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. 2020 Aug 19;223(8):1445–1455. doi: 10.1093/infdis/jiaa516

Orally Administered Bacillus Spores Expressing an Extracellular Vesicle-Derived Tetraspanin Protect Hamsters Against Challenge Infection With Carcinogenic Human Liver Fluke

Wuttipong Phumrattanaprapin 1, Sujittra Chaiyadet 2, Paul J Brindley 3, Mark Pearson 4, Michael J Smout 4, Alex Loukas 4,#,✉, Thewarach Laha 1,#
PMCID: PMC8064041  PMID: 32813017

Abstract

Background

The human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer.

Methods

We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae.

Results

Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group.

Conclusions

These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans.

Keywords: oral vaccine, Bacillus subtilis, spore, Opisthorchis viverrini, tetraspanin, IgA

Infection with the liver fluke Opisthorchis viverrini is a risk factor for bile duct cancer in Southeast Asia. We describe an oral vaccine that interrupts host-parasite crosstalk via extracellular vesicles and induces partial protection in an animal model of opisthorchiasis.

The carcinogenic liver fluke, Opisthorchis viverrini, infects approximately 10 million people in Southeast Asia (Thailand, Lao People's Democratic Republic, Cambodia, Myanmar, and Southern Vietnam) due to the consumption of raw or undercooked cyprinoid fish containing the infective metacercariae [1]. Infection with O. viverrini is recognized by the World Health Organization as group 1 carcinogen [2]. The mechanisms by which O. viverrini drives cholangiocarcinogenesis are multifactorial and include chronic inflammation of the bile duct epithelium, hepatobiliary damage, and bile duct obstruction caused by resident flukes [3, 4], and secretion by the flukes of excretory-secretory molecules and vesicles that are known to stimulate cholangiocyte proliferation, antiapoptosis, and DNA damage [3, 5–7]. The infection rate is persistently high because the tradition of raw/fermented fish consumption persists despite health education campaigns [8].

An alternative to mass drug administration for the prevention of opisthorchiasis and associated hepatobiliary pathologies is the development of a vaccine. Of the subunit vaccines that show promise for human fluke infections in animal models, one of the most promising antigen targets is the tetraspanins (TSPs). TSPs are transmembrane proteins found in many organisms [9, 10] and are present in abundance in the tegument of platyhelminths [11, 12], including O. viverrini [6, 12–16]. In O. viverrini, TSPs have known roles in tegument biogenesis and maintenance of cell membrane integrity [13]. Moreover, TSPs are abundant in the membrane of extracellular vesicles (EVs) that are secreted by O. viverrini and internalized by surrounding host cholangiocytes [6, 13]. The large extracellular loop (LEL) of O. viverrini recombinant TSPs has been expressed as recombinant vaccines and displayed partial efficacy in a hamster model of opisthorchiasis when delivered parenterally [17–19].

Oral vaccination offers distinct advantages over parenteral administration due to its ability to induce both systemic and mucosal immune responses, as well as the ease of administration, which does not require sterile needles and syringes or well-trained personnel [20]. An oral vaccine might induce a protective humoral immune response at the site of final residence (biliary tree) and could limit infection intensity and associated pathology. Several oral vaccine formulations have been described for helminth infections, including the use of recombinant Bacillus subtilis spores that are resistant to the harsh environment of the gastrointestinal tract (GIT) [21, 22]. In the context of vaccines, B. subtilis spores have been adopted as a vehicle to carry recombinant antigen in immunization regimens due to their resistance to low pH and noxious chemicals; in addition, they can be stored for long periods at room temperature [23]. Spore coat proteins are produced as fusions with recombinant antigens presented on the spore surface. B. subtilis spores are surrounded by a coat with 2 major layers, the inner and the outer coat [24–26]. Preferred fusion partners are outer-coat proteins such as CotB [27–29], CotG [29–32], and CotC [29, 33, 34]. Indeed, CotC has been used as a fusion partner for the expression of antigens from the related liver fluke, Clonorchis sinensis, where they induced partial protection in a rat challenge model [33, 35]. The ability to protect O. viverrini vaccine antigens against the hostile environment of the GIT and to improve their delivery across the bile duct mucosa is a valid strategy to achieve sustainable control of opisthorchiasis.

