Recombinant HcGAPDH Protein Expressed on Probiotic Bacillus subtilis Spores Protects Sheep from Haemonchus contortus Infection by Inducing both Humoral and Cell-Mediated Responses

Ethics approval.Animal use was approved by Zhejiang University Experimental Animals Ethics Committee (permit ZJU20160239). All animals were cared for in accordance with guidelines for care and use of laboratory animals set by the same committee.

Parasite and animals.H. contortus Zhejiang strain was kept in the Veterinary Parasitology Laboratory, Zhejiang University, and maintained by serial passage in helminth-free sheep. Infective L3 larvae (iL3s) were obtained by incubation of eggs for 14 days at 28°C.

Six-week-old female BALB/c mice were purchased from the Zhejiang Academy of Medical Science (Hangzhou, China). The mice were raised in a sterilized room with the temperature set at 26 to 27°C, with a 12-h daily light cycle, and fed sterilized food and water. Six-month-old female sheep were purchased from the Miemieyang Animal Husbandry Co., Ltd. (Huzhou, China). All sheep were housed indoor with a natural daily light cycle. They were provided with hay and whole corn as food three times a day, with the same quantity for each group, and water ad libitum. Rabbits were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) at the age of 6 months.

Sheep abomasal microbiota.Six-month-old female sheep were each orally infected with 5,000 H. contortus iL3s suspended in 1 ml of PBS. They were euthanized at 14, 31, or 62 days postinfection (dpi). The control sheep received 1 ml of plain PBS and were sacrificed at 62 dpi. These sheep were housed in separated areas of the same building within the Miemieyang Animal Husbandry Co., Ltd., to minimize cross-contamination. A portion (10 ml) of abomasum fluids was collected from each sheep within 20 min of euthanasia. The abomasal fluids were centrifuged at 5,000 × g for 5 min at 4°C. The supernatants were centrifuged at 12,000 × g for 10 min at 4°C, and the pellet of the second centrifugation was analyzed for 16S rRNA sequences by using an Illumina MiSeq platform (Sangon Biotech, China) to obtain the abomasal microbiota of each sheep. Raw sequences have been deposited in the Sequence Read Archive database under project number SRP217048. Python v1.2.2 was used to analyze both heatmap and community taxonomic system composition. LEfSe v1.1.0 was used to analyze taxonomic cladogram. Krona v2.6.1 was used for hierarchical analyses.

Plasmid construction.The 1,023-bp coding sequence (CDS) of HcGAPDH was amplified from the total cDNA of H. contortus by PCR using primers previously described (10). PCR products were sequenced in both directions (BioSune, China) after being cloned to the pET-32a vector (TaKaRa, China), resulting in the pET32a-HcGAPDH plasmid.

To generate a recombinant spore carrying the CotB-HcGAPDH, genomic DNA of B. subtilis strain 168 was used as the template to amplify the CotB gene of 1,088 bp, which included the promoter of 263 bp and a partial N-terminal CDS of 825 bp. The PCR primers are listed in Table S1. PCR products were cloned into the pMD 18-T vector (TaKaRa, China). The CotB and HcGAPDH genes were fused in order and cloned into the pDG364 vector (pDG364-CotB-HcGAPDH). The CotB-HcGAPDH was amplified by PCR with primers (Table S1). The fused CotB-HcGAPDH was subcloned into E. coli-B. subtilis shuttle vector pDG364 (Miaolingbio, China), resulting in pDG364-CotB-HcGAPDH plasmid. The control plasmid pDG364-CotB was constructed by cloning the CotB gene to the pDG364 vector. All cloned DNAs were confirmed without mutations by sequencing (BioSune, China).

