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A Single Immunization with Spike-Functionalized Ferritin Vaccines
Elicits Neutralizing Antibody Responses against SARS-CoV‑2 in Mice
Abigail E. Powell, Kaiming Zhang, Mrinmoy Sanyal, Shaogeng Tang, Payton A. Weidenbacher,
Shanshan Li, Tho D. Pham, John E. Pak, Wah Chiu, and Peter S. Kim*

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ABSTRACT: The development of a safe and eﬀective SARSCoV-2 vaccine is a public health priority. We designed subunit
vaccine candidates using self-assembling ferritin nanoparticles
displaying one of two multimerized SARS-CoV-2 spikes: fulllength ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion(SΔC-Fer). Ferritin is an attractive nanoparticle platform forproduction of vaccines, and ferritin-based vaccines have been
investigated in humans in two separate clinical trials. We conﬁrmed
proper folding and antigenicity of spike on the surface of ferritin bycryo-EM and binding to conformation-speciﬁc monoclonal
antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus
assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients.
Additionally, a single dose of SΔC-Fer elicited signiﬁcantly higher neutralizing responses as compared to immunization with the
spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔCFer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentationof SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy forsingle-dose vaccination against COVID-19.

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INTRODUCTION
The emergence of SARS-CoV-2 in the human population in2019 has caused a rapidly growing pandemic that has disrupted
nearly all global infrastructures. To date, there have been over
63 million conﬁrmed cases of COVID-19 and nearly 1.5
million deaths worldwide.1 While some nations have controlled
viral spread through social distancing, widespread testing, andcontact tracing, many nations struggle to contain the growing
number of cases and are still experiencing extensive
community spread. Additionally, the introduction of SARSCoV-2 into low-resource settings will lead to severe and lasting
impacts on economic and healthcare systems. Long-term
control of the pandemic will require one or more eﬀective
vaccines that can be made widely available across the globe.
The primary viral target for protective antibody-based
vaccines against COVID-19 is the SARS-CoV-2 spike, atrimeric surface glycoprotein responsible for viral entry.2,3
Importantly, COVID-19 patients have been shown to elicit
robust neutralizing antibody responses directed at the SARSCoV-2 spike, which suggests that this antigen could bepromising in the context of a protective vaccine.4,5 The spike
protein is produced as a single polypeptide and cleaved to form
the S1 and S2 subunits, which are responsible for receptor
binding (S1) and fusion with the host cell membrane (S2).3,6,7
The receptor binding domain (RBD) is a 25 kDa domain of S1© XXXX The Authors. Published byAmerican Chemical Societythat recognizes the SARS-CoV-2 human receptor, angiotensin
converting enzyme 2 (ACE2), and can form a functionallyfolded domain when expressed separately from the rest ofS1.8−10A vast array of vaccination platforms are being employed forthe development of a safe and eﬀective SARS-CoV-2
vaccine.11−19 Several vaccine candidates are currently being
investigated in Phase 3 clinical trials including two mRNAbased vaccines and two virally vectored vaccines.20−22
Importantly, the mRNA vaccine candidates rely on lipid−
nanoparticle encapsulation and require long-term cold-chain
storage (−20 to −80 °C),20,21 leading to extensive logisticalchallenges for distribution and administration. Virus-based
vaccines including inactivated, live-attenuated, and recombinant viral vaccines can produce robust immune responses, but
are known to induce oﬀ-target vector-directed immune
responses23,24 and can be associated with more frequent side
eﬀects and adverse events.25,26
Received: October 16, 2020

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Figure 1. Construct design for SARS-CoV-2 spike-functionalized ferritin nanoparticles. All constructs are based on the Wuhan-Hu-1 amino acidsequence (GenBank MN9089473) of SARS-CoV-2 spike. Spike-functionalized ferritin constructs were made by fusing spike ectodomain (residues
1−1213) or spikeΔC (residues 1−1143) to the H. pylori ferritin subunit separated by an SGG linker. A structural representation based on the spiketrimer cryo-EM structure (PDB 6VXX) and the H. pylori ferritin crystal structure (PDB 3BVE) depicts the 24-subunit particle displaying spike orspikeΔC on the surface. The estimated size of the spike-functionalized ferritin particles based on structural data is ∼300 Å. The S-GCN4 and SΔCGCN4 trimer constructs were made by fusing either the full-length spike residues (1−1213) or spikeΔC (1−1137) to a modiﬁed GCN4
trimerization domain followed by a hexahistidine tag. A structural representation of the spike trimers based on the cryo-EM structure (PDB 6VXX)
is shown with an estimate length of ∼100 Å. The RBD spans residues 319−541 of the spike protein and is preceded by the native signal peptide(not shown) and followed by a hexahistidine tag.

