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Vaccine potential of hemocyanin from Oncomelania hupensis against Schistosoma Japonicum
Parasitology International 60 (2011) 242–246
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Vaccine potential of hemocyanin from Oncomelania hupensis against Schistosoma Japonicum Daoyi Guo 1, Hua Wang 1, Dan Zeng, Xun Li, Xiaolin Fan ⁎, Yongdong Li ⁎ Key Laboratory of Organo-Pharmaceutical Chemistry, Jiangxi Province, Gannan Normal University, Ganzhou 341000, PR China
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Article history: Received 26 August 2010 Received in revised form 21 February 2011 Accepted 19 March 2011 Available online 5 April 2011 Keywords: Immunization Vaccine Oncomelania hupensis Hemocyanin Carbohydrate epitopes Schistosoma japonicum
a b s t r a c t Hemocyanin, a giant oxygen transport protein which is usually found in many arthropods and mollusks was isolated and purified from Oncomelania hupensis. In this study, we showed that Oncomelania hupensis hemocyanin (OhH) shared carbohydrate epitopes with different developmental stages of Schistosoma japonicum (Cercaria, Schistosomulum, Adult worm and Egg) and exhibited serological cross-reaction with these stages of S. japonicum immune sera, which had a potential for use in diagnostic and therapeutic studies of schistosomasis. OhH was used as a vaccine in combination with Freund's adjuvant to evaluate the induction of immune responses and protection against S. japonicum infection in mice. Mice immunized with OhH induced a Th1 type of immune responses. Strong protection against S. japonicum were observed in adult worm and egg burdens after 42 days post-challenge, which showed a significant worm reduction of 52.5% and egg reduction of 69.2% compared to the control groups, respectively. These results indicated that OhH was a potential candidate to compose an anti-schistosome vaccine. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis is a severe parasitic disease, infecting 200 million people mainly in tropical and developing countries and resulting in more than 250,000 deaths per year [1]. The Effective control and treatment rely on praziquantel, a single drug which remains the mainstay of current medical treatment following infection [2], however, the widespread dependence on this single compound for schistosomiasis treatment may hasten the selection of drug-resistant parasites [3–5]. In fact, the emergence of schistosome strains displaying low drug susceptibility to praziquantel have been detected [6–8]. Therefore, a component for cost-effective long-term control of this disease, a schistosomiasis protective vaccine would provide a significant addition to current methods [9–13]. So far, vaccines against schistosome are not yet available for schistosomiasis even including the irradiated cercariae, which regarded as the most effective vaccine candidate in numerous promising vaccines [14,15]. A major problem that has hindered schistosomiasis vaccine research and development lies in the identification and selection of potential protective antigens [16–18]. Schistosomes have a complex life cycle including two hosts: a definitive host, normally mammalian and an intermediate snail
host. During its life cycle, the parasite generates a large array of carbohydrates which have been shown to be major targets of the host immune system [19–23]. In addition, carbohydrate epitopes are also major targets of sera from C57BL/6 J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni, raising the possibility that such carbohydrate antigens might be useful targets for the development of new vaccines [24]. Despite its capacity to infect numerous mammal hosts, Schistosoma japonicum is highly host specific with respect to its intermediate host, using only the gastropod mollusc Oncomelania hupensis. It is a primary factor for the epidemiology of schistosomiasis and its distribution area is consistent with the endemic area of schistosomiasis. Our previous research indicates that the hemocyanin from Oncomelania hupensis (OhH) exhibited serological cross-reaction with S. japonicum infection serum [25]. In this study, we found that OhH shared carbohydrate epitopes with different developmental stages of S. japonicum including cercaria, schistosomulum, adult worm and egg, which have a potential for use in diagnostic and therapeutic studies of schistosomiasis. In addition, we also evaluated the potential of OhH in combination with Freund's adjuvant to provide protection against S. japonicum infection in mice, and the types of immune response were also identified. 2. Materials and methods
Abbreviations: OhH, Oncomelania hupensis hemocyanin; S. japonicum, Schistosoma japonicum. ⁎ Corresponding authors. Tel.: + 86 797 8393536; fax: + 86 797 8393536. E-mail addresses: [email protected] (X. Fan), [email protected] (Y. Li). 1 Both authors contributed equally to this work. 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.03.005
2.1. Mice and parasites and reagents BALB/c female mice, six–eight weeks old, were purchased from the Center of Experimental Animals, Jiangxi University of Traditional
D. Guo et al. / Parasitology International 60 (2011) 242–246
