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Citation: Dan Shan, Xiaoyan Tang, Renqiang Liu, Dan Pan, Xijun Wang, Jinying Ge, Zhiyuan Wen, Zhigao Bu. Immunogenicity of a Recombinant VSV-Vectored SARS-CoV Vaccine Induced Robust Immunity in Rhesus Monkeys after Single-Dose Immunization [J].VIROLOGICA SINICA.

Immunogenicity of a Recombinant VSV-Vectored SARS-CoV Vaccine Induced Robust Immunity in Rhesus Monkeys after Single-Dose Immunization

Short Title: VSV∆G-SARS Is a Rapid Response SARS Vaccine Candidate

  • Corresponding author: Zhiyuan Wen, wenzhiyuan@caas.cn, ORCID: http://orcid.org/0000-0003-3112-0243
    Zhigao Bu, buzhigao@caas.cn, ORCID: http://orcid.org/0000-0001-9242-4211
  • Received Date: 23 February 2021
    Accepted Date: 04 August 2021
    Published Date: 08 October 2021
  • Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31th 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVƤG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVƤG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVƤG-SARS is safe and effective in mice at a dose of 1106 TCID50. More importantly, only a single-dose immunization of 2107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVƤG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.


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    Immunogenicity of a Recombinant VSV-Vectored SARS-CoV Vaccine Induced Robust Immunity in Rhesus Monkeys after Single-Dose Immunization

      Corresponding author: Zhiyuan Wen, wenzhiyuan@caas.cn
      Corresponding author: Zhigao Bu, buzhigao@caas.cn
    • 1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
    • 2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225127, China

    Abstract: 

    Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31th 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVƤG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVƤG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVƤG-SARS is safe and effective in mice at a dose of 1106 TCID50. More importantly, only a single-dose immunization of 2107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVƤG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.

    • Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV), which belongs to the family Coronaviridae, genus β-coronavirus (Guan et al. 2003). Since its initial identification in Guangdong Province of China in November 2002, SARS-CoV has resulted in 8096 infections and 774 deaths (mortality rate: ~10%) in 29 countries by December 31st 2003 (Drosten et al. 2003; Ksiazek et al. 2003; WHO 2004). With no more SARS-CoV infected cases reported, World Health Organization (WHO) has declared that SARS-CoV is eradicated in human populations in 2004 (WHO 2004). However, the COVID-19 pneumonia outbreak underlines the importance of the prevention and control of SARS-CoV and the SARS-like CoVs.

      The SARS-CoV spike (S) protein is one of the most immunogenic antigens for vaccine development, which induces neutralization antibodies to block virus-binding and stimulates host immune responses against infections (Du et al. 2009). A variety of S protein-specific vaccines have been proven effective in animal models, such as inactivated vaccines, DNA vaccines and virus-like particles (VLPs) vaccines (Woo et al. 2005; Qin et al. 2006; Liu et al. 2011). However, some of these strategies demand multiple doses to elicit favorable immune responses. And some strategies have other disadvantages, including uncertain expenditure costs and the possible requirement for appropriate vaccine adjuvants (Graham et al. 2013). Once SARS break out again, a rapid response vaccine is urgently needed.

      Vesicular stomatitis virus (VSV), which causes a self-limited disease in humans and many other species, has been used as a desired attenuated vector to develop commercial vaccines (Lawson et al. 1995). Multiple recombinant VSV-based vaccines can induce rapid robust immune responses in animal models, such as Ebola, Marburg and Influenza vaccines. More importantly, VSV-based vaccines can be used as promising post-exposure prophylaxis against high-risk virus infections (Ryder et al. 2015; Coller et al. 2017; Marzi et al. 2018; Monath et al. 2019). VSV-based vaccine expressing the Ebola virus (EBOV) glycoprotein, has successfully completed a phase Ⅲ clinical trial during the West African EBOV epidemic (Gsell et al. 2017; Henao-Restrepo et al. 2017). A VSV-based chimeric virus expressing Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) protein was constructed by our lab, which has been proven to rapidly induce good immune response in rhesus monkeys (Liu et al. 2018). During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a recombinant replication-competent VSV-based vaccine candidate expressing the SARS-CoV-2 S protein was constructed and proved to protect against severe SARS-CoV-2 infection, supporting its further development as a vaccine (Case et al. 2020; Yahalom-Ronen et al. 2020). Though some similar studies about VSV-vectored vaccines against SARS-CoV-2 have been performed in different kinds of experimental animals, taking significant differences between SARS-CoV and SARS-CoV-2 into consideration, it is necessary to develop recombinant VSV-vectored vaccines especially for SARS-CoV.