Expression of recombinant proteins.The recombinant vector pET32a-HcGAPDH was transformed into E. coli. BL21 bacteria. The transformants were induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) after being cultured until the optical density at 600 nm (OD₆₀₀) reached 0.6 at 37°C. The cells were then broken apart by sonication after centrifugation at 8,000 × g for 10 min and resuspension in a buffer (0.01% digitonin, 10 mM PIPES [pH 6.8], 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, and 5 mM EDTA) with proteinase inhibitors. Soluble His-tagged HcGAPDH was purified from bacterial lysate using a HisTrap column (GE Healthcare Life Sciences). HcGAPDH purity was checked by SDS-PAGE gel stained with Coomassie blue. The anti-HcGAPDH rabbit polyclonal antibodies (rAb) were prepared according to the previous method (16). The purified protein HcGAPDH and anti-HcGAPDH rAb were stored at −80°C.

The linearized pDG364-CotB-HcGAPDH plasmid was transformed into the genome of B. subtilis strain 168 by electroporation (39) at the location of amylase E gene by homologous recombination. B. subtilis spores were generated in 4 liters of DSM with 25 μg/ml chloramphenicol at 37°C for sporulation of the recombinant B. subtilis rBS^CotB-HcG or rBS^CotB, as previously described (22). The spores were purified by treatment with 4 mg/ml lysozyme, followed by washing in 1 M NaCl and 1 M KCl solution with 1 mM phenylmethylsulfonyl fluoride under stringent conditions, as described previously (19). The resultant preparations were then treated at 68°C for 1 h in water to remove any residual sporangial cells. The spores were kept at −80°C at a concentration of 1 × 10¹² CFU/ml in PBS (pH 7.4) until use.

SDS-PAGE and Western blotting.Spores of the recombinant B. subtilis rBS^CotB-HcG transfectant were generated as previously described (22). The spores were harvested and analyzed for the presence of HcGAPDH protein by SDS-PAGE. Further, spore coat proteins were extracted from the spores at 48 h of bacterial incubation in DSM using SDS-DTT extraction buffer (0.5% SDS, 0.1 M dithiothreitol, 0.1 M NaCl) as previously described (39). The extracted proteins were subjected to 12% SDS-PAGE, followed by transfer onto a polyvinylidene fluoride filter (Sigma, Germany). The latter was blocked overnight at 4°C in 5% skimmed milk in PBST (PBS with 0.05% [vol/vol] Tween 20), followed by incubation in anti-HcGAPDH rAb (1:1,000 in PBST) as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 in PBST) as the secondary antibody. The signal was detected by using enhanced chemiluminescence (Beyotime Biotechnology, China).

Immunofluorescence and flow cytometry assay.For immunofluorescence, 5-ml portions of sporulation cultures at 24, 48, or 72 h of incubation were harvested and processed as previously described (10). Spores were blocked with 5% bovine serum albumin for 2 h at 4°C, followed by incubation with anti-HcGAPDH rAb (1:2,000 in PBST) for 2 h at room temperature. Naive preimmunized rabbit sera (1:2,000 in PBST) were used as a negative control. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Invitrogen, 1:500 in PBST) was used as the secondary antibody. Spores were observed and photographed under a fluorescence microscope (Olympus BX51, Japan) equipped with an Olympus camera (Olympus Micro DP72, Japan).

For flow cytometry, 10⁵ purified spores were washed three times in PBS and then incubated with anti-HcGAPDH rAb (1:500 in PBST) at 37°C for 2 h. Naive rabbit sera (1:500 in PBST) was used as a negative control. After being washed three times in PBS, the spores were incubated with FITC-conjugated goat anti-rabbit IgG (1:500 in PBST; Invitrogen) at 37°C for 1 h. The spores were finally resuspended in 1 ml of PBS after three washes, and a minimum of 10⁴ spores were examined by using an FC500 MPL flow cytometer (Beckman Coulter). Expression of the CotB-HcGAPDH fusion protein was analyzed using FlowJo software (TreeStar).