Chinese Medicine. Infected snails were purchased from Hunan Institute of Parasitic Diseases and cercariae of S. japonicum (Chinese mainland strain) were prepared by exposing infected snails to light for 1 h to induce shedding [26]. Cercarial numbers and viability were determined by using a light microscope prior to infection. The peroxidase-conjugated rabbit anti-mouse IgG was purchased from Beijing Biosynthesis Biotechnology Co., LTD, China. The peroxidaseconjugated goat anti-Mouse IgG1and IgG2a antibody were purchased from Bethyl laboratories, INC (USA). 2.2. Preparation of OhH protein The OhH was prepared as previously described by our group [25]. Briefly, Hemolymph of Oncomelania hupensis was collected from the foot muscles of the snails and PMSF(phenylmethylsulfonyl fluoride, 1 mM) was used as a protease inhibitor [27]. Hemocytes and other cells were removed by centrifugation at 8000 g for 20 min at 4 °C. The hemocyanin was precipitated for 6 h at 4 °C using 33% ammonium sulfate then centrifuged at 10,000 g for 1 h. The clear supernatant was discarded and final blue pellet was suspended in Tris buffer. Hemocyanin was pelleted again in a ultracentrifuge of 180,000 g for 3 h at 4 °C. The blue pellet was resuspended in “stabilizing buffer” of 0.05 M Tris, 5 mM CaCl2, 5 mM MgCl2 and 1 mM phenylmethylsulfonyl fluoride, pH 7.0 and stored at 4 °C. 2.3. Anti- schistosome antibodies and cross-reaction The four schistosome antigens prepared with the methods of Lu et al. [28] were derived from those stages that are in intimate contact with the host, namely the infecting cercariae, schistosomulum, adult worms and eggs. Briefly, cercariae were collected from infected snails using the light induction method. To obtain schistosomula, adult worms and eggs for antigen preparation, rabbit were infected with S. japonicum cercariae. Rabbits were portally perfused to recover schistosomula and adult worms on day 12, 42, respectively. Eggs were collected from rabbit's liver. The infected livers in 1% saline were homogenized in a Waring blender. The resulting suspensions were passed through stainless steel filters (80, 120, and 300 mesh counts per inch, in turn), and spun at low speed to sediment the eggs. The four schistosome antigens were prepared by homogenizing the different stages of schistosome in phosphate-buffered saline (PBS) .The schistosome homogenate was centrifuged at 4 °C and the supernatant was collected as antigens and stored at −70 °C. BALB/c mice were injected subcutaneously with 50 μg each schistosome antigens plus Freund's adjuvant (CFA – complete Freund's adjuvant on first immunization and IFA – incomplete Freund's adjuvant on the boosters) at multiple sites of the abdominal area on days 0, 15, respectively. For control group, Freund's adjuvant in PBS was administered using the same immunization protocol. Blood samples from mice were collected at 2 weeks after the second immunization and used for cross-reaction studies. 96-well microtiter plates (Maxisorb, Nunc) were used for ELISA test. Plates were coated with OhH (50 ng) with or without defucosylation by hydrogen fluoride (HF) treatment in carbonate bicarbonate buffer [29], pH 9.6 for 12–16 h at 4 °C, then blocked for 2 h at room temperature with 200 μl /well of PBST (phosphate buffer saline, pH 7.2 with 0.05% Tween-20) plus 1% BSA. Each serum was added per well after dilution of 1:2000 in PBST and then incubated for 1 h at room temperature. Platebound antibody was detected by peroxidase-conjugated rabbit antimouse IgG diluted in PBST 1:1000. The 100 μl tetramethylbenzidine substrate was added. The reaction was stopped with 50 μl per well of 2 M sulfuric acid solution. The OD of each well was read at 450 nm.
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the nape of the neck with 25 μg of OhH on days 0, 15 and 30. The OhH was used in mice immunization with Freund's adjuvant (CFA – complete Freund's adjuvant on first immunization and IFA – incomplete Freund's adjuvant on the boosters). For the control group, Freund's adjuvant in PBS was administered by using the same immunization protocol. 2.5. Challenge infection and worm and egg burden recovery Each mouse was challenged with 40 ± 2 S. japonicum cercaria by the abdomen penetration method two weeks after boosting. 42 days after challenge, all mice (10 in each group) were euthanized, then the abdominal aorta was perfused using PBS after the cutting of hepatic portal, and the worm burdens were determined. The mice livers were each weighed and minced and digested in 10 ml of 5% KOH at 37 °C for 24 h after which schistosome eggs released from the infected livers were counted in 50 μl aliquots. Five aliquots were counted for per liver samples aided by light microscopic examination [30]. The level of protection of vaccinated group was calculated as a percentage based on the reduction in worm burdens or liver eggs per gram of liver, compared with the control group. 2.6. Measurement of specific anti-OhH antibodies Following immunization, sera of ten mice from each vaccinated or control group were collected at two-week intervals. The measurement of specific anti-OhH antibodies was performed by using indirect ELISA. 96-well microtiter plates (Maxisorb, Nunc) were coated with OhH (50 ng ) overnight at 4 °C, blocked with PBST plus 1% BSA for 2 h at room temperature, washed three times with PBST, incubated with serum diluted 1:2000 with PBST for 1 h at room temperature. Plates were washed three times with PBST and incubated with peroxidaseconjugated rabbit anti-mouse IgG, diluted in PBST 1:1000, and peroxidase-conjugated goat anti-mouse IgG1 and IgG2a diluted in PBST 1:10,000. The 100 μl tetramethylbenzidine substrate was added. The reaction was stopped with 50 μl per well of 2 M sulfuric acid solution. The OD of each well was read at 450 nm. 2.7. Statistical analysis Statistical analyses were performed with Student's t test and a P b 0.05 was considered to be statistically significant. 3. Results 3.1. OhH shares carbohydrate epitopes with the different developmental stages of S. japonicum By indirect ELISA, OhH exhibited serological cross-reaction with the different developmental stages of S. japonicum immune serum (Fig. 1). Treatment of OhH after defucosylation by hydrogen fluoride (HF), all the serological cross-reaction observably descended. This indicated OhH mostly shared carbohydrate epitopes with the different developmental stages of S. japonicum and had a potential for use in diagnostic and therapeutic studies of schistosomiasis. 3.2. Analysis of IgG responses to OhH following immunization To investigate the presence of specific anti-OhH IgG antibodies, sera from ten vaccinated animals of each group were tested by ELISA. 30 days after the second immunization, it showed obviously higher levels of IgG in OhH vaccinated groups compared to the control group (Fig. 2).
