) from the 1st week to 5th week. On the 1st and 2nd week, IgG levels of the Oil group were higher than that of RGPL group. Afterwards, on the 4th and 5th week, it turned out that IgG levels of RGPL group were higher than that of Oil group. During the time investigated, PCV-2-specific IgG level of RGPL group kept at a relatively high level (Fig. 2). The production of IgG1, IgG2a, IgG2b and IgG3 were also determined and compared within the 5 weeks (Fig. 3). There was
significant increase of IgG1 level at the 5th week, whereas nearly no change was observed during the first two time points. The change of IgG2a was just the opposite. A second vaccination (boost) considerably enhanced the IgG2a levels, and significant increase was observed at the beginning, and then went down with hardly change in the following two time points. The RGPL–PCV-2 formulation elicited a strong IgG1 and IgG2a response at different time points. It’s interesting to find that during the time points investigated, the production of IgG2b and IgG3 were only slightly changes.
3.3. RGPL promote production of IFN-, IL-4, TNF-˛ and IL-17 in serum of mouse Levels of IFN-␥ production were significantly increased in mice after the second boost with RGPL. Two weeks after the second vaccination, the level of IFN-␥ went up and then decreased gradually. In contract, the production of IL-4 was low at the beginning, while 4–5 weeks after the second boost it was enhanced sharply (Fig. 3). Levels of IL-17 and TNF-␣ in the serum were also significantly increased in mice boosted with RGPL. Changes of IL-17 and TNF-␣
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Fig. 4. Spleen histological changes following immunized with different adjuvants. Spleens of different groups were collected 5 weeks after booster injection in order to investigate spleen changes in mouse immunized with different adjuvants. The slides were photographed at ×100 and ×200 respectively. Different degree hyperplasia was found in different groups. The white pulp (WP), margin zone (MZ) and red pulp (RP) were shown. H&E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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were not that obvious, both of which have been at a relatively high level during all the time points investigated (Fig. 3). 3.4. Spleen histology Histological analyses were carried out in order to investigate spleen change in mouse immunized with different adjuvants. The slides were photographed at ×100 and ×200 respectively. Different degree hyperplasia was found in different groups. As for the RGPL group, the growth center of the white pulp (WP) increased, and there was a significant increased number of lymphocytes in the margin zone (MZ) and around central arteries. Besides, there was also a large number of plasma cells in spleen red pulp (RP). 3.5. RGPL stimulated the phagocytic activity and cytokines production of murine peritoneal macrophages Due to the physical characteristics of liposomes, they are prone to be internalized by scavenger cells, such as macrophages (M). To provide further evidence that immune-stimulating effect of RGP was enhanced after being encapsulated in liposome, the effects on the phagocytic activity and cytokines production of murine peritoneal macrophages were investigated. Macrophages were incubated with RGPL, RGP, BL, LPS (as a positive control), or with medium alone (as a negative control) for 48 h and then harvested for further investigation (Fig. 4). Phagocytic activity of macrophages was analyzed by flow cytometry. The flow cytometry data were expressed as percent of positive fluorescence cells in total cells. The phagocytic activity of RGPL-induced macrophages was 1.2-fold greater than the control cells, and about 1-fold greater than RGP group (Fig. 5). The cytokine contents in the culture supernatant were measured by ELISA. As a result, RGPL-treated macrophages exhibited significantly higher levels of IL-1, IFN-␥, IL-6, IL-12, and TNF-␣, which were about 1.2- to 2-fold, compared to the blank control (medium) (P < 0.05) (Fig. 6). A dose-dependent increase in IL-1 production was observed (Fig. 6A). Similar result was also found in IFN-␥, IL-12, and TNF-␣ production (Fig. 6B, D and E). Besides, RGPL-treated macrophages produced higher levels of IL-1, IFN-␥, IL-6, IL-12, and TNF-␣ than RGP-treated macrophages under the same concentration. 4. Discussion Vaccination, considered as one of the most significant achievements in medicine, remains the most cost-effective way to prevent infectious diseases [35–37]. As nanoparticle-based vaccines are promising modalities as the immune system reacts more vigorously to vaccines presented in a particulate form compared to soluble ones [38,39]. In the past decade, nanoparticle-based vaccine delivery platforms, such as liposomes, were of great scientific interest for exploiting as an innovative strategy for vaccine and adjuvant development [40,41]. At the same time, polysaccharides of herbal plants were proved to enhance humoral and cell-mediated immune responses [42–45]. More importantly, most polysaccharides from herbal plants are typically less immunogenic, more tolerable, non-toxic, and biodegradable; they are an unlimited natural resource and low cost to manufacture [46–48]. RGP is extracted from a therapeutic Scrophulariaceae herb, whose name is R. glutinosa. Our previous study also showed that the immunological enhancement of RGP was significantly enhanced after encapsulation with liposome [30]. Currently, RGPL was further investigated as PCV-2 vaccine adjuvant. During the 30 days stability test, the particle size and the PDI value were both of slight
Fig. 5. The phagocytic activity of macrophages stimulated with RGPL or RGP (200 g/mL). The flow cytometry data were expressed as percent of positive fluorescence cells in total cells.