Analysis of the production and structure of the recombinant spores.The purified WT and recombinant rBS^CotB-HcG spores were collected and fixed in 3% glutaraldehyde overnight at 4°C, followed by dehydration in gradient ethanol at 50, 70, 90, and 100%. After subsequent critical point drying and sputter coating, the samples were processed and photographed under a SU-70 scanning electron microscope (Hitachi, Japan). For transmission electron microscopy, the spores were fixed in glutaraldehyde overnight at 4°C, followed by incubation in 4% osmium tetroxide for 2 h. Next, they were dehydrated in gradient ethanol (50, 70, 90, and 100%) and embedded in epoxy resin, and the ultrathin sections were mounted on a 230-mesh copper mesh stained with 1% uranyl acetate-lead citrate. The spores were observed and photographed under a H-9500 transmission electron microscope (Hitachi, Japan).

To investigate whether the production of spores of the recombinant rBS^CotB-HcG was different from that of the wild type, both strains were individually inoculated in 1 liter of DSM and then cultured at 37°C with constant shaking at 140 r/min. The numbers of viable bacteria and spores were then quantified as previously described (19).

Animal experiments.Six-week-old female BALB/c mice were administered 100 μl of PBS (Ctrl), WT spores at 1 × 10¹⁰ CFU (WT), rBS^CotB spores at 1 × 10¹⁰ CFU (rBS^CotB), or rBS^CotB-HcG spores at 10⁶, 10⁸, or 10¹⁰ CFU (rBS^CotB-HcG) per mouse by oral gavage. There were 20 mice in each group. The mice in all groups, including Ctrl mice, were originally immunized with three doses applied daily on three consecutive days, followed by two boosts at a 1-week interval. Each boost was administered the same way as the original immunization. Mice in the HcGAPDH group were each subcutaneously immunized with 200 μg of purified HcGAPDH emulsified in the complete Freund’s adjuvant, followed by two boosts with 100 μg of HcGAPDH emulsified in the incomplete Freund’s adjuvant 1 week apart. All mice were euthanized at week 5 after the last boosting. Lymphocytes were isolated from spleens and cultured for the extraction of total RNA.

Thirty-six 6-month-old female sheep were assigned to six groups with six animals per group; each group was assigned control or individual treatment. Each group of sheep was kept in a separated pen housed indoors in the same building. All sheep were provided with food three times a day, with same quantity for each group, and water of the same source ad libitum. The entire experiment was conducted in Huzhou, China, between September and November 2018. The temperature was ranged from 32 to 25°C (maximum) to 15 to 30°C (minimum) for this period. Each sheep was administered 1 ml of PBS as the control (Ctrl), the WT spores (WT) at 1 × 10¹² CFU per sheep (Hc+WT), or rBS^CotB-HcG spores at 1 × 10⁸, 10¹⁰, or 10¹² CFU per sheep (Hc+rBS^CotB-HcG) by oral gavage, depending upon its assignment. All sheep except the Ctrl animals were individually challenged with 5,000 H. contortus iL3s 1 week later. Serum samples were collected from the jugular vein of each animal every 2 weeks. All sheep were sacrificed at 2 months postinfection with H. contortus iL3s. PBLs were isolated at 5 dpi using a sheep peripheral blood lymphocyte separation kit (Sangon Biotech, China). Body weight was obtained using a floor scale (Ohaus D52P150RTL2ZH; Shanghai, China) with a sensitivity of 0.01 kg. The body weight gain of each sheep was recorded as the difference in body weight (kg) between weeks 11 and 0. Weight loss recovery of vaccine-immunized sheep versus unimmunized controls (Hc) was calculated as follows: (body weight loss of Hc group-body weight loss of individual treatment)/body weight loss of Hc group × 100%. The eggs per gram (EPG) value was assayed at 14 dpi according to the modified McMaster method (29). The EPG reduction rate was calculated by: (average EPG in control – average EPG in treatment)/average EPG in control × 100%. The numbers of H. contortus adult worms from the abomasum in sheep were counted as described previously (40, 41) after euthanasia at week 11. The worm reduction rate was calculated as follows: (average worm count in control – average worm count in treatment)/average worm count in control × 100%.