2.4. Immunization of mice with OhH
3.3. IgG1/IgG2a production after OhH vaccination
Six–eight weeks old female BALB/c mice were divided into two groups of ten mice each. BALB/c mice were subcutaneously injected in
To determine the type of immune response induced after vaccination, the subclasses of IgG1 and IgG2a were measured. Mice
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OhH Defucosylation of OhH
0.8
1.2
0.7 1.0
0.6 0.5
0.8
OD450nm
OD450nm
PBS(IgG1) OhH(IgG1) OhH(IgG2a) PBS(IgG2a)
0.4 0.3 0.2
0.6
0.4
0.1 0.2 0.0 PBS
Cerearia
Schistosomulum Adult worm
Egg
Vaccination serum
0.0
Fig. 1. OhH with or without defucosylation by hydrogen fluoride (HF) was used for cross-reaction studies with the vaccination serum of cercaria, adult worm, schistosomulum, egg or PBS. OD values (450 nm) are shown as mean ± S.D.
immunized with OhH in the presence of Freund's adjuvant showed statistically significant higher levels of IgG1 and IgG2a compared to the control group (Fig. 3). Additionally, IgG1/IgG2a ratio was reduced at days 30 and 45 that parallels with elevated anti-OhH IgG2a production. The trend of IgG1/IgG2a ratio observed in mice immunized with OhH can lead us to speculate that a Th1 type of immune response was induced following vaccination. 3.4. OhH vaccination conferred protective immunity Two weeks after the third immunization, all groups were challenged with S. japonicum cercariae. 42 days later, all mice were euthanized, and adult worm reduction rate and egg reduction rate in liver were determined. As shown in Table 1, mice vaccinated with OhH in double independent trials showed an average of 52.5% reduction in adult worm burdens and 69.2% reduction in liver egg burdens compared with the control group. 4. Discussion Schistosomes of the different developmental stages survive for years in the host bloodstream despite a strong immune response means they can deploy complex defensive and offensive strategies
0.6
PBS OhH
OD450nm
0.5
15
30
45
Days Fig. 3. Antibody isotype (IgG1,IgG2a) responses in mice vaccinated with OhH or PBS. Sera were collected at days of 15, 30, and 45 after immunization and assayed by ELISA for measurement of anti-OhH antibody isotype (IgG1, IgG2a). OD values (450 nm) are shown as mean ± S.D.
which intervene to host responses ineffective. From the initial penetration of cercariae and migration of schistosome to the portal system, the adaptive developmental changes have been produced, which provide distinct capabilities of evasion from the immune attack. Thus, a successful vaccine need to be a multifunctional antigen which is capable of combat for different stages of the schistosome or a cocktail of antigens rather than a single recombinant protein [31]. Due to the complex life cycle of schistosome and the multiple evasion ways from the host immune system combat, different categories and sources-derived vaccines may be necessary, for instance, vaccines corresponding to proteins produced by different stages of schistosomes, DNA vaccines, carbohydrate vaccines and so on. There are studies that show that keyhole limpet hemocyanin (KLH) shares carbohydrate epitopes with Schistosoma mansoni [29,32–34] and this knowledge has been used extensively for the diagnosis of schistosomiasis [35–37]. In view of these shared epitopes, some study investigated KLH as a vaccine against schistosomiasis [38,39], and on the other hand one literature showed that rabbit antibody against keyhole limpet hemocyanin (KLH) failed to protect recipient mice [40]. These molluscan hemocyanins appear to have different oligosaccharide structures, which very likely is the reason why the immunostimulatory properties of these hemocyanins are different [41–44]. Published literature on other hemocyanins which share carbohydrate epitopes with schistosome are rather scanty. Our results showed that the OhH exhibited serological cross-reaction with the several developmental stages of S. japonicum immune serum.
0.4 Table 1 Protective effects of immunization with OhH on S. japonicum challenge in BALB/c mice. All results were presented as mean ± S.D. Percentages in worm and liver egg reduction were compared with the control groups.
