Ethical permission
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.
Cells and viruses
AAV-293 and Vero cells 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 propagated and titrated in Vero cells.
Experiment animals
6-8-week-old female BALB/c mice were purchased from the Chinese Experimental Animal Resources Research Institute for Food and Drug Control (Beijing, China). Outbred healthy Boer goats (6 months old) were purchased from local self-supporting farmers in Fuyang, China.
Construction of plasmids and recovery of recombinant viruses
The VSV-IN-G gene was amplified by reverse transcription-PCR (RT-PCR) from the purified viral (VSV-IN) RNA using the following two primers: 5´-CGGAATTCEcoR ⅠGCCACC(kozak sequence)ATGAAGTGCCTTTTGTA-3´ and 5´-ATTTGCGGCCGCNot ITTACTTTCCAAGTCGGTTC-3´.
The VSV-NJ-G gene was amplified by RT-PCR from the purified viral (VSV-NJ) RNA using the following primers: 5´-CGGAATTCEcoR ⅠGCCACC(kozak sequence)ATGTTGTCTTATCTAATCTTTGCA-3´ and 5´-ATTTGCGGCCGCNot ITTAACGGAAATGAGCCATTTCCACG-3´. The gene for the VSV-IN-G-NJ-G fusion protein was amplified by overlapping PCR of polypeptides Gly-Gly-Gly-Gly-Ser and the following two primers (containing the linkers´ gene sequence): 5´-GGTGGAGGTGGAAGClinkerATGTTGTCTTATCTAATC-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 and ligated with the 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 plasmids linearized with PacI restriction enzymes were co-transferred into an AAV-293 cell monolayer in six-well tissue culture plates (Costar, Corning, NY, USA) using Lipofectamine-2000 reagent (Invitrogen, USA) as per manufacturer’s instruction. The three rAds constructed in this study were named rAd-IN, rAd-NJ, and rAd-IN-NJ.
The three rAds inoculated into the AAV-293 cells were serially propagated to 20 generations. The CPE was observed and recorded three days post-incubation, and TCID50 was calculated using the Reed-Muench method.
Detection of expressing G protein by western blotting and IFA
AAV-293 cells were infected with rAd-IN, rAd-NJ and rAd-IN-NJ. Mouse anti-VSV-IN-G protein McAb, goat anti-VSV-NJ-G protein PcAb were used to detect the expression of the VSV-IN-G protein and VSV-NJ-G in rAds by western blotting as previously described [27].
For the IFA, Vero cells infected with rAd-IN-NJ were fixed with paraformaldehyde for 30 min at 4℃. After washing three times with PBST, the cells were incubated with anti-VSV-IN-G protein mouse McAb (1:2000) or anti-VSV-NJ goat PcAb (1:500). Following fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG or FITC- rabbit anti-goat IgG were applied and incubated for 30 min at 37℃. The cells were then washed three times and visualized under a fluorescent microscope.
Immunization of mice and goat and sample collection
Mice: In order to evaluate the immune response to the rAds, the study selected 50 six-eight week-old female BALB/c mice. Mice were randomly divided into five groups, with 10 mice per group. The three groups of mice were respectively inoculated subcutaneously three times at two-week intervals with 108 TCID50 rAd-IN, rAd-NJ, or rAd-IN-NJ. Two mice groups were respectively inoculated with the wild-type adenovirus and PBS, which were used as the negative control.
After 0, 2, 4, and 6 weeks of the first inoculation, blood was collected from the retrobulbar plexus 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 -20℃ for future use to detect specific antibody levels using a neutralization test. At the end of the experiments, the mice were euthanized using CO2 as previously described [28]. During the bleeding from retrobulbar plexus, the mice were anesthetized using phenobarbital.
Goats: 20 outbred healthy Boer goats (six months old) were divided randomly into five groups, with four goats per group, and housed in separate rooms. All goats were negative for VSV infection as assessed by neutralizing antibodies (titers<1:2). Groups 1, 2, and 3 were subcutaneously injected twice at three-week intervals with 108 TCID50/mL rAd-IN, rAd-NJ, or rAd-IN-NJ. Groups 4 and 5 were inoculated subcutaneously with 108 TCID50/mL wtAd or 1 mL PBS as negative controls. On 0, 3, 6, 9, and 12 weeks post-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 stored at -20℃ for future use to detect specific antibody levels using a neutralization test. At the end of the experiment, the goats were continued to feed.
Determination of neutralizing antibody titers
Sera were collected and incubated at 56℃ for 30 min to inactivate complements. The sera were diluted five-fold with DMEM, and then serially diluted two-fold in DMEM. Serial dilutions of the serum were incubated with 200 TCID50 of wild-type viruses (VSV-IN or VSV-NJ) for 1 h at 37℃. After incubation, samples were added to Vero cells in quadruplicate assays in 96-well plates, and incubated at 37℃ for three days. The CPE was observed and recorded three days after incubation. The 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 in this study.
Lymphocyte proliferation assay
The lymphocyte proliferation assay for mice was conducted two weeks after the third immunization. Spleens were aseptically removed from three mice from each group and splenocyte suspensions were prepared. 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 a positive control, and the medium was used as a negative control. The plates were incubated at 37℃ in 5% CO2 for three days. After three days, the proliferative response was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation kit (Promega, Madison, WI, USA), following the manufacturer’s instructions.
The lymphocyte proliferation assay for goats was conducted at week 9 after the first immunization. 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 previously [27].
Statistical analysis
The data generated in this study were analyzed using a one-way ANOVA method of the GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). A p values less than 0.05 were considered statistically significant.