Subunit vaccines, in which a protein antigen from thepathogen is used to elicit a protective antibody response, are an
attractive option for an accessible SARS-CoV-2 vaccine forreasons including safety, manufacturing scalability, and ease of
distribution to low- and middle-income nations.26 Thoughtypically less immunogenic than virus-based vaccines, theimmunogenicity of subunit vaccinations can be signiﬁcantly
increased by formulation with adjuvants.25 It has also been
demonstrated that multivalent presentation of antigens
markedly enhances the immune response,27,28 and several
nanoparticle-based platforms have been utilized to multimerizeantigens of interest to improve the antibody response to
subunit vaccine candidates.27−30 Furthermore, two recent
studies have shown that the multivalent presentation of the
SARS-CoV-2 RBD31 as well as the spike ectodomain32 using
various multimerization platforms elicits better neutralizing
antibody responses than nonmultimerized forms of the same
antigens.
One such multimerization platform, Helicobacter pylori
ferritin, has been used to display antigens from inﬂuenza,33,34
HIV-1,35,36 and Epstein−Barr virus,30 among others.37,38 H.
pylori ferritin self-assembles into 24-subunit particles with eight
3-fold axes of symmetry.39 Fusion of a single protomer of aviral glycoprotein to the N-terminal region of an H. pylori
ferritin subunit facilitates assembly of a protein nanoparticle
that displays eight copies of a trimeric antigen on the surface atthe 3-fold axes.33,39 Display of antigens on ferritin generally
elicits a more robust neutralizing antibody response against thetarget pathogen as compared to immunization with the antigen
alone.30,33,35 Importantly, two inﬂuenza-functionalized ferritin
vaccines have been shown to be safe and immunogenic inclinical trials (NCT03186781 and NCT03814720),33,34 androbust pipelines have been established for large-scale
manufacturing of ferritin-based vaccines.40

Here, we fused the full-length spike ectodomain (residues
1−1213) to H. pylori ferritin (denoted S-Fer; Figure 1) todetermine the eﬀect of antigen multimerization on elicitationof antibodies against SARS-CoV-2. Additionally, we designed asecond nanoparticle construct in which we deleted the Cterminal 70 residues of the ectodomain and expressed thistruncated spike (residues 1−1143) on ferritin (SΔC-Fer).
These C-terminal residues are unresolved in the cryo-EM
structures of the spike trimer,3,7 and it has been suggested thatthey have extensive conformational ﬂexibility based on electron
microscopy of soluble trimers and viral particles.41−43 Additionally, this region of the spike contains an immunodominantlinear epitope, as determined via analysis of convalescent serafrom COVID-19 patients.44,45 We therefore hypothesized that
deleting these residues would more readily facilitate formationof spike ferritin particles and could inﬂuence immunogenicity.
As points of comparison, we also expressed and puriﬁed three
additional antigens: spike trimer containing a GCN4-based
trimerization domain either in full-length or SΔC form
(denoted S-GCN4 and SΔC-GCN4)46 and monomeric
receptor binding domain (RBD) (Figure 1).
After expressing and purifying the S-Fer and SΔC-Fer
nanoparticles, we conﬁrmed that they were stable, homogeneous, and properly folded using biophysical, structural, andbinding analyses including size-exclusion chromatography
multiangle light scattering (SEC-MALS), cryoelectron microscopy (cryo-EM), and biolayer interferometry (BLI). To assess
the immune response in vivo, we immunized mice andcharacterized the antibody responses to the spike ferritin
particles versus S-GCN4, SΔC-GCN4, and RBD. After a single
dose, mice immunized with SΔC-Fer exhibited a signiﬁcantly
higher neutralizing antibody response than all nonferritingroups as determined using a spike-pseudotyped lentiviralassay.47,48 Importantly, immunization with a single dose of SFer or SΔC-Fer elicited at least 2-fold higher neutralizing titers
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Figure 2. Spike ferritin nanoparticles can be expressed in mammalian cell culture and puriﬁed to homogeneity. (A) Scheme for expressing andpurifying spike ferritin nanoparticle antigens in mammalian cells. Spike ferritin particle subunits are encoded in a single plasmid that is transfected
into the Expi293F suspension human cell line. Expi293F cells are harvested, and culture supernatant is buﬀer exchanged and puriﬁed via anion
exchange chromatography. Protein-containing fractions are identiﬁed via Western blot, pooled, and puriﬁed by size-exclusion chromatography(SRT SEC-1000). Puriﬁed nanoparticles are assessed using biophysical characterization methods including SDS-PAGE, analytical size-exclusion
chromatography, and BLI follow by in vivo characterization of the immune responses elicited in mice. (B) SEC-MALS UV A280 (left) and lightscattering signals (right) from analysis of spike-based ferritin antigens using an SRT SEC-1000 size-exclusion column. A single prominent peak inboth the UV and light-scattering traces conﬁrms that spike ferritin nanoparticle preparations are homogeneous and do not aggregate.