0.3 0.2
Groups
0.1 0.0 15
30
45
Days Fig. 2. Antibody IgG responses in mice vaccinated with OhH or PBS. Sera of ten immunized mice were collected at days of 15, 30, and 45 after immunization and assayed by ELISA for measurement of anti-OhH antibody IgG. OD values (450 nm) are shown as mean ± S.D.
1st Experiment PBS + CFA/IFA OhH + CFA/IFA 2st Experiment PBS + CFA/IFA OhH + CFA/IFA a
Number of worm recovered
Egg numbers of liver tissue (n/g × 103)
Worm reduction (%)
Liver egg reduction (%)
31.8 ± 3.0 14.6 ± 2.1
35.5 ± 5.5 10.5 ± 2.2
54.1a
70.4a
28.7 ± 5.5 13.8 ± 2.5
31.6 ± 6.1 10.1 ± 2.5
51.9a
68a
Statistically significant compared to the control group (p b 0.05). The two independent experiments showed a average 52.5% reduction in adult worm burdens and a average 69.2% reduction in liver egg burdens compared with the control group.
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Further research indicates that OhH shared carbohydrate epitopes with these different life-cycle stages of S. japonicum and had a potential for use in diagnostic and vaccination against this disease. Development of this type of vaccines, for instance, OhH, is a new way to create broad-range protection against different stages of parasite. To evaluate the protective efficacy of the OhH molecule against S. japonicum infection, BALB/c mice were challenged two weeks after the last boost and 42 days later adult worm and egg burdens were analyzed. Mice immunized with OhH successfully mounted a specific anti-OhH IgG antibody response. Furthermore, we investigated the type of immune response generated within this vaccinated group. In general the relative production of IgG1 and IgG2a antibody isotypes is used as an indicator of the pattern of immune response produced. Here, we found that OhH immunization induced the production of both IgG1 and IgG2a anti-OhH antibodies in BALB/c mice and showed reduced IgG1/IgG2a ratio at days 30 and 45 after the first immunization. This finding suggests a tendency of a Th1 type of immune response induced by OhH vaccination. Efforts have been made in schistosomiasis to obtain insight into the types of immune response involved in the protection. Several antigens had a tendency to induce a Th2 type of immune response failed to engender protection in the mouse model [45,46]. In contrast, Th1 antigens induced partial protection against infection [47,48]. In this study, strong protection against S. japonicum were observed in adult worm and egg burdens after 42 days post-challenge, which showed a significant worm reduction of 52.5% and egg reduction of 69.2% compared to the control groups, respectively. These results indicated that OhH was a potential candidate to compose an anti-schistosome vaccine. Acknowledgments This work was supported by the National Science Foundation of China (30860073, 50727803 and 50968002), the National Key Technologies R&D Program (2009BAI78B01 and 2009BAI78B02), the National Science Foundation of Jiangxi Province (2008GQH0017 and 2008GQY0102), and the Foundation of Jiangxi Educational Committee (GJJ10236). References [1] van der Werf MJ, de Vlas SJ, Brooke S, Looman CW, Nagelkerke NJ, Habbema JD, et al. Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa. Acta Trop 2003;86:125–39. [2] Harder A. Chemotherapeutic approaches to schistosomes: current knowledge and outlook. Parasitol Res 2002;88:395–7. [3] Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Mem Inst Oswaldo Cruz 1998;93(Suppl 1):95–101. [4] Utzinger J, Zhou XN, Chen M, GBergquist R. Conquering schistosomiasis in China: the long march. Acta Trop 2005;96:69–96. [5] Sayed AA, Simeonov A, Thomas CJ, Inglese J, Williams Austin CP, Williams DL. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat Med 2008;14:407–12. [6] Cioli, Pica-Mattoccia D, Praziquantel L. Parasitol Res 2003;90(Supp 1):S3–9. [7] Fallon PG, Sturrock RF, Niang AC, Doenhoff MJ. Short report: diminished susceptibility to praziquantel in a Senegal isolate of Schistosoma mansoni. Am J Trop Med Hyg 1995;53:61–2. [8] Ismail M, Metwally A, Farghaly A, Bruce J, Tao L, F. Bennett JL. Characterization of isolates of Schistosoma mansoni from Egyptian villagers that tolerate high doses of praziquantel. Am J Trop Med Hyg 1996;55:214–8. [9] Bergquist NR. Schistosomiasis: from risk assessment to control. Trends Parasitol 2002;18:309–14. [10] Pacifico LG, Fonseca CT, Chiari L, Oliveira SC. Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine. Immunobiology 2006;211:97–104. [11] Pierrot C, Wilson S, Lallet H, Lafitte S, Jones FM, Daher W, et al. Identification of a novel antigen of Schistosoma mansoni shared with Plasmodium falciparum and evaluation of different cross-reactive antibody subclasses induced by human schistosomiasis and malaria. Infect Immun 2006;74:3347–54. [12] Tran MH, Pearson MS, Bethony JM, Smyth DJ, Jones MK, Duke M, et al. Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis. Nat Med 2006;12:835–40.
