Design of the multi-epitope bivalent vaccine LTB-ETBM
Based on our previous study, the dominant epitopes of each protein were identified by performing specific ELISA, lymphocyte proliferation, flow cytometry and ELISpot assays. The four epitopes with the highest specificity in these assays were EMY1627-13, EMY16236-48, TSP333-42 and TSP380-90 (Pang et al., 2020).
The theoretically optimal sequence consisting of the intra-molecule adjuvant LTB (GenBank: AAL55672.1), linkers, and tandem copies of the Th and B cell epitopes named ETBM was established. The LTB-ETBM sequence was submitted to GenBank (accession number: MT731963). The sequence was analyzed using bioinformatics software for modeling and prediction. For all details, please see our preliminary study(Guo et al., 2014).
Construction, expression, and purification of the LTB-ETBM
To construct the fusion protein LTB-ETBM, a DNA fragment LTB-ETBM was synthesized after reverse translation and codon-optimization. The synthesized LTB-ETBM gene was cloned into the plasmid pCzn1 after digestion (NdeI and XbaI) and connection, generating the expression vector pCzn1-LTB-ETBM. The recombinant plasmid was transformed into ArcticExpress competent cells (DE3). The fusion protein LTB-ETBM was purified by Ni2+-IDA-Sepharose CL-6B (Genscript, Nanjing, China), and measured by 12% SDS-PAGE. Methods of LTB-ETBM purification were followed by the HUPO proteomics standard initiative (http://www.psidev.info/miape) and publication guidelines. The purified protein was concentrated using a dialysis bag. It is stored at -80℃ for later use.
Immunization and infection
The BALB/c mice (SPF, male, 4-6 weeks, n=6) were purchased from Beijing vital river laboratory animal technology company (Beijing, China). The animal experiments on E. multilocularis were approved by the Animal Ethical and Experimental Committee of Qinghai University (QHDX-2019-09).The mice were immunized with 0.5 mg/mL of LTB-ETBM, rLTB (recombinant LTB purified in our lab from E. coli BL21 ArcticExpress competent cells [DE3] transformed with the expression vector pCzn1-LTB), or phosphate buffer solution (PBS) with the same volume of complete Freund’s adjuvant (Sigma, St. Louis, USA) for the first vaccination and incomplete Freund’s adjuvant (Sigma, St. Louis, USA) for the second and third vaccinations. The last booster vaccination consisted of the fusion protein without adjuvant. The mice antisera were collected after the last booster on the fifth day. It is stored at -80℃ for later use.
E.m. protoscoleces were isolated and preserved in our lab as described previously (Li et al., 2018). E.m. protoscoleces were isolated as follows: mice were sacrificed and aseptically separated the cysts from the abdomen and liver. The cysts were cut into pieces and ground through 300-μm nylon mesh and 900-μm nylon mesh in turn. Protoscoleces were suspended in normal saline (1000 protoscoleces/200 μl) after being obtained on the mesh at the last filtration.
The protocol of vaccine protective effect was performed as previously(Boubaker et al., 2015; Li et al., 2018). The mice were vaccinated with LTB-ETBM, rLTB or PBS, with six mice in each group, and the protocol was the same as for immunization. After two weeks, all mice were challenged with protoscoleces (intraperitoneally, 200 μl of normal saline suspension). Vaccinated mice were maintained for four months before the investigation of E.m. infection. The cysts (including subcutaneous, abdominal and thoracic cysts as well as cysts from the inside or surface of the liver) were carefully stripped and weighed. The mice antisera were collected and stored at -80℃ for later use.
The protocol for vaccine therapeutic effect was performed as previously(Li et al., 2018). To establish the E.m.-infected mouse model, mice were challenged with protoscoleces (intraperitoneally, 200 μl with normal saline suspension). After four months, three mice were killed to determine whether they were successfully infected with E. multilocularis. The infected mice were subcutaneously injected monthly for 4 months with 0.5 mg/mL LTB-ETBM in PBS emulsified with the same volume of Freund’s adjuvant or 15μg CpG. The rLTB and PBS follows the same protocol as the control group. All mice were sacrificed and aseptically separated the cysts for evaluation of E.m. infection after two weeks. The cysts (including subcutaneous, abdominal and thoracic cysts and cysts from the inside or surface of the liver) were carefully stripped and weighed. The mice antisera were collected and stored at -80℃ for later use.
