key: cord-0985346-f43035nc
authors: Tioni, Mariana F.; Jordan, Robert; Pena, Angie Silva; Garg, Aditya; Wu, Danlu; Phan, Shannon I.; Cheng, Xing; Greenhouse, Jack; Orekov, Tatyana; Valentin, Daniel; Kar, Swagata; Pessaint, Laurent; Andersen, Hanne; Stobart, Christopher C.; Bloodworth, Melissa H.; Peebles, R. Stokes; Liu, Yang; Xie, Xuping; Shi, Pei-Yong; Moore, Martin L.; Tang, Roderick S.
title: One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2
date: 2021-08-06
journal: bioRxiv
DOI: 10.1101/2021.07.16.452733
sha: 13ec99d67567a10dd6af96509954ba5e566a91b8
doc_id: 985346
cord_uid: f43035nc

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200- fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase 1 clinical trials as a single-dose intranasal COVID-19 vaccine.

COronaVIrus Disease 2019 (COVID-19) is the latest virus pandemic to afflict humanity. The disease began its spread in the late months of 2019 1,2 , and by March 11, 2020, 118,000 people across 114 countries were infected, at which time it was declared a pandemic by the World Health Organization (WHO) 3 . COVID-19 is a respiratory disease often leading to pneumonia, caused by the highly transmissible Severe Acute Respiratory Syndrome Coronavirus 2 4 . The overall mortality rate is approximately 2% and the disease is especially severe in the elderly and in patients with serious underlying medical conditions, such as heart or lung disease and diabetes. As of July 30, 2021, there were 193,553,009 confirmed cases of SARS-CoV-2 infection with a total of 4,200,412 deaths worldwide (WHO dashboard, https://covid19.who.int/). SARS-CoV-2 is an enveloped RNA virus that relies on its surface glycoprotein, spike, for entry into host cells 5, 6 . The spike protein is a type I fusion protein that forms a trimer that protrudes on the viral membrane, giving the virus its characteristic crownlike appearance under electron microscopy 7, 8 . The angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor for SARS-CoV-2 spike 5, 9 . Disrupting the interaction of ACE2 and the receptor binding domain (RBD) of spike is at the core of vaccine design and therapeutics. Currently, three COVID-19 vaccines are approved for emergency use in the United States 10 . The three vaccines are based on the SARS-CoV-2 spike protein, and their high level of efficacy has validated spike as a protective antigen.

All the emergency use authorization (EUA) vaccines currently in use are delivered intramuscularly, and none is live attenuated. Live attenuated vaccines (LAVs) are often administered by the same route of entry as the pathogen they target, and LAVs replicate in the host, mimicking natural infection without causing disease. For respiratory viruses, intranasal LAVs generate mucosal immunity at the site of infection, blocking the pathogen at the earliest phases of infection thus helping control systemic spread 11 . In the case of influenza infection, 4 LAV induces better mucosal immunoglobulin A (IgA) and cell-mediated immunity relative to other vaccine types, eliciting a longer-lasting and broader immune response that more closely resembles natural immunity 12 . Furthermore, comparison of intramuscular vs intranasal vaccines against SARS-CoV in mice showed that serum IgA was only induced following intranasal vaccination 13 , and only intranasal vaccination provided protection in both upper and lower respiratory tracts 14 . For SARS-CoV-2 in particular, the early antibody response is dominated by IgA and mucosal IgA is highly neutralizing 15 , underscoring the importance of developing an intranasal vaccine capable of eliciting mucosal immunity. According to the WHO vaccine tracker, there are currently 108 vaccines in clinical trials, only 4 of which are intranasal replicating vaccines 16 .

Here we describe the rational design, generation, and preclinical evaluation of a novel COVID-19 vaccine candidate. MV-014-212 is a live attenuated recombinant vaccine strain derived from the human respiratory syncytial virus (RSV) vaccine candidate OE4 17 

