key: cord-0950999-034r6cft
authors: Sun, Weina; McCroskery, Stephen; Liu, Wen-Chun; Leist, Sarah R.; Liu, Yonghong; Albrecht, Randy A.; Slamanig, Stefan; Oliva, Justine; Amanat, Fatima; Schäfer, Alexandra; Dinnon, Kenneth H.; Innis, Bruce L.; García-Sastre, Adolfo; Krammer, Florian; Baric, Ralph S.; Palese, Peter
title: A Newcastle Disease Virus (NDV) Expressing a Membrane-Anchored Spike as a Cost-Effective Inactivated SARS-CoV-2 Vaccine
date: 2020-12-17
journal: Vaccines (Basel)
DOI: 10.3390/vaccines8040771
sha: e5f2383a0bcb815533da1b21d4868bd6e25f53f1
doc_id: 950999
cord_uid: 034r6cft

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.

In this study, we investigated NDV-S as an inactivated SARS-CoV-2 vaccine candidate with and without an adjuvant in mice and hamsters. We found that the S-F chimera expressed by the NDV vector is very stable, with no measurable loss of stability after 3 weeks of 4 °C storage in allantoic fluid. The beta-propiolactone (BPL)-inactivated NDV-S vaccine is immunogenic, inducing high titers of S-specific antibodies in both animal models. Furthermore, the effects of a clinical-stage investigational liposomal suspension adjuvant (R-enantiomer of the cationic lipid DOTAP (R-DOTAP)) [11] [12] [13] [14] , as well as an MF-59-like oil-in-water emulsion adjuvant (AddaVax), were also evaluated in mice. Both adjuvants were shown to achieve dose sparing (>10 fold) in mice. The vaccinated animals displayed less weight loss and significantly reduced viral loads in the lungs compared to the vector-only control animals after being challenged with a mouse-adapted SARS-CoV-2 strain. This is encouraging as the existing global egg-based production capacity for inactivated influenza virus vaccines could be utilized immediately to rapidly produce an egg-based NDV-S vaccine with minimal modifications to their production pipelines. Most importantly, this class of products is amenable to large-scale production at a low cost [15] [16] [17] . Alternatively, the NDV-S and other chimeric NDV vaccines could also be manufactured in cultured cells such as Vero cells [18] .

Animal studies were performed in accordance with protocols (PROTO202000098 and CEIRS program, 13-0386 PRYR II-IACUC-2013-1408) approved by the Institutional Animal Care and Use Committee (IACUC) at the Icahn School of Medicine at Mount Sinai. All animals were housed in a 

Animal studies were performed in accordance with protocols (PROTO202000098 and CEIRS program, 13-0386 PRYR II-IACUC-2013-1408) approved by the Institutional Animal Care and Use Committee (IACUC) at the Icahn School of Medicine at Mount Sinai. All animals were housed in a temperature-controlled biosafety level 2 (BSL-2) animal facility in the Annenberg building and Icahn building. All efforts were made to minimize animal suffering.

The construction of the NDV_LS/L289A_S-F rescue plasmid has been described in a previous study [10] . Briefly, the sequence of the ectodomain of the S without the polybasic cleavage site ( 682 RRAR 685 to A) was amplified from the pCAGGS plasmid encoding the codon-optimized nucleotide sequence of the S gene (GenBank: MN908947.3) of a SARS-CoV-2 isolate (Wuhan-Hu-1/2020) by a polymerase chain reaction (PCR) [19] , using primers containing the gene end (GE), gene start (GS), and Kozak sequences at the 5' end [20] . The nucleotide sequence of the transmembrane domain (TM) and the cytoplasmic tail (CT) of the NDV_LaSota fusion (F) protein was codon-optimized for mammalian cells and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) (gBlock). The amplified S ectodomain was fused to the TM/CT of F through a GS linker (GGGGS). Additional nucleotides were added at the 3' end to follow the "rule of six" of the paramyxovirus genome. The S-F gene was inserted between the P and M gene of the pNDV_LaSota (LS) L289A mutant (NDV_LS/L289A) antigenomic cDNA by in-Fusion cloning (Takara Bio USA Inc., Mountain View, CA, USA). This NDV_LS/L289A mutant is currently being used in a Phase I oncolytic NDV trial (NCT0413532). The recombination product was transformed into NEB ® Stable Competent Escherichia. coli (New England Biolabs Inc., Ipswich, MA, USA) to generate the NDV_LS/L289A_S-F rescue plasmid. The plasmid was purified using the PureLink TM HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific, Waltham, MA, USA).