context of immunization;50,51 thus, both vaccine and serology
eﬀorts involving soluble SARS-CoV-2 spike have utilized astabilized form of the trimer.
For comparison, we also produced two spike ectodomain
proteins fused to a trimeric coiled-coil, GCN4-pIQI46 (denoted
S-GCN4 and SΔC-GCN4; Figure 1). SΔC-GCN4 contained adeletion of residues 1138−1143 (which were present in SΔCFer) to eliminate a short helical segment prior to the start of
the GCN4-pIQI coiled-coil. A similar truncated version of the
spike trimer was recently utilized for antibody discovery.5 We
included the SARS-CoV-2 spike receptor binding domain(RBD; Figure 1) in our antigen panel because there is interest
in employing the RBD as a potential vaccine candidate.49,52,53
Spike Ferritin Nanoparticles Can Be Expressed in
Mammalian Cells and Puriﬁed to Homogeneity. The
SARS-CoV-2 spike is a heavily glycosylated trimeric proteinthat can be challenging to produce recombinantly.3,49,54
Production of recombinant spike ectodomain is commonly
done in mammalian cell culture systems in which the protein isglycosylated during synthesis.54 We expressed the spike ferritin
nanoparticles using human Expi293F cells. Since the ferritin
subunit facilitates self-assembly of the nanoparticles, production of puriﬁed spike-functionalized particles was achieved bytransfecting a single plasmid followed by a two-step chromatographic puriﬁcation (Figure 2A). After Expi293F transfection,
we monitored expression levels of the four spike-based
antigens (ferritins and trimers) with Western blots of cell
culture supernatants probed with SARS-CoV-2 reactive
monoclonal antibodies (mAbs) CR3022,55−57 CB6,58 andCOVA2-155 (Figure S1A). Although fusing spike to ferritin

as those observed in plasma from convalescent COVID-19
patients. Taken together, these results provide insights into thedevelopment of SARS-CoV-2 subunit vaccines and demonstrate that spike-functionalized ferritin nanoparticles elicit anenhanced antibody response compared to the spike trimers or
RBD alone.

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RESULTS
Design of Spike-Functionalized Ferritin Nanoparticles. To design functionalized nanoparticles displaying the
spike protein, we generated a fusion protein containing the
spike ectodomain followed by H. pylori ferritin, a 19 kDa
protein that self-assembles into a 24-subunit protein-based
nanoparticle (Figure 1). Given the 3-fold symmetrical axes on
the ferritin particle, fusion of a protomer to the N-terminus of
this domain creates a functionalized particle that displays eight
trimers on the surface.
We designed two versions of the spike-functionalized
nanoparticle: one containing the full-length ectodomain(residues 1−1213; S-Fer) and one in which the C-terminusof the ectodomain was truncated (residues 1−1143; SΔC-Fer)(Figure 1). We sought to investigate whether deletion of thisregion would inﬂuence expression levels, protein stability, and/or the immune response to spike. All spike antigens contained
a mutated furin cleavage site (RRAR mutated to a single
alanine) plus two proline mutations at residues 986 and 987,
which stabilize the spike trimer in the prefusion conformation.3,49 Previous work has shown that stabilization of theprefusion conformation of other coronavirus spikes enhances
protein expression and leads to greater neutralizing titers in theC