      Here, we constructed a chimeric recombinant VSV (designated as VSVΔG-SARS), in which the VSV glycoprotein (G) gene was replaced with the spike (S) gene of SARS-CoV. Only a single-dose immunization with VSVΔG-SARS generated high-level S protein-specific neutralizing antibodies and cellular immune responses in rhesus monkeys. Overall, our data show that VSVΔG-SARS can protect against SARS-CoV infection, supporting its further development as a vaccine. This study paves the way for further development of a VSV-vectored CoVs vaccine.

    • Hamster Syrian kidney cells (BSR cells), BSR-T7/5 and Vero E6 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Oregon, USA) at 37 ℃ in a 5% CO2 incubator.

    • The VSVΔG-eGFP pseudo-virus system described before was modified to construct the recombinant virus VSVΔG-SARS and VSVΔG-eGFP-SARS (Liu et al. 2018). SARS-CoV S gene (GenBank accession No. AAP13441.1) was chemically synthesized and cloned into the pVSVΔG-eGFP plasmid, which contains VSV full-length genomic cDNA without G gene. The final plasmid was designated as pVSVΔG-eGFP-SARS. The pVSVΔG-SARS plasmid was also prepared, in which the eGFP gene was deleted from the pVSVΔG-eGFP-SARS plasmid. The pVSVΔG-eGFP plasmid or the pVSVΔG-eGFP-SARS plasmid, together with plasmids encoding the N, P, and L proteins were transfected into BSR-T7/5 cells. At 96 hours (h) post-transfection, the supernatant was collected and serially passaged on Vero E6 cells, until obvious green fluorescence or cytopathic effect (CPE) was observed. The rescued viruses were plaque purified and further passaged to generate viral stocks. The viral titer of the recombinant VSV viral stock was as high as 2×107 TCID50/mL.

    • Multi-step growth experiments were conducted to evaluate the kinetics of the recombinant viruses. Confluent monolayers of Vero E6 in 6-well plates were respectively infected with native VSV, VSVΔG-SARS or VSVΔG-eGFP-SARS at an MOI = 0.01. The supernatant was harvested at different time intervals (0, 12, 24, 36, 48, 60, 72, 84 and 96h) post infection. Supernatants of each time were separately collected and preserved at -80 ℃. The viral titers were determined by the index of the highest dilution titer (Tissue culture infective dose, TCID50/mL).

    • For Western blot assay, infected Vero E6 cells were collected and lysed at 72h post-infection with the recombinant viruses at an MOI = 1. Samples were electrophoresed in SDS-10% polyacrylamide gels, and the proteins were transferred onto nitrocellulose membranes. Western blot detection was performed with mouse anti-S serum at a dilution of 1:1000 (Invitrogen, Oregon, USA) and HRP-labeled goat ant50i-mouse IgG at a dilution of 1:3000 (Genscript, Nanjing, China). The bands were visualized with Azure C300-C500 (Cycloud, Beijing, China).

      For indirect immunofluorescence assay, Vero E6 cells were planted onto 24-well plates and infected with the recombinant viruses at an MOI = 1. At 36h post-infection, the infected cells were fixed and subjected to indirect immunofluorescence with mouse anti-S serum as the primary antibody at a dilution of 1:100 (Invitrogen, Oregon, USA), and Alexa Fluor 568-conjugated goat anti-mouse IgG as the secondary antibody at a dilution of 1:2000 (Invitrogen, Oregon, USA). Cell nuclei were stained with Hoechst 33342 (Invitrogen, Oregon, USA). The Staining cells were analyzed with LSM880-ZEISS confocal laser scanning microscopy with fast Airyscan (Carl Zeiss, Germany).

