Cells and viruses
AAV-293, Vero and BHK-21cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin at 37 ℃ in 5% CO2. VSV-IN and VSV-NJ stocks were grown and titrated in Vero cells.
Construction Of Plasmids And Recovery Of Recombinant Viruses
The G gene of VSV-IN (VSV-IN-G) was amplified by reverse transcription (RT)-PCR from purified viral (VSV-IN) RNA using the following two primers: 5´-CGGAATTCEcoR ⅠGCCACC(kozak sequence)ATGAAGTGCCTTTTGTA-3´ and 5´-ATTTGCGGCCGCNot ITTACTTTCCAAGTCGGTTC-3´.
The G gene of VSV-NJ (VSV-NJ-G)was amplified by RT-PCR from purified viral (VSV-NJ) RNA using the following primers: 5´-CGGAATTCEcoR ⅠGCCACC (kozak sequence) ATGTTGTCTTATCTAATCTTTGCA-3´ and 5´-ATTTGCGGCCGCNot ITTAACGGAAATGAGCCATTTCCACG-3´. We amplified the fusion G genes of VSV-IN and VSV-NJ(VSV-IN-G-NJ-G༉by overlapping PCR for polypeptides Gly-Gly-Gly-Gly-Ser and the two primers (containing the linkers´ gene sequence) were 5´-GGTGGAGGTGGAAGClinker ATGTTGTCTTATCTAATC-3´and 5´-GCTTCCACCTCCACClinkerCTTTCCAAGTCGGTTC-3´.
The target genes (VSV-IN-G, VSV-NJ-G, VSV-IN-G-NJ-G) and the adenovirus shuttle vector (pacAd-CMV K-NpA) were digested with restriction enzymes EcoR I and Not I. Then the fragments were cloned into the shuttle vector ligated with T4 DNA ligase to construct the recombinant shuttle plasmids containing the target genes (pAd-VSV-IN-G, pAd-VSV-NJ-G, pAd-VSV-IN-G-NJ-G). The recombinant shuttle plasmids and adenovirus backbone plasmid linearized with PacI restriction enzymes were co-transferred to AAV-293 cell monolayer in 6-well tissue culture plates (Costar, Corning, NY, USA) using Lipofectamine-2000 reagent (Invitrogen, USA). Briefly, 2 µg of each recombinant shuttle plasmid were diluted in 250 µL of OPTI-MEM (Invitrogen) and mixed gently. Meanwhile, 10 µL of Lipofectamine-2000 was mixed with 250 µL OPTI-MEM for 5 min. The two dilutions were then combined, incubated for 20 min, and then slowly added to 80%~90% confluent monolayer of AAV-293 cells that had been prewashed twice with OPTI-MEM. After 4 h of incubation at 37 ℃, 5% CO2, the transfection medium was removed, and 2 mL DMEM containing 10% FBS was added to each well, then incubated for 7 ~ 15d. When the AAV-293 cells showed cytopathic effect (CPE), the cell culture medium was collected. The three kinds of rAd were named with rAd-IN, rAd-NJ and rAd-IN-NJ.
The three rAds inoculated into the AAV-293 cells were serially propagated to 20 generations. The rAds of 5th ,10th ,15th, and 20th generations were collected to extract viral DNA using AxyPrepTMBody Fluid Viral DNA / RNA Miniprep Kit, and then identified by PCR. In addition, the rAds of 5th ,10th ,15th, and 20th generations were diluted 10-fold with DMEM containing 2% FBS, and then 100 µL was inoculated in AAV-293 cells. The cells were incubated at 37 ℃, 5% CO2 for 3d. The cytopathic effect (CPE) was observed and recorded on 3 d post-incubation, and TCID50 was calculated by the method of Read-Muenels.
