key: cord-0887378-pyw4nbxa
authors: Laczkó, Dorottya; Hogan, Michael J.; Toulmin, Sushila A.; Hicks, Philip; Lederer, Katlyn; Gaudette, Brian T.; Castaño, Diana; Amanat, Fatima; Muramatsu, Hiromi; Oguin, Thomas H.; Ojha, Amrita; Zhang, Lizhou; Mu, Zekun; Parks, Robert; Manzoni, Tomaz B.; Roper, Brianne; Strohmeier, Shirin; Tombácz, István; Arwood, Leslee; Nachbagauer, Raffael; Karikó, Katalin; Greenhouse, Jack; Pessaint, Laurent; Porto, Maciel; Putman-Taylor, Tammy; Strasbaugh, Amanda; Campbell, Tracey-Ann; Lin, Paulo J.C.; Tam, Ying K.; Sempowski, Gregory D.; Farzan, Michael; Choe, Hyeryun; Saunders, Kevin O.; Haynes, Barton F.; Andersen, Hanne; Eisenlohr, Laurence C.; Weissman, Drew; Krammer, Florian; Bates, Paul; Allman, David; Locci, Michela; Pardi, Norbert
title: A single immunization with nucleoside-modified mRNA vaccines elicits strong cellular and humoral immune responses against SARS-CoV-2 in mice
date: 2020-07-30
journal: Immunity
DOI: 10.1016/j.immuni.2020.07.019
sha: 19ee89a91a073f7d38a062ed3ee6fc16321983dd
doc_id: 887378
cord_uid: pyw4nbxa

Summary SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Δfurin mRNA-LNP CD4 + CD8 + Strong Th1 response CD4 + CD8 + Strong Th1 response

Long-lived plasma cells Strong neutralizing antibody response INTRODUCTION SARS-CoV-2, the causative agent of COVID-19, causes significant mortality and morbidity worldwide and was declared a pandemic by the World Health Organization in March, 2020 (Cucinotta and Vanelli, 2020) . The rapid spread of the virus has caused not only a significant health care burden but also an economic crisis. Governments around the world have introduced strict social distancing measures to keep transmission under control. However, a vaccine will ultimately be required to fully suppress the SARS-CoV-2 pandemic. A wide variety of vaccine designs are being developed against SARS-CoV-2, and there has been promising preclinical and clinical efficacy data published for at least four different vaccine candidates Jackson et al., 2020; Smith et al., 2020; Yu et al., 2020) .

Messenger RNA (mRNA)-based vaccines have recently demonstrated great promise in the fight against infectious diseases (Alameh et al., 2020) . One of the most widely studied of these vaccine platforms uses antigen-encoding nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNP) (Pardi et al., 2015) .

Nucleoside-modified mRNA-LNP vaccines have induced protective immune responses against various pathogens in preclinical studies (Awasthi et al., 2019; Espeseth et al., 2020; Freyn et al., 2020; Meyer et al., 2018; Pardi et al., 2018a; Pardi et al., 2017; Pardi et al., 2018c; Richner et al., 2017; Roth et al., 2019) , in many cases after administration of a single dose (Freyn et al., 2020; Pardi et al., 2018a; Pardi et al., 2017; Pardi et al., 2018c) . This vaccine type has been shown to elicit particularly strong CD4 + T cell, germinal center B cell, and long-lived plasma cell responses associated with durable, protective neutralizing antibody responses (Lindgren et al., 2017; Pardi et al., 2018a) .

Of note, a very limited amount of published human clinical data is available on the safety and efficacy of mRNA-LNPs (Bahl et al., 2017; Feldman et al., 2019) , and no mRNA vaccines for humans have been licensed to date.

In this study, we assessed the immunogenicity of two nucleoside-modified mRNA-LNP vaccines targeting the spike (S) glycoprotein of SARS-CoV-2: one encoding the full length S protein with deleted furin cleavage site and the other encoding the S protein receptor binding domain (RBD). We evaluated immune responses after a single intramuscular (i.m.) injection with the SARS-CoV-2 mRNA-LNP or control vaccines in BALB/c mice. We found that both mRNA vaccines induced potent CD4 + and CD8 + T cell responses in the spleen and lung. Moreover, we measured strong long-lived plasma cell and memory B cell responses associated with high titers of neutralizing antibodies.