Herein, we generated recombinant B. subtilis spores expressing the LEL of Ov-TSP-2 (Ov-TSP-2-LEL) fused to CotC and orally immunized hamsters followed by challenge infection with O. viverrini metacercariae. Hamsters generated serum and bile immunoglobulin G (IgG) and immunoglobulin A (IgA) responses, and serum antibodies blocked the in vitro uptake of O. viverrini EVs by human cholangiocytes, the cells that line the bile duct epithelium. Moreover, fluke and fecal egg numbers recovered from vaccinated hamsters were significantly reduced, and surviving flukes were stunted compared to flukes recovered from control hamsters that were vaccinated with the CotC protein alone.

METHODS

Preparation and Optimization of Recombinant B. subtilis WB800N Spores Expressing Ov-TSP-2-LEL

The CotC-Ov-TSP-2 fusion protein was prepared by transforming B. subtilis WB800N as previously described by us [18]. Briefly, the sequence of the complete coding sequence of CotC from B. subtilis (GenBank accession number X05680.1) was fused to the cDNA sequence of Ov-TSP-2-LEL (GenBank accession number JQ678707.1), resulting in formation of CotC-Ov-TSP-2-LEL. The fused cDNA was inserted into the pHT01 plasmid WB800N (Figure 1A). The pHT01-CotC-Ov-TSP-2-LEL and pHT01-CotC recombinant plasmids were verified by gene sequencing prior to transformation of B. subtilis (MoBiTec) following the manufacturer’s instructions [36] with some modification. The transformants were induced to sporulate in TM media as described [35, 37]. In brief, a fresh Luria-Bertani agar culture of transformed B. subtilis WB800N was washed with normal saline solution (NSS) onto the surface of a Roux bottle (Sigma-Aldrich) containing 250 mL of sporulation medium containing 5 μg/mL chloramphenicol and incubated at 35ºC for 5 days with shaking. The sporulation culture was centrifuged at 10 000g for 10 minutes, resuspended in 50 mL of NSS, and centrifuged as before. The pellet of recombinant spores was purified using 4 mg/mL lysozyme (to break residual sporangial cells) and washed sequentially with 1 M NaCl, 1 M HCl, and NSS (twice), and then phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM to inhibit proteolysis, followed by a final incubation at 65ºC for 1 hour. After purification, protein expression on recombinant spores was visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using anti-Ov-TSP-2 rabbit serum as described [13]. Expression of recombinant protein on the spore surface was also assessed using immunofluorescence microscopy. Slides were incubated with purified IgG from anti-Ov-TSP-2 rabbit serum [13] (2 mg/mL) overnight at 4ºC. The corresponding control samples were incubated with phosphate-buffered saline (PBS). Slides then were treated with Alexa Fluor 488-labeled goat anti-rabbit IgG (Invitrogen) (1:400 in PBS) as previously described [18]. Purified recombinant spore numbers were calculated by direct counting in a Burker chamber under an optical microscope (40× magnification) and aliquoted as 5 × 109 spores per 0.5 mL NSS (equal to 1 dose) and stored at −20°C until use for immunization. The predicted topology of recombinant spores expressing Ov-TSP-2-LEL on the surface is presented in Figure 1B.

Figure 1.

Figure 1.

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A, Construction of recombinant plasmid pHT01-CotC-Ov-TSP-2-LEL. B, Schematic representation of a recombinant Bacillus subtilis spore expressing the Ov-TSP-2-LEL-CotC fusion protein on its surface. Abbreviations: CotC, coat protein C; LEL, large extracellular loop; Ov, Opisthorchis viverrini; TSP, tetraspanin.

Preparation of O. viverrini Metacercariae

O. viverrini metacercariae were obtained and prepared from cyprinid fishes as described [17] and stored in sterile NSS at 4°C until use.

