A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters

Abstract

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Keywords: SARS-CoV-2; VSV; mRNA cap methyltransferase; vaccine.

FIG 1

Recovery and characterization of mtdVSVs expressing SARS-CoV-2 S proteins. (A) Strategy for insertion of SARS-CoV-2 S and its variants into the VSV genome. The codon-optimized full-length S, S1, RBD1, and RBD2 genes were amplified by PCR and inserted into the same position at the gene junction between G and L into the genome of the VSV Indian strain. Domain structure of the S protein. SP, signal peptide; RBD, receptor-binding domain; RBM, receptor-binding motif; FP, fusion peptide; HR, heptad repeat; CH, central helix; TM, transmembrane domain; CT, cytoplasmic tail. Organization of the negative-sense VSV genome. N, nucleocapsid gene; P, phosphoprotein gene; M, matrix protein gene; G, glycoprotein; L, large polymerase gene. A star indicates the D1762A mutation in the MTase catalytic site in the L protein. (B) Plaque morphology of rVSV expressing SARS-CoV-2 S antigens. The plaques for rVSV and rVSV-S_CoV-1 were developed after 24 h of incubation in Vero CCL-81 cells, whereas the plaques for all other viruses were developed at 48 h of incubation. An average number of 20 plaques for each virus is indicated. (C) Single-step growth curve. Confluent BSRT7 cells in 6-well plates were infected with each virus at a multiplicity of infection (MOI) of 1.0. After 1 h of absorption, fresh Dulbecco’s modified Eagle’s medium (DMEM) with 2% fetal bovine serum (FBS) was added. Aliquots (50 μl) of cell culture supernatants were harvested at the indicated time points, and virus titers were determined by plaque assay. Data are geometric mean titers (GMTs) ± standard deviation from n = 3 biologically independent experiments.

FIG 2

SARS-CoV-2 S and its truncations are highly expressed by mtdVSV. (A) Analysis of S protein expression by mtdVSV using SARS-CoV-1 antibody. BSRT7 cells in 6-well plates were infected with each recombinant virus at an MOI of 3.0. At 40 h postinfection, cells were lysed in 300 μl of lysis buffer, and 10 μl of lysate was analyzed by SDS-PAGE and blotted with anti-SARS-CoV-1 S protein antibody (top) or β-actin antibody (bottom). (B) Analysis of S protein expression by pCI using SARS-CoV-1 antibody. 293T cells were transfected with 2 μg of each plasmid. At 48 h posttransfection, cell lysates were collected for Western blot analysis using anti-SARS-CoV-1 S protein antibody. (C and D) Analysis of the expression of S and its truncations by mtdVSV using SARS-CoV-2 antibody. BSRT7 cells in 6-well plates were infected with each recombinant virus at an MOI of 1.0. At 20 h or 28 h postinfection, cells were lysed in 300 μl of lysis buffer, and 10 μl of lysate or supernatant was analyzed by SDS-PAGE and blotted with anti-SARS-CoV-2 S protein antibody (top), VSV G antibody (middle), or β-actin antibody (bottom). Western blots shown are the representatives of three independent experiments. (E and F) Analysis of the incorporation of S into VSV virions. Cell culture supernatants were collected from 10 confluent T150 flasks of BSRT7 cells were infected by rVSV-D1762A or rVSV-D1762A-S. Cell debris were removed by centrifugation at 10,000 × g for 5 min. Virus particles were purified through 10% sucrose cushion (E, left), followed by 20 to 50% sucrose gradient purification (F, left). Aliquots consisting of 3 μg of total protein from the 10% sucrose cushion (E, left) and 10 μg from the sucrose gradient purification (F, left) were analyzed by SDS-PAGE and stained with Coomassie blue. Duplicated samples were also blotted with anti-SARS-CoV-2 S protein antibody (E and F, right). SDS-PAGE and Western blots shown are the representatives of two independent experiments.