BSRT7 cells stably expressing the T7 polymerase were kindly provided by Dr. Benhur Lee at ISMMS. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Gaithersburg, MA, USA) containing 10% (vol/vol) fetal bovine serum (FBS), 100 unit/mL of penicillin, and 100 µg/mL of streptomycin (P/S; Gibco) at 37 • C with 5% CO 2 . SARS-CoV-2 isolate USA-WA1/2020 (WA-1, BEI Resources NR-52281) used for hamster challenge was propagated in Vero E6 cells (ATCC CRL-1586) in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 2% fetal bovine serum (FBS), 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM Non-Essential Amino Acids, 1 mM Sodium Pyruvate, and 10 mM HEPES at 37 • C. All experiments with live SARS-CoV-2 were performed in the Centers for Disease Control and Prevention (CDC)/US Department of Agriculture (USDA)-approved biosafety level 3 (BSL-3) biocontainment facility of the Global Health and Emerging Pathogens Institute at the Icahn School of Medicine at Mount Sinai, in accordance with institutional biosafety requirements.

To rescue NDV_LS/L289A_S-F, six-well plates of BSRT7 cells were seeded 3 × 10 5 cells per well the day before transfection. The next day, 4 µg of pNDV_LS/L289A_S-F, 2 µg of pTM1-NP, 1 µg of pTM1-P, 1 µg of pTM1-L, and 2 µg of pCI-T7opt were re-suspended in 250 µL of Opti-MEM (Gibco, Gaithersburg, MA, USA). The plasmid cocktail was then gently mixed with 30 µL of TransIT LT1 transfection reagent (Mirus) [10] . The mixture was incubated at room temperature (RT) for 30 min. Toward the end of the incubation, the growth medium of each well was replaced with 1 mL of Opti-MEM. The transfection complex was added dropwise to each well and the plates were incubated at 37 • C with 5% CO 2 . The supernatant and cells from transfected wells were harvested at 48 h post-transfection, and briefly homogenized by several strokes using an insulin syringe. Two hundred microliters of the homogenized mixture was injected into the allantoic cavity of 8-to 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. The eggs were incubated at 37 • C for 3 days, before being cooled at 4 • C overnight. The allantoic fluid was collected and clarified by centrifugation. The rescue of NDV was determined by a hemagglutination (HA) assay using 0.5% chicken or turkey red blood cells. The RNA of the positive samples was extracted and treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA). A reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify the transgene. The sequences of the transgenes were confirmed by Sanger Sequencing (Genewiz, South Plainfield, NJ USA). Recombinant DNA experiments were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Biosafety Committee (IBC).

Before concentrating the virus, allantoic fluids were clarified by centrifugation at 3441× g using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 4 • C for 30 min to remove debris. Live virus in the allantoic fluid was pelleted through a 20% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) by ultra-centrifugation in a Beckman L7-65 ultracentrifuge at 25,000 rpm for two hours at 4 • C using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Supernatants were aspirated off and the pellets were re-suspended in PBS (pH 7.4). The protein content was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA). To prepare inactivated concentrated viruses, one part of 0.5 M disodium phosphate (DSP) was mixed with 38 parts of the allantoic fluid to stabilize the pH. One part of 2% beta-propiolactone (BPL) was added dropwise to the mixture during shaking, which gave a final concentration of 0.05% BPL. The treated allantoic fluid was mixed thoroughly and incubated on ice for 30 min. The mixture was then placed in a 37 • C water bath shaken every 15 min for two hours. The inactivated allantoic fluid was clarified by centrifugation at 3441× g for 30 min. The loss of infectivity was confirmed by the lack of growth (determined by the HA assay) of the virus (1:1000 dilution in PBS) in 10-day-old embryonated chicken eggs that were inoculated. The inactivated viruses were concentrated as described above.