change. Also RGPL showed good controlled released effect. Its stability laid a good foundation for its function as vaccine adjuvant in vivo. PCV-2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV-2 infection has been accepted as an important strategy in the prophylaxis of PMWS to overcome the limitation of oil adjuvant, which is the main adjuvant used at present. In this study, RGPL encapsulated with PCV-2 enhanced a long-lasting immune response. RGPL encapsulated with PCV-2 was able to generate comparable level of PCV-2 specific IgG to oil (206 adjuvant). Furthermore, RGPL encapsulated with PCV-2 significantly increased the production of Th1 and Th2 associated IgG subtypes and cytokines (Fig. 3). The dynamic balance and mutual adjustment between Th1 and Th2 played an important role in maintaining normal immunologic
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Fig. 6. RGPL induced IL-1, IFN-␥, IL-6, IL-12, and TNF-␣ production in C57BL/6J peritoneal macrophages. Macrophages were cultured for 48 h in the presence of RGPL, RGP, BL, LPS, or with culture medium. At the end of the incubation time, the culture medium was collected for cytokine analysis by ELISA. A–D bars in the figure without the same superscripts differ significantly (P < 0.05), comparing unstimulated (medium) versus stimulated groups.
function of organism. As is known to all, Th1 and Th2 cytokines are the key regulators of Ig isotype switching. In mice, Th1 cytokines, such as IFN-␥, not only stimulate CTL response but also directly promote IgG2a production. On the other hand, IgG1 is predominantly regulated by Th2 cytokines, such as IL-4 [49]. In the present study, Th1 and Th2 immune response were investigated at 7, 21, 35 days after second immunization. IgG1 was lower at first two time points while raised sharply at the 35th day after the second immunization. It’s interesting that the production of IgG2a was just the opposite, which was much higher at the beginning and turned to be much lower at the 35th day. Similar tendency was found in IFN-␥ and IL-4 production (Fig. 3.). The level of IFN-␥ was consistent with that of IgG2a, while IL-4 production was consistent with IgG1 production. The changes of IgG3 and IgG2b were not that obvious within the same time points. As a whole, the results might indicate that Th1 immune response played dominant role at the early time after vaccination, while later Th2 immune response did. The spleen represents an important and convenient lymphoid organ to study the complex interplay between cells of the innate and adaptive
immune response [50]. Histological analysis of spleen might help to explain the enhanced immune response of mouse immunized with RGPL. Macrophages, typical phagocytic cells, are derived from peripheral blood monocytes and function as professional antigen presenting cells (APCs) and as effector cells in humoral and cellular immunity [51]. They play an important role in body defenses, exert protective and pathogenic activities and have a function in both innate and adaptive immunity [52]. The effect of RGPL on macrophages in vitro was therefore investigated. As a scavenger cell, phagocytic activity is of great importance. According to the result, RGPL significantly improved phagocytic activity of macrophages, which might contribute to its good performance of engulfing pathogens and antigen presentation. Liposomal adjuvanticity is promoted by receptor mediated targeting to macrophages or the presence of co-adjuvants including cytokines. In this study, we investigated the functional activation of macrophages by RGPL. Therefore, the production of cytokines contribute to immunostimulating activities by RGPL-activated of
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macrophages were analyzed. Activated macrophages produce several cytokines, such as IL-1, and TNF-␣, which are critical for successful defense against invading pathogens, especially intracellular pathogens [53,54]. IL-12 plays an important role in the differentiation of the Th1 cell population and is one of the key cytokines that stimulate Th1 cell-mediated immune responses [55]. Compared with negative control (medium) group, RGPL-treated macrophages secreted significant higher concentration of IL-1, and TNF-␣ (Fig. 6A and E). Results also showed that RGPL exhibited better ability on activation of macrophages compared with RGP. Moreover, RGPL could also upregulate the production of IFN␥, IL-6, and IL-12 so as to regulate innate and adaptive immunity (Fig. 6B–D). 