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Figure 3. Cryo-EM and BLI conﬁrm that spike proteins are presented on the particle surface with mAb epitopes intact. (A) Representative motioncorrected cryo-EM micrograph of the SΔC-Fer nanoparticles. Circles indicate representative particles that were picked for further analysis.
Micrographs demonstrate that particles are approximately 300 Å. (B) Reference-free 2D class averages of SΔC-Fer. 2D class averages conﬁrm thepresence of both ferritin particles and the display of spike on the surface seen as density surrounding the particles. (C) Reconstructed cryo-EM mapof the SΔC-Fer nanoparticle in two views. A single spike trimer on the surface is highlighted with each protomer of the trimer shown in a diﬀerent
color. (D) BLI binding of SARS-CoV-2 mAbs to puriﬁed spike antigens. Antigens were diluted to 100 nM monomer concentration (100 nM RBD,
33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Fer and SΔC-Fer 24-mer ferritin particle). Binding of all antigens to three SARS-CoV-2
reactive mAbs indicates that spike ferritin nanoparticles display epitopes similarly to the RBD and spike trimers. Curves were ﬁtted with anassociation/dissociation nonlinear regression, and ﬁts are represented with dashed black lines; kon values for each binding reaction are shown in
Figure S4A. Both S-Fer and SΔC-Fer exhibited a slight increase in signal during the dissociation step, perhaps due to rearrangements of theparticles on the BLI sensor tip due to the extensive avidity present on the multimerized particles. Lack of binding to an oﬀ-target Ebola-speciﬁc
antibody (ADI-15731) is presented in Figure S4B. Binding experiments were performed in at least duplicate; a representative trace and ﬁt are
shown from one replicate.

increases the size of the antigen from ∼450 kDa (trimer) to ∼4
MDa (ferritin), the expression levels for all spike proteins werecomparable (Figure S1A). Nanoparticles can sometimes
remain intact during SDS-PAGE analysis, even underdenaturing conditions, which can lead to retention of somesample within the well, causing the intensity of the monomer
band to appear weaker. Therefore, for a more quantitative
estimate of the protein levels in Expi cell culture supernatant,
we quantiﬁed the expression levels of each spike antigen using
a dot blot of unpuriﬁed cell culture supernatant blotted with
SARS-CoV-2 mAb CR3022. We made standard curves of
puriﬁed antigen and compared these curves to expression levels
from cell culture supernatants from a set (n = 5) of small-scaleprotein expressions (Figure S1B).59 This analysis indicatedsimilar expression trends to those observed via Western blot
and further conﬁrmed that fusion of spike to H. pylori ferritin
did not impact expression levels. Interestingly, it also suggeststhat fusion of SΔC to ferritin enhances the levels of protein
expression under these conditions.
Since the spike ferritin particles lack an aﬃnity puriﬁcation
tag, we puriﬁed them to homogeneity using anion exchange
followed by size-exclusion chromatography (Figure 2A). We

