key: cord-0913484-4qa6mdzz
authors: Joyce, M. Gordon; Chen, Wei-Hung; Sankhala, Rajeshwer S.; Hajduczki, Agnes; Thomas, Paul V.; Choe, Misook; Martinez, Elizabeth; Chang, William; Peterson, Caroline E.; Morrison, Elaine B.; Smith, Clayton; Chen, Rita E.; Ahmed, Aslaa; Wieczorek, Lindsay; Anderson, Alexander; Case, James Brett; Li, Yifan; Oertel, Therese; Rosado, Lorean; Ganesh, Akshaya; Whalen, Connor; Carmen, Joshua M.; Mendez-Rivera, Letzibeth; Karch, Christopher; Gohain, Neelakshi; Villar, Zuzana; McCurdy, David; Beck, Zoltan; Kim, Jiae; Shrivastava, Shikha; Jobe, Ousman; Dussupt, Vincent; Molnar, Sebastian; Tran, Ursula; Kannadka, Chandrika B.; Soman, Sandrine; Kuklis, Caitlin; Zemil, Michelle; Khanh, Htet; Wu, Weimin; Cole, Matthew A.; Duso, Debra K.; Kummer, Larry W.; Lang, Tricia J.; Muncil, Shania E.; Currier, Jeffrey R.; Krebs, Shelly J.; Polonis, Victoria R.; Rajan, Saravanan; McTamney, Patrick M.; Esser, Mark T.; Reiley, William W.; Rolland, Morgane; de Val, Natalia; Diamond, Michael S.; Gromowski, Gregory D.; Matyas, Gary R.; Rao, Mangala; Michael, Nelson L.; Modjarrad, Kayvon
title: SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity
date: 2021-12-08
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.110143
sha: 0644283af6c43add6c66a2962fc91d6322f4d904
doc_id: 913484
cord_uid: 4qa6mdzz

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion SARS-CoV-2 Spike (S), S1 and RBD. These immunogens induce robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A Spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations, and also show durable and potent neutralization against circulating VoC. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2 blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

To date, seven coronaviruses (CoV) are known to cause disease in humans. Three of these, SARS-CoV-1, 

SARS-CoV-2 is easily transmitted by humans and created a pandemic, infecting over 100 million people, 63 causing over 2 million deaths to date, and resulting in an urgent need for protective and durable vaccines.

A rapid vaccine development effort led to the evaluation of hundreds of SARS-CoV-2 vaccine candidates 76 COVID-19 vaccines. S is a class I fusion glycoprotein consisting of a S1 attachment subunit and S2 fusion 77 subunit that remain non-covalently associated in a metastable, heterotrimeric S on the virion surface (Walls 

Due to the unknown parameters of SARS-CoV-2 vaccine durability, specific age-or population-87 needs, emergence of SARS-CoV-2 variants of concern (VoC) (Wibmer et al., 2020) , and the constant threat 

(residues 12-303), and (iv) S1 (residues 12-696) ( Figure 1 , Figure S1 , and Table S1 ). For the S-ferritin 141 nanoparticle designs, a short linker to the ferritin molecule was used to utilize the natural three-fold axis,

for display of eight Spikes. For the other designs, a short region of bullfrog ferritin was utilized to allow 143 equidistant distribution of the 24 S-domain molecules on the ferritin surface ( Figure 1 ). Our overall 144 approach was to compare the immunogen structures, antigenicity, and immunogenicity elicited by these 145 different immunogens with the goal to identify the best immunogen to take forward into further 146 development.

The first design category, S-ferritin nanoparticle designs were based on a modified S with 

(v) addition of heterogenous trimerization domains (GCN4, or foldon), or (vi) signal peptide sequence 154 ( Figure 1B , and Table S1 ).

The second design category, RBD-ferritin nanoparticle designs used the SARS-CoV-2 RBD

(residues 331-527) ( Figure 1C ) connected to the bullfrog-H. pylori chimeric ferritin (Kanekiyo et al., 2015) of the intact S molecule. These regions were iteratively mutated to reduce hydrophobicity, and increase 160 stability of the RFN molecules, expression levels, and antigenicity and immunogenicity.

