Virus-like particles vaccine containing Clonorchis sinensis tegumental protein induces partial protection against Clonorchis sinensis infection

Background Human clonorchiasis, caused by the infection of Clonorchis sinensis, is one of the major health problems in Southeast Asia. However, vaccine efficacy against C. sinensis infection remains largely unknown. Methods In this study, for the first time, we generated virus-like particles (VLPs) vaccine containing the C. sinensis tegumental protein 22.3 kDa (CsTP 22.3) and the influenza matrix protein (M1) as a core protein, and investigated the vaccine efficacy in Sprague-Dawley rats. Results Intranasal immunization of VLPs vaccine induced C. sinensis-specific IgG, IgG2a and IgG2c in the sera and IgA responses in the feces and intestines. Notably, upon challenge infection with C. sinensis metacercariae, significantly lower adult worm loads (70.2%) were measured in the liver of rats immunized with VLPs, compared to those of naïve rats. Furthermore, VLPs immunization induced antibody secreting cells (ASC) responses and CD4+/CD8+ T cell responses in the spleen. Conclusions Our results indicated that VLPs vaccine containing C. sinensis CsTP 22.3 kDa provided partial protection against C. sisnensis infection. Thus, VLPs could be a potential vaccine candidate against C. sinensis. Electronic supplementary material The online version of this article (10.1186/s13071-017-2526-5) contains supplementary material, which is available to authorized users.


Background
Human clonorchiasis is one of the most important foodborne zoonosis, caused by Clonorchis sinensis infection from the consumption of raw or undercooked freshwater fish infected with C. sinensis metacercariae. Clonorchiasis is mainly prevalent in Southeast Asia, including Korea, China, East Russia, Taiwan and northern Vietnam, causing pyogenic cholangitis, cholelithiasis, cholecystitis and hepatic fibrosis, and even cholagiocarcinoma in humans [1][2][3][4]. Thus, C. sinensis has been classified as a group 1 biocarcinogens by the International Agency of Cancer Research, and clonorchiasis is included in control program of neglected tropical diseases by WHO [2,5]. It is estimated that 200 million people are at risk of infection, 15-20 million people are infected with C. sinensis worldwide and, among them, 1.4 million people are currently infected with this fluke in South Korea [2,6]. The development of vaccines would have a significant impact towards the ultimate goal of disease elimination.
Virus-like particles (VLPs) are recombinant vaccines, displaying promising results in preclinical and clinical studies in terms of both safety and efficacy [16]. VLPs are morphologically similar to live viruses, but lack viral genetic materials and therefore cannot replicate, which is advantageous for safety [17][18][19]. In this study, we, for the first time, generated VLPs vaccine containing C. sinensis tegumental protein (CsTP22.3). We found that C. sinensis VLPs vaccine elicited C. sinensis-specific IgG, IgG subclass and IgA antibody responses, antibody secreting cells (ASC) and CD4+/CD8+ T cell responses, resulting in protection against C. sinensis infection in a rat model.

Cells, viruses, parasites, antibodies and animals
Spodoptera frugiperda SF9 insect cells were maintained in suspension in serum-free SF900II medium (Invitrogen, Carlsbad, USA) at 27°C. HEp-2 cells were obtained from ATCC. HEp-2 cells were grown in tissue culture flasks in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), penicillin and streptomycin at 37°C with 5% CO 2 . MDCK cells were infected with influenza virus (A/California/04/09) to obtain influenza virus total RNA. Monoclonal mouse anti-RSV fusion protein (131-2A) (Millipore, Burlington, USA) was used in virus plaque assay. Mouse monoclonal antibody to influenza A virus M1 (Abcam, Cambridge, UK) was used in western blot. HRP-conjugated goat anti-rat immunoglobulins G (IgG), IgG1, IgG2a, IgG2b and IgG2c (Southern Biotech, Birmingham, USA) were used for secondary antibodies. Sprague-Dawley (SD) rats (female, 8 weeks old) and New Zealand white rabbits (male, 2-4 month old) were purchased from Samyook Animal Center, Osan City, Kyonggi-do, Korea. White rabbits were infected with Clonorchis sinensis metacercariae to generate C. sinensis adult worms. Clonorchis sinensis metacercariae were collected from the freshwater fish Pseudorasbora parva by digesting muscles with pepsin-HCl, followed by filtration through layers of gauze.

