A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike

Significance Measles virus (MeV) vaccine is one of the safest and most efficient vaccines with a track record in children. Here, we generated a panel of rMeV-based vaccines with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S antigens inserted near 3′ of the MeV genome. The rMeV expressing a soluble stabilized, prefusion spike (preS) is much more potent in triggering SARS-CoV-2–specific neutralizing antibody than rMeV-based full-length S vaccine candidate. A single dose of rMeV-preS is sufficient to induce high levels of SARS-CoV-2 antibody in animals. Furthermore, rMeV-preS induces high levels of Th1-biased immunity. Hamsters immunized with rMeV-preS were completely protected against SARS-CoV-2 challenge. Our results demonstrate rMeV-preS is a safe and highly efficacious bivalent vaccine candidate for SARS-CoV-2 and MeV.

Rapid assembly of the full-length genomic cDNA of MeV by yeast-based recombination system. The full-length genomic cDNA of Edmonston strain of measles vaccine was assembled into pYES2 vector. The pYES2 vector was modified to insert a yeast replication origin from the plasmid pYES1L (Invitrogen), a T7 RNA polymerase promoter, a hepatitis delta virus ribozyme (HDVRz) sequence, and a T7 terminator (1, 2). The full-length cDNA clone of MeV was constructed using six overlapping fragments (designated from A to F) by using yeast recombination system. Briefly, 100 ng of pYES2 vector was mixed with 200 ng of each MeV DNA fragment in PEG/LiAc solution, and the ligation products were transformed into MaV 203 competent yeast cells by heat-shock and plated on SD/Uraagar plates. After incubation for 3 days at 30℃, individual colony was picked, cultured in SD/Urabroth at 30℃ overnight for plasmid mini-prep.
For initial screening, the connection regions between fragments were amplified by PCR and sequenced. The positive plasmid was then transformed into TOP10 competent cells, and plasmid DNA was verified by restriction enzyme digestion, PCR analysis, and sequenced to confirm that no additional mutations were introduced during the assembly. The final plasmid was designated as pMeV-SARS-CoV-2 (Fig.S1). Primers used in this study was listed in Table S1. Using this method, SARS-CoV-2 full-length S (S), a stabilized prefusion S (preS) with deletion of the furin cleavage site, two proline mutations, and a foldon trimerization domain (3), S with deletion of the transmembrane domain and cytoplasmic tail (S-dTM), S1 subunit, and three different length of RBDs (RBD1, RBD2, and RBD3) containing MeV gene start and gene end sequences were inserting into the gene junction between P and M genes in the MeV genome. These plasmids were named pYES2-S, preS, S-dTM, S1, RBD1, RBD2, and RBD3. All the constructs were confirmed by sequencing. All the S genes and S truncations used in this study were codon optimized for mammalian cells expression.

Recovery of recombinant MeV (rMeV) expressing SARS-CoV-2 S antigens.
Recovery of rMeV from the infectious clone was carried out as described previously (2,4). Briefly, plasmid encoding the full-length genome of MeV Edmonston strain with S, preS, S-dTM, S1, or RBDs, and support plasmids encoding MeV ribonucleocapsid complex (pN, pP, and pL) were cotransfected into HEp-2 cells infected with a recombinant modified vaccinia Ankara virus (MVA-T7) expressing T7 RNA polymerase (kindly provided by Dr. Bernard Moss) (5). At day 4 posttransfection, cells and supernatants were collected, and co-cultured with 90% confluent Vero CCL81 cells. At day 4, the recovered recombinant virus was further amplified in Vero CCL81 cells. Subsequently, the viruses were plaque purified as described previously (6,7). Individual plaques were isolated, and seed stocks were amplified in Vero CCL81 cells. The viral titer was determined by a plaque assay performed in Vero CCL81 cells.