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Vaccine potential of hemocyanin from Oncomelania hupensis against Schistosoma Japonicum Daoyi Guo 1, Hua Wang 1, Dan Zeng, Xun Li, Xiaolin Fan ⁎, Yongdong Li ⁎ Key Laboratory of Organo-Pharmaceutical Chemistry, Jiangxi Province, Gannan Normal University, Ganzhou 341000, PR China
a r t i c l e
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Article history: Received 26 August 2010 Received in revised form 21 February 2011 Accepted 19 March 2011 Available online 5 April 2011 Keywords: Immunization Vaccine Oncomelania hupensis Hemocyanin Carbohydrate epitopes Schistosoma japonicum
a b s t r a c t Hemocyanin, a giant oxygen transport protein which is usually found in many arthropods and mollusks was isolated and purified from Oncomelania hupensis. In this study, we showed that Oncomelania hupensis hemocyanin (OhH) shared carbohydrate epitopes with different developmental stages of Schistosoma japonicum (Cercaria, Schistosomulum, Adult worm and Egg) and exhibited serological cross-reaction with these stages of S. japonicum immune sera, which had a potential for use in diagnostic and therapeutic studies of schistosomasis. OhH was used as a vaccine in combination with Freund's adjuvant to evaluate the induction of immune responses and protection against S. japonicum infection in mice. Mice immunized with OhH induced a Th1 type of immune responses. Strong protection against S. japonicum were observed in adult worm and egg burdens after 42 days post-challenge, which showed a significant worm reduction of 52.5% and egg reduction of 69.2% compared to the control groups, respectively. These results indicated that OhH was a potential candidate to compose an anti-schistosome vaccine. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis is a severe parasitic disease, infecting 200 million people mainly in tropical and developing countries and resulting in more than 250,000 deaths per year [1]. The Effective control and treatment rely on praziquantel, a single drug which remains the mainstay of current medical treatment following infection [2], however, the widespread dependence on this single compound for schistosomiasis treatment may hasten the selection of drug-resistant parasites [3–5]. In fact, the emergence of schistosome strains displaying low drug susceptibility to praziquantel have been detected [6–8]. Therefore, a component for cost-effective long-term control of this disease, a schistosomiasis protective vaccine would provide a significant addition to current methods [9–13]. So far, vaccines against schistosome are not yet available for schistosomiasis even including the irradiated cercariae, which regarded as the most effective vaccine candidate in numerous promising vaccines [14,15]. A major problem that has hindered schistosomiasis vaccine research and development lies in the identification and selection of potential protective antigens [16–18]. Schistosomes have a complex life cycle including two hosts: a definitive host, normally mammalian and an intermediate snail
host. During its life cycle, the parasite generates a large array of carbohydrates which have been shown to be major targets of the host immune system [19–23]. In addition, carbohydrate epitopes are also major targets of sera from C57BL/6 J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni, raising the possibility that such carbohydrate antigens might be useful targets for the development of new vaccines [24]. Despite its capacity to infect numerous mammal hosts, Schistosoma japonicum is highly host specific with respect to its intermediate host, using only the gastropod mollusc Oncomelania hupensis. It is a primary factor for the epidemiology of schistosomiasis and its distribution area is consistent with the endemic area of schistosomiasis. Our previous research indicates that the hemocyanin from Oncomelania hupensis (OhH) exhibited serological cross-reaction with S. japonicum infection serum [25]. In this study, we found that OhH shared carbohydrate epitopes with different developmental stages of S. japonicum including cercaria, schistosomulum, adult worm and egg, which have a potential for use in diagnostic and therapeutic studies of schistosomiasis. In addition, we also evaluated the potential of OhH in combination with Freund's adjuvant to provide protection against S. japonicum infection in mice, and the types of immune response were also identified. 2. Materials and methods
Abbreviations: OhH, Oncomelania hupensis hemocyanin; S. japonicum, Schistosoma japonicum. ⁎ Corresponding authors. Tel.: + 86 797 8393536; fax: + 86 797 8393536. E-mail addresses: [email protected] (X. Fan), [email protected] (Y. Li). 1 Both authors contributed equally to this work. 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.03.005
2.1. Mice and parasites and reagents BALB/c female mice, six–eight weeks old, were purchased from the Center of Experimental Animals, Jiangxi University of Traditional
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Chinese Medicine. Infected snails were purchased from Hunan Institute of Parasitic Diseases and cercariae of S. japonicum (Chinese mainland strain) were prepared by exposing infected snails to light for 1 h to induce shedding [26]. Cercarial numbers and viability were determined by using a light microscope prior to infection. The peroxidase-conjugated rabbit anti-mouse IgG was purchased from Beijing Biosynthesis Biotechnology Co., LTD, China. The peroxidaseconjugated goat anti-Mouse IgG1and IgG2a antibody were purchased from Bethyl laboratories, INC (USA). 