Preparation of C. sinensis antigen
Clonorchis sinensis were collected from rabbit liver and the excretory-secretory antigen (ES Ag) of C. sinensis obtained as described previously [20]. Clonorchis sinensis adults were cultured in RPMI 1640 medium supplemented with antibiotics at 37°C in the presence of 5% CO 2 . Culture medium was collected and centrifuged at 4°C and 1000× rpm for 30 min, and the supernatants were lyophilized at -20°C. Protein concentration was determined, and samples were stored at -70°C until use.
Construction of recombinant baculovirus (rBV) expressing C. sinensis tegumental protein (CsTP22.3) or influenza M1 Total RNA was extracted from C. sinensis adults using RNeasy Mini Kit (Qiagen, Valencia, USA) and complementary DNA (cDNA) was synthesized. The RNA was reverse transcribed to cDNA using a Prime Script 1st strand cDNA synthesis kit according to the manufacturer's instructions (Takara, Otsu, Japan). cDNA was used as a template to amplify the complete coding sequence of CsTP22.3 by polymerase chain reaction (PCR). The primers were designed according to the nucleotide sequence of CsTP22.3 in GenBank (accession number: EF077216.1): forward (5′-AAA GAA TTC ACC ATG TGC GCA CTT TCC TCA CA-3′) and reverse (5′-TTA CTC GAG TCA TAC CCA CGG TGT CTT CCA-3′) with EcoRI and XhoI restriction enzyme sites (underlined). PCR products were inserted into the pFast-Bac vector (Invitrogen, Carlsbad, USA). For influenza M1 gene cloning, all procedures were followed as described previously [21]. The recombinant plasmid was transformed into E. coli DH5-alpha and transferred into a DH10-Bac. All clones were confirmed by DNA sequencing and recombinant plasmid DNAs were stored at -20°C until used.

Generation of rBV and virus-like particles (VLPs)
Generation of recombinant baculovirus (rBV) was obtained using a Bac-to-Bac expression system (Invitrogen), according to the manufacturer's instructions. Briefly, DNA plasmids containing CsTP 22.3 or M1 genes were transfected into SF9 cells using cellfectin II (Invitrogen). VLPs were produced by co-infecting SF9 cells with rBV expressing CsTP22.3 and influenza matrix 1 (M1). To collect the supernatants, cell culture was centrifuged at 6000 rpm for 20 min at 4°C 2-3 days post-infection. The supernatant was pelleted using ultra-centrifugation. The pelleted VLPs were purified by 20-30-60% sucrose gradient at 30,000× rpm for 1 h at 4°C. The VLP bands between 30 and 60% were collected and pelleted at 28,000× rpm for 40 min at 4°C. To re-suspend the VLPs, they were incubated in PBS overnight at 4°C. Protein concentration was determined using BCA Assay Kit (Sigma-Aldrich, St. Louis, USA).

Characterization of VLPs
Western blots and electron microscopy were used to characterize the VLPs. For western blot analysis, rat serum that was infected with C. sinensis was used to probe CsTP 22.3 protein. For M1 protein detection, monoclonal mouse anti-M1 antibody was used. HRPconjugated goat anti-mouse IgG was used as secondary antibody. VLPs were negatively stained using phosphotungstic acid (pH 7.0) and transmission electron microscopy (JEOL 2100, JEOL USA, Inc.; Peabody, MA, USA) was performed at 200 kV to characterize the VLP morphology [22].

VLPs immunization, challenge infection and sample collection
Female Sprague-Dawley rats aged 8 weeks were divided into 3 groups: naïve control, C. sinensis infection control (Naïve + Cha) and VLP immunized plus challenge infected (Immu + Cha). Each group contained 12 rats. Rats were immunized intranasally twice with VLPs (200 μg/rat) at 4-week intervals. At 4 weeks after boost immunization, naïve or immunized rats were challenge infected with 50 C. sinensis metacercariae. Blood samples were collected by puncture of the retro-orbital plexus at 1 week and 4 weeks after prime, boost and challenge infection. Serum samples were collected and stored at -20°C. Feces and intestines were collected at 1 or 4 weeks after challenge infection. Individual rat feces, liver and small intestines were collected as described previously [20]. The collected intestine was incubated in 0.85% saline at 37°C for 1 h. The intestinal mucus was collected and centrifuged at 2000× rpm for 10 min. The supernatant was stored at -70°C until use. The experiment was repeated twice.

