The human liver fluke, Opisthorchis viverrini
, resides in the bile ducts within the biliary tree, where it establishes chronic infection that drives long-term inflammation, hepatobiliary damage, and bile duct obstruction, leading to cholangiocarcinoma (CCA) [1
]. Humans become infected by ingesting the infective stage metacercariae from raw or undercooked cyprinoid fish dishes [2
]. We recently showed that subunit vaccines targeting tetraspanins (TSPs) on the surface of fluke-secreted extracellular vesicles (EVs) confer protection in a hamster challenge model of O. viverrini
infection, and that the antibodies against the TSPs present on the EV surface block the uptake of fluke EVs by the host bile duct epithelial cells [4
TSPs are a family of transmembrane proteins which interact with other transmembrane proteins to form a web that stabilizes membranes [7
]. Structurally, the tetraspanin protein includes two extracellular loops, a short extracellular loop (SEL) and a large extracellular loop (LEL), both of which decorate the extracellular surface of the cell membrane. TSPs are abundant on the surface of the tegument of the fluke, and EVs, derived from the tegumental surface, are actively internalized by adjacent cholangiocytes, the epithelial cells lining the bile duct [8
-TSP-2 shows promise as a vaccine candidate in that it confers protection to vaccinated hamsters against challenge infection with O. viverrini
metacercariae when administered in an adjuvanted form via the parenteral route [4
]. In addition, oral administration to hamsters of spores of recombinant Bacillus subtilis
, expressing Ov
-TSP-2-LEL on the surface of the bacilli, induced serum and bile IgG and IgA responses, and anti-Ov
-TSP-2 IgG blocked the uptake of O. viverrini
-EVs) in a human cholangiocyte cell line. Moreover, vaccination provided up to 56% reductions in both worm and egg burdens after challenge infection [10
To understand the role of antibodies against Ov-TSP-2 in conferring protection against O. viverrini infection, we raised two monoclonal antibodies (mAbs) to recombinant Ov-TSP-2-LEL, both of which significantly blocked the uptake of Ov-EVs by human cholangiocytes in vitro, and one of which, after passive transfer to hamsters, conferred highly significant protection against challenge infection with O. viverrini compared to a control mAb.
2. Materials and Methods
2.1. Ethics Statement
Vertebrate 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. Monoclonal antibody production was approved by the James Cook University Animal Care and Use Committee (A2629).
2.2. Preparation of Recombinant Ov-TSP-2-LEL Antigen
The recombinant protein corresponding to the large extracellular loop (LEL) of the Ov
-TSP-2 (GenBank accession JQ678707.1) was produced as a fusion protein with thioredoxin (TRX) using the plasmid pET32a+ (Novagen, Madison, WI, USA), which was expressed by Escherichia coli
strain BL21DE3. The recombinant fusion protein was purified with a Ni2+
affinity column as previously described [8
]. The purified protein was dialyzed into PBS before the treatment of mice.
2.3. Mouse Immunization
Five-week old male BALB/c mice were immunized a total of 5 times with 50 μL of recombinant Ov-TSP-2-LEL at a concentration of 1 mg/mL in an equal volume of Freund’s complete (first immunization) or incomplete (second, third, and fourth immunizations) adjuvants administered subcutaneously at two-week intervals. The fifth (final) immunization was undertaken with 50 μg of recombinant Ov-TSP-2-LEL without adjuvant. Serum samples were collected 2 days prior to the third, fourth, and fifth immunizations for the analysis of antibody titers. Mice were housed in the specified pathogen-free animal facility at James Cook University, Cairns, Queensland, Australia.