The third design category, RBD-NTD-ferritin molecules were based on addition of optimized RBD 162 molecules in series with an NTD-ferritin construct (residues 12-303) linked to the bullfrog-H. pylori 163 chimeric ferritin molecule. The in-series, but reversed RBD-NTD design ensured distal displacement of the 164 RBD molecule from the ferritin molecule ( Figure 1E ), promoting immune recognition of the RBD molecule 165 with potential benefits for the production and stability of the nanoparticle.

The fourth design category, S1-ferritin design (residues 12 -676) ( Figure 1F , and Table S1 ) was 167 designed based on the MERS S1 immunogen which elicited protective immune responses (Wang et al., 168 2015). Subsequent designs focused on inclusion of a short region of SARS-CoV-2 S2 (residues [689] [690] [691] [692] [693] [694] [695] [696] 169 either using the connecting region that overlaps with the furin site, or by use of a short glycine-rich linker 170 sequence ( Figure 1F ) to enable formation of the S1-ferritin nanoparticle ( Figure 1G ).

Characterization of SARS-CoV-2 S-domain ferritin nanoparticles 173 174

Ten S-ferritin constructs (Table S1) 

SpFN having the highest binding ( Figure 3A and Figure S1 ).

Initial test expression of RBD-ferritin constructs at 37 o C using either 293F or Expi293F cells level of binding was approximately twice that seen for the S-ferritin constructs, indicative of the exposed 205 and accessible nature of the RBD epitopes ( Figure 1D and 2F).

Due to the initial difficulty with S1-ferritin nanoparticle constructs, we developed a set of in the RBD molecule to reduce surface hydrophobicity ( Figure S1 ). Antigenic analysis of these constructs 212 showed that pCoV146 displayed robust antibody-binding ( Figure 2F ).

The initial S1-ferritin construct, pCoV68 (residues 12-676) yielded very low protein expression 214 levels, even with reduced expression temperatures( Figure S3A ). However, using the structure of the S-2P 

Additionally, although the coiled coil was unresolved, the distances between the density for S and ferritin the NTD domain and a more disordered layer for the RBD domain ( Figure 4C ). This particle was 237 approximately 9nm larger in 2D and 3D than the single domain RFN molecule. The reconstruction of the 238 S1-ferritin fusion pCoV111 revealed a surprisingly ordered S1 density compared to the flexible RBD-

NTD fusion, perhaps due to geometric constraints on the surface of the ferritin particle ( Figure 4D) . A 240 density similar in shape to the S1 domain in the closed S2P trimer was resolved although it was slightly 241 more compact, likely due to both overall flexibility of the S1 on the ferritin surface and RBD flexibility.

To evaluate the immunogenicity of the SARS-CoV-2 ferritin-nanoparticles, we utilized two strains of mice 246 (C57BL/6, and BALB/c), two adjuvants (ALFQ, and Alhydrogel  ), and immunized mice three-times 247 intramuscularly at 3-week intervals using a 10 μg dose. In total, we assessed 14 immunogens, two S-ferritin 248 immunogens, seven RBD-ferritin immunogens, one S1-ferritin construct, and four RBD-NTD ferritin 249 immunogens (Table S3) . We also assessed two control proteins (S-2P, and RBD), to allow comparison to 

All four categories of immunogens elicited robust SARS-CoV-2 immune responses. Immune 263 responses seen in C57BL/6 mice were greater than for BALB/c mice after a single immunization, whereas 264 binding and neutralizing antibody titers were comparable after a second or third immunization ( Figure 5 ).

In all cases, the third immunization did not substantively increase the antibody levels induced by the S-266 domain ferritin nanoparticles. In all cases tested, ALFQ was superior to Alhydrogel  as an adjuvant for 267 elicitation of binding and neutralizing responses ( Figure 5 and Figure S4 and Table S4 ). In addition,

Alhydrogel  led to a skewed antibody isotype immune response that was TH2 in nature, as opposed to the 269 balanced immune response seen with ALFQ adjuvanted animals ( Figure 5D ). The IgG2a/c:IgG1 ratio 

The SpFN immunogen elicited a rapid RBD-binding, and pseudovirus neutralizing antibody 274 response with ID50 geometric mean titer (GMT) >10,000 in C57BL/6 mice and >1,000 after a single 275 immunization ( Figure 5C and Figure S4 ). This rapid neutralizing immune response after one immunization Table 4 ).