used SEC-MALS to assess sample quality and homogeneity
following puriﬁcation.60 No evidence of aggregation was
detected from the S-Fer or the SΔC-Fer samples via ultraviolet
absorbance (A280) or light scattering signals (Figure 2B).
Additionally, we determined that a freeze−thaw cycle does not
perturb particle formation or cause sample aggregation (Figure
S2A). We used the light scattering and refractive index datafrom spike nanoparticles to determine (Figure S2B) molecularweights of 4.2 MDa (S-Fer) and 3.1 MDa (SΔC-Fer). These
values are close to those expected from the amino acidsequences and the additional mass61 expected from ∼20
predicted N-linked glycosylation sites (Figure S2B). Taken
together, these experiments conﬁrm that functionalization of
H. pylori ferritin with the SARS-CoV-2 spike did not perturb
assembly of the nanoparticles.
We also produced the S-GCN4, SΔC-GCN4, and RBD in
human Expi293F cells. We puriﬁed these samples using
NiNTA puriﬁcation followed by size-exclusion chromatography and conﬁrmed sample purity and homogeneity using
SEC-MALS (Figure S2C). We observed a main peak and asecondary peak for the S-GCN4 sample, which has also been
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Figure 4. Immunization with SΔC-Fer nanoparticles elicits a stronger neutralizing response than immunization with nonferritin groups in mice.
(A) Immunization schedule including a priming dose with 10 μg of antigen at day 0 and a boost with 10 μg of antigen at day 21. Serum wascollected on days 0, 21, and 28. Both doses were adjuvanted with 10 μg of Quil-A and 10 μg of MPLA in a total volume of 100 μL per mouseadministered via subcutaneous injection. (B) ELISA binding titers to both the RBD and full-length spike ectodomain after a single dose of antigen
demonstrate that all groups elicited a SARS-CoV-2-directed antibody response following immunization. Each point represents the EC50 titer from asingle animal; each bar represents the mean EC50 titer from the group (n = 10 mice per group). Error bars represent standard deviation. Points withsignal less than EC50 1:100 dilution are placed at the limit of quantitation for the assay. (C) S-Fer and SΔC-Fer antigens elicit stronger neutralizing
antibody responses than spike trimers alone or RBD, as indicated by spike-pseudotyped lentivirus neutralizing titers after a single dose of antigen.
Immunization with a single dose of S-Fer or SΔC-Fer elicits neutralizing responses that are at least 2-fold greater on average than those found inplasma from 20 convalescent COVID-19 patients (CCP). Each point represents the IC50 titer from a single animal or patient; each bar represents
the mean IC50 titer from each group (n = 10 per group, with the exception of CCP which is n = 20). Error bars represent standard deviation.
Samples with neutralizing activity that was undetectable at 1:50 dilution or with an IC50 less than 1:100 dilution are placed at the limit ofquantitation. (D) ELISA binding titers to the RBD and spike after two doses of antigen show that the SARS-CoV-2-speciﬁc response against bothantigens was boosted in all groups. Groups and error are as deﬁned in part B. (E) Spike-pseudotyped lentivirus neutralization following two dosesof antigen indicates that although all groups had a neutralizing response following two doses, animals immunized with SΔC-Fer have the highest
neutralizing titers, and these are signiﬁcantly greater than S-GCN4 and SΔC-GCN4. Groups and error are as deﬁned in part C. Statisticalcomparisons for panels B−E were performed using Kruskal−Wallis ANOVA followed by Dunn’s multiple comparisons. All p values are represented
as follows: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Mean titers with standard deviation and values from pairwise