Antibody response by ELISA
Clonorchis sinensis-specific antibodies (IgG, IgG1, IgG2a, IgG2b and IgG2c) were determined in sera and C. sinensis-specific IgA antibody in feces, liver and intestines by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 4 μg/ ml of C. sinensis ES antigen at 4°C overnight. Clonorchis sinensis ES antigen was found to contain CsTP22.3 by SDS-PAGE (Additional file 1: Figure S1a). The plates were washed and then blocked with 0.2% gelatin in PBST for 2 h at 37°C. After washing, diluted sera (1:100), feces (1:50) or intestine (1:50) samples were added and incubated for 2 h at 37°C. Antibody responses were detected using the HRP-conjugated goat anti-rat secondary antibody (IgG, IgG1, IgG2a, IgG2b and IgG2c). The substrate O-phenylenediamine in citrate-phosphate buffer (pH 5.0), containing 0.03% H 2 O 2 , was used to develop color and stopped with 2N H 2 SO 4 . The optical density at 490 nm was measured with an ELISA reader.

Analysis of antibody-secreting cell response in vitro
Clonorchis sinensis-specific antibody-producing cells were determined in the spleen in vitro as described previously [20]. HRP-conjugated secondary goat anti-rat antibodies (IgG, IgG1, IgG2b, and IgG2c) were used and the substrate O-phenylenediamine (Zymed, San Francisco, USA) was used; the optical density was measured at 490 nm. The levels of parasite-specific antibodies secreted into the culture media and bound to the coated antigens were determined as previously described [21].

Flow cytometry analysis
To perform cell phenotype analysis, single cell suspensions from the spleen were isolated from homogenized tissues. For cell phenotype analysis, 1 × 10 6 splenocytes were stained with surface marker antibodies including CD3e-PE-Cy5, CD4-FITC, CD8a-PE (BD Biosciences, Franklin Lakes, USA). Stained cells were acquired on a BD FACS-Calibur and BD AcuriC6 and analyzed using Flow Jo software and BD AcuriC6 software.

Liver histopathology
Individual liver lobes harvested from rats at week 4 post-challenge were inflated and fixed with 10% neutral buffered formalin. The liver tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E, 200× magnification) to assess histological changes as described [23]. At least eight sections per rat were obtained for histopathology analysis.

Worm burden counts in the liver
Rats were challenged with 50 C. sinensis metacercariae and after 1 month flukes were recovered from the bile duct of the rats and counted as described previously [20,24]. The protection rate was calculated as described previously [25].

Statistics
All parameters were recorded for individuals within groups. Statistical comparisons of data were carried out using the Student's t-test and one-way ANOVA of PC-SAS 9.3. P-values < 0.05 were considered to be significant.

Constructs generation
Clonorchis sinensis CsTP 22.3 gene was amplified by PCR and the influenza M1 gene was amplified by RT-PCR with primers containing restriction enzyme sites (Additional file 2: Figure S2a

Production and characterization of VLPs
VLPs were produced by co-infected CsTP 22.3 with influenza M1 recombinant baculoviruses (Fig. 1a-c). The components of generated VLPs were confirmed by western blot (Fig. 1d). VLPs were reacted with rat sera that were infected with C. sinensis. Influenza M1, used as VLPs core protein, was detected by monoclonal mouse anti-M1 antibody. VLPs exhibited spherical shapes under microscopy and spikes were observed on the surface of the VLPs (Fig. 1e).

