Recombinant adenovirus expressing vesicular stomatitis virus G proteins induce both humoral and cell-mediated immune responses in mice and goats

Background Vesicular stomatitis (VS) is an acute, highly contagious and economically important zoonotic disease caused by the vesicular stomatitis virus (VSV). There is a need for effective and safe stable recombinant vaccine for the control of the disease. The human type 5 replication-defective adenovirus expression vector is a good way to construct recombinant vaccines. Results Three recombinant adenoviruses (rAd) were successfully constructed that expressed the VSV Indiana serotype glycoprotein (VSV-IN-G), VSV New Jersey serotype glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]) with potentiality to induce protective immunity. G proteins were successfully expressed with good immunogenicity. The rAds could induce the production of VSV antibodies in mice, and VSV neutralizing antibodies in goats, respectively. The neutralizing antibody titers could reach 1:32 in mice and 1:64 in goats. The rAds induced strong lymphocyte proliferation in mice and goats, which was significantly higher compared to the negative control groups. Conclusions The three rAds constructed in the study expressed VSV-G proteins and induced both humoral and cellular immune responses in mice and goats. These results lay the foundation for further studies on the use of rAds in vaccines expressing VSV-G. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-020-02740-6.

neutralizing antibodies in infected animals. These virus can be identified by neutralization and complement fixation tests [7,8]. Although these two serotypes are morphologically and pathologically similar, in infected animals they generate distinct neutralizing antibodies [9]. The cross-protection between the two serotypes is poor; thus, different vaccines are required for each serotype [10,11]. From 2004 to 2006, 751 VS outbreaks caused by VSV-NJ were reported in the US [12]. These VS outbreaks also spread from the US to Mexico from 2005 to 2011; however, no new variant strain was produced [13]. As VS can be prevented and controlled by vaccination, some advancements have been made with regard to developing vaccines against VSV. However, further efforts are still needed in developing new vaccines [10,11,14].
The recombinant adenovirus (rAd) vector has been widely used to achieve transient inducible expression [15]. The adenovirus vector can either be replicationdefective or replicative [16]. In the replicationdefective adenovirus vector, all structural genes are removed, and only the vital genes for cis-acting elements and the packaging signal sequence are retained; therefore, its cytotoxicity is significantly reduced. Foreign sequences of up to 37 Kb can be inserted into this vector. In addition, the cytomegalovirus (CMV) promoter is also inserted into this vector to enable the efficient expression of foreign genes. This expression system is currently the main vector used for rAd vector vaccine research [17].
The vesicular stomatitis virus glycoprotein (VSV-G) contains glycosylation sites and the antigenic determinant [18]. The VSV-G present as protrusions on the virus envelope is strain-specific and capable to stimulate the production of neutralizing antibodies. VSV-G is the preferred antigen for VSV vaccine research [19]. Effective vaccines currently available against VSV are mostly live attenuated. Since attenuated virus has the risks of virulence reversion, a stable vaccine is therefore in need. In this study we assessed the potential of the VSV-G gene as a vaccine candidate by using a replication-defective human adenovirus type 5 expression vector expressing the VSV-IN glycoprotein (VSV-IN-G), VSV-NJ glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]). Furthermore, we evaluated the immunogenicity of these rAd vectors expressing VSV-G in mice and goats.

Generation and identification of recombinant adenovirus
The three rAds named rAd-IN, rAd-NJ, rAd-IN-NJ were replicated in AAV-293 cells. After a 10-day incubation period, the cytopathic effect (CPE) was observed in the infected cells (Fig. 1). The three rAds were serially propagated to four generation and the rAds were collected to extract the viral DNA. The VSV-IN-G gene (1536 bp), VSV-NJ-G gene (1554 bp) and the VSV-IN-G-NJ-G gene (3100 bp) were amplified by PCR using specific primers (Fig. 2).