MV-014-212 is a novel, live attenuated, recombinant vaccine against SARS-CoV-2, based on the backbone of the human respiratory syncytial virus (RSV) (Fig. 1) . The G and F proteins of RSV were replaced by a chimeric protein consisting of the ectodomain and transmembrane (TM) domains of SARS-CoV-2 spike (USA-WA1/2020) and the cytoplasmic tail of RSV F (strain line 19) . The rational design of this chimera was based on previous observations of the need for the homologous TM of spike in coronavirus infectious particle production [18] [19] [20] and the reported importance of the cytoplasmic tail of RSV F in the production of RSV progeny 21 . The sequence of amino acids at the junction between spike and F proteins is shown in Fig. 1 . Notably, the chimeric spike/RSV F protein retains functionality as MV-014-212 growth relies on it for attachment and fusion with the host cell. Moreover, since this is the only surface protein of MV-014-212, the possibility of negative interference due to preexisting immunity against RSV is eliminated. Various chimeric spike constructs that differed in the junction position were assessed for growth in Vero cells ( Supplementary Fig. S1 ). In particular, a construct with the entire native SARS-CoV-2 spike was evaluated (MV-014-300, Fig. S1 ). While this construct could be rescued, it did not propagate productively in cell culture, demonstrating that the cytoplasmic tail of the F protein contributes to viral growth of the chimeric virus expressing spike. Of the constructs expressing different chimeric spike/RSV F fusion proteins, MV-014-212 was selected for further evaluation based on the ease of rescue and its ability to grow to acceptable titers for preclinical and clinical studies.

The RSV backbone used to generate MV-014-212 was attenuated for replication in primary cells by codon deoptimization of the genes encoding the proteins NS1 and NS2 that suppress host innate immunity 22 . In addition, the short hydrophobic glycoprotein SH was deleted ( Fig. 1) to further attenuate the virus in vivo and increase transcription of downstream genes 17 .

To facilitate the development of a microneutralization assay, a reporter virus derived from MV-014-212 was constructed by inserting the gene encoding the fluorescent mKate2 protein 23, 24 upstream of the NS1 gene (MVK-014-212, Fig. 1 ).

The recombinant virus constructs were electroporated into Vero cells and infectious virus was rescued and propagated for further characterization 24 . In MV-014-212, CPE is observed as the formation of polynucleated bodies or syncytia and eventual cell detachment (Fig. 2a) . The electroporated cells were expanded until the CPE was extensive and the virus stock was harvested as a total cell lysate. The titers obtained for MV-014-212 and MVK-014-212 were comparable and within the range 1-5x10 5 PFU/mL.

The SARS-CoV-2 spike protein contains a cleavage site between the S1 and S2 domains that is processed by furin-like proteases 25 (Fig. 1) . As for other coronaviruses, the S1 and S2 subunits of SARS-CoV-2 spike are believed to remain noncovalently bound in the prefusion conformation after cleavage 26, 27 . To determine if the chimeric spike protein encoded by MV-014-212 is expressed and proteolytically processed, virus stocks prepared from lysates of infected Vero cells were analyzed on western blots and probed with polyclonal antiserum against SARS-CoV-2 spike protein. Both MV-014-212 and MVK-014-212 viruses express the full length and cleaved forms of the chimeric spike protein (Fig. 2b) , consistent with partial cleavage at the S1-S2 junction, with the expected apparent sizes 28, 29 (Supplementary Fig. S2 ).

Multicycle growth kinetics of MV-014-212 was compared to wild-type (wt) recombinant RSV A2 in Vero cells (Fig. 2c) . MV-014-212 exhibited delayed growth kinetics relative to RSV A2,

showing an initial lag phase of approximately 12 hours. Both viruses reached their peak titers at 7 approximately one order of magnitude lower than that of RSV A2. To determine if the insertion of the mKate2 gene affected replication kinetics of MVK-014-212, Vero cells were infected with MV-014-212 or MVK-014-212. The growth kinetics of MVK-014-212 was similar to that of MV-014-212, reaching comparable peak titers by 72 hpi (Fig. 2d) . These data are consistent with a report that insertion of mKate2 in the first gene position did not significantly attenuate RSV A2line 19F in vitro 24 .

To evaluate the short-term thermal stability of MV-014-212, aliquots of the viral stock were incubated at different temperatures for a period of 6 hours and the amount of infectious virus after the incubation was determined by plaque assay. Two stocks of MV-014-212 prepared in different excipients were compared in this study (Fig. 2e) . The results demonstrate that MV-014-212 is stable for at least 6 hours in either excipient at -80 °C and room temperature.

The genetic stability of MV-014-212 was examined by serial passaging in Vero cells.

Subconfluent Vero cells were infected in triplicate with an aliquot of MV-014-212 and passaged for 10 consecutive passages ( Supplementary Fig. S3 ). Viral RNA was isolated from passages 0 and 10 and amplified by reverse transcription polymerase chain reaction (RT-PCR). The sequence of the entire coding regions of the viral genome was determined by Sanger sequencing. The results showed that for all three lineages there were no variations detected at passage 10 relative to the starting stock (passage 0). Thus, the vaccine candidate was highly genetically stable in vitro.