5. Conclusion The present study reported the RGPL as a simple and potent antigen delivery system that could encapsulate PCV-2 antigen with high efficiency and great stability. More importantly, the particle based adjuvant dramatically induced strong and durable specific antibody response, with comparable amounts of PCV-2-specific IgG to oil. Moreover, RGPL nanoparticles were less proinflammatory than the oil in the injection sites. RGPL also offers the optimal balance between humoral and cellular immune responses and strong induction of Th1- and Th2-associated cytokines, which changed over time. The more potent adjuvant activity of RGPL may be partially attributed to their ability to protecting antigen from degradation, increasing the phagocytic activity of macrophages and upregulating the production of IFN-␥, IL-6, and IL-12. Hence, RGPL encapsulated with PCV-2 has the potential to be developed into a more effective and safer adjuvant to formulate new vaccines and reformulate existing oil vaccine adjuvant. Acknowledgments The project was supported by National Natural Science Foundation of China (Grant No. 31372472), Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303046, 201403051) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all other staff in the Institute of Traditional Chinese Veterinary Medicine of Nanjing Agricultural University for their assistance in the experiments. References [1] A.E. Gregory, R. Titball, D. Williamson, Front. Cell. Infect. Microbiol. 3 (2013) 13. [2] L. Zhao, A. Seth, N. Wibowo, C.X. Zhao, N. Mitter, C. Yu, A.P. Middelberg, Vaccine 32 (2014) 327–337. [3] E. Riet, A. Ainai, T. Suzuki, G. Kersten, H. Hasegawa, Adv. Drug Deliv. Rev. 74 (2014) 28–34. [4] J.J. Moon, H. Suh, A. Bershteyn, M.T. Stephan, H. Liu, B. Huang, Nat. Mater. 10 (2011) 243–251. [5] S. De Koker, B.N. Lambrecht, M.A. Willart, Y. Van Kooyk, J. Grooten, C. Vervaet, Chem. Soc. Rev. 40 (2011) 320–339. [6] S.T. Reddy, A.J. Van der Vlies, E. Simeoni, V. Angeli, G.J. Randolph, C.P. O’Neil, Nat. Biotechnol. 25 (2007) 1159–1164. [7] Y.J. Kwon, E. James, N. Shastri, J.M. Frechet, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 18264–18268. [8] S.P. Kasturi, I. Skountzou, R.A. Albrecht, D. Koutsonanos, T. Hua, H.I. Nakaya, Nature 470 (2011) 543–547. [9] D. Christensen, K.S. Korsholm, I. Rosenkrands, T. Lindenstrom, P. Andersen, E.M. Agger, Expert Rev. Vaccines 6 (2007) 785–796.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
The enhanced immune response of PCV-2 vaccine using Rehmannia glutinosa polysaccharide liposome as an adjuvant Yee Huang, Zhenguang Liu, Ruonan Bo, Jie Xing, Li Luo, Sisi Zhen, Yale Niu, Yuanliang Hu, Jiaguo Liu, Yi Wu, Deyun Wang ∗ Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e
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Article history: Received 5 January 2016 Received in revised form 27 January 2016 Accepted 1 February 2016 Available online 3 February 2016 Keywords: Rehmannia glutinosa polysaccharide liposome Adjuvant PCV-2
a b s t r a c t Liposomes, one kind of vaccine adjuvants, have been demonstrated as effective adjuvants and vaccine delivery system. Immunization against PCV-2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV-2 infection. Inactivated vaccines represent a complex mixture of viral antigens closely resembling the native virion. In the present study, PCV-2 attenuated antigen was encapsulated within Rehmannia glutinosa polysaccharide liposome, instead of oil adjuvant which is the mainstream adjuvant. Our results showed that RGPL could elicit a strong IgG response and significantly increased the production of Th1 and Th2 associated IgG subtypes and cytokines. R. glutinosa polysaccharide liposome showed excellent particle stability. In vitro, R. glutinosa polysaccharide liposome could also significantly promote phagocytic activity of macrophage and the levels of cytokines it produced. Overall, the results demonstrated that R. glutinosa polysaccharide liposome has the potential to be developed into a more effective and safer vaccine adjuvant. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Vaccine plays an important role in protecting food-producing animals against pathogens, as well as in preventing and controlling infectious diseases. With the development of modern vaccines, a variety of materials have been investigated as vaccine adjuvants. Over the past 20 years there has been increasing interest in the use of nanoparticles as vaccine adjuvants [1,2], as well as carriers for the targeted delivery of antigens to immune competent cells [3]. Formulating protein antigens in nanoparticles has indeed emerged as one of the most promising strategies to augment both humoral and cellular immune responses to vaccine antigens [4–8]. Liposomes, one kind of vaccine adjuvants, have been demonstrated as an effective adjuvant and vaccine delivery system [9]. The adjuvanticity of them can be attributable to their capability of protecting antigen from degradation [10], forming a depot at the site of injection [11], and facilitating Ag uptake [12]. Moreover, liposomes also demonstrate intrinsic immunostimulatory proper-
Abbreviations: RGPL, Rehmannia glutinosa polysaccharide liposome; RGP, Rehmannia glutinosa polysaccharide; PCV-2, porcine circovirus type 2; BL, blank liposome; PBS, phosphate buffered saline. ∗ Corresponding author. Fax: +86 25 84398669. E-mail address: [email protected] (D. Wang). http://dx.doi.org/10.1016/j.ijbiomac.2016.02.003 0141-8130/© 2016 Elsevier B.V. All rights reserved.