283 neutralization ID50 GMT >10,000 in both mice strains after two immunizations ( Figure 5A -C). We 284 assessed seven RBD-ferritin immunogen designs in immunogenicity studies (Table S3) were comparable or exceeded that seen for the SpFN_1B-06-PL immunogen. Of note, the RBD-ferritin 289 immunogens elicited substantial S binding responses that were highly comparable to that of other 290 immunogens that contained additional S domains ( Figure 5B ). When comparing RBD-ferritin elicited 291 immune responses to the control protein RBD, the differences were striking. Using similar mouse strains, 292 adjuvants, and performing the experiments contemporaneously, it took three immunizations to elicit 293 detectable neutralizing antibody titers using ALFQ as an adjuvant. In the Alhydrogel-adjuvanted study 294 groups, we did not detect neutralizing titers even after the third immunization.

In a pattern similar to that seen for the RBD-ferritin immunogens, both the RBD-NTD-ferritin and 296 the S1-ferritin immunogens elicited binding responses, and detectable pseudovirus neutralization after a 297 single immunization that were increased by the second immunization to give ID50 GMT values >10,000, 298 and ID80 GMT titers ~5,000 ( Figure 5A -C and Figure S4 ).

Given the rapid induction of immune responses after a single immunization by SpFN_1B-06-PL,

we further characterized this immunogen in a dose-ranging study ( Figure S5 and Figure 5D and 5E). In 

with all our immunogenicity assays ( Figure 5D and Figure S5G ). At this dose, the immune response was 306 comparable to that seen for the typical 10 μg dose. In addition, we assessed both the 10 and 0.08 μg 

Analysis of the mouse sera for binding or neutralization of SARS-CoV-1 showed that RFN-338 immunized mice elicited the highest SARS-CoV-1 RBD binding response ( Figure 6B and S6).

In addition, to RBD binding, we also observed SARS-CoV-1 ACE2-RBD inhibitory activity with the 340 SpFN-immunized mice ( Figure S5 ). We further assessed the SpFN-or RFN-immunized mouse sera for 341 neutralization against SARS-CoV-1 using the pseudovirus assay ( Figure 6C ). We observed robust 342 neutralization levels with ID50 > 1,000 for the SpFN_1B06-PL or the RFN_131 immunized animals. In 

Animal weight was measured twice daily for 14 days after challenge, and animals that lost > 25% 365 weight during the study were euthanized. All animals that received the highest amount of antibody (470 366 μg SpFN-derived, or 370 μg RFN-derived) showed serum neutralization ID50 GMT titers of 3,424 and 367 2,356 respectively at day 0 immediately prior to virus inoculation ( Figure 7C ). All animals in these two 368 groups showed minimal weight loss ( Figure 7D ) and all survived the study ( Figure 7E ). In the two groups lines. Three hydrophobic regions of the RBD which were mutated for nanoparticle 767 immunogen design are shown in light green surface, with residues in stick representation. 768

The ACE2 binding site contains two of these regions, while a third hydrophobic patch near 769 the C-terminus of the RBD is typically buried by S2 and part of S1 in the context of the 770 trimer molecule. displaying RBD and NTD epitopes is depicted and colored according to the schematic. 777

Truncation points, linkers, and alterations made to the native spike sequence are indicated 778 on the primary structure. 779 (F) S1-Ferritin immunogen design. The SARS-CoV-2 S1 forms a hydrophobic collar around 780 the N-terminal β-sheet of S2 (residues 689-676). S1-ferritin immunogen design required 781 inclusion of this short stretch of S2 (colored cyan) attached by a linker. Terminal residues 782 of the structured portions of S1 and S2 are labeled. 783