Detection of expressing G protein by western blot and IFA
AAV-293 cells infected with rAds were washed twice with phosphate -buffered saline (PBS:137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM K2HPO4, pH7.2) and then lysed in 4x SDS-PAGE sample buffer. For western blot analysis, 100 µg purified rAds were loaded on 10% SDS-PAGE. Separated proteins were electro-phoretically transferred to a nitrocellulose membrane. The nonspecific antibody binding sites were blocked for 12 h at 4℃ with a blocking solution containing 5% skim-milk in PBST (PBS and 0.05% Tween-20), then membranes were washed with PBST three times. Then the one membrane was reacted for 1 h at 37℃ with mouse anti-VSV-IN-G protein McAb diluted (1:5000) in PBST containing 5% skim-milk incubated for 1 h at 37℃. with PBST for 3 times, it was treated with peroxidase-conjugated goat anti-mouse IgG diluted (1:2000) in PBST containing 5% skim-milk incubated for 1 h at 37℃ to detect the expression of the VSV-IN- G protein in rAd-IN or rAd-IN-NJ. Goat anti-VSV-NJ-G protein PcAb (1:1000) and peroxidase-conjugated rabbit anti-goat IgG (1:2000) were used to detect the expression of the VSV-NJ-G protein in rAd-NJ or rAd-IN-NJ. 3,3-Diaminobenzidine tetra hydrochloride was used as the substrate for membrane development.
For indirect immunofluorescence assay (IFA), Vero cells were seeded into six-well tissue culture plates (Costar Corning Inc., Corning, NY) at 2 × 105 concentration of cells every well. When the cells reached approximately 80 ~ 90% confluence, the culture medium was removed and the cells were washed with PBS (pH 7.2) three times. Then 500 µL DMEM containing 2 × 105 PFU of recombinant adenovirus were added into the six-well tissue culture plates incubated for 1 h at 37℃, 5% CO2. After removing the virus, fresh medium was added and cultures were incubated at 37℃, 5% CO2 for IFA. At 48 h incubation, the cells were washed with PBS three times and then fixed with 4% paraformaldehyde 500 µL /mL for 30 min at 4℃. Then the paraformaldehyde was removed and the cells were washed three times with PBST. The rAd-IN and rAd-VSV-IN-NJ were detected using anti-VSV-IN-G protein mouse McAb (1:2000) followed by fluorescein isocyanate conjugated goat anti-mouse IgG (1:200) for 1 h at room temperature. rAd-NJ and rAd-IN-NJ were detected with Anti-VSV-NJ goat PcAb (1:500) and peroxidase-conjugated rabbit anti-goat IgG using the same method. Then the cells were visualized under a fluorescent microscope.
Immunization And Sample Collection
rAd antigen preparation: Stock antigens were prepared from AAV-293 cells infected with the rAd-IN, rAd-NJ, rAd-IN-NJ. AAV-293 cells were infected with rAds of 10 MOI for 2 d incubation at 37℃, 5% CO2. After two days, the cells were collected and repeatedly frozen and thawed at -20℃/room temperature three times. Then the rAds was obtained from the supernatant content centrifuged at 4℃, 3000 rpm for 10 min. The TCID50 of the rAds were determined by the method of Read-Muenels. In order to evaluate the immune effects of the rAds, the study selected 50 6-8-week-old female BALB/c mice, which were purchased from Chinese Experimental Animal Resources Research Institute for food and drug control (Beijing, China) and divided randomly into five groups, each group of 10. The three groups of mice were respectively inoculated subcutaneously three times at 2-week intervals with 108 TCID50 of rAds. At last, two groups of mice were inoculated with wildtype adenovirus and PBS, and the PBS group was used as the negative control.
After 0, 2, 4, 6 weeks of the first inoculation, the blood was collected from the orbital cavity of the mice. The serum was collected from the blood incubated at 4℃ and centrifuged at 4℃, 3000 rpm for 10 min. Then the serum was stored at -80℃ for future use to detect the specific antibody levels by neutralization test. At the end of the experiments, the mice were euthanized by CO2. The procedure of CO2 euthanasia was referenced by previously published article [34]. Briefly, the mice home cage was placed in a transparent polycarbonate euthanasia chamber (44 cm × 23.5 cm × 21 cm). The chamber was covered with an acrylic lid with ports for CO2 gas inlet and outlet. Compressed CO2 gas was provided from a cylinder (Weiler Welding, Moraine, OH) and controlled by the regulator (Western Medica, Westlake, OH). The flow rate of CO2 was 1.7 L per minute. Before turning off the CO2, death was confirmed when the complete cessation of breathing of mice was observed for at least 3 minutes.