ties. They can encapsulate one or more ingredients either within the aqueous core or integrated within the lipid bilayer [13,14]. Currently, there are about 12 liposome-based drugs approved for clinical use and more are in various of clinical trials, such as delivery of surface antigens derived from the hepatitis A or influenza virus and Inflexal V [15]. Porcine circovirus type 2 (PCV-2) is considered the causative agent of the post-weaning multisystemic wasting syndrome (PMWS), one of the major disease that has a significant threat to the global swine industry. The control of PCV-2 disease is based on management strategies, control of co-infections, and mainly on vaccination. Immunization against PCV-2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV-2 infection. Over the years, various PCV-2 vaccines, including DNA vaccines [16,17] and subunit vaccines [18], have been described for the control of PCV-2 infections. DNA vaccination is not suitable for clinical application because of the possibility of carcinogenesis [19]. Subunit vaccination is safer but not suitable for farmers in developing countries due to its high cost. Inactivated vaccines were made from PCV-2 infected cell cultures which were lack of infectivity but still maintain good immunogenicity. They are theoretically advantageous because they represent a complex mixture of viral antigens closely resembling the native virion. Without the help of appropriate adjuvants, immune response induced by vaccine would be suboptimal. So it’s of great impor-
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tance to develop new types of vaccine adjuvant. The study of heral medicine has become a hotspot nowadays [20,21]. Our previous study demonstrated that liposome encapsulated with herbal active ingredient, such as gypenosides [22], propolis flavonoids [23] salidroside [24] and glycyrrhetinic acid [25], could serve as favorable adjuvant. In the present study, we investigated the adjuvanticity of liposome encapsulated with Rehmannia glutinosa polysaccharide (RGP). R. glutinosa is a natural medicine which has been used in China for thousands of years. According to the record in Chinese medical classics “Shennong’s Herba”, R. glutinosa is basically thought as a drug for nourishing Yin (Yin means negative, dark, and feminine and Yang, positive, bright, and masculine in traditional Chinese medicine, TCM), and enriching the blood and benefitting the marrow, etc. RGP is one of the major active components of R. glutinosa. Thus far, several studies have proved that RGP could enhance the immunity [26–28]. Our previous study also demonstrated that RGP could significantly promote lymphocyte proliferation, increase the concentration of IFN-␥ and IL-2 secreted by T lymphocyte, and promote DCs stimulation on T lymphocyte proliferation and its ability on antigen presentation [29]. Furthermore, when polysaccharide encapsulated within liposome, the immunological enhancement activity would be further improved [30–32]. This study aim to investigate the adjuvanticity of liposome encapsulated with R. glutinosa polysaccharide (RGPL) in vitro and in vivo. PCV-2 attenuated antigen was encapsulated within RGPL. The effect of it as the adjuvant system for PCV-2 vaccine was explored in C57BL/6J mice. Compared with oil adjuvant, RGPL could lead to an enhanced long-lasting immune response, and showed excellent particle stability. In vitro, the results demonstrated that RGPL could significantly promote phagocytic activity of macrophage.
Inc.) for 20 min to form the uniform liposome. Ultimately, the solution was successively filtered using 0.45 m and 0.22 m millipore membrane. The resulting solution was kept at 4 ◦ C for subsequent use. Inactivated PCV-2 antigen was provided by Jiangsu Academy of Agricultural Sciences. The inactivated antigen was used as vaccine antigen and mixed thoroughly with different adjuvants (Vantigen :Vadjuvant = 1:3) respectively on a vortex mixer until a milky suspension was obtained, and then freezing and thawing repeated three times. RGPL adjuvant contained 2 mg RGP and 20 mg lipid in 0.25 mL of vaccine. RGP adjuvant vaccine contained 2 mg RGP in 0.25 mL of vaccine. Blank liposome adjuvant contained 20 mg lipid in 0.25 mL of vaccine. Oil adjuvant (ISA 206 adjuvant) was also provided by Jiangsu Academy of Agricultural Sciences. All the vaccines contained the same amount of inactivated PCV-2 antigen. Mice of the blank control group (BC) were injected with PBS (0.25 mL/mouse, pH 7.4). Encapsulation efficiency (EE) of RGP in the liposome was measured by phonel-sulfate method [33] and the EE was 72.75 ± 0.32%. The free RGP was separated from encapsulated RGP through Sephadex G50 column. Liposome sizes and PDI value were measured at 25 ◦ C using a Zetasizer (Nano-ZS, Mavern Instruments, UK) at determined time intervals. 2.3. Mouse immunization Groups of mice received 0.25 mL of vaccine formulations. Vaccine formulations were administered subcutaneously in the dorsal skinfold, and each mouse received on day 0, and a booster dose was given to each primed mouse 7 days after the first immunization. 2.4. Serum antibody response