African green monkeys (AGMs) are semi-permissive for replication of both wt SARS-CoV-2 [30] [31] [32] [33] and RSV 34 , and therefore constitute an appropriate nonhuman primate model for studying the attenuation and protective immunogenicity of MV-014-212. 8 The AGM study design is depicted in Fig. 3a . On Day 0, AGMs were inoculated via the intranasal (IN) and intratracheal (IT) routes with 1.0 mL of 3×10 5 PFU/mL MV-014-212 or wt RSV A2 at each site for a total dose of 6×10 5 PFU per animal. Due to the semi-permissive nature of the AGM model, IT inoculation was necessary to promote replication of the vaccine and the SARS-CoV-2 challenge virus in the lungs. Animals in the mock group were similarly mock-inoculated with phosphate-buffered saline (PBS). Nasal swabs (NS) and bronchoalveolar lavage (BAL) samples were collected through Day 12 after immunization. Viral shedding in NS and BAL samples was determined by plaque assay using fresh samples that were not frozen at the study site. The results showed that the level of infectious virus in animals inoculated with MV-014-212 and duration of shedding in nasal secretions were lower than in animals inoculated with RSV A2 (Fig. 3b and Supplementary Fig. S4 ). The mean peak titer for RSV A2 was approximately 20-fold higher than that observed for animals inoculated with MV-014-212. These results show that MV-014-212 is attenuated in the upper respiratory tract of AGMs, compared to RSV A2. Low to undetectable virus titers were also observed in the lower respiratory tract of animals inoculated with MV-014-212 or RSV A2 over the course of 12 days. Both viruses replicated at low levels, but peak levels occurred earlier for MV-014-212. In this study, RSV A2 showed 100to 1000-fold lower peak titers in the lower respiratory tract of AGMs compared with wt RSV A2 titers reported in literature [35] [36] [37] [38] , confounding the ability to assess attenuation of MV-014-212 in the lungs. Subsequently, lower titers were also observed in the lungs of cotton rats for the recombinant A2 used in this study (rA2 from Meissa Vaccines Inc., Supplementary Fig. S5 ), relative to biologically derived RSV strains, suggesting that the RSV A2 used in this AGM study was attenuated in lungs. Vaccination with MV-014-212 resulted in increased clearance of SARS-CoV-2 in lungs compared to RSV A2 or mock inoculation with PBS ( Fig. 3c and Supplementary Fig. S6 ). The peak titer of SARS-CoV-2 in BAL samples occurred at Day 2 and was similar in all three treatment groups. Lung titers were undetectable in MV-014-212-vaccinated animals on Days 4 through 10 whereas SARS-CoV-2 was readily measured in animals inoculated with RSV A2 or PBS. Shedding of infectious SARS-CoV-2 in BAL and NS after challenge was quantified by a 10 TCID50 assay ( Supplementary Fig. S7 ). The results show that AGMs vaccinated with MV-014-212 had on average between 100-and 1000-fold less infectious SARS-CoV-2 in BAL, relative to RSV A2 and PBS controls, at peak shedding days. The kinetics of clearance of the MV-014-212 group was also faster, in accordance with the results obtained by sgRNA qPCR assay. In NS, the average peak shedding of the MV-014-212 group was more than 1000-fold lower than that of the RSV A2 group (Fig. S7 ). In the NS of the mock-vaccinated group, however, one of the animals did not have detectable shedding of infectious virus at any of the time points (Fig. S7) .

As a result, the overall average peak shedding was only 30-fold higher in the mock group than that seen in the MV-014-212 vaccinated group.

Taken together, these data show that a single mucosal administration of MV-014-212 protected AGMs from wt SARS-CoV-2 challenge.

SARS-CoV-2 spike-specific serum immunoglobulin G (IgG) and nasal IgA were measured by enzyme-linked immunosorbent assay (ELISA) in sera and NS, respectively, from AGMs immunized with MV-014-212, RSV A2, or PBS on Day 25 post immunization. All animals were seronegative for RSV A2 and SARS-CoV-2 at the start of the study. AGMs inoculated with MV-014-212 produced higher levels of SARS-CoV-2 spike-specific IgG in serum compared to AGMs inoculated with RSV A2 or PBS, which had levels of spike-specific IgG that were close to the limit of detection (Fig. 4a ).