Goats: 20 outbred healthy Boer goats (6 months old) were were purchased from local self-supporting farmers in Fuyang (China) and divided randomly into 5 groups, 4 goats per group, and housed in separate rooms. All goats were negative VSV infection as detected by neutralizing antibodies (titers < 1:2). Groups 1, 2, and 3 were subcutaneously injected twice at 3-week interval with 108 TCID50/mL rAd-IN, rAd-NJ, or rAd-IN-NJ, Group 4 and 5 were inoculated subcutaneously with 108 TCID50/mL wtAd or 1 mL PBS as negative controls. All experiments were approved by the Ethics committee of Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, (Permit number:2015-014). All manipulations were carried out in accordance with the requirements of the Regulations of Experimental Animal Administration of China. On 0, 3, 6, 9, 12 weeks post first inoculation, blood was collected from the vein of each goat. Sera were collecting from the blood incubated at 4℃ and centrifuged at 4℃, 3000 rpm for 10 min. Then, the sera were kept at -20℃ for using to detect the specific antibody levels by neutralization test. At the end of the experiments, the goats were euthanized by intravenous injection of the euthanasia solution, which was composed of 429 mg/kg of sodium pentobarbital and 55 mg/kg of phenytoin based on the weight of the goat [35].
Determination Of Neutralizing Antibody Titers
Sera were collected and incubated at 56℃ for 30 min to inactivate complement. The sera were diluted 5-fold with DMEM, and then serially diluted 2-fold in DMEM. Serial dilutions of the serum were incubated with 200 TCID50 doses of wild-type viruses (VSV-IN or VSV-NJ) for 1 h at 37℃. After incubation, samples were added to BHK-21 cells in quadruplicate assays in 96-well plates, incubated at 37℃ for 3 days. The CPE was observed and recorded after 3d incubation. The virus-neutralizing antibody (VNA) titer was defined as the highest serum dilution that inhibited CPE by at least 50%. A titer equivalent to 10 or higher was considered positive.
Lymphocyte Proliferation Assay
The lymphocyte proliferation assay of the mice was conducted at 2 weeks after the 3rd immunization. Spleens were aseptically removed from three mice every group and splenocyte suspensions were prepared as previously described [20]. The splenocytes were extracted and purified using a Spleno-cyte Extraction kit (TBD Science, Tianjin, China) and seeded in 96-well flat-bottom plates at a density of 2 × 106 cells per well in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) containing 10% FBS. Then 100 µL of medium containing VSV-IN or VSV-NJ was added to each well of splenocytes. Concanavalin A (ConA) (Sigma Aldrich, St. Louis, MO, USA) was used as positive control, and medium was used as negative control. The plates were incubated at 37℃ in 5% CO2 for 3 days. After 3days, the proliferative response was determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, and the Cell Titer 96 Aqueous One Solution Cell Proliferation kit (Promega, Madison, WI, USA), following the manufacturer’s instructions. At the end of the incubation, the OD of the plate wells was read at 490 nm. The lymphocyte proliferation rate was quantified using the stimulation index (SI), which was calculated as the ratio of the OD490 of the stimulated cells to the OD490 of the negative controls.
The lymphocyte proliferation assay of the goats was conducted at week 9 after first immunization. The blood was collected from the jugular vein of goats and was heparinized. Then, peripheral blood mononuclear cells (PBMC) were separated by Filoll-Hypque density gradient centrifugation (TBD Sciences, Tianjin, China) as described by Wang et al. (2013), Cells were resuscitated at 2 × 106/mL with RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 50 mM 2-mercaptoethanol, 100 IU/mL streptomycin, and 100 IU/mL penicillin. The cells were plated in 96-wel plates with 100 µl per well. Subsequently, 100 µL per well of medium with or without inactivated VSV-NJ or VSV-IN were added and mixed. ConA (5 µg/mL, Sigma-Aldrich) was used as a positive control. Uninfected cells cultured only in medium were used as a negative control. Each sample was tested in triplicate. After incubation of the samples at 37℃ in 5% CO2 for 72 h, 20 µL of MTS was added, samples were incubated at 37℃ in 5% CO2 for 4 h. At the end of the incubation, the OD490 was measured. The stimulation index (SI) was calculated as the ratio of the average OD490 value of stimulated cells to average OD490 value of negative control.
Statistical analysis
The data were analyzed with one-way ANOVA in GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA); p values less than 0.05 were considered statistically significant.