2. Materials and methods 2.1. Animals The same quantity of female and male C57BL/6J mice (4week-old, weighing between 18 and 22 g), were purchased from Comparative Medicine Centre of Yangzhou University and acclimatized for 7 days prior to use. The experimental protocol involving animal subjects was approved by the University Ethics Committee for the humane care and use of experimental animals, and each mouse was used once and treated in accordance with the National Institutes of Health guide lines for the care and use of laboratory animals. The temperature was maintained at 23–25 ◦ C under the 12/12 light/dark cycle with ∼60% relative humidity. The animals were fed with mouse pellets and distilled water ad libitum. 2.2. Liposome and vaccine formation RGP (purchased from Shanxi Ciyuan Biotechnology Co., Ltd.) liposomes were prepared as described previously [30]. Briefly, appropriate amounts of soybean phospholipid, cholesterol and Tween-80 (40:5:4, mass ratio) were added into a solution with chloroform (5 mL) and methanol (5 mL). After evaporation the remaining lipid film was dissolved by ethyl ether to obtain a liposomal dispersion. RGP solution was injected into the lipid solution. The resulting mixture was homogenized using an ultrasonic cleaner in ice water to form a stabile W/O emulsion, then it was evaporated to form a colloid using a vacuum and appropriate amounts of PBS (pH 7.4) was added to hydrate for an additional 15 min. The resulting mixture was then homogenized using an ultrasonic cell disintegrator (JY92-II DN, Xinzhi Bio-technology and Science
For serum antibody responses, the mice of all groups (6 mice per group) were bled 1 week after the first injection just before the booster and once again 1–5 weeks post-booster. The serum samples were inactivated at 56 ◦ C for 30 min before adding to 96-Well ELISA Plates (BD Biosciences). Their sera were assayed for antigen specific total IgG and IgG subclasses by ELISA. The plates coated with anti-PCV-2 antibody were provided by Jiangsu Academy of Agricultural Sciences. Plates were washed three times and PBS containing 10% BSA was added (150 L/well). The buffer was then discarded and serum diluents (1:400) added to the wells. Plates were incubated for 30 min at 37 ◦ C. After five times of washing, ZyMaxTM goat-anti-mouse IgG (H + L) HRP conjugate (Zymed) was added to each well and incubated at 37 ◦ C for 30 min. Plates were then washed again and then HRP substrate containing 1 L/mL hydrogen peroxide 30% was added. Plates were incubated at room temperature for 10 min in the dark. The reaction was stopped by addition of Stop Reagent (Sigma). The absorbance was finally measured at a wavelength of 630 nm. For specific IgG subtypes determination in the mouse sera, the homologous antigen coated wells reacted with diluted mouse sera (1:200) were appropriately filled with goat-anti-mouse IgG1, IgG2a, IgG2b and IgG3 followed by HRP-labeled rabbit anti-goat immunoglobulins (Sigma, Israel) and ABTS, HRP chromogenic substrate, respectively. The OD at 450 nm of the content in each well was measured against the blank. 2.5. Cytokine study Serum samples were obtained as described above and stored at –70 ◦ C until use. The presence of IL-4, IFN-␥, IL-12, IL-17 and TNF-␣ in the serum samples were determined using the mouse IL-4, IFN-
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Fig. 1. Stability profiles of RGPL. (A) Changes of particle size when stored at 4 ◦ C for 30 days. (Images are representative of three independent experiments.) (B) Changes of PDI value when stored at 4 ◦ C for 30 days. (Images are representative of three independent experiments.)
RGP, blank liposome (BL) and LPS respectively, and serum-free RPMI1640 medium was added as blank control. After incubation for 48 h, macrophages were harvested for phagocytosis activity analysis using Vybrant Phagocytosis Assay Kit (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. The cells were suspended in Hanks balanced salt solution (HBSS) and incubated with Fluorescein-labeled Escherichia coli K-12 Bioparticles at room temperature for 2 h in dark. After the phagocytotic reaction, the cells were washed twice with PBS to remove excessive E. coli particles. The cell viability was assessed by trypan blue staining, and the phagocytic activity was assessed via flow cytometry where positive florescence indicated the phagocytosing cells.
␥, IL-12, IL-17 and TNF-␣ ELISA kits (BD Biosciences) in accordance with manufacturer’s instructions.
2.7.2. In vitro cytokine production from primary cultured peritoneal macrophages The peritoneal macrophages from C57BL/6J were harvested as above. The cell suspension was plated on 96-well culture plates. After stimulation with RGPL, RGP, BL and LPS respectively, and serum-free RPMI1640 medium for 48 h, the supernatants were collected. The levels of murine IL-6, IFN-␥, IL-12, IL-1 and TNF␣ production in cell culture supernatant were analyzed using ELISA kits (R&D, USA), respectively according to the manufacturer’s instructions.
2.6. Spleen histological analysis
2.8. Statistical analysis
Spleens collected at necropsy were immediately fixed in 4% formaldehyde solution for 24 h, dehydrated in alcohol series and processed for embedding in paraffin wax for subsequent sectioning at 5 mm. Hematoxylin and eosin (H&E)-stained histology slides were subsequently analyzed by a certified histopathologist in a blinded manner using a Coolscope digital light microscope (Nikon Co., Tokyo, Japan).
Data are expressed as mean ± standard errors (S.E.). Duncan and LSD’s multiple range test were used to determine the difference among groups. P-values of less than 0.05 were considered to be statistically significant.