(G) Schematic and 3D model of an S1-Ferritin nanoparticle. A modeled nanoparticle 784 displaying RBD and NTD domains is depicted and colored according to the S1-ferritin 785 schematic with truncation points and domain linkers indicated. 786

See also Figure S1 and Table S1 . an asymmetric unit of non-ferritin density colored and the size of each particle indicated in 816 nanometers. Spike trimer density, is colored in red, and a model of a SARS-CoV-2 trimer 817 based on PDB 6VXX is shown docked into the negative-stain map and colored according to 818 the sequence diagram. 819

(B) Two non-ferritin densities per asymmetric unit were observed for RFN and are 820 highlighted in green. These densities putatively correspond to the receptor-binding domain 821

(RBD) but lack low resolution distinguishing features due to the small, globular shape of 822 these domains. The presence of two densities is likely due to flexibility in the linker and 823 heterogeneity in the RBD pose. 824

(C) Two layers of densities were distinguishable for pCoV146, with the putative N-terminal 825 domain (NTD) density of an asymmetric unit colored blue, proximal to the ferritin and two 826 smaller, more flexible densities corresponding to RBD distal to the ferritin and colored 827 green. 828

(D) An asymmetric unit of non-ferritin density for pCoV111 is colored in orange and a 829 monomer of S1 in the closed trimer state from PDB 6VXX is shown docked into the density 830

with domains colored as in the sequence diagram. 831

See also Figure S2 and carried out using a Mann-Whitney unpaired two-tailed non-parametric test. 851

In panels A -C, all groups at a given study timepoint were compared to each other. Only 852 groups with significant differences are indicated by a bar; all other groups did not show 853 statistically significant differences. P values <0.0001 (****), <0.001 (***), <0.01 (**), or 854 <0.05 (*). 855

See also Figure S4 , S5 and S6, and Immunogens Joyce et al. generate four categories of engineered SARS-CoV-2 ferritin nanoparticle immunogens using structure-based vaccine design that recapitulate the prefusion SARS-CoV-2 Spike, S1, and RBD. These immunogens induce robust and protective neutralizing antibody responses against SARS-CoV-2, and elicit potent neutralization against variants of concern, and the heterologous SARS-CoV-1.

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Thermo Fisher Scientific) at 37°C and 1050 5% CO2. 1051 1052 Mouse strains 1053 Female BALB/c mice, C57BL/6, and hACE2 K18 Tg mice mice aged 6-to 8-weeks were speed for 1 min, mixed on a roller for 15 minutes, and stored at 4 o C for at least 1 h prior to 1234 immunization

Bottom plates were coated with 1 μg/mL of RBD or S protein (S-2P) 1239 antigen in Dulbecco's PBS, pH 7.4. Plates were incubated at 4°C overnight and blocked with 1240 blocking buffer (PBS containing 0.5% milk and 0.1% Tween 20, pH 7.4), at room temperature 1241 (RT) for 2 h. Individual serum samples were serially diluted 2-fold in blocking buffer and 1242 added to triplicate wells and the plates were incubated for 1 hour (h) at RT. Horseradish 1243 peroxidase (HRP)-conjugated sheep anti-mouse IgG

Site) was added and incubated at RT for an hour, followed by the addition of 2,2'-Azinobis 1245 [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) HRP substrate (KPL) for 1246 1 h at RT. The reaction was stopped by the addition of 1% SDS per well and the absorbance 1247 (A) was measured at 405 nm (A405) using an ELISA reader Spectramax

Antibody positive (anti-RBD mouse cells with a SARS-CoV-2 S plasmid (pcDNA3.4) and an HIV-1 NL4-3 luciferase reporter 1280 plasmid (The reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, 1281 NIAID, NIH: Human Immunodeficiency Virus 1 (HIV-1) NL4-3 ΔEnv Vpr Luciferase Reporter 1282 Vector (pNL4-3.Luc.R-E-), ARP-3418, contributed by Dr. Nathaniel Landau and Aaron 1283 Diamond). The SARS-CoV-2 S expression plasmid sequence was derived from the Wuhan 1284 seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome (GenBank 1285 accession MN908947), and the SARS-CoV-1 expression