Spike-specific IgA was also detected in the nasal swabs of monkeys inoculated with MV-014-212. There was more than an 8-fold increase in nasal spike-specific IgA in the MV-014-212vaccinated animals 25 days after vaccination (Fig. 4b) . In contrast, RSV A2 or mock-vaccinated animals did not show a significant change in spike-specific IgA. Supplementary Fig. S8 ), the microneutralization assay showed that the NT50 of the AGM sera were approximately 4-fold lower than the human convalescent pool (Fig. 5c) . Lastly, the serum of one AGM vaccinated with MV-014-212 (the animal showing the highest titers in Fig. 5C ), was used in a conventional 50% plaque-reduction neutralization assay with SARS-CoV-2 in BSL-3 containment 39, 40 . In this assay, the neutralizing titers (PRNT50) against SARS-CoV-2 virus or the two variants of concern, B.1.351 and B.1.17, were determined (Fig. 5d) . The results showed that vaccination with MV-12 014-212 elicits neutralizing activity against the wt strain and both variants of concern (the preimmune serum titers were below the limit of detection for all the virus strains tested, data not shown). The neutralization titer of B.1.351 was equivalent to that of the wt strain and B.1.1.7

showed slightly higher neutralizing titers. Taken together, the data suggested that MV-014-212 elicited modest serum neutralizing antibody titers in AGMs, and there was cross-neutralization against the variants B.1.351 and B.1.1.7.

Mouse models of vaccine-associated enhanced respiratory disease (VAERD) suggest that an imbalance in type 1 (Th1) and type 2 (Th2) T helper cell immunity with a skewing towards Th2 response contributes to enhanced lung pathology following challenge 41 . To assess the balance of Th1 and Th2 immunity generated after vaccination with MV-014-212, transgenic mice expressing human ACE2 receptor were inoculated with a single dose of MV-014-212 or PBS by the intranasal route. A control group received an intramuscular prime and boost vaccination with SARS-CoV-2 spike protein formulated in alum, which has been shown to skew immunity towards a Th2 response 42 . On Day 28, serum was collected to measure total spike-specific IgG, IgG2a, and IgG1 by ELISA. In addition, spleens were collected and the number of splenocytes expressing interferon-γ (IFNγ) or interleukin (IL)-5 were measured by ELISpot assay. The ratio of IgG2a/IgG1 and the ratio of cells producing IFNγ/IL-5 are indicators of Th1-biased cellular immune response 42, 43 .

The results showed that MV-014-212 induced spike-reactive splenocytes as measured by ELISpot assay (Fig. 6a) . Importantly, MV-014-212 induced higher numbers of splenocytes expressing IFNγ relative to IL-5 when cell suspensions were stimulated with a spike peptide pool, suggesting that vaccination with MV-014-212 produced a Th1-biased immune response.

The ratio of IFNγ-producing cells to IL-5-producing cells in the MV-014-212 group was more 13 than one order of magnitude higher than in the group vaccinated with alum-adjuvanted spike protein (Fig. 6b) . Consistent with the ELISpot data, the ratios of IgG2a/IgG1 detected in serum were higher in the animals vaccinated with MV-014-212 than the control group vaccinated with alum-adjuvanted spike ( Fig. 6c and 6d) . These data suggest that intranasal vaccination with live attenuated, recombinant MV-014-212 induced a Th1-biased antiviral immune response. AGMs were selected as the model for evaluating the attenuation and immunogenicity of MV-014-212 because they support both RSV 34 and SARS-CoV-2 [30] [31] [32] [33] replication. The level of SARS-CoV-2 detected in the mock-treated AGMs in this study was as high or higher than the levels reported for the mock groups in other vaccine studies [44] [45] [46] , allowing comparison of the results between different monkey species. 15 The use of a semi-permissive animal model for the evaluation of the LAV candidate MV-014-212 constitutes a limitation of this study, since LAVs rely on replication to elicit a robust immune response. The semi-permissiveness of AGMs to RSV and SARS-CoV-2 would result in lower replication of MV-014-212 and a more modest serum neutralizing antibody response. In a more permissive host, like humans, MV-014-212 is expected to replicate to higher titers, potentially resulting in higher immunogenicity than that observed in AGMs.

Remarkably, immunization of AGMs with MV-014-212 resulted in both mucosal and systemic antibody responses. There was approximately 100-fold more spike-specific total serum IgG in MV-014-212-vaccinated AGMs compared to AGMs receiving wt RSV A2 or PBS inoculations.

Spike-specific IgA was also detected in nasal swabs of MV-014-212-immunized animals. There was approximately an 8-fold increase in IgA concentration 25 days following vaccination with MV-014-212. In an experimental human challenge study, low RSV F-specific mucosal IgA was a better predictor for susceptibility to RSV challenge in seropositive adults than serum IgG and neutralizing antibody levels 47 . Indeed, spike RBD-specific dimeric serum IgA was shown to be more potent at neutralizing SARS-CoV-2 than monomeric IgG 48 This response was also observed in a luciferase-based pseudovirus assay and in a conventional plaque reduction assay with SARS-CoV-2. The plaque reduction assay also detected a neutralizing response against 2 variants of concern, B. 