The allantoic fluid containing the NDV_LS/L289A_S-F virus was harvested and clarified by centrifugation. The clarified allantoic fluid was aliquoted into 15 mL volumes. Week (wk) 0 allantoic fluid was concentrated immediately after centrifugation as described above, through a 20% sucrose cushion. The pelleted virus was re-suspended in 300 µL phosphate buffered saline (PBS) and stored at −80 • C. The other three aliquots of the allantoic fluid were maintained at 4 • C to test the stability of the S-F construct. Week 1, 2, and 3 samples were collected consecutively on a weekly basis, and concentrated virus was prepared in 300 µL PBS using the same method. The protein content of the concentrated virus from wk 0, 1, 2, and 3 was determined using the BCA assay after one freeze-thaw from −80 • C. One microgram of each concentrated virus preparation was resolved in 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA). The S-F protein and the NDV hemagglutinin-neuraminidase (HN) protein were detected by Western blot.

Concentrated live or inactivated virus samples were mixed with Novex™ Tris-Glycine SDS Sample Buffer (2X) (Thermo Fisher Scientific, Waltham, MA, USA) with NuPAGE™ Sample Reducing Agent (10X) (Thermo Fisher Scientific, Waltham, MA, USA). One or two micrograms of the concentrated viruses was heated at 95 • C for 5 min, before being resolved on 4-20% SDS-PAGE (Bio-Rad, Hercules, CA, USA), using the Novex™ Sharp Pre-stained Protein Standard (Thermo Fisher Scientific, Waltham, MA, USA) as the protein marker. To perform Western blots, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (GE healthcare, Chicago, IL USA). The membrane was blocked with 5% non-fat dry milk in PBS containing 0.1% v/v Tween 20 (PBST) for 1 h at room temperature (RT). The membrane was washed with PBST on a shaker three times (10 min at RT each time) and incubated with an S-specific mouse monoclonal antibody 2B3E5 (provided by Dr. Thomas Moran at ISMMS) or an HN-specific mouse monoclonal antibody 8H2 (MCA2822, Bio-Rad, Hercules, CA, USA) diluted in PBST containing 1% bovine serum albumin (BSA) overnight at 4 • C. The membranes were then washed with PBST on a shaker three times (10 min at RT each time) and incubated with secondary sheep anti-mouse IgG linked with horseradish peroxidase (HRP) diluted (1:2000) in PBST containing 5% non-fat dry milk. The secondary antibody was discarded and the membranes were washed with PBST on a shaker three times (10 min at RT each time). Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added to the membrane, and the blots were imaged using the Bio-Rad Universal Hood Ii Molecular imager (Bio-Rad, Hercules, CA, USA) and processed by Image Lab Software (Bio-Rad, Hercules, CA, USA).

Seven-week-old female BALB/cJ mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in this study. Experiments were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Mice were divided into 10 groups (n = 5) receiving the inactivated virus without or with an adjuvant at three different doses intramuscularly. The vaccination followed a prime-boost regimen with a two-week interval. Specifically, group 1, group 2, and group 3 received 5, 10, and 20 µg inactivated NDV-S vaccine (total protein) without the adjuvant, respectively; group 4, group 5, and group 6 received low doses of 0.2, 1, and 5 µg inactivated NDV-S vaccine, respectively, combined with 300 µg/mouse of R-DOTAP (PDS Biotechnology); group 7, group 8, and group 9 mice received 0.2, 1, and 5 µg inactivated NDV-S vaccine, respectively, with 50 µL/mouse of AddaVax (Invivogen) as the adjuvant; and group 10 received 20 µg inactivated wild type (WT) NDV as the vector-only control. The SARS-CoV-2 challenge was performed at the University of North Carolina by Dr. Ralph Baric's group in a biosafety level 3 (BSL-3) facility. Mice were intranasally (i.n.) challenged 19 days after the boost using a mouse-adapted SARS-CoV-2 strain at 7.5 × 10 4 plaque forming units (PFU) [2, 21] . Weight loss was monitored for 4 days.

Eight-week-old female golden Syrian hamsters were used in this study. Experiments were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Four groups (n = 8) of hamsters were included. The inactivated vaccines were given intramuscularly following a prime-boost regimen with a two-week interval. Group 1 received 10 µg of inactivated NDV-S vaccine, group 2 received 5 µg of inactivated NDV-S vaccine combined with 50 µL of AddaVax per hamster, and group 3 hamsters received 10 µg of inactivated WT NDV as the vector-only control. A healthy control group receiving no vaccine was also included. Twenty-four days after the boost, hamsters were challenged intranasally with 10 4 PFU of the USA-WA1/2020 SARS-CoV-2 strain in a biosafety level 3 (BSL-3) facility. Weight loss was monitored for 5 days.