Vaccination, Challenge, and Specimen Collection

Animal protocols were approved by the Animal Ethics Committee of Khon Kaen University (approval number ACUC-KKU-121/62) according to the Ethics of Animal Experimentation of the National Research Council of Thailand. Fifteen male golden Syrian hamsters (Mesocricetus auratus) reared at the animal facility of the Faculty of Medicine, Khon Kaen University, were randomly divided into 3 equal groups designated CotC-Ov-TSP-2-LEL, CotC (vehicle control group), and NSS (negative control group). The time course of recombinant spore immunization, fluke challenge infection, and specimen collection is shown in Figure 2. Hamsters were immunized with a total of 9 doses of recombinant spores at week 0 (day 1, 2, and 3), week 2 (day 15, 16, and 17), and week 4 (day 29, 30, and 31). Each dose of CotC or CotC-Ov-TSP-2-LEL was orally administrated with 5 × 109 spores in 500 µL NSS, and the NSS group was orally administered with 500 µL of NSS alone. Spore dose was guided by similar studies where hamsters were vaccinated and challenged with the related liver fluke C. sinensis [33, 35]. All hamster immunizations were conducted using an orogastric feeding tube. Two weeks after the final immunization (week 6), all orally immunized hamsters (n = 5 per group) were challenged with 50 living O. viverrini metacercariae through orogastric administration.

Figure 2.

Figure 2.

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Schematic representation of the hamster vaccination and challenge regimen.

Fecal Egg Counts and Worm Recovery

Feces were collected 1 week before euthanasia at week 13. A modified formalin-ether acetate concentration technique method was used to determine the number of eggs per gram feces.

Livers containing O. viverrini adult flukes were removed from hamsters at necropsy, dissected in NSS, and adult flukes gently removed by squeezing the tissue to push flukes out of the bile ducts. Bile was collected from the gallbladder using a 1-mL syringe. Bile was used to determine anti-Ov-TSP-2-LEL–specific IgG and IgA antibody responses. Livers were dissected in NSS to determine adult worm burdens. To measure worm length, 7–8 worms from each individual hamster were randomly selected, washed with NSS, and fixed in prewarmed 10% formalin. Worms were photographed under microscopy and measured using NIS-Element software (Nikon).

Detection of Hamster Anti-Ov-TSP-2-LEL–Specific IgG and IgA

Detection of hamster anti-Ov-TSP-2-LEL IgG in sera and bile by enzyme-linked immunosorbent assay (ELISA) was carried out as described previously [18]. In brief, 96-well microtiter plates (Thermo Fisher Scientific) were coated with recombinant Ov-TSP-2-LEL expressed in Escherichia coli and purified using nickel-nitrilotriacetic acid (NTA) chromatography [13] (2 μg/mL) overnight at 4°C. Plates were washed 3 times with PBS 0.05% Tween-20 (PBST) and then blocked with 200 μL of 5% skim milk in coating buffer for 2 hours at 37°C. Sera (100 μL, 1:500 in PBST/2% skim milk) or bile (1:50 in PBST/2% skim milk) were added and incubated for 1.5 hours at 37°C. The plates were washed with PBST and probed with 100 μL of anti-hamster IgG-horseradish peroxidase (HRP; BioRad; diluted 1:1000 in PBST for both serum and bile) and anti-mouse IgA-HRP (Invitrogen; diluted 1:500 in PBST for bile). After washing, the plates were developed with 3,3',5,5'-tetramethylbenzidine (TMB; Thermo Fisher Scientific) and the reaction was stopped with 2 M H2SO4. The colorimetric reaction was read at 450 nm on a Spectra Max microplate reader (Molecular Devices). We also used western blot to show recognition of purified recombinant Ov-TSP-2 fused to E. coli thioredoxin [13] using sera from immunized hamsters. For western blotting, 2 μg of recombinant LEL-Ov-TSP-2 [13] was subjected to 15% SDS-PAGE and transblotted onto a nitrocellulose membrane (Mini Trans-Blot Cell; Bio-Rad) that was washed with PBST and treated with 5% skim milk in PBST for 2 hours at ambient temperature. Membrane was incubated with immunized hamster serum (1:100 in 1% skim milk in PBST) overnight at 4°C, followed by incubation with goat anti-hamster IgG-HRP (Thermo Fisher Scientific) diluted 1:1000 in 1% skim milk in PBST at ambient temperature for 2 hours. Immunoreactive protein was visualized using the enhanced chemiluminescence method (Luminata Forte Western HRP substrate; Merck Millipore).