    • BSR cells were seeded at 24 well plate. Lipofectamine2000 (Invitrogen, Oregon, USA) was used for transfection of the BSR cells with the cloned vectors pCAGGs-hACE2-Flag and pCAGGs-Flag according to the manual instructions. After 24h post transfection, pCAGGs-transfected BSR cells and human ACE2-transfected BSR cells were infected with VSVΔG-eGFP-SARS at an MOI = 0.05. At 36h post-infection, the infected cells were stained with anti-Flag antibodies (Genscript, Nanjing, China) and Hoechst 33342 and observed with inverted fluorescence microscope.

    • Immunoelectron microscopy method was conducted to show the morphology of the recombinant viruses as described (Ge et al. 2011). The high-speed (10, 000 ×g) centrifuged recombinant viruses were adsorbed for 20 min by flotation onto a freshly discharged 200 mesh palladium carbon-coated nickel grids (Electron Microscopy Sciences, Hatfield, UK). For immunoelectron microscopy, mouse anti-SARS S antibody at a dilution of 1:100 (Invitrogen, Oregon, USA) was used as primary antibody, and 10 nm gold particle-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, USA) was used as the secondary antibody. Viral particles were examined under a model H7500 transmission electron microscope (Hitachi High Technologies, Schaumburg, USA) at 80 kV. Images were obtained by using an XR100 digital camera system (Advanced Microscopy Techniques, Danvers, USA).

    • For mouse immunizations, sixteen 6-week-old female Balb/c mice were randomly divided into two groups. In group 1, intramuscular (i.m) immunizations were performed with VSVΔG-SARS at a dose of 1×106 TCID50 in 0.1 mL via hind limb muscle injection. In group 2, i.m immunizations were performed with 0.1 mL PBS as a control. The mice received the booster dose injection three weeks post the prime immunization. Blood samples were collected at 21 days after the prime and boost immunizations.

      For monkey immunizations, five 2-year-old male rhesus monkeys were intramuscularly immunized with VSVΔG-SARS at a dose of 2×107 TCID50 via hind limb muscle injection under anesthesia. All monkeys were housed in separate cages in a Biosafety level-3 laboratory, and supplied with a specialized monkey puffed diet, various fresh fruits and adequate drinking water. Blood samples were collected at day 0, 10, 21, 28, and 42 post-immunizations.

    • SARS-CoV neutralization assay was adapted from our previously published protocol (Liu et al. 2017). Briefly, mouse or monkey serum samples were heat-inactivated at 56 ℃ for 30 min before use. Vero E6 cells were seeded in 96-well plates and grown into confluent monolayer overnight. Serum samples were two-fold serially diluted in DMEM (from 1:2 to 1:28), VSVΔG-eGFP-SARS were diluted to 1×102 TCID50 per well and added on top of serum dilutions. The virus-serum mix was incubated at 37 ℃ for 2h. The infected cells with virus-serum mixtures were scored for the inhibition of expressing GFP at 36h post-infection. The titer of neutralizing antibody is defined as the reciprocal of the highest dilution, at which the percentage of GFP expression is less than or equal to 50%.