The inferior lung lobes of mice were collected and homogenized in 1 mL PBS. Upper right (UR) and lower right (LR) lung lobes of hamsters were harvested at day 2 and day 5 post-infection. Each lung lobe of hamsters was homogenized in 1 mL PBS. A plaque assay was performed to measure the viral titer in the lung homogenates, as described previously [2, 10, 21] . Geometric mean titers of plaque forming units (PFU) per lobe (mice) or per mL (hamsters) were calculated using GraphPad Prism 7.0.

Mice were bled pre-boost and 11 days after the boost. Hamsters were bled pre-boost and 26 days after the boost. Sera were isolated by low-speed centrifugation. ELISAs were performed as described previously [19] . Briefly, Immulon 4 HBX 96-well ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 2 µg/mL of recombinant trimeric S protein produced in insect cells (50 µL per well) in coating buffer (SeraCare Life Sciences Inc., Milford, MA, USA) overnight at 4 • C [19] . The next day, all plates were washed three times with 220 µL PBS containing 0.1% (v/v) Tween-20 (PBST) and blocked in 220 µL blocking solution (3% goat serum, 0.5% non-fat dried milk powder, 96.5% PBST) for 1 h at RT. Both mouse sera and hamster sera were three-fold serially diluted in blocking solution starting at 1:30, followed by a 2 h incubation at RT. ELISA plates were washed three times with PBST and incubated in 50 µL per well of sheep anti-mouse IgG-horseradish peroxidase (HRP) conjugated antibody (GE Healthcare, Chicago, IL USA) or goat anti-hamster IgG-HRP conjugated antibody (Invitrogen, Carlsbad, CA, USA) diluted (1:3000) in blocking solution. Plates were washed three times with PBST and 100 µL of o-phenylenediamine dihydrochloride (SigmaFast OPD; Sigma, St. Louis, MO, USA) substrate was added per well. After developing the plates for 10 min, 50 µL of 3 M hydrochloric acid (HCl) was added to each well to stop the reactions. The optical density (OD) was measured at 492 nm on a Synergy 4 plate reader (BioTek, Winooski, VT, USA) or equivalents. An average of OD values for blank wells plus three standard deviations was used to set a cutoff for plate blank outliers. A cutoff value was established for each plate that was used for calculating the endpoint titers. The endpoint titers of serum IgG responses were graphed using GraphPad Prism 7.0. 

All neutralization assays were performed using Vero E6 cells in the biosafety level 3 (BSL-3) facility following institutional guidelines, as described previously [19, 22] . Serum samples were heat-inactivated at 56 • C for 60 min prior to use. Pooled sera in technical duplicates were serially diluted three-fold, starting at 1:20 dilution. The cells were fixed with 100 µL 10% formaldehyde per well for 24 h, before being taken out of the BSL-3 facility. A cell-based ELISA using an anti-NP antibody (1C7), kindly provided by Dr. Thomas Moran at ISMMS, was performed in a BSL-2 biosafety cabinet, as previously described [19, 22] . The OD of 492 nm was measured on a Biotek SynergyH1 Microplate Reader. Non-linear regression curve fit analysis (the top and bottom constraints were set at 100% and 0%, respectively) over the dilution curve was performed to calculate 50% of inhibitory dilution (ID 50 ) of the serum using GraphPad Prism 7.0.

The statistical analysis was performed using GraphPad Prism 7.0. The statistical difference in lung viral titers was determined using the Kruskal-Wallis test with Dunn's correction for multiple comparisons.