O. viverrini EV Internalization by Human Biliary Epithelial Cells

O. viverrini EVs (1.25 μg, 5 × 107 vesicles) were labeled with PKH67 (Sigma-Aldrich) following the manufacturer’s instructions as described previously [17] and incubated with pooled hamster preimmunization serum or sera from hamsters vaccinated with spores expressing Ov-TSP-2-LEL or CotC at a dilution of 1:2.5 for 1 hour at room temperature as described elsewhere [6]. O. viverrini EV-antibody complexes were washed with PBS using Amicon Ultra 100-kDa cutoff purification columns (Merk Millipore) and cultured with the H69 normal human biliary cell line at 37°C with 5% CO2 for 2 hours. Nuclei were stained with 2 μg/mL of Hoechst (Invitrogen) for 15 minutes. Fluorescence images were captured using a Carl Zeiss confocal microscope (LSM800) at 200× original magnification. Thirty cells from 2 biological replicates were analyzed for fluorescence intensity using imageJ version 1.52a.

Statistical Analysis

Experimental values are expressed as mean ± standard deviation (SD). Data were analyzed using 1-way analysis of variance (ANOVA) and 2-way ANOVA using GraphPad Prism software version 8.3.0 (www.graphpad.com). P values ≤ .05 were considered as statistically significant.

RESULTS

Oral Vaccination of Hamsters Induces Serum and Bile Antibody Responses Against Ov-TSP-2-LEL

Sera were collected 3 separate times at preimmunization, postimmunization (1 day before challenge) and at termination (week 14). Hamsters immunized with recombinant spores expressing Ov-TSP-2-LEL had significantly higher serum IgG levels against Ov-TSP-2-LEL postimmunization and postchallenge compared to preimmunization (P < .01). In addition, at prechallenge, anti-Ov-TSP-2-LEL serum IgG levels of hamsters immunized with CotC-Ov-TSP-2-LEL were significantly higher than levels of hamsters immunized with CotC alone or NSS hamsters (P < .01). On the other hand, serum IgG levels against Ov-TSP-2-LEL in hamsters orally immunized with CotC and NSS hamsters were not significantly different between preimmunization and prechallenge serum samples. The total serum IgG levels against Ov-TSP-2-LEL in NSS, CotC, and CotC-Ov-TSP-2-LEL groups were significantly higher postchallenge compared to preimmunization and prechallenge levels (P < .05, P < .001, and P < .01, respectively; Figure 3A).

Figure 3.

Figure 3.

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Serum IgG, bile IgG, and bile IgA levels in immunized hamsters determined by ELISA and western blotting. A, Serum IgG against recombinant Ov-TSP-2-LEL expressed in Escherichia coli and purified using nickel-NTA chromatography was measured from hamsters that were orally immunized with Bacillus subtilis spores expressing Ov-TSP-2-LEL, control spores (CotC), and negative control group (NSS) at preimmunization, prechallenge, and postchallenge. Bile IgG (B) and bile IgA (C) levels were measured in hamsters that were orally immunized with B. subtilis spores expressing Ov-TSP-2-LEL, control spores (CotC), and negative control group (NSS) postchallenge. Results represent the mean absorbance measured at 450 nm for each group. D, Western blot of recombinant purified Ov-TSP-2-LEL fused to E. coli thioredoxin separated by 15% SDS-PAGE, transferred to PVDF membrane and probed with hamster postvaccination sera followed by HRP-conjugated goat anti-hamster IgG and visualized with enhanced chemiluminescence. Lane 1, protein molecular mass standards; lane 2, serum from a hamster immunized with spores transformed with pHT01-CotC; lane 3, serum from a control hamster immunized with NSS; lanes 4–7, immune sera from 4 randomly selected hamsters immunized with spores transformed with pHT01-CotC-Ov-TSP-2-LEL. Error bars denote standard deviation of the mean. Abbreviations: CotC, coat protein C; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IgA, immunoglobulin A; IgG, immunoglobulin G; LEL, large extracellular loop; NSS, normal saline solution; NTA, nitrilotriacetic acid; OD, optical density; Ov, Opisthorchis viverrini; PVDF, polyvinylidene fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TSP, tetraspanin.