Importantly, our nucleoside-modified (1-methylpseudouridine-containing) SARS-CoV-2 mRNA-LNP vaccines are very similar to clinical vaccine candidates utilized by Moderna Therapeutics (Jackson et al., 2020) and BioNTech RNA Pharmaceuticals in partnership with Pfizer Inc. (Mulligan et al., 2020) ; thus, we believe that the studies described within this manuscript may inform ongoing clinical trials and the design of future human trials.

We designed and produced mRNAs encoding three potential SARS-CoV-2 vaccine antigens: full length S protein (wild type; WT), full length S protein with a deleted furin cleavage site (∆furin), and a short construct encoding the soluble RBD of S protein. The ∆furin mutant was included as a potential way to stabilize the full length S and to maintain the covalent association of the S1 and S2 subunits (Kirchdoerfer et al., 2016) , while the RBD was investigated as it is a critical target of neutralizing antibodies against SARS-CoV-2. Protein expression from mRNAs was confirmed by in vitro cell transfection studies. RBD protein secretion was demonstrated by ELISA using supernatant from RBD mRNA-transfected 293F cells ( Figure 1A ). As the full length WT and ∆furin S proteins contain the transmembrane domain, they were expressed on the surface of transfected 293F cells. Thus, we used flow cytometry to assess binding of full length WT and ∆furin S proteins by an anti-RBD monoclonal antibody, D001, and a human ACE2-Fc (hACE2-Fc) fusion protein. Interestingly, we found that the full length ∆furin S protein showed higher binding capacity to D001 and hACE2-Fc compared to its WT counterpart, indicating that it may be a better vaccine antigen, due either to higher expression or favorable antigenicity ( Figure 1B ). Therefore, we selected the full length ∆furin construct to evaluate in immunization studies along with RBD.

BALB/c mice were injected with a single i.m. dose of 30 µg of mRNA-LNPs encoding full length ∆furin, RBD, or firefly luciferase (Luc, negative control) mRNA-LNPs, and S protein-specific CD4 + and CD8 + T cell responses were evaluated after 10 days by intracellular cytokine staining (Figures 2 and S1-2). Both spike mRNA constructs elicited antigen-specific, polyfunctional CD8 + ( Figure 2A ) and CD4 + ( Figure 2B ) T cells expressing type 1 (Th1) immune response cytokines (IFN-γ, TNF, and IL-2) as well as CD8 + T cells with cytotoxic markers (granzyme B + CD107a + ) ( Figure 2C ) in both the spleen and lungs. These responses were particularly robust in the lungs, especially for CD8 + T cells. We also noted that the vast majority of the CD8 + T cell response in BALB/c mice was directed at epitopes in the N-terminal half of the S protein, while CD4 + T cells recognized epitopes in both halves of the protein ( Figure S2A , B). As S proteinspecific lung-infiltrating T cell responses may contribute to SARS-CoV-2 vaccine protection as seen with SARS-CoV-1 (Zhao et al., 2016) , we next examined whether vaccine-induced lung T cells were truly infiltrating into the lung parenchyma. We performed intravenous (i.v.) labeling with a CD45-specific antibody in order to differentiate between vascular (i.v. label-positive) and tissue-infiltrating (i.v. labelnegative) lung CD4 + and CD8 + T cells (Figures 2D-G and S1C, S2C, D). SARS-CoV-2 mRNA-LNP vaccines elicited significant increases in activated (CD69 + or PD-1 + ) and

antigen-experienced (CD44 + CD62L -) CD8 + and CD4 + T cells that were tissue-infiltrating, with comparatively modest increases in the vasculature, suggesting that activated vaccine-induced T cells readily exit the vasculature and enter the lung parenchyma D) . Of note, in each of the above assays, we found that the full length ∆furin vaccine induced greater T cell responses compared to the RBD vaccine; this might be explained by the presence of additional T cell epitopes in the longer protein product produced by the full length ∆furin construct.