FIG 3

Attenuation and immunogenicity of rVSVs expressing SARS-CoV-2 antigens in IFNAR1^−/− mice. (A) Immunization schedule in IFNAR1^−/− mice. IFNAR1^−/− mice (n = 5) were inoculated intramuscularly with phosphate-buffered saline (PBS) or 1.0 × 10⁶ PFU of each of the rVSV-based vaccine candidates. Blood was collected from each mouse at weeks 2, 4, 6, and 10 for antibody detection. (B) Body weight changes after mtdVSV inoculation. Body weight was monitored every 3 to 4 days until week 6 postinoculation. (C) Measurement of SARS-CoV-2 RBD-specific antibody by enzyme-limited immunosorbent assay (ELISA). Highly purified RBD protein was used as the coating antigen for the ELISA. The dotted line indicates the detectable level at the lowest dilution. (D) Measurement of SARS-CoV-2 S-specific antibody by ELISA. Highly purified prefusion S (preS) protein was used as the coating antigen for the ELISA. The dotted line indicates the detectable level at the lowest dilution. Data are expressed as the geometric mean titers (GMTs) of five mice in each group ± standard deviation. Data were analyzed using two-way analysis of variance (ANOVA) (*, P < 0.05; **, P < 0.01).

FIG 4

Attenuation and immunogenicity of rVSVs expressing SARS-CoV-2 antigens in BALB/c mice. (A) Immunization schedule in BALB/c mice. BALB/c mice (n = 5) were inoculated intramuscularly with PBS or with 1.0 × 10⁶ PFU of each of the rVSV-based vaccine candidates, or with 50 μg of plasmid DNA vaccine. Two weeks later, animals were boosted with same construct at the same dose. Blood was collected from each mouse at weeks 2, 4, 6, and 8 for antibody detection. (B) Body weight changes after mtdVSV inoculation. Body weight of selected groups was monitored every 3 to 4 days until week 6 postinoculation. (C and D) Measurement of SARS-CoV-2 RBD-specific antibody by ELISA at weeks 2 (C) and 4 (D). Highly purified RBD protein was used as the coating antigen for the ELISA. The dotted line indicates the detectable level at the lowest dilution. (E) Comparison by ELISA of antibody response between rVSV-D1762A-S and pCI-S. Highly purified RBD protein was used as the coating antigen for the ELISA. Data are expressed as the geometric mean titers (GMTs) of five mice in each group ± standard deviation. Data were analyzed using Student’s t test (*, P < 0.05; **, P < 0.01.

FIG 5

Attenuation and immunogenicity of rVSVs expressing SARS-CoV-2 antigens in C57BL/6J mice. (A) Replication of VSV in C57BL/6J mice. C57BL/6J mice (n = 5, female) were inoculated intranasally with PBS or with 1.0 × 10⁶ PFU of rVSV, rVSV-D1762A, or rVSV-D1762A-S. VSV titer in lungs (B) and brains (C). At day 4 after inoculation, mice were sacrificed, and lungs and brains were collected for virus titer determination by plaque assay. Viral titers are the geometric mean titer (GMT) of 5 animals ± standard deviation. The limit of detection (LoD) is 2.0 log₁₀ PFU per gram of tissue (dotted line). VSV genomic RNA copies in lungs (D) and brains (E). Total RNA was extracted from each lung and brain sample. VSV genomic RNA copies were quantified by real-time reverse transcription-PCR (RT-PCR) using primers annealing to the VSV leader sequence and the N gene. Black bars indicate GMT of 5 mice in each group. The dotted line indicates the detection limit. (F) Immunization schedule in C57BL/6J mice. C57BL/6J mice (n = 10, 5 male and 5 female) were inoculated (half subcutaneously and half intranasally) with PBS or with 1.0 × 10⁶ PFU of each of the mtdVSV-based vaccine candidates. Two weeks later, animals were boosted with same virus at the same dose. Blood was collected from each mouse at week 4 for antibody detection. (G) Measurement of SARS-CoV-2 S-specific antibody by ELISA. Highly purified prefusion S protein was used as the coating antigen for the ELISA. The dotted line indicates the detectable level at the lowest dilution. (H) Measurement of SARS-CoV-2-specific neutralizing antibodies (NAbs) by plaque reduction/neutralization test (PRNT). Antibody titer was determined by PRNT. The 50% plaque reduction/neutralization titer (PRNT₅₀) was calculated from each serum sample. The dotted line indicates the detectable level at the lowest dilution. (I) Measurement of NAbs by a lentivirus-based pseudotype neutralization assay. The 50% inhibitory concentration (IC₅₀) from each serum sample was calculated. Ten sera from the rVSV-D1762A-S and rVSV-D1762A-S1 groups and 5 sera from rVSV-D1762A group were used for this assay. Data are expressed as the geometric mean titers (GMTs) of 5 mice in each group ± standard deviation. Data were analyzed using one-way ANOVA (*, P < 0.05; ****, P < 0.0001).