Fig. 2. RGPL encapsulated with PCV-2 induced a long-lasting and strong PCV2-specific antibody response. Bars shown are mean ± SE (n = 6), and differences between groups are determined using one-way ANOVA analysis. * P < 0.05, ** P < 0.001.
3. Results 3.1. Stability of RGPL
2.7. Peritoneal macrophages 2.7.1. Phagocytic activity assay Mice peritoneal macrophages were obtained following the method of Kuan et al. [34], with some modifications. Peritoneal macrophages were obtained from C57BL/6J mice 4 days after intraperitoneal injection of 1 mL 3% thioglycollate medium (Sigma–Aldrich St. Louis, MO) by lavage of the peritoneal cavity with PBS. The cells were washed and suspended in RPMI1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Collected macrophages were plated on 6-well culture plates at a density of 2.0 × 106 cells/ml and cultured for 4–6 h at 37 ◦ C with 5% CO2 humidified air. The non-adherent cells were subsequently washed off by PBS, and the remaining adherent monolayer cells were further cultured and stimulated with RGPL,
Particle size and PDI value were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK). Liposomes either co-encapsulated with RGP and antigen(RGPL–PCV), or encapsulated with RGP alone (RGPL) or antigen alone (BL–PCV) were all included for comparison. A few important observations could be made from Fig. 1A and B. The particle size of RGPL was decreased after being co-encapsulated with PCV-2. The changes of BL were the same. As for PDI value, RGPL–PCV was lower than RGPL, which indicated that homogeneity of RGPL–PCV was improved compared that of RGPL. During the 30 days, the particle size of RGPL was increased by 94.6 nm, from 170.8 nm to 265.4 nm. Whereas that of RGPL–PCV only increased by 51.8 nm, from 138.7 nm to 190.5 nm. Change of RGPL–PCV is nearly half of that of RGPL. The similar results were obtained from
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Fig. 3. The effect of RGPL on PCV-2-induced immune responses in mice. The production of IgG subtypes (IgG1, IgG2a, IgG2b and IgG3) and cytokines (IFN-␥, IL-4, TNF-␣ and IL-17) in serum was measured using ELISA. Bars shown are mean ± SE (n = 6), and differences between groups are determined using one-way ANOVA analysis. * P < 0.05, ** P < 0.001. * Compared with BC group, # compared with oil group.
the changes of BL–PCV and BL. The results may indicate that adding the antigen contributed to the stability of RGPL. 3.2. RGPL enhances the antibody response to vaccine antigens Mice were injected subcutaneously with different adjuvants coencapsulated with PCV-2 inactivated antigen. The PCV-2-specific antibody responses (IgG and its subtypes IgG1, IgG2a, IgG2b, IgG3) were measured at weekly intervals from 0 to 5 weeks after two s.c. vaccinations. Levels of PCV-2-specific IgG after two injections were significantly increased for the mice in RGPL group (RGPL adjuvant with PCV-2), which was significantly higher than that of RGP group (RGP adjuvant with PCV-2), Oil group (206 adjuvant with PCV-2) and blank control (BC) group (PBS) from the 1st week to 5th week. On the 1st and 2nd week, IgG levels of the Oil group were higher than that of RGPL group. Afterwards, on the 4th and 5th week, it turned out that IgG levels of RGPL group were higher than that of Oil group. During the time investigated, PCV-2-specific IgG level of RGPL group kept at a relatively high level (Fig. 2). The production of IgG1, IgG2a, IgG2b and IgG3 were also determined and compared within the 5 weeks (Fig. 3). There was
significant increase of IgG1 level at the 5th week, whereas nearly no change was observed during the first two time points. The change of IgG2a was just the opposite. A second vaccination (boost) considerably enhanced the IgG2a levels, and significant increase was observed at the beginning, and then went down with hardly change in the following two time points. The RGPL–PCV-2 formulation elicited a strong IgG1 and IgG2a response at different time points. It’s interesting to find that during the time points investigated, the production of IgG2b and IgG3 were only slightly changes.