Assay equivalency for SARS-CoV-2 was established by 1300 participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey (SNACS) run by the 1301 Virology Quality Assurance Program and External Quality Assurance Program Oversite 1302 Laboratory (EQAPOL) at the Duke Human Vaccine Institute, sponsored through programs 1303 supported by the National Institute of Allergy and Infectious Diseases, Division of AIDS. 1304 1305 SARS-CoV-2 authentic virus neutralization assay 1306 The neutralization assay has been described in detail previously (Case et al., 2020). Briefly, 1307 SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 was obtained from the Centers for Disease 1308 Control and Prevention. Virus was passaged once in Vero CCL81 cells (ATCC) and titrated by 1309 focus-forming assay on Vero E6 cells. Mouse sera were serially diluted and incubated with 1310 100 focus-forming units of SARS-CoV-2 for 1 h at 37°C. Serum-virus mixtures were then 1311 added to Vero E6 cells in 96-well plates and incubated for 1 h at 37°C. Cells were overlayed 1312 with 1% (w/v) methylcellulose in MEM. After 30 h, cells were fixed with 4% PFA in PBS for 1313 20 minutes at room temperature then washed and stained overnight at 4°C with 1 µg/ml of 1314 CR3022

K18-hACE2 transgenic mice 1322 All research in this study involving animals was conducted in compliance with the Animal 1323 Welfare Act, and other federal statutes and regulations relating to animals and experiments