RT-PCR verification of SARS-CoV-2 gene.
To characterize the insertion of SARS-CoV-2 genes, viral RNA was extracted from rMeVs by using a RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions. SARS-CoV-2 S, preS, S-dTM, S1, RBD1, RBD2, and RBD3 genes were amplified by a One Step RT-PCR kit (Qiagen) using primers annealing to MeV P gene and MeV M gene. The amplified products were analyzed on 1% agarose gel electrophoresis and sequenced. Primers used for RT-PCR and sequencing of pYES2-SARS-CoV-2 are listed in Table S1.
Multi-step growth curves. Confluent monolayers of Vero CCL81 cells in 12-well-plates were infected with individual viruses at a multiplicity of infection (MOI) of 0.01. After 1 h of absorption, the inoculum was removed, the cells were washed twice with Dulbecco's modified Eagle's medium (DMEM), fresh DMEM (supplemented with 2% fetal bovine serum) was added, and the infected cells were incubated at 37°C. The cell culture fluid and cell lysates were harvested and combined at the indicated intervals, and virus titers were determined by plaque assay in Vero CCL81 cells.
MeV and SARS-CoV-2 plaque assays. MeV and SARS-CoV-2 plaque assay was performed on Vero CCL81 and Vero-E6 cells in 12-well plates, respectively. For MeV, confluent Vero CCL81 cells in 12-well plates were infected with serial dilutions of rMeV or rMeV expressing SARS-CoV-2 antigen in DMEM. Similar procedure was used for SARS-CoV-2 plaque assay. After absorption for 1 h at 37 °C, cells were washed three times with DMEM and overlaid with 2 ml of DMEM containing low-melting agarose (0.25% w/v). After incubation at 37°C for 4-5 days (MeV) or 2 days (SARS-CoV-2), cells were fixed with 4% paraformaldehyde for 2 h. The overlays were removed, and the plaques were visualized after staining by crystal violet. The diameter of plaques for each virus were measured using Image J Software.
Preparation of large stock of rMeVs. T150 flasks of Vero CCL81 cells were infected with individual rMeV at a MOI of 0.1. When extensive CPEs were observed at day 3 or 4, the supernatants were harvested and kept on ice. Cell pellets were subjected to three freeze-thaw cycles in 0.5 ml of fresh DMEM with 10% trehalose (8). The two portions of supernatants were combined and the virus titers were determined by plaque assay in Vero CCL81 cells.
Detection of SARS-CoV-2 S antigen by Western blot. Vero CCL-81 cells were infected with parental rMeV or rMeV expressing SARS-CoV-2 S antigens as described above. At the indicated times post-infection, cell culture medium was harvested and clarified at 5,000 g for 15 min. In the meantime, cells were lysed in RIPA buffer (Abcam, ab156034). Proteins were separated by 12% SDS-PAGE and transferred to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham) in a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). The blot was probed with rabbit anti-SARS-CoV-2 S or RBD antibody at a dilution of 1:2,000, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz) at a dilution of 1:5,000. The blot was developed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and exposed to Kodak BioMax MR film. were randomly divided into 9 groups, with 5 cotton rats per group (n=5). Cotton rats in groups 1-9 were inoculated subcutaneously with PBS, 4×10 5 PFU of each of Edmonston vaccine strain (parental rMeV, rMeV-S, rMeV-preS, rMeV-S1, rMeV-RBD1, rMeV-RBD2, or rMeV-RBD3).
Four weeks later, cotton rats were boosted with 2×10 6 PFU of each virus at the same immunization route. After inoculation, the animals were evaluated twice every day for any possible abnormal reaction. Blood samples were collected from each cotton rat at weeks 4, 6, and 8 by retro-orbital bleeding, and the serum was isolated for antibody detection.
Immunogenicity in IFNAR -/--hCD46 transgenic mice. IFNAR -/--hCD46 transgenic mice that are deficient for type I IFN receptor and transgenically express human CD46 (11,12) were bred in-house under SPF conditions. Twenty-one four-week-old female IFNAR1 -/--hCD46 mice were randomly divided into 4 groups (n=5, or 6). Mice in groups 1-3 were immunized with 8×10 5 PFU (half subcutaneous and half intranasal) of parental rMeV, rMeV-preS, or rMeV-S1. Mice in group 4 served as normal controls (unimmunized and unchallenged controls). Two weeks later, mice were boosted with 6×10 5 PFU of each virus (half subcutaneous and half intranasal). After inoculation, the animals were evaluated twice every day for safety. Blood samples were collected from each mouse at weeks 3 by facial vein bleeding, and the serum was isolated for antibody detection. At week 3 post-immunization, spleens were isolated from each mouse for a T cell assay.
Comparison of single and booster immunization of rMeV-preS in IFNAR -/mice. 4-weekold IFNAR -/mice female IFNAR1 -/mice were randomly divided into 3 groups (n=5, or 6). Mice in groups 1 were immunized with 8×10 5 PFU of rMeV-preS (half subcutaneous and half intranasal). Mice in group 2 were immunized with 8×10 5 PFU of rMeV-preS (half subcutaneous and half intranasal) and were boosted at the same dose at the same route 4 weeks later. Mice in group 3 were immunized with 8×10 5 PFU of rMeV and served as controls. At weeks 7 and 8, blood samples were collected from each mouse by facial vein bleeding, and the serum was isolated for detection of S-specific antibody by ELISA.
Immunization and challenge experiment in Golden Syrian hamsters. We selected 2 vaccine candidates (rMeV-preS and rMeV-S1) for immunization and challenge experiments in Golden Syrian hamsters. Forty 4-week-old female Golden Syrian hamsters were initially housed in BSL2 animal facility and randomly divided into 4 groups (n =10). Group 1 received 8×10 5 PFU of rMeV-preS, Group 2 received 8×10 5 PFU of rMeV-S1, Group 3 received 8×10 5 PFU of parental rMeV, and Group 4 received PBS. Three weeks later, hamsters in each group were boosted with the respective rMeV strain. All immunizations were done by combination of subcutaneous and intranasal routes (4×10 5 PFU for subcutaneous and 4×10 5 PFU for intranasal inoculation). At weeks 2, 4, and 6 post-immunization, blood was collected from each hamster for antibody detection. At week 4 post-booster immunization, animals of groups 1-3 were transferred into BSL3 facility and challenged intranasally with 10 5 PFU of SARS-CoV-2. Hamsters in group 4 were inoculated with DMEM and served as unimmunized unchallenged controls. After challenge, clinical sign and body weight of each hamsters were monitored daily. At day 4 post-challenge, 5 hamsters in each group were euthanized, left lung, nasal turbinate, brain, liver, and spleen were collected for detection of SARS-CoV-2 and viral RNA. In addition, the right lung was preserved in 4% (vol/vol) phosphate-buffered formaldehyde for histology and immunohistochemistry (IHC).
At day 12 post-challenge, the remaining 5 hamsters were terminated, and tissues were collected and processed as described above. Events were collected on a BD LSRFortessa X-20 flow cytometer following compensation with UltraComp eBeads (Invitrogen). Data were analyzed using FlowJo v10 (Tree Star).