    • The level of antibodies against SARS-CoV S protein was measured using an enzyme-linked immunosorbent assay (ELISA) as described before (Kong et al. 2012). Briefly, Vero E6 cells were infected with recombinant Newcastle disease virus expressing SARS-CoV S protein at an MOI = 0.1. The clarified ultrasonicated supernatant after detergent extraction was collected after 48h post-infection and coated in 96-well ELISA plates overnight at 4 ℃. The wells were washed and blocked for 1h at 37 ℃ with PBST containing 5% skim milk. Next, the mouse or monkey serum samples were 2-fold serially diluted and incubated at 37 ℃ for 1h. The plates were then washed three times with PBST, and anti-mouse IgG, IgG1 or IgG2a-peroxidase antibody (Southernbiotech, AL, USA) or anti-monkey IgG-peroxidase antibody (Southernbiotech, AL, USA) at a dilution of 1:5000 was added. After 1h of incubation 37 ℃, the plates were washed five times with PBST, and the color reaction was developed with 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) liquid substrate (Sigma, USA). The developing process was stopped after 15 min at 37 ℃ with 2 mol/L H2SO4, and the plates were read at OD450 with a Model 680 microplate reader (Bio-Rad). A standard curve was generated by coating mouse IgG at known concentrations, and a linear equation was built based on the IgG concentration and their OD values. The concentration of SARS-specific IgG in each sample was tested in duplicate and the mean absorbance was calculated according to the linear equation.

    • Enzyme-linked immune-spot (ELISPOT) assay was carried out as described before (Liu et al. 2018). A synthetic overlapping peptide pool spans the whole SARS-CoV S protein, which consisted of 249 peptides (15-mers with 10 amino acids overlapped, designed for CD8+ T cells) (Supplementary Table S1). Briefly, peripheral blood mononuclear cells (PBMCs) were separated from monkey blood at 10 days after immunization. Millipore 96-well HTS HA sterile plates (Millipore, USA) were pre-coated with purified mouse anti-human IFN-γ (BD Pharmingen, San Diego, USA), and blocked with RPMI 1640 medium with 10% FBS. Each 15-mers peptide from the whole SARS-CoV S protein synthetic overlapping peptide pool was diluted and added into the well. A total of 5×105 monkey PBMCs from the same group were pooled, added into the well and mixed with peptides. The monkey PBMCs were added with irrelevant peptides as negative control, and added 10 ng/mL phorbol myristate acetate (PMA) (Sigma, USA) with 1 µmol/L Ionomycin (Sigma, USA) as positive control (PMA+iono). After incubation at 37 ℃ for 24h, the plates were vigorously washed with PBS, and incubated with biotin-labeled mouse anti-human IFN-γ at a dilution of 1:3000 (BD Pharmingen, San Diego, USA) for 1h. After the final wash with PBS, AEC substrate (BD Pharmingen, San Diego, USA) was added to develop spots. The number of spots was counted by ElISPOT Reader (AID, Germany) and analyzed by GraphPad Prism 6 (GraphPad Software, CA, USA).

    • Statistical analyses were performed using one-way ANOVA with Bonferroni's multiple comparison tests. All P value < 0.05 was considered statistically significant. ***, P < 0.001. **, P < 0.01. *, P < 0.05.

    • As expected, the recombinant VSVΔG-SARS and VSVΔG-eGFP-SARS were successfully generated and rescued by using an established VSV reverse genetics system, in which the SARS-CoV spike (S) gene was inserted between the M and L gene instead of the VSV G gene (Fig. 1A). EGFP expression was observed in VSVΔG-eGFP-SARS-infected Vero E6 cells (Fig. 1C). Indirect confocal immunofluorescence staining and Western blot confirmed the expression of the S protein in VSVΔG-eGFP-SARS and VSVΔG-SARS-infected Vero E6 cells, which indicated the incorporation of S protein into the viral particles (Fig. 1B and 1C).

      Figure 1.  Generation of VSVΔG-SARS and VSVΔG-eGFP-SARS and expression of S protein. A Schematic representation of the genome structure in native VSV and the recombinant viruses. B Western blot analysis of S protein expression in recombinant VSVs-infected Vero E6 cells. C Indirect immunofluorescence staining of S protein expression in VSVΔG-SARS and VSVΔG-eGFP-SARS infected Vero E6 cells.