2.4. Hybridoma Generation
Hybridomas were generated by fusing SP2/0 myeloma cells and splenocytes from a vaccinated mouse (Figure 1
) 3 days after the final immunization. Briefly, the mouse with the highest antibody titer against recombinant Ov
-TSP-2-LEL was euthanized and the spleen was immediately removed aseptically and carefully disaggregated using a cell strainer to form a single-cell suspension, followed by 3 washes using a serum-free medium (SFM) (Thermo Fisher Scientific, Waltham, MA, USA). The cells were mixed at a ratio of 1:5 viable parental myeloma cells to each viable splenocyte, and carefully fused using 1 mL of polyethylene glycol (PEG) (Sigma-Aldrich, St. Louis, MO, USA) for 1 min. The fused cells were resuspended in SFM and incubated in a water bath at 37 °C for 15 min, and then centrifuged at 400× g
for 7 min at room temperature to eliminate the PEG. The supernatant was discarded and the fused cells were gently resuspended in 10 mL of 10% serum containing medium (SCM) (10% of fetal bovine serum in SFM), placed in a T-75 cm2
tissue culture flask (Sigma-Aldrich, St. Louis, MO, USA) containing 20 mL of 10% SCM (total culture volume is 30 mL), and incubated at 37 °C and 5% CO2
overnight. The fused cell suspension was removed from the flask, centrifuged, and directly transferred to a bottle containing 90 mL of ClonaCellTM
-HY Medium D (STEMCELL Technologies, Waterbeach, Cambridge, UK) and mixed thoroughly. The Medium D-containing fused cells were carefully transferred to 10 × 100 mm Petri dishes. The dishes containing fused cells were incubated at 37 °C and 5% CO2
for 14 days without disturbance. After 14 days, colonies detected on each Petri dish were transferred into an individual well of a 96-well tissue culture plate (Sigma-Aldrich, St. Louis, MO, USA) containing 200 µL of selection media (5% of fetal bovine serum in SFM with 1× hypoxanthine, aminopterin, and thymidine (HAT) (Sigma-Aldrich, St. Louis, MO, USA), and the plates were incubated at 37 °C and 5% CO2
for 3–4 days prior to preliminary screening by indirect ELISA.
2.5. Expansion and Production of Monoclonal Antibodies
Hybridomas in positive wells of 96-well tissue culture plates were expanded into 24 well-plates and then the supernatant was screened by indirect ELISA, using recombinant Ov
-TSP-2-LEL and anti-mouse antibodies against IgG (Sigma-Aldrich, St. Louis, MO, USA) and IgM (Sigma-Aldrich, St. Louis, MO, USA). We did not detect any IgG mAbs, however, clones that generated an IgM signal by ELISA were expanded to a T-25 cm2
tissue culture flask (Sigma-Aldrich, St. Louis, MO, USA). After several subcloning and screening rounds, the stable, confirmed positive hybridoma cell lines were transferred to T-75 cm2
tissue culture flasks (Sigma-Aldrich, St. Louis, MO, USA). For upscaling of hybridoma cells for mAb collection, we used HYPERFlask®
M cell culture vessels (High Yield PERformance Flask, Corning Inc., Corning, NY, USA) following the manufacturer’s instructions, with some modification [11
]. Briefly, cells in T-75 cm2
tissue culture flasks were counted using a hemocytometer. At least 1 × 107
cells in 560 mL of SFM with 1X HAT and 1X Nutridoma-SP (Sigma-Aldrich, St. Louis, MO, USA), were transferred to HYPERFlask®
M cell culture vessels (Corning Inc., Corning, NY, USA). After 14 days of incubation, the supernatants were separated from the cells by filtration using a 0.22 μm Stericup®
-GP quick release sterile vacuum filtration system (Sigma-Aldrich, St. Louis, MO, USA) and transferred to a new 1 L glass bottle prior to the purification of mAbs from the cell supernatant.
2.6. Purification of Monoclonal Antibodies
The cell supernatants containing IgM mAbs were purified using a HiTrap®
IgM Purification HP (GE Healthcare Bio-Sciences, Uppsala, Sweden) attached to an AKTATM
start chromatography system (GE Healthcare Bio-Sciences, Uppsala, Sweden) following the manufacturer’s instructions as previously described [12
]. Briefly, the cell supernatant was precipitated with saturated (NH4
to a final concentration of 0.8 M. The ammonium sulfate-containing supernatant was filtered through a 0.45 µm filter immediately before applying it to the column. Before applying the sample to the column, the column was washed with 5 mL of distilled water to remove ethanol, then the column was equilibrated with 5 mL of binding buffer (20 mM sodium phosphate and 0.8 M (NH4
with pH 7.5) at a flow rate of 1 mL/min. The sample was applied to the column at a flow rate of 1 mL/min. The unbound sample was washed out using 15 mL of binding buffer with a flow rate of 1 mL/min until the absorbance reached a steady baseline. IgM mAbs were eluted using 12 mL of elution buffer (20 mM sodium phosphate with pH 7.5). After elution, the column was regenerated and washed with 7 mL of wash buffer (20 mM sodium phosphate, pH 7.5 with 30% isopropanol) and re-equilibrated with 5 mL of binding buffer, prior to the subsequent purification, or washed with distilled water then maintained in ethanol for longer term storage.