Detection of SARS-CoV-2-specific antibody by ELISA.
Ninety-six-well plates were first coated with 50 µl of highly purified prefusion SARS-CoV-2 preS protein (8 µg/ml, in 50 mM Na2CO3 buffer, pH 9.6) per well at 4°C overnight, and then blocked with Bovine Serum Albumin (BSA, 1% W/V in PBS, 100 µl/well) at 37°C for 2 h. Subsequently, individual serum samples were tested for S-specific Ab on antigen-coated plates. Briefly, serum samples were 2-fold serially diluted and added to S protein-coated wells (100 µl/well). After 2 h of incubation at room temperature, the plates were washed three times with phosphate-buffered saline containing 0.05% Tween (PBST), followed by incubation with 100 µl of horseradish peroxidase (HRP)-conjugated secondary Abs After 1 h of incubation at 37°C, the virus-serum mixtures were removed and the cell monolayers were covered with 1 ml of Eagle's minimal essential media (MEM) containing 0.25% agarose, 0.12% sodium bicarbonate (NaHCO3), 2% FBS, 25mM HEPES, 2mM L-Glutamine, 100µg/ml of streptomycin, and 100U/ml penicillin. Then, the cells were incubated for another 2 days and then fixed with 4% formaldehyde. The plaques were counted; serum dilution with 50% plaque reduction were calculated as the SARS-CoV-2-specific neutralizing antibody titers.

Determination of SARS-CoV-2 titer in hamster tissues.
After SARS-CoV-2 challenge, left lung, nasal turbinate, brain, liver, and spleen was collected. Organs were weighed and homogenized by hand with a mortar and pestle (Golden, CO) in 1mL of sterile PBS. Each sample was subjected to 10-fold serial dilutions. The initial dilution of each tissue sample is 1: 10 StepOne real-time PCR system (Applied Biosystems). A standard curve was generated using a plasmid encoding the nucleocapsid (N) gene or full-length genome of SARS-CoV-2 plasmid.
Amplification cycles used were 2 min at 95°C, and 40 cycles of 15 s at 95°C, and 1 min at 60°C.
The threshold for detection of fluorescence above the background was set within the exponential phase of the amplification curves. For each assay, 10-fold dilutions of standard plasmid or viral RNA were generated, and negative-control samples and double-distilled water (ddH2O) were included in each assay. After real-time qPCR, the Ct value from each sample was converted into log10 viral RNA copies/mg tissue according to the standard curve. The RNA copies were calculated with the following formula: RNA copies/mg tissue = Log10 [Ct-converted copies/µl×10(2µl from 20µl total cDNA) ×25 (2µl from 50µl total RNA) ×10 (100µl from 1ml homogenized tissue) / tissue weight (mg)]. The LoD is set as the maximum value of the normal control group. The exact log10 RNA copies/mg was reported for each sample.

Quantification of cytokine in lungs of hamsters. Total RNA was extracted from lungs of Golden
Syrian hamsters, and IFN-α1, IFN-γ, IL-1b, IL-2, IL-6, TNF, and CXCL10 mRNAs were quantified by real-time RT-PCR (14,15). GAPDH mRNA was used as internal controls. The cytokine mRNA of each group was expressed as fold-change in gene expression compared to normal animals (unimmunized and unchallenged) after normalization. Primers used for RT-qPCR were listed in Table S2.   week-old IFNAR -/mice female IFNAR1 -/mice were randomly divided into 3 groups (n=5, or 6). Mice in groups 1 were immunized with 8×10 5 PFU of rMeV-preS (half subcutaneous and half intranasal). Mice in group 2 were immunized with 8×10 5 PFU of rMeV-preS (half subcutaneous and half intranasal) and were boosted at the same dose at the same route 4 weeks later. Mice in group 3 were immunized with 8×10 5 PFU of rMeV and served as controls. At weeks 7 and 8, blood samples were collected from each mouse by facial vein bleeding, and the serum was isolated for detection of S-specific antibody by ELISA. (B) Measurement of SARS-CoV-2 S-specific antibody by ELISA. Highly purified preS protein was used as the coating antigen for the ELISA. Dot line indicates the detectable level at the lowest dilution. Data are expressed as the geometric mean titers (GMT) of 5 or 6 mice ± standard deviation. Data were analyzed using Student's t-test (**P < 0.01; ns indicates no significant difference).