VLPs immunization induced significantly higher levels of mucosal IgG and IgA antibody responses
Rats were intranasally immunized with VLPs and challenge infected with C. sinensis metacercariae. To determine whether mucosal immunity can be induced, rat feces and intestines were collected before and after challenge infections. As shown in Fig. 4a, b, significantly higher levels of IgA antibodies were observed in feces, in which IgA antibody responses were higher after challenge infection (Fig. 4b, χ 2 = 8.0563, df = 2, P = 0.0178), compared to that before challenge infection ( Fig. 4a; significant differences: χ 2 = 8.1563, df = 2, P = 0.012,). Intestine IgA antibody responses also were much higher in immunized rats compared to unimmunized control after challenge infection ( Fig. 4c; significant differences: χ 2 = 7.2, df = 2, P = 0.0273). Liver IgG and IgA antibody responses were much higher, compared to those before challenge infections (Fig. 4d, e; significant differences: χ 2 = 6.1434, df = 2, P = 0.032; χ 2 = 6.5, df = 2, P = 0.04). These results indicate that intranasal immunization with VLPs in rats induced significantly higher levels of mucosal IgG and IgA antibody responses, which might also contribute to the protection in immunized rats.

VLPs immunization significantly reduced inflammation responses in the liver
Histopathological change upon C. sinensis infection was assessed to evaluate inflammation status in the liver. As seen in Fig. 7a, heavier periductal infiltration of inflammatory cells, such as plasma cells, lymphocytes and mononuclear cells, was found in unimmunized rats upon challenge infection [ Fig. 7a(a) compared to naïve rats ( Fig. 7a(b)]. Notably, VLPs immunized rats [ Fig. 7a(c)] showed less infiltration of inflammatory cells compared to unimmunized naïve rats upon challenge infection. Unimmunized naïve rats showed significantly enlarged liver duct upon challenge [ Fig. 7a(d) compared to naïve (Fig. 7a(e)) and immunized (Fig. 7a(f ))]. These results indicate that VLPs immunization significantly reduced liver inflammation following C. sinensis metacercariae challenge infection.
VLPs immunization significantly reduced liver C. sinensis worm loads following challenge infection Liver adult worm loads following infection are the most prominent indicator to assess vaccine protective efficacy. Immunized and naïve rats were infected with C. sinensis metacercariae (50/rat) at 4 weeks after boost, and liver worm burden was determined at week 4 post-infection. As shown in Fig. 7b (significant differences: t (44) = 4.46, P = 0.021), significantly decreased worm loads were detected in VLPs immunized rats compared to unimmunized rat control (70.2%). The result indicates that VLPs