3.3. RGPL promote production of IFN-, IL-4, TNF-˛ and IL-17 in serum of mouse Levels of IFN-␥ production were significantly increased in mice after the second boost with RGPL. Two weeks after the second vaccination, the level of IFN-␥ went up and then decreased gradually. In contract, the production of IL-4 was low at the beginning, while 4–5 weeks after the second boost it was enhanced sharply (Fig. 3). Levels of IL-17 and TNF-␣ in the serum were also significantly increased in mice boosted with RGPL. Changes of IL-17 and TNF-␣
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Fig. 4. Spleen histological changes following immunized with different adjuvants. Spleens of different groups were collected 5 weeks after booster injection in order to investigate spleen changes in mouse immunized with different adjuvants. The slides were photographed at ×100 and ×200 respectively. Different degree hyperplasia was found in different groups. The white pulp (WP), margin zone (MZ) and red pulp (RP) were shown. H&E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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were not that obvious, both of which have been at a relatively high level during all the time points investigated (Fig. 3). 3.4. Spleen histology Histological analyses were carried out in order to investigate spleen change in mouse immunized with different adjuvants. The slides were photographed at ×100 and ×200 respectively. Different degree hyperplasia was found in different groups. As for the RGPL group, the growth center of the white pulp (WP) increased, and there was a significant increased number of lymphocytes in the margin zone (MZ) and around central arteries. Besides, there was also a large number of plasma cells in spleen red pulp (RP). 3.5. RGPL stimulated the phagocytic activity and cytokines production of murine peritoneal macrophages Due to the physical characteristics of liposomes, they are prone to be internalized by scavenger cells, such as macrophages (M). To provide further evidence that immune-stimulating effect of RGP was enhanced after being encapsulated in liposome, the effects on the phagocytic activity and cytokines production of murine peritoneal macrophages were investigated. Macrophages were incubated with RGPL, RGP, BL, LPS (as a positive control), or with medium alone (as a negative control) for 48 h and then harvested for further investigation (Fig. 4). Phagocytic activity of macrophages was analyzed by flow cytometry. The flow cytometry data were expressed as percent of positive fluorescence cells in total cells. The phagocytic activity of RGPL-induced macrophages was 1.2-fold greater than the control cells, and about 1-fold greater than RGP group (Fig. 5). The cytokine contents in the culture supernatant were measured by ELISA. As a result, RGPL-treated macrophages exhibited significantly higher levels of IL-1, IFN-␥, IL-6, IL-12, and TNF-␣, which were about 1.2- to 2-fold, compared to the blank control (medium) (P < 0.05) (Fig. 6). A dose-dependent increase in IL-1 production was observed (Fig. 6A). Similar result was also found in IFN-␥, IL-12, and TNF-␣ production (Fig. 6B, D and E). Besides, RGPL-treated macrophages produced higher levels of IL-1, IFN-␥, IL-6, IL-12, and TNF-␣ than RGP-treated macrophages under the same concentration. 4. Discussion Vaccination, considered as one of the most significant achievements in medicine, remains the most cost-effective way to prevent infectious diseases [35–37]. As nanoparticle-based vaccines are promising modalities as the immune system reacts more vigorously to vaccines presented in a particulate form compared to soluble ones [38,39]. In the past decade, nanoparticle-based vaccine delivery platforms, such as liposomes, were of great scientific interest for exploiting as an innovative strategy for vaccine and adjuvant development [40,41]. At the same time, polysaccharides of herbal plants were proved to enhance humoral and cell-mediated immune responses [42–45]. More importantly, most polysaccharides from herbal plants are typically less immunogenic, more tolerable, non-toxic, and biodegradable; they are an unlimited natural resource and low cost to manufacture [46–48]. RGP is extracted from a therapeutic Scrophulariaceae herb, whose name is R. glutinosa. Our previous study also showed that the immunological enhancement of RGP was significantly enhanced after encapsulation with liposome [30]. Currently, RGPL was further investigated as PCV-2 vaccine adjuvant. During the 30 days stability test, the particle size and the PDI value were both of slight
Fig. 5. The phagocytic activity of macrophages stimulated with RGPL or RGP (200 g/mL). The flow cytometry data were expressed as percent of positive fluorescence cells in total cells.
change. Also RGPL showed good controlled released effect. Its stability laid a good foundation for its function as vaccine adjuvant in vivo. PCV-2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV-2 infection has been accepted as an important strategy in the prophylaxis of PMWS to overcome the limitation of oil adjuvant, which is the main adjuvant used at present. In this study, RGPL encapsulated with PCV-2 enhanced a long-lasting immune response. RGPL encapsulated with PCV-2 was able to generate comparable level of PCV-2 specific IgG to oil (206 adjuvant). Furthermore, RGPL encapsulated with PCV-2 significantly increased the production of Th1 and Th2 associated IgG subtypes and cytokines (Fig. 3). The dynamic balance and mutual adjustment between Th1 and Th2 played an important role in maintaining normal immunologic
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Fig. 6. RGPL induced IL-1, IFN-␥, IL-6, IL-12, and TNF-␣ production in C57BL/6J peritoneal macrophages. Macrophages were cultured for 48 h in the presence of RGPL, RGP, BL, LPS, or with culture medium. At the end of the incubation time, the culture medium was collected for cytokine analysis by ELISA. A–D bars in the figure without the same superscripts differ significantly (P < 0.05), comparing unstimulated (medium) versus stimulated groups.