Mice were immunized i.m. with a single dose of 30 µg of full length ∆furin, RBD, and Luc mRNA-LNPs and antibody responses were evaluated. Both SARS-CoV-2 vaccines induced high levels of S protein-specific IgG by four weeks post immunization, and IgG titers further increased by week 9 ( Figure 3A ). Using a vesicular stomatitis virus (VSV)based pseudovirus neutralization assay, we demonstrated that nucleoside-modified SARS-CoV-2 mRNA-LNP vaccines induced high and sustained levels of neutralizing antibodies after administration of a single vaccine dose, with week 9 sera showing higher neutralization activity than week 4 ( Figure 3B ). Importantly, induction of antibodies with high neutralization titers was also demonstrated by microneutralization assay using live SARS-CoV-2 with week 9 post immunization samples ( Figure 3C ).

Both assays indicated that the full length ∆furin mRNA-LNPs generated slightly higher levels of neutralizing antibodies than the RBD vaccine at 9 weeks post immunization ( Figure 3B , C). Importantly, the levels of neutralization we observed are similar to those mediated by SARS-CoV-2 immune human convalescent plasma samples analyzed by the same laboratory in the same assay format .

is a potential serious concern for several vaccines including those for Zika and dengue viruses and coronaviruses (Smatti et al., 2018) . Thus, we investigated whether or not the SARS-CoV-2 mRNA vaccine-elicited antibodies induced ADE of infection in HEK293T cells expressing mouse FcγR1 (mFcγR1-293T cells). We demonstrated that none of the mRNA-vaccinated mouse immune sera mediated SARS-CoV-2 ADE under in vitro conditions. As a positive control to validate the mFcγR1-293T cells as generally susceptible to ADE, we show robust ADE of Zika virus (ZIKV) infection by sera derived from ZIKV-infected mice ( Figure 3D ) using the same system. Although ADE assays are typically conducted in the absence of the bona fide viral entry receptor, to examine whether the viral receptor affects ADE efficiency, cells expressing hACE2 as well as mFcγR1 were also used in ADE assays. SARS-CoV-2 pseudovirus infection of hACE2/mFcγR1-293T cells was efficiently neutralized as expected by sera derived from mice vaccinated with the full length ∆furin or RBD mRNAs at low dilutions ( Figure S3 ), and there was no enhanced infection observed at any serum dilution. As in the mFcγR1-293T cells, ZIKV-immune mouse sera mediated robust ADE in the hACE2/mFcγR1-293T cells. These results demonstrate that neither the full length ∆furin nor RBD mRNA-LNP vaccines generate antibodies with mFcγR1-dependent ADE activity in vitro.

Most successful vaccine approaches rely on the generation of memory B cells (MBC) and long-lived plasma cells (LLPC) (Sallusto et al., 2010) . Figure 4J ). We found that ASCs primarily produce antigenspecific IgG1, IgG2a and IgG2b.

The rapid spread of COVID-19 has caused a global health tragedy and significant economic loss. Safe, effective, and widely available vaccines could end the pandemic;