Anti-Ov-TSP-2-LEL IgG levels in the bile of hamsters immunized with recombinant spores expressing Ov-TSP-2-LEL were significantly higher (OD450, 0.36 ± 0.18) than those of the NSS control group (OD450, 0.11 ± 0.03; P < .05) but there was no significant difference detected when compared to hamsters immunized with CotC-expressing spores (OD450, 0.22 ± 0.14; Figure 3B). Anti-Ov-TSP-2-LEL IgA levels in the bile of hamsters immunized with recombinant spores expressing Ov-TSP-2-LEL were significantly higher than the levels in hamsters immunized with CotC and NSS hamsters (P < .0001; Figure 3C). IgA levels in serum in response to vaccination were negligible and not significantly different between vaccinated and control groups (not shown).

We confirmed specific immunoreactivity by western blot of a 29-kDa purified recombinant fusion protein consisting of Ov-TSP-2-LEL fused to thioredoxin and purified using nickel-NTA chromatography [13] using IgG antibodies from hamsters vaccinated with CotC-Ov-TSP-2-LEL. Antisera raised to CotC-expressing spores and NSS sera did not recognize the recombinant TSP-2 fusion protein (Figure 3D).

Oral Vaccination of Hamsters With Spores Expressing Ov-TSP-2-LEL Induces Partial Protection Against O. viverrini Challenge Infection

The worm burden of hamsters orally immunized with recombinant spores expressing Ov-TSP-2-LEL was significantly lower (15.2 ± 3.7) at necropsy than those from hamsters that were orally immunized with CotC-expressing spores (34.6 ± 4.98) and the NSS group (37.4 ± 3.91) (P < .0001). No significant differences were observed between the CotC and NSS control groups (Figure 4A).

Figure 4.

Figure 4.

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Vaccinated hamsters harbored significantly fewer worms and passed significantly fewer fecal eggs than the control animals. Adult worms were collected from whole livers of vaccinated hamsters and dissected in NSS to allow removal of Opisthorchis viverrini adult flukes from the bile ducts. A, Worm burdens in hamsters orally immunized with Bacillus subtilis spores expressing Ov-TSP-2-LEL, control spores (CotC), and negative control group (NSS). B, Number of eggs per gram of feces, mean ± SD, from each vaccinated hamster are shown. Abbreviations: CotC, coat protein C; LEL, large extracellular loop; NSS, normal saline solution; Ov, Opisthorchis viverrini; TSP, tetraspanin.

Eggs per gram feces of hamsters immunized with recombinant spores containing CotC-Ov-TSP-2-LEL were significantly lower than those of hamsters that were orally immunized with CotC-expressing spores and the NSS group (P < .05) at week 13. No significant differences were observed between the CotC and NSS control groups (Figure 4B).

Worms Recovered From Hamsters Vaccinated With Ov-TSP-2-LEL Are Stunted

The body length of recovered worms from hamsters immunized with recombinant spores expressing Ov-TSP-2-LEL was significantly shorter than worms recovered from the CotC group and NSS groups (P < .001 and P < .01, respectively; Figure 5A). The average length of examined worms from hamsters orally immunized with recombinant spores expressing Ov-TSP-2-LEL was 3.6 ± 0.49 mm, while the average length of worms from CotC group and NSS group was 4.25 ± 0.61 and 4.22 ± 0.8 mm, respectively. A representation of the morphology of the worms from each group is shown in Figure 5B.

Figure 5.

Figure 5.