Following release of the SARS-CoV-2 sequence on Jan 10 th 2020, initial RBD-Ferritin and S1-1070Ferritin immunogens were designed (Table S1) RBD-Ferritin designs were generated by assessment of the hydrophobic surface of 1083 the SARS-CoV-2 RBD surface and determining surface accessible mutations that reduced the 1084 hydrophobic surface. S1-Ferritin designs were creating using the PDB ID: 6VXX and 1085including a short region of the S2 domain, which interacts with S1. Spike-Ferritin designs 1086were created by modeling the coiled coil region between S residues 1140 and 1158 and 1087increasing the coil-coil interaction either by mutagenesis, or by increasing the length of the 1088 interaction region. RBD-NTD-Ferritin designs utilized RBD constructs with improved 1089properties in the context of RBD-Ferritin, which were fused to the N-terminus of NTD (12 -1090 303)-Ferritin by a short 6 amino-acid linker. 1091 1092 DNA plasmid construction and preparation 1093 SARS-CoV-2 S-domain ferritin constructs were derived from the Wuhan-Hu-1 strain genome 1094 sequence (GenBank MN9089473), to include the following domains: RBD subunit (residues 1095 J o u r n a l P r e -p r o o f 25 331 -527), NTD subunit (residues 12 -303), RBD subunit linked to NTD subunit (residues 1096 331 -527 linked to residues 12 -303 with a short GSG linker), S1 domain (residues 12 -696) 1097and S ectodomain (residues 12 -1158). Constructs were modified to incorporate a N-1098terminal hexa-histadine tag (his) for purification of the RBD-Ferritin and RBD-NTD-Ferritin 1099constructs. 1100An S-2P construct was used as a template to generate the set of Spike ferritin 1101 nanoparticles. Subsequent designs involving small deletions, additions and point mutations 1102were generated using a modified QuikChange site-directed mutagenesis protocol (Agilent). 1103RBD-ferritin and S1-ferritin constructs were synthesized by GenScript. For some of the RBD-1104 ferritin constructs, gene segments (gBlocks) were synthesized by Integrated DNA 1105Technologies to encode various linker regions between RBD and ferritin. Gene segments 1106were stitched together with RBD-and ferritin-encoding PCR products using overlap 1107 extension PCR and were re-subcloned into the CMVR vector. The His-tagged SARS-CoV-2 1108 RBD molecule was generated by amplifying the RBD domain from the RBD-Ferritin plasmid 1109 while encoding the 3' purification tag and subcloned into the CMVR vector. The NTD protein 1110 subunit was generated in a similar manner, by amplifying the NTD domain from the S1-1111Ferritin construct. Purified proteins were deposited at 0.02-0.08 mg/ml on carbon-coated copper grids and 1163 stained with 0.75% uranyl formate. Grids were imaged using a FEI T20 operating at 200 kV 1164with an Eagle 4K CCD using SerialEM or using a Thermo Scientific Talos L120C operating at  1165 120 kV with Thermo Scientific Ceta using EPU. All image processing steps were done using 1166 RELION 3.0.8, RELION 3.1.1, and/or cisTEM-1.0.0-beta. Particles were picked either 1167 manually or using templates generated from manually picked 2D class averages. CTF 1168 estimation was done with CTFFIND 4.1.13 and used for 2D classification. 3D reconstructions 1169were generated using an initial reference generated from a corresponding synthetic atomic 1170 model with a low pass filter of 80-100 angstroms to remove distinguishable features or from 1171 a similar construct also low pass filtered to 80-100 angstroms. For all 3D reconstructions, O 1172 symmetry was enforced, and no explicit mask was used. Visual analysis and figure  1173 generation was conducted using UCSF Chimera and ChimeraX. 1174 1175Dynamic Light Scattering 1176Spike-domain ferritin nanoparticle hydrodynamic diameter was determined at 25°C using a 1177Malvern Zetasizer Nano S (Malvern, Worcestershire, UK) equipped with a 633-nm laser. 1178Samples were assessed accounting for the viscosity of their respective buffers. 1179 1180Octet Biolayer Interferometry binding and ACE2 inhibition assays 1181All biosensors were hydrated in PBS prior to use. All assay steps were performed at 30°C 1182with agitation set at 1,000 rpm in the Octet RED96 instrument (FortéBio). Biosensors were 1183 equilibrated in assay buffer (PBS) for 15 seconds before loading of IgG antibodies (30 µg/ml 1184 diluted in PBS). SARS-COV-2 antibodies were immobilized onto AHC biosensors (FortéBio)  1185 for 100 seconds, followed by a brief baseline in assay buffer for 15 s. Immobilized antibodies 1186were then dipped in various antigens for 100-200 s followed by dissociation for 20-100 s. 1187 carried out as follows. HIS1K biosensors(FortéBio) were equilibrated in assay buffer for 15 1189 s before loading of His-tagged RBD (30 μg/ml diluted in PBS) for 120 seconds. After briefly 1190 dipping in assay buffer (15 seconds in PBS), the biosensors were dipped in mouse sera (100-1191 fold dilution) for 180 seconds followed by dissociation for 60 seconds. 