thus, generating such vaccines is an urgent unmet clinical need. Currently, multiple SARS-CoV-2 vaccine candidates are under development using conventional immunogens (live attenuated virus, inactivated virus, adjuvanted protein subunit vaccines), as well as viral and genetic (DNA and mRNA) approaches (Amanat and Krammer, 2020) . mRNA-based vaccines are among the lead candidates because they have been shown to be generally highly effective in small and large animal models (Pardi et al., 2018b) and have additional beneficial features over other vaccine platforms. First, mRNA is non-infectious, cannot integrate into the genome, and incorporation of modified nucleosides into the mRNA sequence along with proper purification (Baiersdorfer et al., 2019; Kariko et al., 2005; Kariko et al., 2008; Weissman et al., 2013) can decrease its inflammatory capacity; thus mRNA vaccines may have a stronger safety profile than virus-based (potentially integrating, replicating, or contagious) vaccine platforms. Secondly, mRNA vaccine antigens can be very rapidly designed and produced, as illustrated by Moderna Therapeutics who produced a SARS-CoV-2 nucleoside-modified mRNA-LNP vaccine for human use in just 42 days after obtaining the sequence of the target antigen. Thirdly, nucleoside-modified mRNA-LNP vaccines have been demonstrated to induce extremely potent and protective immune responses against other viruses after administration of a single dose in animal models (Pardi et al., 2018a; Pardi et al., 2017; Pardi et al., 2018c) . If reproducible in humans, this one-time immunization feature of the vaccine platform would be particularly important in a pandemic or epidemic setting, by inducing immune protection after a single point-of-care visit.

Despite active research, no peer-reviewed preclinical studies have been published on nucleoside-modified SARS-CoV-2 mRNA-LNP vaccines to date. Here, we provide a detailed evaluation of two nucleoside-modified SARS-CoV-2 mRNA-LNP vaccine formulations (full length ∆furin and RBD) in mice. Animals were immunized only once in all experiments presented in this study. Both the full length ∆furin and RBD mRNA vaccines induced potent CD4 + and CD8 + T cell responses, particularly in the lungs. The production of Th1 cytokines was strong, with approximately one half of detectable S protein-specific CD4 + T cells producing the hallmark Th1 cytokine IFN-γ, along with a high frequency of IFN-γ-producing CD8 + T cells. This result is noteworthy, as a major safety consideration of SARS-CoV-2 vaccine design is the elicitation of a strong Th1-biased immune response, instead of a Th2-biased response that might induce vaccine-associated enhanced respiratory disease (VAERD) (Graham, 2020) . A potential protective role of lung-homing T cells has been suggested by the SARS-CoV-1 literature (Zhao et al., 2016) and prompted us to examine T cell responses in the lung.

Interestingly, we found that S protein-specific CD4 + and CD8 + T cell responses were substantially higher in the lung compared to the spleen, leading us to hypothesize that T cells raised by this format of nucleoside-modified mRNA-LNP vaccine may preferentially home to the lungs, or at least to mucosal surfaces in general. Probing further, we found that a large fraction of these cells could be detected in the lung parenchyma, indicating that these cells might be well positioned to contribute to immune protection. Future studies should examine the role of T cells in immune protection from SARS-CoV-2 infection and diseases, particularly at later time points.

important to achieve durable protective humoral immune responses (Sallusto et al., 2010) . We found that both SARS-CoV-2 mRNA-LNP vaccines elicited potent LLPC and MBC responses, as well as the rapid generation of neutralizing antibodies that persisted at a high level until at least week 9 after immunization. Antibody-dependent enhancement (ADE) of disease results from antibody-mediated enhancement of infection, which has been described for some flaviviruses (Dowd and Pierson, 2011) and has been raised as a potential concern for coronaviruses. ADE has only been and systemic reactions, were clearly dose-dependent, and were typically worse following the second immunization. A second nucleoside-modified mRNA-LNP vaccine platform (also similar or identical to ours), encoding the SARS-CoV-2 spike receptor binding domain and developed by Pfizer/BioNTech, was recently shared as a pre-print and showed similar dose-dependent immunogenicity and adverse events (Mulligan et al., 2020) . The data presented here could potentially inform the evaluation of these two vaccines in humans. For example, our data prompt the question of whether these vaccines would induce T cells that home to the airways in humans and whether these might contribute to protection from COVID-19 disease or otherwise alter the inflammatory environment during SARS-CoV-2 infection. Finally, our observation of strong CD8 + T cell responses with cytotoxic markers suggests a potentially important way that mRNA-LNP vaccines may differ from protein subunit and inactivated virus vaccines, which are not expected to generate strong CD8 + T cell responses. However, it remains to be seen whether CD8 + T cell responses, lung-resident or otherwise, contribute meaningfully to immune protection in SARS-CoV-2 animal models or in humans.