2.7. Screening of Hybridoma Cells by Indirect ELISA
The supernatants of wells and flasks containing hybridomas and purified IgM mAbs were screened for mAb reactivity by indirect ELISA against the recombinant Ov
-TSP-2-LEL antigen used to immunize the mice, as described previously, with some modification [5
]. Briefly, 96-well microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with recombinant Ov
-TSP-2-LEL expressed in Escherichia coli
and purified using nickel-NTA chromatography [8
] at a final concentration of 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 PBST for 2 h at room temperature. After washing with PBST, 100 μL of each supernatant or purified IgM mAb (1 mg/mL) was added in duplicate, incubated for 1.5 h at room temperature and then washed with PBST. The plates were probed with 100 μL/well of anti-mouse IgG-HRP (BioRad; diluted 1:5000 in PBST) or anti-mouse IgM μ-chain specific-HRP (Sigma-Aldrich, St. Louis, MO, USA; diluted 1:5000 in PBST) and incubated for 1 h at room temperature. After washing, the plates were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) (Thermo Fisher Scientific, Waltham, MA, USA) and the reaction was stopped with 2 M of H2
. The colorimetric reaction was read at 450 nm on a SpectraMax microplate reader (Molecular Devices, San Jose, CA, USA). Pre-immunization mouse sera and the sera from mice that received the full course of immunization (100 μL, diluted 1:1000 in PBST) were used as negative and positive controls, respectively.
2.8. Preparation of O. viverrini Metacercariae
metacercariae were prepared as described in [4
]. Briefly, cyprinid fishes, from natural sources, were homogenized with an electric blender and then pepsin solution (0.25% pepsin powder, 15% HCl in normal saline solution (NSS)) was added at a ratio of 1:3 volume by volume, followed by incubation at 37 °C for 1 h in a water bath. The digested solution was filtered through 1000, 300, and 106 μm steel sieve meshes. The filtered content obtained by filtering with the 106 μm mesh sieve was washed and repeatedly sedimented with NSS until clear. Sediments were examined for metacercariae under a dissecting microscope. O. viverrini
metacercariae were collected and stored in sterile NSS at 4 °C until use.
2.9. Passive Immunization, Challenge, and Specimen Collection
Fifteen male golden Syrian hamsters (Mesocricetus auratus
) 6–8 weeks of age, reared at the animal facility of the Faculty of Medicine, Khon Kaen University, were randomly divided into three groups. Groups 1 and 2 received intraperitoneal administration of the anti-Ov
-TSP-2 mAbs designated 1D6 and 3F5. Group 3 was vaccinated with a commercially purchased mouse monoclonal IgM isotype control (Thermo Fisher Scientific, Waltham, MA, USA). The time course of passive immunization, fluke challenge infection, and specimen collection is shown in Figure 2
. The hamsters were intraperitoneally immunized with a single 200 μg dose of 1D6, 3F5, or control IgM in 500 μL of dH2
O one day before challenge (day −1). The following day (day 0), all immunized hamsters (n
= 5 per group) were challenged with 50 O. viverrini
metacercariae through orogastric administration. Eight weeks later, all hamsters were euthanized. At necropsy, their livers were collected for worm counts. The sera of hamsters were collected three times at pre-immunization (day −1), post-immunization/pre-challenge (day 0), and post-challenge at necropsy (week 8 post-infection).