comparisons between groups can be found in Tables S1−S3.
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and puriﬁcation conditions of spike trimers.62 These
populations may correspond to conformational populationsof the trimer and have been suggested to potentially be related
to RBDs on diﬀerent protomers in “up” and “down” states.3,7,62
Structural and Functional Analysis Demonstrates
That Spike-Functionalized Nanoparticles Are Stably
Folded and Properly Display Epitopes of Interest. To
conﬁrm that spike is displayed on the surface of ferritin, we
performed cryo-EM on both S-Fer and SΔC-Fer. Cryo-EM
raw images showed observable densities around ferritin
particles (Figure 3A and Figure S3A), indicating proper
formation of the nanoparticles and display of spike on thesurface. The two-dimensional class averages further showed thedensities of spike surrounding ferritin (Figure 3B and Figure
S3B). We chose to perform additional data collection andimage processing of the SΔC-Fer particles since they had moredeﬁned spike density than the S-Fer particles. Using singleparticle analysis, we determined the three-dimensional (3D)
structure of the SΔC-Fer complex from ∼60 000 particles andachieved overall resolutions of 13.5 and 23.6 Å, with andwithout octahedral symmetry applied, respectively (Figure
S3C−E). The overall shapes of the two cryo-EM maps werevery similar, with a cross-correlation coeﬃcient of 0.99 (Figure
S3D,E). The map obtained with octahedral symmetry can beseen in two diﬀerent views in Figure 3C. One representative
trimer on the surface is highlighted, with each protomer
indicated in a separate color to demonstrate the display oftrimeric spike on the surface of ferritin.
The smearing of the spike protein densities (Figure 3B and
Figure S3B) is likely due to the ﬂexibility of spike on theferritin surface resulting from the Ser-Gly-Gly linker (Figure1). This conclusion predicts that density for the ferritin core of
the nanoparticles would not be smeared. Indeed, when singleparticle analysis of the SΔC-Fer cryo-EM data set focused on
only the ferritin core, excluding spike-speciﬁc density, we wereable to obtain a 3.7 Å resolution structure of the ferritin core of
SΔC-Fer (Figure S3F).
Since we aimed to use the S-Fer and SΔC-Fer nanoparticles
as antigens for eliciting SARS-CoV-2-directed antibodies, we
conﬁrmed that they displayed properly folded spike that could
recognize mAbs and ACE2. We used BLI (Figure 3D) toassess binding of three SARS-CoV-2 mAbs (CR3022, COVA215, and CB6) in IgG form to S-Fer and SΔC-Fer. We
performed the BLI experiments at a set concentration of 100
nM monomer for each antigen which corresponds to 100 nM
RBD monomer, 33.3 nM S-GCN4 and SΔC-GCN4 trimer,and 4.2 nM S-Fer and SΔC-Fer 24-mer ferritin nanoparticle.
Thus, observed diﬀerences in the magnitude of binding arelargely due to diﬀerences in molar concentration of antigen.
We ﬁt the BLI data using an association/dissociation nonlinear
regression and extracted kon values for each mAb binding eachantigen (Figure S4A). We could not obtain koff values for asubset of the binding interactions due to the undetectabledissociation of IgGs from the highly avid spike ferritin
nanoparticle samples (Figure 3D). Thus, we did not determine
KD values for mAbs binding to each antigen but used the kon
values as a metric to compare binding (Figure S4A). The mAbs
bound the functionalized nanoparticles similarly to both the
spike trimers and the RBD (Figure 3D and Figure S4A),
conﬁrming that display of the spike on ferritin nanoparticles
does not perturb or occlude critical conformation-speciﬁc
epitopes. We observed minimal binding when a nontarget mAb(anti-Ebola glycoprotein mAb, ADI-1573163) was loaded on