1192 SARS-CoV-2 and SARS-CoV-1 RBD hACE2 inhibition assays were carried out as 1193follows. SARS-CoV-2 or SARS-CoV-1 RBD (30 μg/ml diluted in PBS) was immobilized on 1194 HIS1K biosensors (FortéBio) for 180 seconds followed by baseline equilibration for 30 s. 1195Serum was allowed to occur for 180 s followed by baseline equilibration (30 s). ACE2 protein 1196(30 μg/ml) was the allowed to bind for 120 s. Percent inhibition (PI) of RBD binding to ACE2 1197 by serum was determined using the equation: PI = 100 − [(ACE2 binding in the presence of 1198 mouse serum)) ⁄ (mouse serum binding in the absence of competitor mAb)] × 100. 1199 1200Mouse immunization 1201All research in this study involving animals was conducted in compliance with the Animal 1202Welfare Act, and other federal statutes and regulations relating to animals and experiments 1203involving animals and adhered to the principles stated in the Guide for the Care and Use of 1204Laboratory Animals, NRC Publication, 1996 edition. The research protocol was approved by 1205 the Institutional Animal Care and Use Committee of WRAIR. BALB/c and C57BL/6 mice were 1206 obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in the animal 1207 facility of WRAIR and cared for in accordance with local, state, federal, and institutional 1208 policies in a National Institutes of Health American Association for Accreditation of 1209Laboratory Animal Care-accredited facility. 1210 C57BL/6 or BALB/c mice (n=10/group) were immunized intramuscularly with 10 µg 1211 of immunogen (unless stated) adjuvanted with either ALFQ or Alhydrogel® in alternating 1212 caudal thigh muscles three times, at 3-week intervals; blood was collected 2 weeks before 1213 the first immunization, the day of the first immunization, and 2 weeks following each 1214immunization, and at week 10 (Table S1 ). For immunogen SpFN_1B-06-PL, mice were 1215 immunized with reduced doses of protein adjuvanted with ALFQ with immunization 1216 schedule, site of injections, and timing of bleeds as described. Mice were randomly assigned 1217to experimental groups and were not pre-screened or selected based on size or other gross 1218 physical characteristics. Serum was stored at 4°C or -80°C until analysis. Antibody responses 1219were analyzed by Octet Biolayer Interferometry, enzyme-linked immunosorbent assay 1220 (ELISA), pseudovirus neutralization assay, and live-virus neutralization assay. 1221 1222Immunogen-Adjuvant preparation 1223Purified research grade nanoparticle immunogens were formulated in PBS with 5% glycerol 1224 at 1 mg/ml and subsequently diluted with dPBS (Quality Biological) to provide 10 μg or 1225 lower amount per 50 μl dose upon mixing with adjuvant. ALFQ (1.5X) liposomes, containing 1226 600 µg/mL 3D-PHAD and 300 μg ug/mL QS-21, were gently mixed by slow speed vortex 1227 prior to use. Antigen was added to the ALFQ, vortexed at a slow speed for 1 minute, mixed 1228 on a roller for 15 minutes, and stored at 4 o C for 1 h prior to immunization. Spike-Ferritin 1229nanoparticle immunogens were formulated with ALFQ to contain 20 μg 3D-PHAD and 10 μg 1230 QS21 per 50 μl dose. Alhydrogel® stock (10 mg/ml aluminum (GMP grade; Brenntag)) was 1231 diluted to 900 µg/mL (1.5X) with DPBS and gently mixed. Appropriate volume and 1232concentration of antigen was added to the diluted Alhydrogel® before being vortexed at low 1233 mAb; BEI resources) and negative controls were included on each plate. The results are 1250 expressed as end point titers, defined as the reciprocal dilution that gives an absorbance 1251 value that equals twice the background value (antigen-coated wells that did not contain the 1252 test sera, but had all other components added). 1253Mouse isotype ELISA were performed using a similar approach as above, but with the 1254 following differences. Only Spike protein (S-2P) was used to coat the wells. Figure S6 . 1342For the passive immunization study, on day -1, K18-hACE2 mice were injected 1343 intravenously with purified IgG from C57BL/6 vaccinated mice. On study day 0, all mice were 1344 inoculated with 1.25x10 4 PFU of SARS-CoV-2 USA-WA1/2020 via intranasal instillation. All 1345 mice were monitored for clinical symptoms and body weight twice daily, every 12 hours, 1346from study day 0 to study day 14. Mice were euthanized if they displayed any signs of pain 1347 or distress as indicated by the failure to move after stimulated or inappetence, or if mice have 1348 greater than 25% weight loss compared to their study day 0 body weight. assessed with a Kruskal-Wallis test followed by a Dunn's post-test. Authentic virus 1357 neutralization comparisons between the two dose groups at each time point were carried 1358 out using a Mann-Whitney unpaired two-tailed non-parametric test. SARS-CoV-1 1359 pseudovirus neutralization titers elicited by SpFN and RFN immunogens were compared 1360 using a Mann-Whitney unpaired two-tailed non-parametric test. Mouse challenge groups 1361were carried out using a Mantel-Cox test followed by Bonferroni correction. Serum 1362IgG2a/c:IgG1 ratios were assessed using a Mann-Whitney unpaired two-tailed non-1363 parametric test. P values are summarized within figures as: <0.0001 (****), <0.001 (***), 1364<0.01 (**), or <0.05 (*). All statistical analyses were conducted using GraphPad Prism v8.0, 1365 for serum IgG endpoint titers, pseudovirus and authentic neutralization titers, biolayer 1366 interferometry binding responses, and body weight loss of mice in challenge assessments. 1367