FIG 6

mtdVSV-based SARS-CoV-2 vaccines induce strong Th1-biased T-cell immune responses. The 5 female mice from each group shown in Fig. 5 were terminated at week 4 postimmunization for the T-cell assays. (A) Enzyme-linked immunosorbent spot assay (ELISPOT) quantification of IFN-γ-producing T cells. Spot-forming cells (SFC) were quantified after the cells were stimulated by peptides representing N (S1 peptides, pink) and C (S2 peptides, green) termini of the SARS-CoV-2 spike protein. A peptide pool of covering N protein SARS-CoV-2 (N peptide) was used as a control. Data are means of 5 mice ± standard deviation. *, P < 0.05; **, P < 0.01, determined by unpaired t test. (B) Intracellular cytokine staining of T cells in vaccinated mice. Splenocytes of 4 rVSV-D1762A-S (left) or 4 rVSV-D1762A-S1 (right) vaccinated mice were stimulated ex vivo for 5 h with pools of N, S1, or S2 peptides (5 μg/ml each), followed by staining for intracellular cytokines and analysis using multicolor flow cytometry. Frequencies of CD8⁺ T cells expressing cytokines represent CD8⁺ T cells expressing IFN-γ, TNF-α, IL-2, IL-17a, IL-21, IL-4, or IL-10. Data are means of 5 mice ± standard deviation and were analyzed using an unpaired t test (*, P < 0.05). (C) Flow plots showing T cells producing the three Th1 cytokines. Splenocytes of rVSV-D1762A-S-vaccinated (upper) and rVSV-D1762A-S1-vaccinated (lower) mice were stimulated with either S1 peptides or no peptides (one representative plot). CD8⁺ T cells were gated to show the frequencies of antigen-specific T cells producing IFN-γ, TNF-α, or both cytokines. Additionally, CD8⁺ T cells expressing IL-2 are shown in pink.

FIG 7

The mtdVSV-based SARS-CoV-2 vaccine is highly immunogenic in Golden Syrian hamsters. (A) Immunization schedule in hamsters. Four-week-old female Golden Syrian hamsters (n = 5) were immunized (half subcutaneously and half intranasally) with 1.0 × 10⁶ PFU rVSV-D1762A-S, rVSV-D1762A-S1, or rVSV-D1762A, or with PBS. Hamsters were boosted 3 weeks later. At weeks 2, 4, and 6, sera were collected for antibody detection. At week 7, hamsters were challenged with 1.0 × 10⁵ PFU SARS-CoV-2. Unimmunized, unchallenged controls were inoculated with DMEM. (B) Measurement of SARS-CoV-2 S-specific antibody. Highly purified preS protein was used as a coating antigen for ELISA. The dotted line indicates the detectable level at the lowest dilution. (C) Measurement of SARS-CoV-2-specific NAbs by PRNT. Antibody titer was determined by a plaque reduction/neutralization test. Human convalescent-phase sera from recovered COVID-19 patients were used as side-by-side controls. The PRNT₅₀ was calculated from each serum sample. The dotted line indicates the detectable level at the lowest dilution. (D) Measurement of NAbs by a lentivirus-based pseudotype neutralization assay. The IC₅₀ from each serum sample was calculated. Data are expressed as the geometric mean titers (GMTs) of 5 hamsters. Data were analyzed using two-way ANOVA or Student’s t test (*, P < 0.05; ****, P < 0.0001).