2.10. Fecal Egg Counts and Worm Recovery
Hamster feces were collected at week 7, after the challenge with O. viverrini metacercariae, for egg counts. A modified formalin-ether acetate concentration technique (FECT) was used to determine the number of eggs per gram of feces (EPG). Whole livers were removed from hamsters at necropsy, dissected in NSS, and adult flukes were gently removed by squeezing the tissue to obtain the flukes from the bile ducts to determine adult worm burdens. To measure worm length, a total of 40, 30, and 20 worms from the control IgM group, 1D6, and 3F5, respectively (less worms were recovered from the two test groups), were randomly selected, washed three times with NSS and fixed in pre-warmed 10% formalin. The worms were photographed under microscopy and the worm length was measured using NIS Element software (Nikon, Japan).
2.11. Detection of Hamster Anti-Ov-TSP-2-LEL-Specific IgG, IgM, and IgA
-TSP-2-LEL-specific IgG, IgM, and IgA in sera were measured by ELISA, as described, with some modification, in [10
]. Briefly, 96-well microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 100 μL of recombinant Ov
-TSP-2-LEL expressed in E. coli
and purified using nickel-NTA chromatography [8
] (2 μg/mL) overnight at 4 °C in 0.05 M of Na2
with a pH of 9.6 (coating buffer). The plates were washed and blocked as described above. One hundred μL of sera (1:500 in PBST and 2% skim milk) was added and incubated for 1.5 h at room temperature. The plates were washed with PBST and probed with 100 μL of anti-hamster IgG-HRP (BioRad, Hercules, CA, USA; diluted 1:2000 in PBST), HRP-conjugated anti-IgM (μ-chain specific) (Sigma-Aldrich, St. Louis, MO, USA, diluted 1:2000 in PBST), and anti-IgA-HRP (Invitrogen, Carlsbad, CA, US; diluted 1:500 in PBST) was then added and incubated for 1 h at room temperature. After, the washing plates were developed using TMB. Pre-immunization hamster sera and the sera from hamsters that received the full course of oral immunization of recombinant Bacillus subtilis
spores expressing Ov
-TSP-2-LEL, as described previously [10
] (100 μL, diluted 1:1000 in PBST), served as negative and positive controls, respectively.
2.12. O. viverrini EV Internalization by Human Biliary Epithelial Cells
A total of 1.25 μg (5 × 107
vesicles) of O. viverrini
EVs were labeled with PKH67 (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer’s instructions as described previously [4
], and incubated with pooled pre-vaccination serum at a dilution of 1:2.5 and 1.25 μg of 1D6, 3F5, or the control IgM at a dilution of 1:1 for 1 h at room temperature with periodic mixing [9
]. After incubation, EV-antibody complexes were washed with PBS using Amicon Ultra 100 kDa cut-off purification columns (Merck Millipore, Burlington, MA, USA) and cultured with the H69 normal human biliary cell line [13
] at 37 °C with 5% CO2
for 2 h. The nuclei were stained with 2 μg/mL of Hoechst (Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature. Fluorescence images were captured by confocal microscopy (Carl Zeiss LSM800, Dublin, CA, USA) at 200× original magnification. Thirty cells from two biological replicates were analyzed for fluorescence intensity using ImageJ version 1.52a.
2.13. Statistical Analysis
Experimental values were expressed as mean ± standard deviation (SD). The data were analyzed using one-way analysis of variance (ANOVA) and two-way ANOVA using GraphPad Prism 9 software version 9.1.1 (GraphPad Software Inc., San Diego, CA, USA), and an unpaired t-test was employed to compare the two normally distributed groups. A p values of ≤ 0.05 was considered to be statistically significant.
Here, we showed that hamsters passively immunized with an anti-Ov
-TSP-2-LEL IgM mAb had reduced worm and egg burdens after challenge with O. viverrini
metacercariae. Circulating mAb was readily detected in the sera of vaccinated hamsters one day post-intraperitoneal immunization, and we hypothesize that transferred antibodies readily bound to parasite tegument and secreted EVs expressing Ov
-TSP-2 as the worms migrated through the tissues en route to the bile ducts. These findings indicate that Ov
-TSP-2-LEL is a promising vaccine antigen, which is consistent with findings in our earlier studies that showed the efficiency of this tetraspanin fragment as a component of a subunit vaccine in both parenteral and oral form [4
Both 1D6 and 3F5 mAbs almost completely blocked the in vitro uptake of O. viverrini EVs by cholangiocytes, but only 3F5 provided significant protection against challenge infection. Since we did not assess increasing quantities of the mAbs by passive transfer followed by challenge infection, we cannot exclude that 1D6 may confer protection at a higher concentration than that tested here. It is also unclear whether the two mAbs bind to different epitopes. We did assess the binding of the mAbs by ELISA to overlapping 13-mer synthesized peptides corresponding to Ov-TSP-2 LEL, but the data were unconvincing, possibly due to the reduced specificity of antigen binding displayed by IgM antibodies (not shown).