function of organism. As is known to all, Th1 and Th2 cytokines are the key regulators of Ig isotype switching. In mice, Th1 cytokines, such as IFN-␥, not only stimulate CTL response but also directly promote IgG2a production. On the other hand, IgG1 is predominantly regulated by Th2 cytokines, such as IL-4 [49]. In the present study, Th1 and Th2 immune response were investigated at 7, 21, 35 days after second immunization. IgG1 was lower at first two time points while raised sharply at the 35th day after the second immunization. It’s interesting that the production of IgG2a was just the opposite, which was much higher at the beginning and turned to be much lower at the 35th day. Similar tendency was found in IFN-␥ and IL-4 production (Fig. 3.). The level of IFN-␥ was consistent with that of IgG2a, while IL-4 production was consistent with IgG1 production. The changes of IgG3 and IgG2b were not that obvious within the same time points. As a whole, the results might indicate that Th1 immune response played dominant role at the early time after vaccination, while later Th2 immune response did. The spleen represents an important and convenient lymphoid organ to study the complex interplay between cells of the innate and adaptive
immune response [50]. Histological analysis of spleen might help to explain the enhanced immune response of mouse immunized with RGPL. Macrophages, typical phagocytic cells, are derived from peripheral blood monocytes and function as professional antigen presenting cells (APCs) and as effector cells in humoral and cellular immunity [51]. They play an important role in body defenses, exert protective and pathogenic activities and have a function in both innate and adaptive immunity [52]. The effect of RGPL on macrophages in vitro was therefore investigated. As a scavenger cell, phagocytic activity is of great importance. According to the result, RGPL significantly improved phagocytic activity of macrophages, which might contribute to its good performance of engulfing pathogens and antigen presentation. Liposomal adjuvanticity is promoted by receptor mediated targeting to macrophages or the presence of co-adjuvants including cytokines. In this study, we investigated the functional activation of macrophages by RGPL. Therefore, the production of cytokines contribute to immunostimulating activities by RGPL-activated of
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macrophages were analyzed. Activated macrophages produce several cytokines, such as IL-1, and TNF-␣, which are critical for successful defense against invading pathogens, especially intracellular pathogens [53,54]. IL-12 plays an important role in the differentiation of the Th1 cell population and is one of the key cytokines that stimulate Th1 cell-mediated immune responses [55]. Compared with negative control (medium) group, RGPL-treated macrophages secreted significant higher concentration of IL-1, and TNF-␣ (Fig. 6A and E). Results also showed that RGPL exhibited better ability on activation of macrophages compared with RGP. Moreover, RGPL could also upregulate the production of IFN␥, IL-6, and IL-12 so as to regulate innate and adaptive immunity (Fig. 6B–D). 5. Conclusion The present study reported the RGPL as a simple and potent antigen delivery system that could encapsulate PCV-2 antigen with high efficiency and great stability. More importantly, the particle based adjuvant dramatically induced strong and durable specific antibody response, with comparable amounts of PCV-2-specific IgG to oil. Moreover, RGPL nanoparticles were less proinflammatory than the oil in the injection sites. RGPL also offers the optimal balance between humoral and cellular immune responses and strong induction of Th1- and Th2-associated cytokines, which changed over time. The more potent adjuvant activity of RGPL may be partially attributed to their ability to protecting antigen from degradation, increasing the phagocytic activity of macrophages and upregulating the production of IFN-␥, IL-6, and IL-12. Hence, RGPL encapsulated with PCV-2 has the potential to be developed into a more effective and safer adjuvant to formulate new vaccines and reformulate existing oil vaccine adjuvant. Acknowledgments The project was supported by National Natural Science Foundation of China (Grant No. 31372472), Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303046, 201403051) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all other staff in the Institute of Traditional Chinese Veterinary Medicine of Nanjing Agricultural University for their assistance in the experiments. References [1] A.E. Gregory, R. Titball, D. Williamson, Front. Cell. Infect. Microbiol. 3 (2013) 13. [2] L. Zhao, A. Seth, N. Wibowo, C.X. Zhao, N. Mitter, C. Yu, A.P. Middelberg, Vaccine 32 (2014) 327–337. [3] E. Riet, A. Ainai, T. Suzuki, G. Kersten, H. Hasegawa, Adv. Drug Deliv. Rev. 74 (2014) 28–34. [4] J.J. Moon, H. Suh, A. Bershteyn, M.T. Stephan, H. Liu, B. Huang, Nat. Mater. 10 (2011) 243–251. [5] S. De Koker, B.N. Lambrecht, M.A. Willart, Y. Van Kooyk, J. Grooten, C. Vervaet, Chem. Soc. Rev. 40 (2011) 320–339. [6] S.T. Reddy, A.J. Van der Vlies, E. Simeoni, V. Angeli, G.J. Randolph, C.P. O’Neil, Nat. Biotechnol. 25 (2007) 1159–1164. [7] Y.J. Kwon, E. James, N. Shastri, J.M. Frechet, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 18264–18268. [8] S.P. Kasturi, I. Skountzou, R.A. Albrecht, D. Koutsonanos, T. Hua, H.I. Nakaya, Nature 470 (2011) 543–547. [9] D. Christensen, K.S. Korsholm, I. Rosenkrands, T. Lindenstrom, P. Andersen, E.M. Agger, Expert Rev. Vaccines 6 (2007) 785–796.
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