FIG 8

rVSV-D1762A-S provides complete protection against SARS-CoV-2 challenge in Golden Syrian hamsters. (A) Dynamics of hamster body weight changes after SARS-CoV-2 challenge. The body weight for each hamster was measured daily and is expressed as a percentage of the body weight on the challenge day. The average body weight of 5 hamsters (n = 5) in each group is shown. SARS-CoV-2 titer in lungs (B) and nasal turbinate (C). At day 4 after challenge, 5 hamsters from each group were sacrificed, and lungs and nasal turbinates were collected for virus titer determination by plaque assay. Viral titers are the geometric mean titer (GMTs) of 5 animals ± standard deviation. The limit of detection (LoD) is ∼2.7 to 2.8 log₁₀ PFU per gram of tissue (dotted line). SARS-CoV-2 genomic RNA copies in lungs (D), nasal turbinate (E), brain (F), liver (G), and spleen (H). Total RNA was extracted from the homogenized tissue using TRIzol reagent. SARS-CoV-2 genome copies were quantified by real-time RT-PCR using primers annealing to the 5′ end of the genome. SARS-CoV-2 subgenomic RNA copies in lungs (I), nasal turbinate (J), brain (K), liver (L), and spleen (M). SARS-CoV-2 subgenomic RNA copies were quantified by real-time RT-PCR using primers annealing to the N gene at the 3′ end of the genome. Black bars show GMTs of 5 hamsters in each group. The dotted line indicates the detection limit. Data were analyzed using two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 9

rVSV-D1762A-S immunization prevents a cytokine storm in the lungs. Total RNA was extracted from lungs of hamsters terminated at day 4 after challenge with SARS-CoV-2. Hamster IL-6 (A), IFN-γ (B), CXCL10 (C), TNF-α (D), IL-1b (E), IL-2 (F), and IFN-α1 (G) mRNAs were quantified by real-time RT-PCR. GAPDH mRNA was used as an internal control. Data are shown as fold change in gene expression compared to normal animals (unimmunized and unchallenged) after normalization. Data were analyzed using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 10

Lung pathology score after challenge with SARS-CoV-2. Fixed lung tissues from day 4 after SARS-CoV-2 challenge were embedded in paraffin, sectioned at 5 μm, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) for the examination of histological changes under light microscopy. Each slide was quantified based on the severity of histologic changes, which included interstitial pneumonia, alveolar damage, multinucleated giant cells, extensive inflammation, bronchiolitis, pulmonary hemorrhage, mononuclear cell infiltration, and peribronchiolar inflammation. 4 = extremely severe lung pathological changes; 3 = severe lung pathological changes; 2 = moderate lung pathological changes; 1 = mild lung pathological changes; 0 = no pathological changes.

FIG 11

rVSV-D1762A-S immunization protects lung pathology. Hematoxylin and eosin (H&E) staining of lung tissue of hamsters euthanized at day 4 after SARS-CoV-2 challenge is shown. Micrographs with ×1, ×2, ×4, and ×10 magnification of a representative lung section from each group are shown. Scale bars are indicated at the left corner of each image.

FIG 12

rVSV-D1762A-S immunization prevents SARS-CoV-2 antigen expression in the lungs. Immunohistochemistry (IHC) staining of lung sections from hamsters euthanized at day 4 after SARS-CoV-2 challenge is shown. Lung sections were stained with anti-SARS-CoV-2 N antibody. The same lung sections shown in Fig. 11 are presented to show the correlation of pathological change and SARS-CoV-2 antigen distribution. Micrographs with ×1, ×2, ×4, and ×10 magnification are shown. Scale bars are indicated at the left corner of each image.