Research Article

the tip (Figure S4B). We also compared binding of the threemAbs as well as the ectodomain of human ACE2, the SARSCoV-2 receptor, to all antigens using enzyme-linked
immunosorbent assays (ELISAs; Figure S5). These ELISA
results were consistent with those from BLI, indicating that
SARS-CoV-2 and ACE2 bind the spike-functionalized particles
as well as they bind spike trimers and RBD.
Immunization with Spike-Functionalized Nanoparticles Elicits SARS-CoV-2 Neutralizing Antibodies with aSingle Dose. To assess the immune response to S-Fer and
SΔC-Fer nanoparticles versus the other antigens, we immunized 10 mice (n = 5 in two replicate immunizations) with 10
μg of antigen adjuvanted with 10 μg of Quil-A and 10 μg ofmonophosphoryl lipid A (MPLA).64−66 We collected serum atday 21 to assess the response to a single dose of antigen. We
administered a second dose of antigen at day 21 andsubsequently collected serum at day 28 to determine how
the initial response to each antigen was boosted (Figure 4A).
Mean ELISA titers and spike-pseudotyped lentivirus neutralization titers for all antigen groups at day 21 and day 28 can befound in Table S1.
After a single dose (“Prime only”), all groups exhibited a
detectable antibody response against both RBD and spike, as
revealed by ELISA (Figure 4B). To analyze neutralizing titers,
we used lentivirus pseudotyped with SARS-CoV-2 spike47 andassessed inhibition of viral entry into HeLa cells overexpressing
ACE2.48 Neutralization with pseudotyped viruses is a common
way to assess viral inhibition in a BSL2 setting since the SARSCoV-2 replicating pathogen requires a BSL3 facility.47,67 We
validated the neutralization assay using published neutralizing
mAbs against SARS-CoV-2 (Figure S6).
Analysis of antisera from immunized mice revealed that the
only groups with notable neutralizing activity after a single
dose of antigen were those immunized with S-Fer or SΔC-Fer(Figure 4C), underscoring that multivalent presentation
greatly enhances the neutralizing antibody response in asingle-dose regimen of an adjuvanted SARS-CoV-2 subunit
vaccine. As illustrated in Figure 4C, the SΔC-Fer group had
signiﬁcantly higher neutralizing titers than all three nonferritingroups at the day 21 time point, whereas the S-Fer groupelicited signiﬁcantly greater neutralizing responses than SΔCGCN4 and RBD. Importantly, antisera from mice immunizedwith a single dose of either S-Fer or SΔC-Fer had ∼2-foldhigher mean neutralizing titers than those observed in a cohortof 20 convalescent COVID-19 plasma donors (Figure 4C, farright).
The lack of neutralizing titers after a single-dose
immunization in both the RBD and SΔC-GCN4 groups andminimal neutralizing titers in the S-GCN4 group (Figure 4C)
are consistent with recent reports that also found that a single
dose of spike trimer or RBD was insuﬃcient to elicit robust
neutralizing antibodies in mice.31,32,68 Consistent with thelentiviral neutralization results (Figure 4C), the only twogroups with substantial ACE2-competing antibodies (asdetermined by a decrease in ACE2 binding to RBD on
ELISA) were those immunized with spike ferritin nanoparticles(Figure S7, left). ACE2-blocking activity in the antisera of mice
after a single dose of antigen (day 21) appeared to be weak, aseven the nanoparticle groups with the strongest neutralizing
response exhibited minimal blocking at the highest serum
concentration tested (1:50 dilution), and no groups exhibitedany blocking activity at a 1:500 dilution (Figure S7).
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S-GCN4, SΔC-GCN4, and RBD groups, the His-tag on theplated antigen likely did not impact the overall titers for the SGCN4 and RBD groups but may have slightly increased thetiters for the SΔC-GCN4 group.
Though antisera from all groups exhibited neutralizing
activity after a boost, mice immunized with SΔC-Fer exhibitedthe highest neutralizing titers overall and had signiﬁcantly
higher titers than both the S-GCN4 and SΔC-GCN4 groups(Figure 4E). Importantly, the variation in response to thenonferritin groups was also considerably larger than thevariability detected in mice immunized with S-Fer or SΔC-Fer
nanoparticles (Figure 4E), suggesting that the multivalent
presentation of spike facilitated a more consistent immune
response. Furthermore, the neutralizing antibody response
elicited by SΔC-Fer shows dose-dependent trends (Figure
S10A). Though a decrease in dose from 10 to 1 μg led to anincreased variability in the elicitation of neutralizing titers, thepresence of neutralizing antibodies in a subset of micefollowing only 1 μg of SΔC-Fer could have important dosesparing implications. Additionally, neutralizing titers remain
stable even to 20 weeks following a single immunization with
20 μg (Figure S10B), demonstrating that this formulation may
successfully achieve neutralizing antibody levels that persist in
the months following vaccination. These data demonstrate thatthe spike ferritin nanoparticles presented here, particularly
SΔC-Fer, are superior vaccine candidates to spike trimer or
RBD alone.