Western blot analysis of the immunoreactivity of the LTB-ETBM vaccine
Purified EMY162 was separated by 12% SDS-PAGE (Bio-Rad, California, USA) and equilibrated in ice cold transfer buffer, then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA) by 200 mA constant current. The PVDF membrane was incubated with mice polyclonal anti-LTB-ETBM serum (1:2500). The membrane was washed with PBST four times and incubated with HRP-goat anti-mouse IgG (Jackson Immuno Research Lab, West Grove, United States) at a dilution of 1: 10,000. Luminescence ECL detection kits (Thermo Fisher) were used to monitor the positive signals.
Measurement of immunogenicity of the LTB-ETBM vaccine
After the last injection, the anti-serum was analyzed by indirect ELISA. The 96-well plates were coated with EMY162 or TSP3 overnight and blocked with 5% (w/v) bovine serum albumin (BSA) at room temperature for 4 h. After three times washes, 100 μl diluted serum (1:2500) were added to the corresponding well for 1 h at 37℃. The plate was washed with PBST three times, then 100 μl HRP-goat anti-mouse IgG (1:10,000, IgG1 (1:2,000), IgG2a (1:2,000), IgM (1:2,000), IgE (1:2,000) or IgA (Santacruz, Dallas, USA; 1:6,000) was added to the corresponding well for 1 h at 37℃. Then, the substrates were incubated in 100 μl TMB for 10 min at room temperature and the reaction was stopped by the addition of 50 μl 2 M H2SO4. The optical density (OD) was measured at 450 nm by a microplate reader (TECAN, Switzerland).
E.m. whole protein-specific antibodies were measured by an ELISA assay as follows: 96-well microplates were coated with E.m. protoscoleces whole protein (1 μg/well, the protein extraction in accordance with the general protocol) at 4 °C for all night. The plate washed with PBST three times, and then added with the anti-serum (1:500) for 1 h at 37℃. The plate was washed with PBST three times, then 100 μl HRP-goat anti-mouse IgG (1:10,000) was added to the corresponding well for 1 h at 37℃. Then, the substrates were incubated in 100 μl TMB for 10 min at room temperature and the reaction was stopped by the addition of 50 μl 2 M H2SO4. Tests of the ELISA method were described as mentioned above.
Determination of specific antibody production after challenge E.m.
Blood and serum samples were collected from the mice after prophylactic and therapeutic vaccination. The titers of serum specific antibodies against EMY162 were determined by indirect ELISA. The 96-well microplates were coated with EMY162 overnight at 4°C. The sera was diluted to 1:8,000 and 1:4,000, respectively. The HRP-goat anti-mouse IgG, IgG1, IgG2a, IgA, IgM and IgE (IgE were purchased from Jackson Immuno Research Lab., West Grove, United States) at a dilution of 1:10,000 were used as secondary antibodies, respectively.
Evaluation of T lymphocyte responses
The splenocyte proliferation assay was performed according to the protocol previously described . Splenocytes were prepared using 70μm nylon mesh cell strainer (Falcon, Corning, USA) and Lympholyte®-M (Cedarlane, Canada) from mice vaccinated with LTB-ETBM or PBS. The splenocytes were seeded with 2×105 cells/well and cultured in triplicate in a 96-well plate. Subsequently, the cells were stimulated with 2 μg/well LTB-ETBM, EMY162, EMY16236–48, EMY1627–13, TSP380-90, or TSP333-42. The plates were incubated for sixty hours in a cell incubator, then added 20 µl/well of MTS (Promega, Beijing, China). After three hours of incubation, the absorbance was measured at 490 nm. The stimulation index (SI) represents cell proliferation, and the formula is based on our previous study(Li et al., 2018).
Determination of cytokine production
Cytokines (IFN-γ, IL-4, IL-17, and IL-10) in serum were measured by its mouse ELISA kit on the basis of the user guide (R&D Systems, Minneapolis, MN, United States) after immunization, prophylactic and therapeutic vaccine. In our experiment, the PBS control group was immunized with Freund's adjuvant, which would affect the cytokine concentration, so we added a normal control group to indicate the cytokine concentration of vaccine LTB-ETBM.
All statistical analyses were performed using GraphPad Prism 6 software. Data is expressed as mean ± standard deviation (SD). Differences between the two groups were tested using Student’s paired t-tests, and ***p < 0.001, **p < 0.01, *p < 0.05 was considered statistically significant. One‑way analysis of variance was used to make statistical comparisons of the IgG antibodies specific for EMY162, it was applied to compare the differences among groups.