Our study has some limitations, the most critical one being the absence of protective efficacy studies that could assess whether our nucleoside-modified mRNA-LNP vaccines induce protection from SARS-CoV-2 viral replication or disease in an animal model. We were unable to perform these studies in the BALB/c mouse model used here, as BALB/c mice are not susceptible to robust SARS-CoV-2 replication or disease. Instead, transgenic mice carrying the human angiotensin-converting enzyme 2 (hACE2) gene are a more appropriate animal model for future protective efficacy studies for SARS-CoV-2 McCray et al., 2007) . Another suitable mouse model was recently reported (Hassan et al., 2020) , involving the intranasal delivery of an adenoviral vector encoding hACE2 to wild type mice prior to viral challenge. These useful tools were not available as of the time of this publication but are the subject of ongoing and future protection studies.

One key consideration for human SARS-CoV-2 vaccines is to determine the ideal vaccine dose and vaccination schedule. While important, our study was not designed to address this dose optimization question. Additionally, it is unclear how mouse vaccine doses can be extrapolated to human immunization, i.e. whether it is at all proportional to body weight or affected by immunogenetic differences between species. As a result, the dose and schedule will likely need to be optimized through human clinical trials. The major aim of our studies was to provide detailed analyses of immune responses to two nucleoside-modified SARS-CoV-2 mRNA vaccines in mice as a proof-of-principle that may guide the exploration of similar mRNA-LNP vaccines in human clinical trials. We used a single immunization of 30 µg mRNA-LNPs in these studies because we found that this vaccine dose induced durable protective immune responses against other viruses in mice (Pardi et al., 2018a; Pardi et al., 2017; Pardi et al., 2018c See also Figure S4 .

Further information and requests for supporting data, resources, and reagents should be directed to and will be fulfilled upon request by the Lead Contact, Norbert Pardi (pnorbert@pennmedicine.upenn.edu).

Reagents from this study are available upon request.

The source of protein and nucleic acid sequences are indicated in the manuscript and are available from the corresponding author on request.

Mice BALB/c mice aged 8 weeks were purchased from Jackson Laboratory (T cell studies), or Charles River Laboratories (all other studies).

FreeStyle 293 

The investigators faithfully adhered to the "Guide for the Care and Use of Laboratory (Luc) were codon-optimized, synthesized and cloned into the mRNA production plasmid as described (Freyn et al., 2020) . mRNA production and LNP encapsulation was performed as described (Freyn et al., 2020) . Briefly, mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. m1Ψ-5'-triphosphate (TriLink #N-1081) instead of UTP was used to generate modified nucleoside-containing mRNA. Capping of the in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap (TriLink #N-7413). mRNA was purified by cellulose (Sigma-Aldrich # 11363-250G) purification, as described (Baiersdorfer et al., 2019) . All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at -20°C. Cellulosepurified m1Ψ-containing RNAs were encapsulated in LNPs using a self-assembly process as previously described wherein an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing mRNA at acidic pH (Maier et al., 2013) . The RNAloaded particles were characterized and subsequently stored at -80°C at a concentration of 1 µg µl -1 . The mean hydrodynamic diameter of these mRNA-LNP was ~80 nm with a polydispersity index of 0.02-0.06 and an encapsulation efficiency of ~95%. Two or three batches from each mRNA-LNP formulations were used in these studies and we did not observe variability in vaccine efficacy.

293F cells were diluted to 1 x 10 6 cells/ml before transfection. 3 µg mRNA encoding full length WT and ∆furin S protein was transfected into 6 ml of cells. For soluble RBD, 30 ml of cells were transfected with 15 µg mRNA. TransIT-mRNA Transfection Kit (Mirus #MIR 2250) was used for mRNA transfection following the manufacturer's instructions.

Transfected cells were cultured at 37°C with 8% CO 2 and shaking at 130 rpm for 48 hours (for full length WT and ∆furin S protein) or 72 hours (for soluble RBD).