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Liver flukes recovered from hamsters that were orally immunized with CotC-Ov-TSP-2-LEL were significantly stunted in length. A, Body length of 7–8 worms from each hamster from the NSS and CotC, and 5–6 worms from the CotC-Ov-TSP-2-LEL groups were randomly selected and their lengths measured using the NIS-element program. B, Representative photomicrographs of the worms recovered from each group of hamsters. Scale bar denotes 500 µm. Abbreviations: CotC, coat protein C; LEL, large extracellular loop; NSS, normal saline solution; Ov, Opisthorchis viverrini; TSP, tetraspanin. Error bars denote standard deviation of the mean.

Antibodies From Vaccinated Hamsters Block Uptake of O. viverrini EVs Into Human Cholangiocytes

PKH67-labelled O. viverrini EVs were incubated with pooled sera of hamsters orally immunized with recombinant spores expressing Ov-TSP-2-LEL, recombinant spores expressing CotC, and from the NSS control group at a dilution of 1:2.5 before being cultured with H69 cholangiocytes. Uptake of EVs was significantly reduced by sera from hamsters vaccinated with CotC-Ov-TSP-2-LEL compared to sera from CotC-immunized hamsters and NSS hamsters by 80% and 79%, respectively (Figure 6).

Figure 6.

Figure 6.

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Sera from hamsters orally immunized with Bacillus subtilis spores expressing Ov-TSP-2-LEL block Opisthorchis viverrini EV internalization by host cholangiocytes. PKH67-labeled O. viverrini EVs were incubated with hamster pooled prevaccination serum (A), anti-CotC serum (B), and anti-CotC-Ov-TSP-2-LEL serum (C) before coculture with H69 cholangiocytes for 2 hours. D, Fluorescence intensity (CTCF) at 490 nm (green channel) was quantified using ImageJ software. The nuclei were stained blue using Hoechst dye no. 33258. Error bars denote standard deviation of the mean. Abbreviations: CotC, coat protein C; CTCF, corrected total cell fluorescence; EV, extracellular vesicle; LEL, large extracellular loop; Ov, Opisthorchis viverrini; TSP, tetraspanin.

DISCUSSION

In the present study we have shown that oral immunization of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL reduced both worm and egg burden, and that IgG and IgA antibody levels specific to Ov-TSP-2-LEL were significantly raised in serum and in bile of vaccinated hamsters. The development of the adult parasites was significantly hampered in hamsters vaccinated with recombinant B. subtilis spores expressing Ov-TSP-2-LEL, as demonstrated by stunted parasite growth in immunized hamsters.

Successful oral vaccination relies on antigen efficiently penetrating the selectively permeable mucus layer of the intestine and its recognition by antigen presenting cells and subsequent delivery to the Peyer’s patches, where lymphocytes produce effector actions including antibody production, cytokine secretion, and cytotoxicity [38]. IgA plays a potential role as a frontline defense mechanism against liver fluke infection, and our findings show that oral immunization of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL resulted in an IgA response that was readily detected in bile, the fluid surrounding adult flukes in the biliary tract. Moreover, oral vaccination induced a robust IgG response in both serum and bile, notably postchallenge. Oral vaccination of rats with C. sinensis TP22.3 tegumental protein expressed on B. subtilis spores generated a mucosal IgA response that was detected in fecal supernatant and hypothesized to react with the juvenile worm’s tegument and hamper development [33]. Similarly, our results showed a significant increase in Ov-TSP-2-specific bile IgA in vaccinated hamsters compared to control animals.

Although we cannot confirm whether IgG or IgA (or both isotypes) was the most important isotype for generating protective responses, serum from hamsters that were orally immunized with recombinant B. subtilis spores expressing Ov-TSP-2-LEL blocked the uptake of EVs by cholangiocytes in vitro, providing a plausible mechanism by which the vaccination strategy exerted its effect.