2.2. Preparation of OhH protein The OhH was prepared as previously described by our group [25]. Briefly, Hemolymph of Oncomelania hupensis was collected from the foot muscles of the snails and PMSF(phenylmethylsulfonyl fluoride, 1 mM) was used as a protease inhibitor [27]. Hemocytes and other cells were removed by centrifugation at 8000 g for 20 min at 4 °C. The hemocyanin was precipitated for 6 h at 4 °C using 33% ammonium sulfate then centrifuged at 10,000 g for 1 h. The clear supernatant was discarded and final blue pellet was suspended in Tris buffer. Hemocyanin was pelleted again in a ultracentrifuge of 180,000 g for 3 h at 4 °C. The blue pellet was resuspended in “stabilizing buffer” of 0.05 M Tris, 5 mM CaCl2, 5 mM MgCl2 and 1 mM phenylmethylsulfonyl fluoride, pH 7.0 and stored at 4 °C. 2.3. Anti- schistosome antibodies and cross-reaction The four schistosome antigens prepared with the methods of Lu et al. [28] were derived from those stages that are in intimate contact with the host, namely the infecting cercariae, schistosomulum, adult worms and eggs. Briefly, cercariae were collected from infected snails using the light induction method. To obtain schistosomula, adult worms and eggs for antigen preparation, rabbit were infected with S. japonicum cercariae. Rabbits were portally perfused to recover schistosomula and adult worms on day 12, 42, respectively. Eggs were collected from rabbit's liver. The infected livers in 1% saline were homogenized in a Waring blender. The resulting suspensions were passed through stainless steel filters (80, 120, and 300 mesh counts per inch, in turn), and spun at low speed to sediment the eggs. The four schistosome antigens were prepared by homogenizing the different stages of schistosome in phosphate-buffered saline (PBS) .The schistosome homogenate was centrifuged at 4 °C and the supernatant was collected as antigens and stored at −70 °C. BALB/c mice were injected subcutaneously with 50 μg each schistosome antigens plus Freund's adjuvant (CFA – complete Freund's adjuvant on first immunization and IFA – incomplete Freund's adjuvant on the boosters) at multiple sites of the abdominal area on days 0, 15, respectively. For control group, Freund's adjuvant in PBS was administered using the same immunization protocol. Blood samples from mice were collected at 2 weeks after the second immunization and used for cross-reaction studies. 96-well microtiter plates (Maxisorb, Nunc) were used for ELISA test. Plates were coated with OhH (50 ng) with or without defucosylation by hydrogen fluoride (HF) treatment in carbonate bicarbonate buffer [29], pH 9.6 for 12–16 h at 4 °C, then blocked for 2 h at room temperature with 200 μl /well of PBST (phosphate buffer saline, pH 7.2 with 0.05% Tween-20) plus 1% BSA. Each serum was added per well after dilution of 1:2000 in PBST and then incubated for 1 h at room temperature. Platebound antibody was detected by peroxidase-conjugated rabbit antimouse IgG diluted in PBST 1:1000. The 100 μl tetramethylbenzidine substrate was added. The reaction was stopped with 50 μl per well of 2 M sulfuric acid solution. The OD of each well was read at 450 nm.
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the nape of the neck with 25 μg of OhH on days 0, 15 and 30. The OhH was used in mice immunization with Freund's adjuvant (CFA – complete Freund's adjuvant on first immunization and IFA – incomplete Freund's adjuvant on the boosters). For the control group, Freund's adjuvant in PBS was administered by using the same immunization protocol. 2.5. Challenge infection and worm and egg burden recovery Each mouse was challenged with 40 ± 2 S. japonicum cercaria by the abdomen penetration method two weeks after boosting. 42 days after challenge, all mice (10 in each group) were euthanized, then the abdominal aorta was perfused using PBS after the cutting of hepatic portal, and the worm burdens were determined. The mice livers were each weighed and minced and digested in 10 ml of 5% KOH at 37 °C for 24 h after which schistosome eggs released from the infected livers were counted in 50 μl aliquots. Five aliquots were counted for per liver samples aided by light microscopic examination [30]. The level of protection of vaccinated group was calculated as a percentage based on the reduction in worm burdens or liver eggs per gram of liver, compared with the control group. 2.6. Measurement of specific anti-OhH antibodies Following immunization, sera of ten mice from each vaccinated or control group were collected at two-week intervals. The measurement of specific anti-OhH antibodies was performed by using indirect ELISA. 96-well microtiter plates (Maxisorb, Nunc) were coated with OhH (50 ng ) overnight at 4 °C, blocked with PBST plus 1% BSA for 2 h at room temperature, washed three times with PBST, incubated with serum diluted 1:2000 with PBST for 1 h at room temperature. Plates were washed three times with PBST and incubated with peroxidaseconjugated rabbit anti-mouse IgG, diluted in PBST 1:1000, and peroxidase-conjugated goat anti-mouse IgG1 and IgG2a diluted in PBST 1:10,000. The 100 μl tetramethylbenzidine substrate was added. The reaction was stopped with 50 μl per well of 2 M sulfuric acid solution. The OD of each well was read at 450 nm. 2.7. Statistical analysis Statistical analyses were performed with Student's t test and a P b 0.05 was considered to be statistically significant. 3. Results 3.1. OhH shares carbohydrate epitopes with the different developmental stages of S. japonicum By indirect ELISA, OhH exhibited serological cross-reaction with the different developmental stages of S. japonicum immune serum (Fig. 1). Treatment of OhH after defucosylation by hydrogen fluoride (HF), all the serological cross-reaction observably descended. This indicated OhH mostly shared carbohydrate epitopes with the different developmental stages of S. japonicum and had a potential for use in diagnostic and therapeutic studies of schistosomiasis. 3.2. Analysis of IgG responses to OhH following immunization To investigate the presence of specific anti-OhH IgG antibodies, sera from ten vaccinated animals of each group were tested by ELISA. 30 days after the second immunization, it showed obviously higher levels of IgG in OhH vaccinated groups compared to the control group (Fig. 2).