In vitro studies Binding reactivity of anti-RBD chimeric mAb, D001 (Sino Biologicals #40150-D001) and

hACE2-Fc fusion protein to full length S protein constructs (WT and ∆furin) was All flow cytometry data were analyzed with FlowJo software (FlowJo LLC).

The RBD and full-length S proteins were produced in 293F cells, as described previously Stadlbauer et al., 2020) . Briefly, 600 million cells were transfected with 200 µg of purified DNA encoding codon-optimized RBD of SARS-CoV-2 using ExpiFectamine 293 transfection kit (Gibco #A14525). The manufacturer's protocol was followed and cells were harvested on day 3. Cells were spun at 4000g for 10 minutes and sterile-filtered with a 0.22 µm filter. Supernatant was incubated with Ni-NTA resin (Qiagen #30230) for 2 hours. After 2 hours, this mixture was loaded onto columns and the protein was eluted using elution buffer with high amounts of imidazole.

Protein was concentrated using 10 kDa Amicon centrifugal units (Millipore Sigma #UFC901024) and re-constituted in PBS. Concentration was measured using Bradford reagent (Bio-Rad #5000201) and a reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was run to check the integrity of the protein.

MultiScreenHTS IP Filter Plate, 0.45 µm (Millipore Sigma #MSIPS4W10) were coated overnight at 4°C with 2.5 µg/ml recombinant SARS-CoV-2 RBD or Full Spike proteins in bicarbonate buffer (35 mM NaHCO 3 and 15 mM Na 2 CO 3 ). Plates were washed three times with PBS and blocked with complete DMEM for at least 1 hour at 37°C. Single cell suspensions of murine BM cells were diluted serially in complete DMEM with halving dilutions starting at 1 x 10 6 cells. Following overnight incubation at 37°C and 5% CO 2 , plates were washed three times with 0.05% Tween-20 in PBS. Membranes were incubated with IgG-HRP (Jackson ImmunoResearch #115-035-003) diluted in complete DMEM for 2 hours at room temperature. Following incubation with the detection antibody, plates were washed three times with 0.05% Tween-20 in PBS. Spots corresponding to antigen-specific antibody-secreting cells were developed using BD ELISPOT AEC Substrate Set (#551951) and counted using a CTL Immunospot analyzer. Isotype specific ELISPOT plates were coated and incubated with cells as above and then were washed 4x with 0.1% Tween-20 in PBS. Membranes were then incubated with 1:3000 isotype-specific biotinylated antibodies (Southern Biotech #1020-08, #1050-08, #1060-08, #1080.08, #1090-08, #1100-08, Biolegend #400703) for 1 hour. Membranes were then washed 4x and incubated in 1:20,000 streptavidin-alkaline phosphatase (Sigma # E2636) for 30 minutes. Membranes were then washed 4x and incubated with 50 µL BCIP/NBT (Sigma #B1911-100mL) for ~10 minutes or until spots developed at which time reaction was quenched with 100 µl 1M sodium phosphate monobasic solution. Membranes were then dried and counted above.

mRNA-LNPs were diluted in PBS and injected into the gastrocnemius muscle (40 µl injection volume) with a 3/10cc 29½G insulin syringe (Covidien #8881600145).

Blood was collected from the orbital sinus under isoflurane anesthesia. Blood was centrifuged for 5 minutes at 13,000 rpm and the serum was stored at -20°C and used for ELISA, virus neutralization assays, and ADE assays.

Samples from cell transfections Supernatant from 293F cells transfected with RBD-encoding mRNA was harvested 72

hours after transfection and concentrated 60x with Vivaspin 20 kDa molecular weight cut-off concentrator (GE Healthcare #20-9323-60). The expression and binding of soluble RBD were measured by indirect ELISA. RBD samples were added to capture antibody D001 (2 µg/ml)-coated plates for one hour, followed by detection with serum from a SARS-CoV S protein-immunized guinea pig for 1 hour. Serum binding was detected via horseradish peroxidase-conjugated goat anti-guinea pig IgG (Fc) (Jackson ImmunoResearch #106-035-008, used at 1:10,000 To test RBD sample binding to ACE2, plates were first coated with goat anti-human IgG (Fc) antibody (Sigma-Aldrich #I2136) (2 µg/ml), in order to capture the hACE2-mFc construct (5 µg/ml, one hour). Next, the RBD samples were incubated for one hour. The RBD was detected by a rabbit anti-RBD antibody R007 (Sino Biologicals #40150-R007, used at 1:4000) followed by Goat Anti-Rabbit IgG H&L (HRP) (Abcam #97080). The detection of RBD and development procedure were the same as described above.