The stability of the antigen could be of concern as the vaccine needs to be purified and inactivated through a temperature-controlled (~4 • C) process. The final product is often formulated and stored in liquid buffer at 4 • C. To examine the stability of the S-F protein, allantoic fluid containing the NDV-S live virus was aliquoted into equal volumes (15 mL) and stored at 4 • C. Samples were collected weekly (wk 0, 1, 2, and 3) and concentrated through a 20% sucrose cushion. The concentrated virus was re-suspended in equal amounts of PBS. The total protein content of the four aliquots was comparable among the preparations (wk 0: 0.94 mg/mL; wk 1: 1.04 mg/mL; wk 2: 0.9 mg/mL; wk 3: 1.08 mg/mL). The stability of the S-F construct was evaluated by Western blot with the anti-S monoclonal antibody 2B3E5. Compared to the stability of the NDV HN protein, the Spike protein remained stable when kept in allantoic fluid at 4 • C (Figure 2A) . The inactivation by 0.05% BPL was confirmed by the lack of HA activity following inoculation of the inactivated virus into embryonated chicken eggs ( Figure 2B) . Importantly, the inactivation procedure using 0.05% BPL did not cause any loss of antigenicity of the S-F, as evaluated by Western blot ( Figure 2C ). These observations demonstrate that the membrane-anchored S-F chimera expressed by the NDV vector is very stable, without degradation at 4 • C for 3 weeks or when treated with BPL for inactivation.

For a pre-clinical evaluation of the inactivated NDV-S vaccine, the immunogenicity and dose-sparing ability of the adjuvants were investigated in mice. A dose-ranging study of the vaccine in the presence or absence of an adjuvant was evaluated based on the ability to induce antibody/neutralizing antibody responses. After partial purification, the vaccine preparation was administered intramuscularly, following a prime-boost regimen with a 2-week interval. Specifically, for the three unadjuvanted groups, mice were intramuscularly immunized with increasing doses of inactivated NDV-S vaccine at 5, 10, or 20 µg per mouse. Two adjuvants were tested here: A clinical-stage adjuvant, liposomal suspension of the pure R-enantiomer of the cationic lipid DOTAP (R-DOTAP) and the MF59-like oil-in-water emulsion adjuvant AddaVax. Each adjuvant was combined with low doses of NDV-S vaccines at 0.2, 1, and 5 µg. Mice receiving 20 µg of inactivated WT NDV were used as vector-only (negative) controls ( Figure 3A ). Mice were bled pre-boost (2 weeks after prime) and 11 days post-boost to examine antibody responses by ELISAs and micro-neutralization assays ( Figure 3A ) [19, 22] . After one immunization, all vaccination groups developed S-specific antibodies. The boost greatly increased the antibody titers of all NDV-S immunization groups. Immunization with R-DOTAP combined with 5 µg of vaccine induced the highest antibody titer. Immunization with one microgram of vaccine formulated with R-DOTAP or AddaVax and 5 µg of vaccine with AddaVax induced comparable levels of binding antibody, which is also similar to the titers induced by 20 µg of vaccine without an adjuvant. As expected, immunization with the inactivated wild-type NDV virus did not induce S-specific antibody responses ( Figure 3B ). We performed microneutralization assays to determine the neutralizing activity of serum antibodies collected from vaccinated mice. Except for mice immunized with the WT NDV, sera from all mice immunized with the NDV-S vaccine showed neutralizing activity against the SARS-CoV-2 USA-WA1/2020 strain. The neutralization titers induced by the immunization of 1 µg of vaccine with R-DOTAP (ID 50 of~476) and 5 µg of vaccine with AddaVax groups (ID 50 of~515) appeared to be the highest and were comparable to each other. These levels are also in the higher range of human convalescent serum neutralization titers, as measured in our previous studies [19, 23] . Interestingly, although the group receiving 5 µg of vaccine with R-DOTAP developed the most abundant binding antibodies detected by ELISA, these sera were not the most neutralizing ones, suggesting that R-DOTAP might have a different impact on immunogenicity compared to AddaVax. It is possible that with more antigen combined with R-DOTAP, the immune responses were skewed towards the induction of non-neutralizing antibodies ( Figure 3C ). In any case, these results demonstrated that the inactivated NDV-S vaccine expressing the membrane-anchored S-F was immunogenic, inducing potent binding and neutralizing antibodies. Importantly, at least 10-fold dose sparing was achieved with an adjuvant in mice. 

For a pre-clinical evaluation of the inactivated NDV-S vaccine, the immunogenicity and dose- DOTAP might have a different impact on immunogenicity compared to AddaVax. It is possible that with more antigen combined with R-DOTAP, the immune responses were skewed towards the induction of non-neutralizing antibodies ( Figure 3C ). In any case, these results demonstrated that the inactivated NDV-S vaccine expressing the membrane-anchored S-F was immunogenic, inducing potent binding and neutralizing antibodies. Importantly, at least 10-fold dose sparing was achieved with an adjuvant in mice. 