Vero reference cell bank (RCB)1 (WHO Vero RCB 10-87) cells were grown in minimal essential medium (MEM, Gibco, Thermo-Fisher Scientific,) containing 10% fetal bovine serum (FBS, Corning) and 1x Corning Antibiotic/Antimycotic mix consisting of 100 IU/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin, with 0.085 g/L NaCI. RCB2 cells were derived from RCB1 and adapted to grow in serum-free media. RCB2 cells used in this study were grown in serum-free medium OptiPro (Gibco) supplemented with 4 mM of L-glutamine (Gibco). Both Vero cell lines were cultured at 37 °C , 5% CO2, with 95% humidity.

African green monkeys (Chlorocebus aethiops) were obtained from St. Kitts and were of indeterminate age, weighing 3-6 kg. The monkeys were screened and verified to be seronegative for RSV and SARS-CoV-2 by an RSV microneutralization assay and spike SARS-CoV-2 ELISA (BIOQUAL). Animals also underwent a physical examination by the veterinary staff to confirm appropriate health status prior to study. Each AGM was uniquely identified by a tattoo. One male and 3 females were assigned to the MV-014-212 and RSV groups. Two females and 1 male were assigned to the mock group. Cage-side observations included mortality, moribundity, general health, and signs of toxicity. Clinical observations included skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, somatomotor, and behavior patterns. The body weight of each monkey was recorded before the start of the dosing period and at each time of sedation. 

Plaque assays for all the viruses used were performed in 24-well plates on Vero cells. Cells at 70% confluence were inoculated with 100 µL of 10-fold serial dilutions of viral samples (10 -1 to 10 -6 ). Inoculation was carried out at room temperature with gentle rocking for 1 hour before adding 0.75% methylcellulose (Sigma) dissolved in MEM supplemented with 10% FBS (Corning) and 1x Corning Antibiotic/Antimycotic mix. Cells were incubated for 4-5 days at 32 °C before fixing in methanol and immunostaining. For MV-014-212 and MVK-014-212, we used rabbit anti-SARS-CoV-2 spike polyclonal antibody (Sino Biological) and goat anti-rabbit HRPconjugated secondary antibody (Jackson ImmunoResearch). For rA2-mKate2, the reagents used were goat anti-RSV primary antibody (Millipore) and donkey anti-goat HRP-conjugated secondary antibody (Jackson ImmunoResearch). In all cases, the viral plaques were stained with AEC (Sigma). The limit of detection is 1 PFU per well, corresponding to a minimum detectable titer of 100 PFU/mL.

RNA from MV-014-212 samples was extracted using QIAamp ® Viral RNA Mini Kit following the protocol suggested by the manufacturer (Qiagen). The quality and concentration of the extracted RNA were evaluated by gel electrophoresis and UV spectrophotometry. The extracted RNA was used as the template for reverse transcription (RT) using Invitrogen SuperScript ® IV First-Strand Synthesis System using a specific primer or random hexamers. The cDNA 2nd strand was synthesized with the Platinum TM SuperFi TM PCR Master Mix. The purified PCR products were directly sequenced using the BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing reactions were purified using Sephadex G-50 purification and analyzed on ABI 3730xl DNA Analyzer. The sequence traces were assembled using Sequencher software and the assembly was manually confirmed. The RNA sequencing for this study was performed by Avance Biosciences Inc., Houston TX.

Viruses and control recombinant SARS-CoV-2 spike protein (LakePharma) were denatured with Laemmli sample buffer (Alfa Aesar) by heating at 95 °C for 10 minutes. Proteins were separated by SDS-PAGE in a 4%-15% gradient gel and transferred to polyvinylidene fluoride (PVDF) membranes using a transfer apparatus according to the manufacturer's protocol (BIO-RAD).

After transfer, blots were washed in deionized water and probed using the iBind Flex system according to the manufacturer's protocol (Invitrogen, Thermo-Fisher). Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc, Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was diluted in iBind Solution at 1:5000. Blots were washed in deionized water and developed with ECL system (Azure Biosystems) according to manufacturer's protocol. The blots were stripped with Restore Western Blot Stripping Buffer (Thermo-Fisher) and reprobed with goat anti-RSV polyclonal antisera (Sigma-Aldrich) and a monoclonal antibody specific for GAPDH (6C5) protein (Thermo-Fisher).