Production and characterization of recombinant adenovirus
The three rAds were inoculated into the AAV-293 cells and were serially propagated to 20 generations. The 5th, 10th, 15th, and 20th generation rAds were collected to extract the viral DNA, and the VSV-IN-G gene (1536 bp), VSV-NJ-G gene (1554 bp) and the VSV-IN-G-NJ-G gene (3100 bp) were then correctly identified using PCR and DNA sequencing, which matched the expected results (Fig. 3). No band was observed in the control group. The TCID 50 's of the 5th, 10th, 15th, and 20th rAd-IN generations were 10 -8.56 /mL, 10 -8.16 /mL, 10 -8.96 /mL, and 10 -8.34 /mL, respectively. The TCID 50 's of the 5th, 10th, 15th, and 20th rAd-NJ generation were 10 -7.76 /mL, 10 -8.16 /mL, 10 -7.67 /mL, and 10 -8.2 /mL respectively, and the corresponding values for rAd-IN-NJ were 10 -6.96 /mL, 10 -7.16 /mL, 10 -7.67 /mL, and 10 -7.2 /mL suggesting certain level of genetic stability of the rAds. Vero cells were infected with rAd-IN-NJ to detect the expression of VSV-G by immunofluorescence assay (IFA). IFA showed that specific green fluorescence was observed for rAd-IN-NJ, which was not detected in the negative control wild-type Ad (wtAd) (Fig. 6). These results demonstrated that the recombinant fusion G protein could be expressed in-vitro following rAd infection in Vero cells, and that the protein could retain its antigenic reactivity.

Virus neutralizing antibody responses
The mice and goats immunized with rAds containing the VSV-G genes using a micro-neutralization assay suggested that these animals could produce neutralizing antibodies after immunization. The virus-neutralizing antibody (VNA) titers in all the experimental groups were low after the first immunization in mice and goats, but these titers significantly increased after the second immunization. As shown in Tables 1 and 2, VNA was   Tables 3 and 4, the VNA titers with VSV-NJ were equal to or exceeded a ratio of 1:8 after the second immunization in the rAd-NJ or rAd-IN-NJ vaccinated mice and goat groups. In the neutralization assay with VSV-IN, the VNA titer of the rAd-NJ group was lower than those of the rAd-IN or rAd-IN-NJ vaccinated mice and goat groups. Upon conducting the neutralization assay with VSV-NJ, the VNA titers of the rAd-NJ and rAd-IN-NJ groups were higher than that of the rAd-IN vaccinated mice and goat groups. As expected, no neutralization activity against VSV was detected in the pre-immune sera or in the sera of experimental animals injected with wtAd and phosphate-buffered saline (PBS).

Lymphocyte proliferation response
To investigate the cell-mediated immune responses induced by the three rAds, the lymphocyte proliferation responses of mice were analyzed 2 weeks after the third immunization. In case of goats, the lymphocyte proliferation responses were analyzed 6 weeks after the secondary immunization. VSV-IN and VSV-NJ are two different antigens that stimulated lymphocyte proliferation in different experimental groups. The data showed ( Fig. 7 and 8) that mice and goats immunized with the rAds induced significantly stronger lymphocyte proliferation responses by VSV-IN and VSV-NJ compared with the PBS and wtAd control groups. When stimulated by VSV-IN, the lymphocyte proliferative responses in the rAd-IN and rAd-IN-NJ groups were stronger, with a stimulation index (SI) of approximately 2.5. In addition, the SI of the rAd-IN-NJ group was relatively higher than those of the rAd-IN and rAd-NJ groups in mice and goats. There was a significant difference between the rAd groups and the PBS and wtAd control groups (P < 0.05), but there was no significant difference among mice immunized with the different rAds (P > 0.05).