To evaluate vaccine-induced protection, mice were challenged 19 days post-boost using a mouse-adapted SARS-CoV-2 virus ( Figure 3A ) [2, 10, 21] . Weight loss was monitored for 4 days post-infection, at which point the mice were euthanized to assess pulmonary virus titers. Only the negative control group receiving the WT NDV, was observed to lose notable weight (~10%) by day 4 post-infection, while all of the vaccinated groups showed no weight loss ( Figure 4A ). Viral titers in the lung at 4 days post-challenge were also measured. As expected, the negative control group given the WT NDV exhibited the highest viral titer of >10 4 PFU/lobe. Groups receiving 5 µg of unadjuvanted vaccine or 0.2 µg of vaccine with R-DOTAP exhibited detectable but low viral titers in the lung, while all of the other groups were fully protected, showing no viral loads ( Figure 4B ). These results are encouraging as immunization with 0.2 µg of vaccine adjuvanted with AddaVax conferred a level of protection that was equal to that induced by immunization with 10 µg of vaccine without an adjuvant. Although 0.2 µg of vaccine with R-DOTAP did not induce sterilizing immunity, approximately, a 1000-fold reduction of viral titer in the lungs was achieved.

all of the other groups were fully protected, showing no viral loads ( Figure 4B ). These results are encouraging as immunization with 0.2 µg of vaccine adjuvanted with AddaVax conferred a level of protection that was equal to that induced by immunization with 10 µg of vaccine without an adjuvant. Although 0.2 µg of vaccine with R-DOTAP did not induce sterilizing immunity, approximately, a 1000-fold reduction of viral titer in the lungs was achieved. 

Golden Syrian hamsters have been characterized as a useful small animal model for COVID-19 as they are susceptible to SARS-CoV-2 infections and manifest SARS-CoV-2-induced diseases [24, 25] . Here, we conducted a pilot study that assessed the immunogenicity and protective efficacy of the inactivated NDV-S vaccine in hamsters. Female golden Syrian hamsters were immunized by a primeboost regimen with a 2-week interval via the intramuscular administration route. Twenty-four days after the booster immunization, hamsters were intranasally infected with 10 4 PFU of SARS-CoV-2 (USA-WA1/2020) virus. Animals were bled pre-boost and at 2 days post-infection (dpi). Lungs of a subset of animals were harvested at 2 dpi. The lungs of the rest of the animals were harvested at 5 dpi. Four groups of hamsters were included in this pilot study. Group 1 was immunized with 10 µg of inactivated NDV-S vaccine per animal without adjuvants. Group 2 received 5 µg of inactivated NDV-S vaccine with AddaVax as an adjuvant. Group 3 was immunized with 10 µg of inactivated WT NDV as the vector-only negative control group. Group 4, which was not vaccinated and was mock-challenged with PBS, was included as the healthy control group ( Figure 5A ). Serum IgG titers sampled prior to the booster immunization and at 2 dpi were measured by ELISA. One immunization 