      VSVΔG-SARS and VSVΔG-eGFP-SARS grew to similar growth kinetic in Vero E6 cells, whose peak titers both reached 1×106.7 TCID50/mL at 72 h post-infection (Fig. 2). The recombinant viruses showed delayed growth kinetics and lower peak titer as compared with that of VSV. RT-PCR and IFA results demonstrated the stable expression of SARS-CoV S gene after 10 passages of VSVΔG-SARS and VSVΔG-eGFP-SARS in Vero E6 cells (Supplementary Figure S1).

      Figure 2.  Multistep growth properties of recombinant viruses in Vero E6 cells. Vero E6 cells planted on 6-well plate were respectively infected with native VSV, VSVΔG-SARS or VSVΔG-eGFP-SARS at an MOI = 0.01, the supernatant was harvested from 0h to 96h at 12h intervals and flash frozen in −80 ℃. The titer was expressed as the reciprocal of the highest dilution titer (Tissue culture infective dose, TCID50). Data were shown as mean ± SD.

      Figure Supplementary Figure S1.  The stable expression of SARS-CoV S gene in the recombinant viruses. RT-PCR (A) and IFA (B) results demonstrated the stable expression of SARS-CoV S gene after 10 passages of VSVΔG-SARS in Vero E6 cells. RT-PCR assay indicated S gene inserted in the genome of VSVΔG-SARS at passage1 (F1) (lane 1) and 10 (F10) (lane 2), with naive VSV as negative control (lane 3). Indirect immunofluorescence staining (red) indicated S protein expression in VSVΔG-SARS at F1and F10, and the cells were stained with Hoechst 33342 (blue).

    • The results of electron microscopy assay showed that S protein was successfully expressed on the surface of VSVΔG-SARS and VSVΔG-eGFP-SARS particles (Fig. 3, white arrows indicating S protein). Immunoelectron microscopy clearly showed that the recombinant VSVs were bound with gold-labeled anti-S antibodies. These data confirmed the efficient incorporation of S protein into VSVΔG-SARS and VSVΔG-eGFP-SARS.

      Figure 3.  SARS-CoV S protein is incorporation into the surface of the recombinant viral particles. The results of electron and immunoelectron microscopy showed that S protein is efficiently incorporated into the viral particles. White arrows indicate S protein and black arrows indicate VSV G protein.

      Since VSVΔG-SARS and VSVΔG-eGFP-SARS have SARS-CoV S protein instead of VSV G protein, their infection should solely depend on the SARS-CoV receptor, human ACE2 (hACE2). A SARS-CoV non-permissive cell line, hamster Syrian kidney cells (BSR cells), were transfected with pCAGGs-FLAG or pCAGGs-ACE2-FLAG (a eukaryotic plasmid encoding human ACE2 protein) and infected with VSVΔG-eGFP-SARS. Results showed that VSVΔG-eGFP-SARS couldn't infect pCAGGs-FLAG-transfected BSR cells, while hACE2-FLAG-transfected BSR cells were infected by the recombinant virus (Fig. 4), which indicated that the recombinant VSVs use hACE2 as receptors to infect cells. We also observed that syncytia formation in VSVΔG-eGFP-SARS and VSVΔG-SARS infected Vero E6 cells (Fig. 1C and Fig. 4), which indicated that the recombinant viruses rely on S protein fusion mechanism to mediate viral entry.

      Figure 4.  VSVΔG-SARS and VSVΔG-eGFP-SARS utilize human ACE2 as a functional receptor. pCAGGs-Flag transfected BSR cells and pCAGGs-hACE2(human ACE2)-Flag transfected BSR cells were infected with VSVΔG-eGFP-SARS. At 36h post-infection, the infected cells were observed with inverted fluorescence microscope.

    • The safety and immunogenicity of VSVΔG-SARS were evaluated in Balb/c mice at first. Mice were intramuscularly inoculated with 1×105 TCID50 VSVΔG-SARS and daily monitored. All the mice were healthy and did not show any sign of sickness. There was no significant difference between the experimental and the PBS control group mice in body weight gain (Fig. 5A). SARS-CoV neutralizing antibodies and S protein-specific IgG antibodies were detected after the first dose injection, and the antibody levels rose significantly after the booster dose injection (Fig. 5B and 5C-Ⅲ). Furthermore, VSVΔG-SARS induced high-level specific IgG1 (Th2) (Fig. 5C-Ⅰ) and IgG2a (Th1) antibodies (Fig. 5C-Ⅱ) after the second dose.