Discussion
Vesicular stomatitis is a viral disease that primarily affects horses, cattle, and swine. The virus is capable to affect a wide range of host including sheep and goats. Thus, effective vaccination is very important for the prevention and control of VS. Currently, controlling the spread of VS involves isolation of the affected animals, quarantining the affected premise and is mainly dependent on the immunization of susceptible animals. In addition to inactivated vaccines, live attenuated vaccines are widely used for immunization against VSV. However, due to the risk of virulence reversion in such vaccines, heat-stable, safe, and effective VS vaccines need to be developed.
The human type 5 replication-defective adenovirus expression vector is a common vector used in the research of vaccines and genetic therapy. The newly developed heat-stable technology enables adenovirus vector vaccines to be stored at room temperature up to 45°C for 6 months and maintain a steady infection hold. rAd vector vaccines can be inoculated using a variety of methods since they have a wider range of permissive host cells. As effective vaccine vectors both the replicative and non-  replicative adenovirus are long been used to infect a wide varieties of mammalian cells to to expression higher level of recombinant proteins [20][21][22][23][24][25]. Considering these advantages, it is valuable to perform further research for the development of vaccine candidates using an adenovirus vector for the prevention and control of VS.
VSV glycoprotein plays major role in the mechanism of disease production in in many of the natural host including pigs [26]. Therefore, in this study we targeted VSV glycoproteingenes to clone and induce with an aim to induce VSV-G proteins for better immune response. It has been reported that the recombinant VSV-IN containing VSV-NJ-G-IN-G expressed both glycoproteins stably through multiple rounds of replication in pigs and induced neutralizing antibodies against both VSV serotypes [10,26]. In our study, the genes for both VSV-G serotypes were accurately cloned by reverse transcription-PCR (RT-PCR), and the fusion VSV-IN-G-NJ-G gene of 3100 bp was successfully amplified by overlapping PCR using the GlyGlyGlyGlySer polypeptides.
In order to determine the best time to harvest the rAds from the cell culture, we performed numerous rAd optimization experiments, and found that 10-15 days was the appropriate time to harvest the rAds. In addition, PCR was conducted to amplify the target genes from the cell culture supernatants containing rAds to confirm the genetic stability of the VSV-G genes. The western blotting and IFA results suggested that the three target proteins could be expressed in AAV-293 cells and Vero cells, but that rAd could only induce a CPE in AAV-293 cells at 36-48 h after virus infection.
One of the major aim of the study was to evaluate the immunogenicity of the newly constructed rAds. Mice and goats inoculated with rAd-IN, rAd-NJ, or rAd-IN-NJ were subjected to virus neutralization test to detect neutralizing antibody levels in their sera at different time interval. The results of the lymphocyte proliferation test showed that the rAds could elicit lymphocyte proliferation in mice and goats, and was significantly higher compared to the negative control groups (P < 0.05).

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.

Construction of plasmids and recovery of recombinant viruses
The

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 10 8 TCID 50 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°C, and centrifuged at 4°C (3000 rpm for 10 min). Then the serum was stored at − 20°C for future use to detect specific antibody levels using a neutralization test. At the end of the experiments, the mice were euthanized using CO 2 as previously described [28]. During the bleeding from retrobulbar plexus, the mice were anesthetized using phenobarbital.
Goats: 20 outbred healthy Boer goats (6 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 10 8 TCID 50 /mL rAd-IN, rAd-NJ, or rAd-IN-NJ. Groups 4 and 5 were inoculated subcutaneously with 10 8 TCID 50 / 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°C, and centrifuged at 4°C (3000 rpm for 10 min). Then, the sera were stored at − 20°C 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°C 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 TCID 50 of wild-type viruses (VSV-IN or VSV-NJ) for 1 h at 37°C. After incubation, samples were added to Vero cells in quadruplicate assays in 96-well plates, and incubated at 37°C for 3 days. The CPE was observed and recorded 3 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 2 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 Splenocyte Extraction kit (TBD Science, Tianjin, China) and seeded in 96-well flat-bottom plates at a density of 2 × 10 6 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°C in 5% CO 2 for 3 days. After 3 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.