Golden Syrian hamsters have been characterized as a useful small animal model for COVID-19 as they are susceptible to SARS-CoV-2 infections and manifest SARS-CoV-2-induced diseases [24, 25] . Here, we conducted a pilot study that assessed the immunogenicity and protective efficacy of the inactivated NDV-S vaccine in hamsters. Female golden Syrian hamsters were immunized by a prime-boost regimen with a 2-week interval via the intramuscular administration route. Twenty-four days after the booster immunization, hamsters were intranasally infected with 10 4 PFU of SARS-CoV-2 (USA-WA1/2020) virus. Animals were bled pre-boost and at 2 days post-infection (dpi). Lungs of a subset of animals were harvested at 2 dpi. The lungs of the rest of the animals were harvested at 5 dpi. Four groups of hamsters were included in this pilot study. Group 1 was immunized with 10 µg of inactivated NDV-S vaccine per animal without adjuvants. Group 2 received 5 µg of inactivated NDV-S vaccine with AddaVax as an adjuvant. Group 3 was immunized with 10 µg of inactivated WT NDV as the vector-only negative control group. Group 4, which was not vaccinated and was mock-challenged with PBS, was included as the healthy control group ( Figure 5A ). Serum IgG titers sampled prior to the booster immunization and at 2 dpi were measured by ELISA. One immunization with NDV-S vaccine with or without the adjuvant successfully induced spike-specific antibodies. Since there was no seroconversion from infection at 2 dpi indicated by the baseline level of the WT NDV sera, the increase in titers at 2 dpi compared to titers after vaccine priming most likely represents vaccine-induced antibody levels after the boost. As expected, the boost substantially increased the antibody titers in the NDV-S vaccination groups, whereas the WT NDV sera showed negligible binding signals ( Figure 5B ). Nevertheless, we cannot exclude a contribution from a rapid production of S antibodies by vaccine-induced memory B cells after exposure to SARS-CoV-2. Hamsters were challenged and weight loss was monitored for 5 days. The WT NDV group lost up to 15% of its weight by 5 dpi. Animals receiving 10 µg of inactivated NDV-S vaccine lost~10% of their weight by 3 dpi, at which point body weights started to recover. Animals receiving 5 µg of inactivated NDV-S vaccine with AddaVax only lost weight by 2 dpi, at which point body weights started to recover ( Figure 5C ). Viral titers in the upper right (UR) lung lobes and lower right (LR) lung lobes were also measured. The lung lobes were homogenized in 1 mL of PBS. Viral titers in the lung homogenates were measured by a plaque assay. Animals vaccinated with NDV-S with or without adjuvant displayed a substantial reduction of viral titers at 2 dpi, while the viral titers of these two groups at 5 dpi were below the limit of detection ( Figure 5D ).

Hamsters were challenged and weight loss was monitored for 5 days. The WT NDV group lost up to 15% of its weight by 5 dpi. Animals receiving 10 µg of inactivated NDV-S vaccine lost ~10% of their weight by 3 dpi, at which point body weights started to recover. Animals receiving 5 µg of inactivated NDV-S vaccine with AddaVax only lost weight by 2 dpi, at which point body weights started to recover ( Figure 5C ). Viral titers in the upper right (UR) lung lobes and lower right (LR) lung lobes were also measured. The lung lobes were homogenized in 1 mL of PBS. Viral titers in the lung homogenates were measured by a plaque assay. Animals vaccinated with NDV-S with or without adjuvant displayed a substantial reduction of viral titers at 2 dpi, while the viral titers of these two groups at 5 dpi were below the limit of detection ( Figure 5D ). Vaccine-induced serum IgG titers towards the trimeric spike protein were determined by ELISA. Endpoint titers are shown as the readout for ELISA. (C) Weight loss of hamsters challenged with SARS-CoV-2. Weight loss of SARS-CoV-2-infected hamsters was monitored for 5 days. (D) Viral titers in the lungs. Viral titers in the upper right (UR) and lower right (LR) lung lobes of the animals at 2 and 5 dpi were measured by a plaque assay (LoD: limit of detection). Statistical analysis was performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. P-values between groups were shown. 

We have previously reported NDV-based SARS-CoV-2 live vaccines expressing two forms of spike protein (S and S-F) [10] . Since the S-F showed superior incorporation into NDV particles, we investigated its potential to be used as an inactivated vaccine in this study. The NDV-S was found to be very stable when stored at 4 • C for 3 weeks, without degradation of the S-F protein. In mice, we have shown that a total amount of inactivated NDV-S vaccine as low as 0.2 µg could significantly reduce viral titers in the lung when combined with R-DOTAP, by approximately a factor of 1000, while the adjuvant AddaVax conferred even better protection. The NDV-S vaccine at 1 µg with either adjuvant elicited potent neutralizing antibodies and resulted in undetectable viral titers in the lung after SARS-CoV-2 challenge. These pre-clinical results demonstrate that antigen-sparing greater than 10-fold can be achieved in a mouse model, providing a valuable input for clinical trials in humans. In a pilot hamster experiment, the inactivated NDV-S vaccine is also immunogenic, inducing high titers of spike-specific antibodies. Since hamsters are much more susceptible to SARS-CoV-2 infection, the group receiving the WT NDV lost up to 15% of its weight by day 5, while both NDV-S vaccinated groups with or without the adjuvant showed greatly attenuated weight loss and reduced viral titers in the lungs. The AddaVax adjuvant again enhanced vaccine-induced protection, resulting in weight loss at only 2 dpi of the group. We did not evaluate the adjuvant R-DOTAP, as the dosing was not well-determined for this model by the time of this study. However, R-DOTAP and additional adjuvants will be evaluated in combination with the inactivated NDV-S vaccine in future studies. Moreover, the presented studies were highly focused on humoral responses and protection against the SARS-CoV-2 challenge in mice and hamsters as a proof of principle for the inactivated NDV-based vaccine, in which T cells and cytokine responses were not analyzed. It will be important to examine CD8+ T cells, CD4+ T cells, and Th1/Th2 responses in future preclinical studies with and without the adjuvant of choice to evaluate the mechanisms of protection and safety.