At least 570 therapeutic mAbs were studied in clinical trials [14
], and 79 therapeutic mAbs were approved by the United States Food and Drug Administration and are currently on the market, including 30 mAbs for the treatment of cancer [15
]. However, whereas therapeutic mAbs against bacterial and viral infections were approved, there are no antibody-based therapies in use for parasitic infections [16
There are five antibody isotypes in mice and humans: IgG, IgM, IgA, IgE, and IgD. IgM antibodies are either pentameric or hexameric, found in blood, and function similarly to IgG in defending against antigens. IgM is the main antibody produced in an initial attack by a specific bacterial or viral antigen, while IgG is usually produced in later infections caused by the same agent. IgM antibodies were shown to inhibit infectivity of organisms by causing aggregation or agglutination of the pathogen or infected cell, leading to its clearance from the body [18
]. IgM plays an essential role in protective immune responses against numerous parasitic helminths. For example, in a mouse model of human strongyloidiasis, immunization with infective larvae induced protective immunity that could be passively transferred to naïve mice [19
]. Parasite-specific IgG1, IgM, and IgA titers were elevated in immunized mice, but IgM was the only isotype capable of inducing parasite elimination, and this was dependent on cell contact, the presence of granulocytes, and complement activation. Moreover, IgM from immunized mice passively transferred immunity to naïve IL-5 knockout mice, which are deficient in eosinophils [20
], suggesting that neutrophils were the required granulocyte for IgM-dependent killing [21
]. Moreover, an IgM mAb that targeted multiple antigens from the filarial nematode Brugia malayi
, conferred 89% protection when passively transferred to jirds that were challenged with infective larvae, and was thought to function in an antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent manner [22
While IgM mAbs that confer passive protection against liver flukes have not (to the best of our knowledge) been described, they were for other platyhelminth infections, primarily schistosomiasis. Two IgM mAbs against Schistosoma mansoni
28 kDa glutathione S-transferase (GST), one of which had enzyme neutralizing properties, conferred protection against challenge infection when transferred to rats [23
]. Eight mAbs were raised to surface antigens of Schistosoma japonicum
, only one of which was an IgM that conferred protection via passive transfer studies in mice [24
]. An IgG mAb was raised to 78 kDa ES protein-protected mice against challenge infection with the livestock liver fluke Fasciola hepatica
], but to the best of our knowledge, mAbs raised against human liver fluke antigens have only been used to develop diagnostic tools [26
] and were not shown to confer protective efficacy in passive transfer studies.
We found that both serum IgG and IgM levels increased post-challenge in hamsters that received both 3F5 and 1D6 as well as control IgM, indicating that these responses were a result of parasite challenge and not passive transfer of mAb. Due to the fact we passively immunized hamsters with anti-Ov-TSP-2-LEL IgM mAbs, antigen-specific IgG responses were not detectable pre-challenge and were only observed post-challenge as a result of infection. However, as expected, we detected increased antigen-specific IgM levels in serum pre-challenge from hamsters that received 3F5 and 1D6, but not the control IgM. The Ov-TSP-2-specific IgM levels increased again to comparable levels in all three groups post-challenge (including the control IgM group), implying that challenge infection promoted a strong anti-TSP-2 IgM response in all groups.
Both 3F5 and 1D6 mAbs almost completely blocked the uptake of fluke EVs by cholangiocytes in vitro, whereas control IgM had no effect, providing a plausible mechanism by which the vaccination strategy exerted its effect. Others have shown that anti-O. viverrini
IgG, IgM, and IgA were found at significantly higher levels in stools of egg-negative, compared to egg positive, residents in O viverrini
-endemic areas, suggesting that all antibody isotypes contribute to naturally occurring protective responses against opisthorchiasis [30