Corning 96 Well Clear Polystyrene High Bind Stripwell™ Microplates (Corning #2592) were coated with 1 µg/ml purified RBD in PBS overnight at 4°C. The plates were blocked with 2% BSA in PBS for 2 hours and washed four times with wash buffer (0.05% Tween-20 in PBS). Mouse sera were diluted in blocking buffer and incubated for 2 hours at room temperature, followed by four washes. HRP-conjugated anti-mouse secondary antibody (Jackson Immunoresearch #715-035-150) was diluted 1:10,000 in blocking buffer and incubated for 1.5 hours, followed by 4 washes. KPL 2-component TMB Microwell Peroxidase Substrate (Seracare #5120-0050) was applied to the plate and the reaction was stopped with 2 N sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax 190 microplate reader. RBD-specific IgG end-point dilution titer was defined as the highest dilution of serum to give an OD greater than the sum of the background OD plus 0.01 units. All samples were run in technical duplicates.

Production of VSV pseudotype with SARS-CoV-2 S: 293T cells plated 24 hours previously at 5 X 10 6 cells per 10 cm dish were transfected using calcium phosphate (FRNT 50 ) was measured as the greatest serum dilution at which focus count was reduced by at least 50% relative to control cells that were infected with pseudotype virus in the absence of mouse serum. FRNT 50 titers for each sample were measured in two technical replicates performed on separate days.

Neutralization assays with live SARS-CoV-2 (USA-WA1/2020; GenBank: MT020880) were performed in a biosafety level 3 (BSL3) facility with strict adherence to institutional regulations. Twenty thousand Vero.E6 cells per well were seeded in a 96-well cell culture plate one day before the neutralization assay and this protocol has been published earlier wells])*100) whereby 'X' is the read for each well. Non-linear regression curve fit analysis over the dilution curve was performed to calculate IC 50 .

production SARS-CoV-2 PV was produced as previously described (Moore et al., 2004) through 763rd amino acids of the polyprotein of a ZIKV molecular clone (Zhang et al., 2018) with Renilla luciferase with the 2A self-cleaving peptide fused at its C-terminus.

This construct contains the tetracycline-responsive P tight promoter that derives ZIKV RNA transcription. The PV-and VLP-containing culture supernatants were cleared by 0.45 µm filtration and immediately frozen in aliquots at -80°C.

The ability of SARS-CoV-2 immune sera to mediate ADE was measured using HEK293T cells or those stably expressing hACE2 transfected with pCMV-SPORT6-mFcγR1 (Dharmacon #MMM1013-202708624). Mouse immune sera used for ADE assays were obtained 9 weeks post vaccination or from naive mice. Sera of ten mice per vaccine group were pooled in two groups (five per pool) and assessed separately in ADE assays but combined afterwards for data analysis. We have previously shown efficient ZIKV ADE (Shim et al., 2019) , and thus used ZIKV VLP as a positive control in ADE assays. ZIKV immune sera were prepared by intraperitoneally injecting mice with ZIKV (strain PB-81) and bled at 5 weeks post infection. 

GraphPad Prism was used to perform Kruskal-Wallis and Mann-Whitney tests for nonparametric data and one-way or two-way ANOVA corrected for multiple comparisons for parametric data to compare immune responses in vaccinated and control mice. Figure S1 . Gating strategy for intracellular cytokine staining and activation marker staining in CD8 + and CD4 + T cells Pre-immune Week 4

Week 9 **** **** **** **** **** **** **** **** 

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