      Figure 5.  Humoral responses of VSVΔG-SARS-immunized mice. Sixteen mice were intramuscularly immunized with 1×105 TCID50 VSVΔG-SARS or PBS in the same volume. Each vaccinated mouse was daily monitored and weighed during the observation period (A). At 3 weeks after the initial inoculation, all mice were given the booster dose. Neutralizing antibody (B) and S-specific IgG (C) titers were detected by neutralization and ELISA assays. Data were shown as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    • To further evaluate the immunogenicity of the VSVΔG-SARS vaccine in non-human primates, the immunization experiment was performed in rhesus monkeys to monitor their humoral and cellular responses (Fig. 6). The rhesus monkeys were received single dose of 2×107 TCID50 of VSVΔG-SARS via the intramuscular (i.m) route. SARS-CoV neutralizing antibodies and S protein-specific IgG were detected 10-day after immunization, which reached their peak level at day 28 (Fig. 6A and 6B). Moreover, the ELISPOT assay was performed to further evaluate the T cell response in immunized monkeys. The PBMCS from the immunized monkeys were stimulated with a 15-peptide from an overlapping SARS-CoV S peptide pool, which consisted of 249 peptides designed for CD8+ T cells. Results showed that immunized rhesus monkeys produced the IFN-γ secreting T cells specific to the S protein peptide stimulation (Fig. 6C). There are six relatively concentrated peptide groups in the S protein synthetic overlapping peptide pool, which can produce most dominant spots in ELISPOT assay. The six most 'dominant' peptides (Fig. 6C) were selected from six peptide groups to list in Table 1, which induced the highest level of IFN-γ (400 spots/well on average). These peptides were speculated to contain S protein specific CD8+ T cell epitopes in rhesus monkeys.

      Figure 6.  Humoral immune responses and T cell responses of rhesus monkeys to VSVΔG-SARS vaccination. Monkeys were immunized with 2×107 TCID50 of the recombinant virus intramuscularly. Blood samples were collected at the indicated time-points. Neutralizing antibody (A) and serum IgG (B) titers were determined by neutralization and ELISA assays. C ELISPOT assay was performed to evaluate T cell responses in rhesus monkeys. PBMCs from immunized monkeys were stimulated with SARS-CoV S peptide from an overlapping peptide pool, which contained 249 peptides designed for CD8+ T cells. The data of six selected peptides were shown for presentation, which produced most spots in ELISPOT assay. The monkey PBMCs were added with irrelevant peptides as negative control, and added with PMA and ionomycin (PMA+iono) as positive control.

      Peptide # Peptide sequence Amino acid position
      S27 ELCDNPFFAVSKPMG 131–145
      S51 AYFVGYLKPTTFMLK 251–265
      S102 APATVCGPKLSTDLI 506–520
      S162 NKVTLADAGFMKQYG 806–820
      S217 GTSWFITQRNFFSPQ 1081–1095
      S229 HTSPDVDLGDISGIN 1141–1155
      #.Sequences were based on the S gene of SARS-CoV (isolate Urbani, GenBank accession No. AAP13441.1)

      Table 1.  T cell reactive most 'dominant' S protein peptides in monkeys vaccinated with VSVΔG-SARS.