We have shown promising protection by immunization with inactivated NDV-S in both mouse and hamster models. Even though sterilizing immunity might not always be induced, the trade-off for having an affordable and widely available effective vaccine that reduces the symptoms of COVID-19 should be much preferred over a high-cost vaccine that is limited to high-income populations. Most importantly, the egg-based production of an NDV-S vaccine requires only minor modifications to the current inactivated influenza virus vaccine manufacturing process. The cost of inactivated influenza virus vaccines (trivalent and quadrivalent) is in the low-dollar range. Since NDV grows to similarly high titers as the influenza virus, the cost of goods should be similar to that of a monovalent inactivated influenza virus vaccine (a fraction of the cost of a quadrivalent seasonal influenza virus vaccine), or even lower due to dose sparing with an adjuvant that is inexpensive to manufacture.

We conclude that the inactivated NDV-S vaccine administered through the intramuscular route is immunogenic, inducing potent antibody responses targeting the spike protein in mice and hamsters. The vaccine attenuated weight loss and reduced viral loads in the lungs of mice and hamsters after the SARS-CoV-2 challenge. In the presence of an adjuvant, antigen-sparing could be achieved, which would potentially further ensure the low-cost of the vaccine when produced using the existing influenza virus vaccine capacity.

An mRNA Vaccine against SARS-CoV-2-Preliminary Report

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

Rapid development of an inactivated vaccine candidate for SARS-CoV-2

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: A dose-escalation, open-label, non-randomised, first-in-human trial

Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens

Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses

Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine candidate for Middle East respiratory syndrome: An open-label, phase 1 trial

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as vaccine candidate

Trp2 peptide vaccine adjuvanted with (R)-DOTAP inhibits tumor growth in an advanced melanoma model

Immunomodulation to enhance the efficacy of an HPV therapeutic vaccine

Combining R-DOTAP and a particulate antigen delivery platform to trigger dendritic cell activation: Formulation development and in-vitro interaction studies

Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response

Immunogenicity and Reactogenicity of an Inactivated Quadrivalent Influenza Vaccine Administered Intramuscularly to Children 6 to 35 Months of Age in 2012-2013: A Randomized, Double-Blind, Controlled, Multicenter, Multicountry, Clinical Trial

Immunogenicity and safety of an AS03-adjuvanted H7N1 vaccine in adults 65years of age and older: A phase II, observer-blind, randomized

Immunogenicity and Safety of an Inactivated Quadrivalent Influenza Vaccine in US Children 6-35 Months of Age During 2013-2014: Results From A Phase II Randomized Trial

Production of Newcastle disease virus by Vero cells grown on cytodex 1 microcarriers in a 2-litre stirred tank bioreactor

A serological assay to detect SARS-CoV-2 seroconversion in humans

Design and Production of Newcastle Disease Virus for Intratumoral Immunomodulation

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures

An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening

SARS-CoV-2 infection induces robust, neutralizing antibody responses that are stable for at least three months

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development

Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: Implications for disease pathogenesis and transmissibility

We thank Benhur Lee for kindly sharing the BSRT7 cells. We also thank Thomas Moran for the 2B3E5 and 1C7 antibodies.

The Icahn School of Medicine at Mount Sinai has filed patent applications entitled "RECOMBINANT NEWCASTLE DISEASE VIRUS EXPRESSING SARS-COV-2 SPIKE PROTEIN AND USES THEREOF".Vaccines 2020, 8, 771