2.4. Immunization of mice with OhH
3.3. IgG1/IgG2a production after OhH vaccination
Six–eight weeks old female BALB/c mice were divided into two groups of ten mice each. BALB/c mice were subcutaneously injected in
To determine the type of immune response induced after vaccination, the subclasses of IgG1 and IgG2a were measured. Mice
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OhH Defucosylation of OhH
0.8
1.2
0.7 1.0
0.6 0.5
0.8
OD450nm
OD450nm
PBS(IgG1) OhH(IgG1) OhH(IgG2a) PBS(IgG2a)
0.4 0.3 0.2
0.6
0.4
0.1 0.2 0.0 PBS
Cerearia
Schistosomulum Adult worm
Egg
Vaccination serum
0.0
Fig. 1. OhH with or without defucosylation by hydrogen fluoride (HF) was used for cross-reaction studies with the vaccination serum of cercaria, adult worm, schistosomulum, egg or PBS. OD values (450 nm) are shown as mean ± S.D.
immunized with OhH in the presence of Freund's adjuvant showed statistically significant higher levels of IgG1 and IgG2a compared to the control group (Fig. 3). Additionally, IgG1/IgG2a ratio was reduced at days 30 and 45 that parallels with elevated anti-OhH IgG2a production. The trend of IgG1/IgG2a ratio observed in mice immunized with OhH can lead us to speculate that a Th1 type of immune response was induced following vaccination. 3.4. OhH vaccination conferred protective immunity Two weeks after the third immunization, all groups were challenged with S. japonicum cercariae. 42 days later, all mice were euthanized, and adult worm reduction rate and egg reduction rate in liver were determined. As shown in Table 1, mice vaccinated with OhH in double independent trials showed an average of 52.5% reduction in adult worm burdens and 69.2% reduction in liver egg burdens compared with the control group. 4. Discussion Schistosomes of the different developmental stages survive for years in the host bloodstream despite a strong immune response means they can deploy complex defensive and offensive strategies
0.6
PBS OhH
OD450nm
0.5
15
30
45
Days Fig. 3. Antibody isotype (IgG1,IgG2a) responses in mice vaccinated with OhH or PBS. Sera were collected at days of 15, 30, and 45 after immunization and assayed by ELISA for measurement of anti-OhH antibody isotype (IgG1, IgG2a). OD values (450 nm) are shown as mean ± S.D.
which intervene to host responses ineffective. From the initial penetration of cercariae and migration of schistosome to the portal system, the adaptive developmental changes have been produced, which provide distinct capabilities of evasion from the immune attack. Thus, a successful vaccine need to be a multifunctional antigen which is capable of combat for different stages of the schistosome or a cocktail of antigens rather than a single recombinant protein [31]. Due to the complex life cycle of schistosome and the multiple evasion ways from the host immune system combat, different categories and sources-derived vaccines may be necessary, for instance, vaccines corresponding to proteins produced by different stages of schistosomes, DNA vaccines, carbohydrate vaccines and so on. There are studies that show that keyhole limpet hemocyanin (KLH) shares carbohydrate epitopes with Schistosoma mansoni [29,32–34] and this knowledge has been used extensively for the diagnosis of schistosomiasis [35–37]. In view of these shared epitopes, some study investigated KLH as a vaccine against schistosomiasis [38,39], and on the other hand one literature showed that rabbit antibody against keyhole limpet hemocyanin (KLH) failed to protect recipient mice [40]. These molluscan hemocyanins appear to have different oligosaccharide structures, which very likely is the reason why the immunostimulatory properties of these hemocyanins are different [41–44]. Published literature on other hemocyanins which share carbohydrate epitopes with schistosome are rather scanty. Our results showed that the OhH exhibited serological cross-reaction with the several developmental stages of S. japonicum immune serum.
0.4 Table 1 Protective effects of immunization with OhH on S. japonicum challenge in BALB/c mice. All results were presented as mean ± S.D. Percentages in worm and liver egg reduction were compared with the control groups.