NS and BAL samples were collected and stored on ice until assayed for vaccine shedding by plaque assay. Vero cells were seeded in 0.5 mL per well at 1 x 10 5 cells/mL in culture media in 24-well plates. The plates were incubated overnight at 37 °C in a humid incubator containing 5% CO2. The samples were diluted in Dulbecco's modified Eagle medium (DMEM) without serum by adding 30 μL of nasal swab or BAL to 270 μL of DMEM. A total of six 10-fold serial dilutions were prepared in DMEM from 10 -1 to 10 -6 . The media were removed from the 24-well plate and 100 μL of each dilution was added to duplicate wells of the 24-well plate of Vero cells. The plate was incubated at room temperature with constant rocking on a Rocker 35EZ, Model Rocker 35D (Labnet) for 1 hour. At the end of this incubation, 1 mL of methyl cellulose media (MEM supplemented with 10% fetal bovine serum, 1x antibiotic/antimycotic, and 0.75% methyl 23 cellulose) was added to each well. The plate was incubated at 34 °C for 6 days in a humid incubator containing 5% CO2.

The plaques were visualized by immunostaining using RSV or SARS-CoV-2 antibodies. For immunostaining, the methyl cellulose media were aspirated, and the cell monolayers were washed with 1 mL of PBS at room temperature. The PBS was removed, and the cells were fixed by the addition of 1 mL of methanol to each well, and the plate was incubated at room temperature for 15 minutes. The methanol was removed, and cells washed with 1 mL of PBS followed by the addition of 1 mL Blotto solution (5% nonfat dried milk in Tris-buffered saline, Thermo-Fisher). The plates were incubated at room temperature for 1 hour. The Blotto solution was removed, and 0.25 mL of primary goat anti-RSV polyclonal antibodies (Millipore) diluted 1 to 500 in Blotto was added to RSV-infected cells. Cells infected with MV-014-212 were stained with primary rabbit anti-SARS-CoV-2 spike protein polyclonal antisera (Sino Biologicals, Beijing, China). The plates were incubated for 1 hour at room temperature with constant rocking.

Primary antibodies were removed, and wells were washed with 1 mL Blotto solution.

For RSV-infected cells, 0.25 mL of donkey anti-goat HRP-conjugated polyclonal antisera (Jackson ImmunoResearch) diluted 1:250 in Blotto was added to each well. For MV-014-212infected cells, goat anti-rabbit HRP-conjugated polyclonal antisera (Jackson ImmunoResearch) diluted 1:250 in Blotto was added to each well. The pate was incubated for 1 hour at room temperature with constant rocking. After incubation, the secondary antibodies were removed, and the wells washed with 1 mL of PBS. Developing solution was prepared by diluting AEC substrate 1:50 in 1x AEC buffer solution. A total of 0.25 mL of developing solution was added to each well and the plate was incubated at room temperature for 15 to 30 minutes with constant rocking until red immunostained plaques were visible. The developing reaction was terminated by rinsing the plate under tap water. The plaques were enumerated, and titers were calculated.

The standard curve was prepared from frozen RNA stocks and diluted to contain 10 6 to 10 7 copies per 3 μL. Eight 10-fold serial dilutions of control RNA were prepared using RNAse-free water to produce RNA concentrations ranging from 1 to 10 7 copies/reaction. The plate was placed in an Applied Biosystems 7500 Sequence detector and amplified using the following program: 48 °C for 30 minutes, 95 °C for 10 minutes followed by 40 cycles of 95 °C for 15 seconds, and 1 minute at 55 °C. The number of copies of RNA per mL of sample was calculated based upon the standard curve.

Total RNA from tissues was extracted using RNA-STAT 60 (Tel-test "B")/ chloroform followed by precipitation of the RNA and resuspension in RNAse-free water. To detect SARS-CoV-2 sgRNA, a primer set and probe were designed to detect a region of the leader sequence and E gene RNA from SARS-CoV-2. The E gene mRNA is processed during replication to contain a 5' leader sequence that is unique to sgRNA (not packaged into the virion) and therefore can be used to quantify sgRNA. A standard curve was prepared using known quantities of plasmid DNA containing the E gene sequence, including the unique leader sequence, to produce a concentration range from 1 to 10 6 copies/reaction. The PCR reactions were assembled using 45 μL master mix (Bioline) containing 2x buffer, Taq-polymerase, reverse transcriptase, and RNAse inhibitor. The primer pair was added at 2 μM, and 5 μL of the sample RNA was added to The plates were incubated for 20 hours at 37 °C and 5% CO2. The fluorescent foci in each well were counted using a Celigo Image Cytometer (Nexcelom) and converted to % inhibition using the formula below:

where MIN is the average number of foci obtained in the control wells with only cells (no virus) and MAX is the average number of foci from the wells in the control wells with only virus (no serum). L is the number of foci in the sample wells. The resulting curves of inhibition vs. dilution of the sera were fitted using non-linear regression, option "[inhibitor] vs normalized responsevariable slope" in GraphPad Prism (version 9.0.0). From the fitting, half-maximal inhibitory concentration (IC50) was obtained and NT50 was calculated as the reciprocal of IC50.