Discussion
Clonorchis sinensis epidemics are one of the most common zoonoses and remain a major health concern as C. sinensis infection is closely related to cholangiocarcinoma (CCA), fibrosis and other human hepatobiliary diseases [6]. However, protective immunity against C. sinensis infection remains unknown. Study of vaccines against C. sinensis infection would have a significant impact. Virus-like particles vaccines have been successful in their safety and efficacy in virus fields in preclinical and clinical studies [16]. Commercial VLPs vaccines against hepatitis B virus (HBV) and human papilloma virus (HPV) have been escalated the interests in developing new VLP vaccines against respiratory viruses. Recently, the VLPs against parasite infections of Trichinella spiralis and Toxoplasma gondii showed encouraging results [26,27]. The current study investigated for the first time the protective efficacy of VLPs containing the C. sinensis tegumental protein 22.3 kDa (CsTP 22.3) and the influenza matrix protein (M1) as a core protein. We found that intranasal immunization of VLPs vaccine induced C. sinensis-specific IgG, IgG2a and IgG2c in the sera and IgA responses in feces and intestines. Vaccinated rats showed less inflammatory responses in the liver compared to unvaccinated controls. Notably, significantly lower adult worm loads (70.2%) were measured in the liver of rats immunized with VLPs, compared to those of naïve rats, providing partial protection against C. sinensis infection.
The tegument of C. sinensis is critically important in identifying potential antigens for diagnosis and vaccine candidates [14]. Recombinant C. sinensis tegumental protein 22.3 kDa has been shown to confer protection against C. sinensis, reducing worm burden by 44.7% upon challenge infection with C. sinensis metacercariae [14]. In the current study, we developed baculovirus expressing CsTP 22.3, and generated virus-like particles containing CsTP22.3. The VLPs we generated exhibited spherical shapes, and CsTP 22.3 protein exhibited as spikes on VLP surface under electron microscopy (Fig. 1e). Influenza M1 as a core protein in VLPs resembles virions in morphology and size. VLPs contain repetitive, high density displays of C. sinensis surface protein CsTP22.3 that seem to elicit strong T and B cell immune responses [26]. Bacillus subtilis spores displaying TP22.3 elicited TP22.3-specific IgA mucosal immunity, showing 44.7% of protection against Clonorchis sinensis [14]. However, in our current study, VLPs expressing CSTP22.3 protein provided systemic IgG and mucosal IgA antibodies, as well as T cell immune responses, resulting in higher protection. Thus, VLPs vaccination provided higher vaccine efficacy (70.2%) than the previous report.
Induction of mucosal immunity is critical in inducing protection against pathogens in the mucosal organ. Intranasal immunization with inactivated influenza virus (PR8) elicited mucosal IgA antibodies in mucosal organs. Significantly higher levels of virus-specific IgA Fig. 7 Inflammatory responses and worm burden in the liver. Liver tissues were collected from individual rats at week 4 after challenge, and tissue sections were stained with hematoxylin and eosin to assess liver inflammatory responses (200× magnification). Heavier periductal infiltration of inflammatory cells, such as plasma cells, lymphocytes and mononuclear cells was found in unimmunized rats upon challenge infection (b) compared to naïve rats (a) (a vs b). VLPs immunized rats showed less infiltration of inflammatory cells (c) compared to unimmunized rats upon challenge infection (b) (c vs b). Unimmunized (e), naïve (d) and immunized rats (f) showed different observed by naked eye. Clonorchis sinensis worm burden were determined from rats immunized with VLPs and controls (b). Liver tissues were collected from individual rats at week 4 after challenge. Rats immunized with VLPs showed significantly lower levels of worm burden compared to naïve control upon challenge infection (b, *P < 0.05) antibodies were detected in the lung, vagina and intestine (feces), compared to those before immunization [28]. The results indicate that intranasal immunization induced virus-specific IgA antibodies in respiratory tract at the site of vaccination route, as well as the reproductive organs and gastrointestinal tract. Since C. sinensis infection occurs through the mucosa of the gastrointestinal tract, we hypothesize that intranasal immunization route applied in current study will induce mucosal antibody response. As indicated in current study, VLPs vaccinated rats showed significantly higher levels of C. sinensis-specific IgA antibodies in feces and intestine, compared to unimmunized control rats (Fig. 4). Interestingly, after challenge infection, significantly higher levels of C. sinensis-specific IgG or/and IgA antibodies in feces and livers were rapidly expanded compared to those before challenge infection (Fig. 4a-e). Thus, enhanced mucosal IgG and IgA antibodies might be involved in the protection induced by VLP vaccination. Mucosal IgG and IgA antibodies in the bile ducts of liver are known to be associated with the resistance against C. sinensis in rats [20,29].
Rats have four subclasses known as IgG1, IgG2a, IgG2b and IgG2c; IgG1 and IgG2a are Th2-related, and IgG2b and IgG2c are Th1-related [30]. Our analysis of IgG isotypes IgG1, IgG2a, IgG2b and IgG2c antibodies in the sera and isotype antibody-secreting plasma cells in the spleen in VLPs vaccinated rats showed that significantly higher levels of IgG1 and IgG2c antibodies were observed, compared to IgG2a and IgG2b (Figs. 2,3,5) in the sera as well as in the spleen, indicating that VLPs vaccination induced Th1-and Th2-related isotypes.
To investigate other potential components contributing to protection, we determined CD4+ and CD8+ T cells in the spleen. We found that VLP vaccination induced higher levels of CD4+ and CD8+ T cell responses, which might also contribute to the protection against C. sinensis infection. These results are consistent with previous studies showing that significantly higher levels of CD4+ and CD8+ T cell responses induced by vaccination with T. gondii IMC VLPs may be involved in the protection against T. gondii infection [27].
In summary, our results demonstrate that C. sinensis VLPs containing CSTP 22.3 can induce systemic and mucosal IgG, IgA antibodies and CD4+ and CD8+ T cell responses, which can reduce worm burden in the bile duct. These results provide evidence that the VLP format is highly immunogenic and is a promising approach for developing effective prophylactic vaccines.