    • SARS has been a highly threatening zoonotic disease. Although no more SARS cases have been reported in human populations since 2004, given the zoonotic nature and emergence possibility of SARS-CoV or SARS-like CoVs, developing a fast-response vaccine is urgently needed. Here we used the VSV reverse genetics system to successfully generate a recombinant chimeric virus VSVΔG-SARS as a candidate vaccine, in which the glycoprotein (G) gene of VSV is replaced with the spike (S) gene of SARS-CoV. The VSVΔG-SARS maintains the bullet-like shape of the native VSV, and can use S protein on its surface to bind with human ACE2 to infect host cells. Furthermore, the recombinant chimeric virus VSVΔG-SARS can induce high-level neutralizing antibodies and robust T cell responses in rhesus monkeys after a single-dose immunization, which indicates the potential of VSVΔG-SARS as a rapid response vaccine candidate for human use.

      To date, several SARS candidate vaccines have been reported, including inactivated virus vaccines, live-attenuated virus vaccines, subunit vaccines, DNA vaccines and recombinant viral vector vaccines (Bukreyev et al. 2004; He et al. 2004; Zhao et al. 2004; Li et al. 2005; Ge et al. 2006; Graham et al. 2012; Fett et al. 2013). Although these attempts are all worth the efforts, there is a crucial need for an effective response vaccine for the fast spread of diseases caused by SARS-CoV or SARS-like CoVs. Vesicular stomatitis virus (VSV) is a desired attenuated vector for developing commercial vaccines. First, VSV-vectored vaccines can trigger a rapid immune response, but DNA vaccines and subunit vaccines always need to immunize for several times (Zhao et al. 2004; Chen et al. 2005; Li et al. 2005). Our results showed a single-dose VSVΔG-SARS immunization could induce significant neutralizing antibodies and T cell responses in rhesus monkeys. Second, VSV can reach high titers in multiple cell lines, and be easily prepared in large quantity. VSVΔG-SARS grows to high titers in Vero E6, which indicates that its manufacture is technically and economically feasible in developing countries. Although an adenovirus-based vaccine described before was shown to induce SARS-CoV-specific immune responses in the monkeys, costly helper cell lines and high-dose immunizations do not promise its nice application (Gao et al. 2003). Thus, VSVΔG-SARS should be more practicable for applications among human populations for its highly-effectiveness and convenience, especially in the developing countries.

      Recently, SARS-CoV-2, related to SARS-CoV, has resulted in a global pandemic and shutdown of economies around the world (Zhou et al. 2020; Zhu et al. 2020). Some recent studies on the recombinant VSV-vectored vaccines for SARS-CoV-2 were reported (Case et al. 2020; Yahalom-Ronen et al. 2020). The study of VSV-∆G-MERS in our lab showed that intranasal administration did do better than intramuscular injection in monkeys (Liu et al. 2018). However, the advantage of intranasal administration is not obvious in our study, thus these data were not shown in our paper. We deduced that viral stability and the binding ability of receptors in experimental animals could be the reason for that. Systematic studies are needed to study the immunology on VSV-vectored vaccines of different major viral antigen proteins. Our study is just the beginning, in-depth studies are needed to investigate the immunogenicity of VSV-vectored vaccines against coronaviruses. Thus, developing a rapid response SARS vaccine can be an effective means to provide the protection for high-risk populations, which still have deep enlightenment and reference meaning for prevention and control of emerging coronaviruses in the future.

    • We thank Prof. Bernard Moss (University of Tennessee Health Science Center) for providing the VSV∆G-eGFP pseudovirus system. This study was supported by grants from the National Key Research and Development Program of China (2017YFD050 1804).

    • ZW and ZB designed the experiments. DS, XT, RL, DP, XW and JG carried out the experiments. DS, XT and RL analyzed the data. DS and RL wrote the paper. ZW and ZB checked and finalized the manuscript. All authors read and approved the final manuscript.

    • The authors declare that they have no conflict of interest.

    • The whole study was approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS) (approval number 2020-01-01JiPi). All animal experiments were performed in strict accordance with the regulations for the Institutional Animal Care and Ethics Committee (IACEC) of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

    • The online version of this article contains supplementary material, which is available to authorized users.

    • Overlapping peptide pool spanning the SARS-CoV S protein designed for CD8+ T cells.

    Figure (7)  Table (1) Reference (33) Relative (20)

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