SARS-CoV-2 spike ΔCT protein-bearing vesicular stomatitis virus (VSV) pseudotyped particles in which the VSV glycoprotein G gene was replaced by the luciferase reporter gene were purchased from Nexelis, and the assay was performed at Meissa. Starting with a dilution of 1/25, serial 2-fold dilutions of heat-inactivated AGM sera were incubated with the pseudotyped viral particles (3 x 10 5 RLU/well) at 37 °C for 1 hour, after which the mixes were used to infect Vero E6 monolayers in white 96-well plates. The cells were incubated with the infection mixes for 20 ± 2 hours at 37 °C with 5% CO2. The medium was then discarded, and the cells were lysed in ONE-GloTM EX Luciferase Reagent (Promega). The lysis and luciferase reactions were allowed to proceed for 3 minutes at room temperature with shaking at 600 rpm and luminescence was read with a SpectraMax i3D plate reader. The values of luminescence were converted to percentage of inhibition using the equation shown in the previous paragraph where MIN is the average of reads obtained in the cell-only wells and MAX is the average of the reads from the wells of the pseudovirus-only control. L is the sample luminescence value. The inhibition vs dilution curves were fitted using nonlinear regression, option "[inhibitor] vs normalized response-variable slope" in GraphPad Prism (version 9.0.0). From the fitting IC50 was obtained and NT50 was calculated as the reciprocal of IC50.

A conventional 50% plaque-reduction neutralization test (PRNT50) was performed to quantify the serum-mediated virus suppression as previously reported 39 . Briefly, individual sera were 2-fold serially diluted in culture medium with a starting dilution of 1:40 (dilution range of 1:40 to 1:1280). The diluted sera were incubated with 100 PFU of USA-WA1/2020 or mutant SARS-CoV-2. After 1 hour of incubation at 37 °C, the serum-virus mixtures were inoculated onto a monolayer of Vero E6 cells pre-seeded on 6-well plates on the previous day. A minimal serum dilution that suppresses >50% of viral plaques is defined as PRNT50.

All recombinant SARS-CoV-2s with spike mutations 40 were prepared on the genetic background of an infectious cDNA clone derived from clinical strain USA-WA1/2020 62 .

Spike-specific IgG1 and IgG2a ELISA 29 Serum samples from mice were collected on Day -21 and on Day 28 post vaccination to quantify the levels of SARS-CoV-2 spike-specific IgG1 and IgG2a antibodies by ELISA. Purified perfusion-stabilized SARS-CoV-2 spike protein (SARS-CoV-2/human/USA/WA1/2020, from LakePharma) was diluted to 1 µg/mL in PBS and 100 μL was added to each well of a MaxiSorp immuno plate (Thermo-Fisher) and incubated overnight at 4 ºC. The plate was washed 4 times in PBST (PBS + 0.05% Tween 20) and 100 μL of blocking solution (PBST + 2% BSA) was added to each well, and the plate was incubated for 1 hour at room temperature. Serum dilutions were prepared in blocking solution with the first dilution at 1:25 for the IgG1 assay or 1:10-1:100 for the IgG2a assay. SARS-CoV-2 spike IgG1 (Sino Biological) or anti-spike-RBD-mIgG2a (InvivoGen) were diluted in blocking solution and used as standards for the assay.

The blocking solution was removed and 100 μL of diluted antibody added to each well. The plate was incubated at room temperature for 1 hour and then washed 4 times in PBST using the plate washer. Then, 100 μL of HRP-conjugated goat-anti-mouse IgG1 (Thermo-Fisher) or HRPconjugated goat-anti-mouse IgG2a (Thermo-Fisher) secondary antibodies diluted 1:32,000 and 1:1000, respectively, were added to each well and the plate was incubated at room temperature for 1 hour. The plate was washed 4 times in PBST. 100 μL of 1-step ultra TMB-ELISA substrate solution (Thermo-Fisher) was added to each well and the plate incubated for 30 minutes with constant rocking on an orbital shaker. After the incubation period, 100 μL of stop solution (Invitrogen) was added to each well and the plate read on a Spectramax id3 plate reader (Molecular Devices) at 450 nm and 620 nm.