Lymphocyte proliferation assay.As described previously (42), murine splenic lymphocytes were stimulated with LPS (5 μg/ml; Sigma, Germany), ConA (10 μg/ml; Sigma, Germany), or purified HcGAPDH protein (15 μg/ml). The cells were evaluated for proliferation by using an MTT assay kit (Sangon Biotech, China) according to the manufacturer’s instructions. Experiments with sheep PBLs were performed as described for the murine lymphocytes except for using LPS, ConA, and purified HcGAPDH protein at 10, 15, and 25 μg/ml, respectively.

qRT-PCR assay.Total RNA was extracted from PBLs. The cDNA synthesized by using a qPCR-RT kit (Toyobo, Japan) was subjected to quantitative real-time PCR (qPCR) to measure the mRNA levels of cytokines and transcription factors with SYBR green PCR master mix (Applied Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The primers specific for the mouse or sheep TGF-β, IFN-γ, IL-2, IL-12, IL-4, IL-6, IL-10, T-bet, and GATA-3 genes are listed in Table S1 in the supplemental material.

Determination of antibody levels by ELISA.Serum was collected weekly from each mouse after administration of the spores. The intestinal mucus was collected at the week 5 as previously described (26). The levels of anti-HcGAPDH IgG, sIgA, IgG1, and IgG2a were measured by an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates (Bethyl) were coated with 50 μl of purified HcGAPDH dissolved in coating buffer (0.05 M carbonate-bicarbonate [pH 9.6]) at a concentration of 1,000 ng/ml, followed by incubation in 5% skimmed milk in the coating buffer for 18 h at room temperature. After three washes in PBST, the plates were incubated at 37°C for 2 h in 1:400-diluted serum or mucus in PBST. Subsequently, HRP-conjugated goat anti-mouse IgG (1:5,000 dilutions; Abcam, UK), goat anti-mouse IgA (1:5,000 dilutions; Abcam), or goat anti-mouse IgG1 or IgG2a (1:1,000 dilutions; Abcam) was used as the suitable secondary antibody. After 1 h of incubation, the plates were washed again, and 100 μl of the substrate TMB (3,3′,5,5′-tetramethylbenzidine; BD Biosciences) was added. The reaction was stopped by adding 50 μl of 2 M H₂SO₄ after 5 min of incubation in dark, and the plates were read three times at 450 nm in a microplate ELISA reader (Bio-Rad, Japan). Negative-control wells incubated with naive sera were included in each plate. The results are expressed as the average of three OD₄₅₀ values. Anti-HcGAPDH IgG and sIgA levels in ovine serum and mucus, respectively, were similarly analyzed by ELISA. In this case, the secondary antibodies used were HRP-conjugated rabbit anti-sheep IgG and rabbit anti-sheep IgA (1:5,000 dilutions; Abcam), respectively.

HE staining.Abomasal sections (5-μm thick) were prepared from formalin-fixed and paraffin-embedded tissue blocks and subjected to hematoxylin and eosin (HE) staining as described previously (30). The samples were then examined under a microscope (Zeiss, Germany).

Analysis of abomasal microbiota of sheep.The relative abundances of the abomasal microbiota in sheep from the the Ctrl, Hc, Hc+WT, and Hc+rBS^CotB-HcG groups were analyzed by 16S rRNA gene sequencing. Both sampling and sequencing were processed according to a protocol previously described (43).

Statistical analysis.Results are presented as means ± the standard errors of the mean (SEM). One-way analysis of variance was performed. A P value of ≤0.05 was considered statistically significant.

Data availability.All data supporting the findings of this study are available either within the article or in the supplemental material. Raw abomasal microbiota sequences have been deposited in the Sequence Read Archive (SRA) database under project number SRP217048 (the authors could not make this SRA record available at the time of this paper’s publication due to circumstances related to the COVID-19 pandemic, but it will be made accessible as soon as possible after publication).