TSPs have been regarded as candidate vaccine antigens for parenteral administration in schistosomiasis using animal challenge models [39–42] and in phase 1 human clinical trials [43, 44]. Ov-TSP-2 is expressed on the outermost tegument of adult O. viverrini flukes and is enriched in O. viverrini secreted EVs [6, 13, 16]. Moreover, this molecule showed promise when parenterally delivered to hamsters in recombinant and adjuvanted form followed by parasite challenge. It is noteworthy, however, that after parenteral administration, Ov-TSP-2 induced a 34% reduction in worm burdens (P < .01) and 41% egg reduction when compared with the adjuvant control group [17]. Our findings herein show improved efficacy with the orally administered vaccine (56% reduction in both worm and egg burdens, P < .0001) compared to the CotC-spore group.

To protect protein antigens during transit in the intestinal tract, several chemical methods have been used such as formation of nanoparticles with polymer coats and liposome-based formulations [38]. Liposome formulations have been tested as vaccines against numerous parasitic infections. For example, peptides derived from a digestive protease of the gastrointestinal hookworm, Nippostrongylus brasiliensis, were produced in different formulations, including liposomes and self-adjuvanting lipopeptides, and orally administered to mice prior to parasite challenge. Vaccination resulted in very high levels of protection and this approach is now under consideration for development of a human hookworm vaccine [45].

Biological delivery mechanisms such as B. subtilis spores have attracted a lot of attention for vaccine development but have been largely overlooked in helminth infections where most effort has been focused on parenteral delivery. B. subtilis has multiple layers of coat proteins that are resistant to the harsh environment of the GIT [21, 22] and several studies have successfully displayed protection with oral delivery of spore coat-fusion proteins in animal models. One helminth species that has been the focus of a vaccine delivery platform based on Bacillus spores is the liver fluke Clonorchis sinensis, where the approach has enjoyed mixed success [33, 35, 46, 47]. For example, rats that received oral vaccination with C. sinensis TP22.3 spores had significantly reduced worm (44.7%; P < .05) and fecal egg burdens (30.4%; P < .05) compared to controls [33]. Recombinant enolase from C. sinensis has also been tested as a vaccine using B. subtilis spores, and conferred worm and egg reduction rates of 60.07% (P < .001) and 80.67% (P < .001), respectively [35]. B. subtilis-based oral vaccines have also been tested in fish. Grass carp (Ctenopharyngodon idella), an intermediate host of C. sinensis, were orally immunized with a fluke cysteine protease expressed on B. subtilis spores by feeding fish a basal pellet food containing the spores. Vaccinated fish developed an IgG antibody response in serum, bile, and mucus (surface and intestinal) [48], but antihelminth efficacy was not reported in this study.

In summary, we show that oral immunization of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL strongly stimulates the production of IgA antibody in bile and IgG antibody in both serum and bile that confers protection against fluke challenge infection in hamsters. Moreover, antibodies from hamsters immunized with Ov-TSP-2-LEL significantly blocked internalization of O. viverrini EVs by biliary epithelial cells. Our results show for the first time that an oral vaccine against this devastating human pathogen induces a protective immune response against challenge infection in a clinically relevant animal model. However, the efficacy of this oral vaccine requires further optimization in terms of antigen composition (monovalent vs multivalent), spore number per dose, and frequency of dosing. Indeed, inclusion of multiple antigens that are selected in a rational fashion and target distinct parasitism pathways is likely to improve efficacy. Future work should address the relative roles of IgG and IgA in protection at mucosal surfaces and whether antibody blockade of EV cellular uptake is critical in driving protection in vivo. Our findings imply that Ov-TSP-2-LEL might be considered as an orally administered vaccine candidate against opisthorchiasis that potentially induces protection via antibodies that bind to and block subsequent uptake of EVs by host cells, thereby interfering with host-parasite intercellular communication [17, 19].

Notes

Acknowledgments. We thank the laboratory staff of the Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia and the Department of Parasitology, Faculty of Medicine, Khon Kaen University for technical support.

Financial support. This work was supported by the Thailand Research Fund through the Royal Golden Jubilee PhD Program (grant number PHD/0163/2561 to W. P. and T. L.); the National Cancer Institute, National Institutes of Health (grant number 2R01CA164719-06A1 to T. L., A. L., and P. J. B.). A. L. is supported by a senior principal research fellowship (grant number 1117504) and program grant (grant number 1132975) from the National Health and Medical Research Council, Australia.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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