Spleens from vaccinated ACE2 mice were collected on Day 28 post inoculation and stored in DMEM containing 10% FBS on ice until processed. The spleens were homogenized on a sterile petri dish containing medium. The homogenate was filtered through a 100-μm cell strainer and the cell suspension transferred to a sterile tube on ice. The cells were collected by centrifugation The ELISpot assay was performed using a mouse IFNγ/IL-5 Double-Color ELISPOT assay kit (Cell Technology Limited). Murine IFNγ/IL-5 capture solution and 70% ethanol was prepared according to the manufacturer's protocol. The membrane on the plate was activated by addition of 15 μL of 70% ethanol to each well. The plate was incubated for less than 1 minute at room temperature followed by addition of 150 μL PBS. The underdrain was removed to drain the solution in the wells and each well was washed twice with PBS. Murine IFNγ/IL-5 capture solution (80 μL) was added to each well and the plate was sealed with parafilm and incubated at 4 ºC overnight. The capture solution was removed, and the plate washed once with 150 μL PBS. A peptide pool containing peptides of 15 amino acids in length that span the SARS-CoV-2 spike protein (PepMix™ SARS-CoV-2 spike Glycoprotein, JPT Peptide Technologies) were prepared at 10 mg/mL and 100 μL was added to each well. A positive control containing Concanavalin A (Con A) mitogen (10 μg/mL) was added to a separate reaction mixture. The splenocytes were mixed with CTL-Test™ Medium (Cell Technology Limited) to yield a final cell 31 density of 3,000,000 cells/mL and 100 μL/well were added to the plate using large orifice tips.

The plate was incubated at 37 ºC in a humidified incubator containing 9% CO2 for 24 hours. The plates were washed twice with PBS and then twice with 0.05% Tween-PBS at a volume of 200 μL/well for each wash followed by addition of 80 μL/well anti-murine IFNγ/IL-5 detection solution. The plates were incubated at room temperature for 2 hours. The plate was washed 3 times with PBST at 200 μL/well for each wash followed by the addition of 80 μL/well of tertiary solution. The plates were incubated at room temperature for 1 hour. The plate was washed twice with PBST, and then twice with 200 μL/well of distilled water. Blue Developer Solution was added at 80 μL/well and the plate was incubated at room temperature for 15 minutes. The plate was rinsed 3 times in tap water to stop the developing reaction. After the final wash, red developer solution was added at 80 μL/well and the plate was incubated at room temperature for 5-10 minutes. The plate was rinsed 3 times to stop the developing reaction. The plate was air-dried for 24 hours face-down on paper towels on the bench top. The spots on the plate representing splenocytes expressing IFNγ (red) or IL-5 (blue) were quantified using the CTLimmunospot plate reader (ImmunoSpot 7.0.23.2 Analyzer Professional DC\ImmunoSpot 7, Cellular Technology Limited) and software (CTL Switchboard 2.7.2).

Source data are provided with this paper. The sequences of the antigenomes of MV-014-212 and MVK-014-212 are deposited in GenBank with accession numbers MZ695841 and MZ695842.

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We thank Anthony Cook, Renita Brown, Tammy Putmon-Taylor, Tracey-Ann Campbell, Shanai Browne, Zack Flinchbaugh, Elyse Teow and Jason Velasco at Bioqual for animal care support and technical expertise with our African green monkey study. We thank Douglas Haney for biostatistics support and Nexelis for sharing pseudovirus neutralization assay and reagents. We 

CT, cytoplasmic tail; FP, fusion peptide; IFP, internal fusion peptide; HR1 and 2, heptad repeats 1 and 2; NTD, N-terminal domain; RBD, receptor binding domain; S1, subunit S1; S2, subunit S2; S1/S2 and S2', protease cleavage sites; TM, transmembrane domain. LOD is 20 and is indicated as a dashed line.In all cases, the data shown is the average of 2 technical replicates. Statistical analysis was one-way ANOVA. *p < 0.05. AGM, African green monkey; ANOVA, analysis of variance; LOD, limit of detection; RSV, respiratory syncytial virus; wt, wild type. immunoglobulin isotype was determined from standard curves generated with purified SARS-CoV-2 spike specific monoclonal IgG2a or IgG1 antibodies.d Log of the ratio of IgG2a/IgG1 (as shown in c). Statistical analysis is an unpaired t-test. ****p < 0.0001. ACE, angiotensin-converting enzyme; ELISA, enzyme-linked immunosorbent assay; hACE2, human ACE2; IFN, interferon; Ig, immunoglobulin; IL, interleukin; PBS, phosphatebuffered saline; Th, helper T cell.