0.3 0.2
Groups
0.1 0.0 15
30
45
Days Fig. 2. Antibody IgG responses in mice vaccinated with OhH or PBS. Sera of ten immunized mice were collected at days of 15, 30, and 45 after immunization and assayed by ELISA for measurement of anti-OhH antibody IgG. OD values (450 nm) are shown as mean ± S.D.
1st Experiment PBS + CFA/IFA OhH + CFA/IFA 2st Experiment PBS + CFA/IFA OhH + CFA/IFA a
Number of worm recovered
Egg numbers of liver tissue (n/g × 103)
Worm reduction (%)
Liver egg reduction (%)
31.8 ± 3.0 14.6 ± 2.1
35.5 ± 5.5 10.5 ± 2.2
54.1a
70.4a
28.7 ± 5.5 13.8 ± 2.5
31.6 ± 6.1 10.1 ± 2.5
51.9a
68a
Statistically significant compared to the control group (p b 0.05). The two independent experiments showed a average 52.5% reduction in adult worm burdens and a average 69.2% reduction in liver egg burdens compared with the control group.
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Further research indicates that OhH shared carbohydrate epitopes with these different life-cycle stages of S. japonicum and had a potential for use in diagnostic and vaccination against this disease. Development of this type of vaccines, for instance, OhH, is a new way to create broad-range protection against different stages of parasite. To evaluate the protective efficacy of the OhH molecule against S. japonicum infection, BALB/c mice were challenged two weeks after the last boost and 42 days later adult worm and egg burdens were analyzed. Mice immunized with OhH successfully mounted a specific anti-OhH IgG antibody response. Furthermore, we investigated the type of immune response generated within this vaccinated group. In general the relative production of IgG1 and IgG2a antibody isotypes is used as an indicator of the pattern of immune response produced. Here, we found that OhH immunization induced the production of both IgG1 and IgG2a anti-OhH antibodies in BALB/c mice and showed reduced IgG1/IgG2a ratio at days 30 and 45 after the first immunization. This finding suggests a tendency of a Th1 type of immune response induced by OhH vaccination. Efforts have been made in schistosomiasis to obtain insight into the types of immune response involved in the protection. Several antigens had a tendency to induce a Th2 type of immune response failed to engender protection in the mouse model [45,46]. In contrast, Th1 antigens induced partial protection against infection [47,48]. In this study, strong protection against S. japonicum were observed in adult worm and egg burdens after 42 days post-challenge, which showed a significant worm reduction of 52.5% and egg reduction of 69.2% compared to the control groups, respectively. These results indicated that OhH was a potential candidate to compose an anti-schistosome vaccine. Acknowledgments This work was supported by the National Science Foundation of China (30860073, 50727803 and 50968002), the National Key Technologies R&D Program (2009BAI78B01 and 2009BAI78B02), the National Science Foundation of Jiangxi Province (2008GQH0017 and 2008GQY0102), and the Foundation of Jiangxi Educational Committee (GJJ10236). References [1] van der Werf MJ, de Vlas SJ, Brooke S, Looman CW, Nagelkerke NJ, Habbema JD, et al. Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa. Acta Trop 2003;86:125–39. [2] Harder A. Chemotherapeutic approaches to schistosomes: current knowledge and outlook. Parasitol Res 2002;88:395–7. [3] Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Mem Inst Oswaldo Cruz 1998;93(Suppl 1):95–101. [4] Utzinger J, Zhou XN, Chen M, GBergquist R. Conquering schistosomiasis in China: the long march. Acta Trop 2005;96:69–96. [5] Sayed AA, Simeonov A, Thomas CJ, Inglese J, Williams Austin CP, Williams DL. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat Med 2008;14:407–12. [6] Cioli, Pica-Mattoccia D, Praziquantel L. Parasitol Res 2003;90(Supp 1):S3–9. [7] Fallon PG, Sturrock RF, Niang AC, Doenhoff MJ. Short report: diminished susceptibility to praziquantel in a Senegal isolate of Schistosoma mansoni. Am J Trop Med Hyg 1995;53:61–2. [8] Ismail M, Metwally A, Farghaly A, Bruce J, Tao L, F. Bennett JL. Characterization of isolates of Schistosoma mansoni from Egyptian villagers that tolerate high doses of praziquantel. Am J Trop Med Hyg 1996;55:214–8. [9] Bergquist NR. Schistosomiasis: from risk assessment to control. Trends Parasitol 2002;18:309–14. [10] Pacifico LG, Fonseca CT, Chiari L, Oliveira SC. Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine. Immunobiology 2006;211:97–104. [11] Pierrot C, Wilson S, Lallet H, Lafitte S, Jones FM, Daher W, et al. Identification of a novel antigen of Schistosoma mansoni shared with Plasmodium falciparum and evaluation of different cross-reactive antibody subclasses induced by human schistosomiasis and malaria. Infect Immun 2006;74:3347–54. [12] Tran MH, Pearson MS, Bethony JM, Smyth DJ, Jones MK, Duke M, et al. Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis. Nat Med 2006;12:835–40.
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