To evaluate the eﬀect of boosting on the immune responses
to these antigens, we administered a second dose of antigen on
day 21 and collected serum at day 28 (“Prime + Boost”). We
analyzed the post-boost serum for RBD and spike titers, ACE2
blocking activity, and neutralization potency. The immune
response to all antigens was increased, and antigen-speciﬁctiters for both RBD and spike were similar among groups after
the second dose (Figure 4D). ACE2 blocking activity wasnotably enhanced after the boost, and all ﬁve antigen groups
had nearly complete ACE2 blocking at 1:50 serum dilution atthat time point (Figure S7, right). While all antigen groups also
had partial ACE2 blocking activity at 1:500 serum dilution, thelevels of ACE2 blocking at this dilution did not relate to overall
trends in neutralizing titers (compare Figure 4E and FigureS7). This discordance indicates that ACE2-blocking activity
may not correlate with neutralizing titers for some antigens andsuggests that neutralizing epitopes other than the ACE2
binding site exist on the SARS-CoV-2 spike (see refs 5 and69).
To assess the polarization of the immune response following
two doses of antigen, we quantiﬁed the levels IgG1, IgG2a, andIgG2b titers for RBD-speciﬁc antibodies (Figure S8). IgG
subclasses can provide information regarding whether a given
response is biased toward a Th1- or Th2-like response.70−73
Adjuvants are known to play a major role in inﬂuencing thepolarization of vaccine immune responses, and prior work has
shown that Quil-A and MPLA adjuvants can lead to a Th1biased response.70,71 Some vaccine candidates against SARSCoV were found to cause pulmonary immunopathology
potentially due to Th2-type responses in small animalmodels,74−77 and thus one focus for SARS-CoV-2 vaccine
development has been to favor a Th1-type response.72,73 A
comparison of the ratio of IgG2a/IgG1 antibody titers among
antigen groups demonstrated that, except for the RBD, theantigens exhibit a balanced IgG2a and IgG1 response, which
was slightly skewed toward IgG2a for both the SΔC-Fer and SGCN4 groups (Figure S8A,B). Notably, the RBD elicited a
substantially higher IgG1 response as compared to both IgG2aand IgG2b, suggesting that this antigen in combination with
Quil-A/MPLA caused a Th2-biased immune response (Figure
S8B,C). IgM responses did not substantially diﬀer among
antigen groups at the day 28 time point and were lower than
IgG isotypes (Figure S8D), and at the same time point serum
IgA levels were undetectable (data not shown).
Given that each antigen contains a non-SARS-CoV-2
fragment, we wanted to assess the oﬀ-target antibody responses
directed at either the H. pylori ferritin, the GCN4 trimerization
domain, or the His-tag. We observed anti-H. pylori ferritin
responses in the S-Fer and SΔC-Fer groups that were similar
and were equivalent to or less than anti-RBD and anti-spike
titers from the same groups as determined by ELISA (Figure
S9, left). The S-GCN4 and SΔC-GCN4 groups had similaranti-GCN4 responses (Figure S9, middle), suggesting that theH. pylori ferritin and GCN4 domains are equivalently
immunogenic. Interestingly, when we probed the anti-His-tag
response speciﬁcally, we observed that the SΔC-GCN4 group
had a notably higher His-tag response as compared to either
the S-GCN4 or the RBD group, even though all three antigens
contained His-tags (Figure S9, right). This could suggest that
this antigen either was less structurally stable or had otherantigenic features that altered the overall immunogenicity tocause a higher tag-directed response. Additionally, thisindicates that, for the RBD- and spike-speciﬁc titers from the■

DISCUSSION
Global management of the COVID-19 pandemic depends not
only upon the development of safe and protective vaccine
candidates but also on the rapid production and deployment ofbillions of doses. Given these extensive manufacturing
challenges, vaccination strategies requiring only a single dose
could be critical to achieving worldwide immunization against
SARS-CoV-2. Toward this end, we designed and characterized
two vaccine candidates based on ferritin nanoparticles (S-Ferand SΔC-Fer) that display multiple copies of the SARS-CoV-2
spike and can be readily expressed in mammalian cells. These
proteins can be puriﬁed using routine methods and a
production scheme similar to that for soluble spike trimers.
Immunization with the spike nanoparticles elicited neutralizing antibodies in mice after a single dose, whereas
immunization with the RBD or spike trimers elicited little tono neutralizing titers with the same dose (Figure 4C).
Interestingly, deletion of the C-terminal portion of the spikeectodomain enhanced the neutralizing antibody response
speciﬁcally in the context of the nanoparticle (Figure 4C).
After a second dose, all antigen groups elicited neutralizing
antibodies; however, the SΔC-Fer immunized mice had thehighest overall neutralizing titers and, importantly, hadsigniﬁcantly higher titers than both the S-GCN4 and SΔCGCN4 trimer groups (Figure 4E). These results provide
important insight into the use of spike ferritin nanoparticles as
an eﬀective single-dose vaccine against SARS-CoV-2.
Ferritin nanoparticles are of signiﬁcant interest fordeveloping new vaccines because they self-assemble into stablestructures that display trimeric proteins on 3-fold symmetryaxes.39 Unlike nanoparticle platforms that require postpuriﬁcation conjugation of an antigen to a carrier or scaﬀold,78
here, spike-functionalized nanoparticles were produced bytransfecting a single plasmid encoding the spike fused to theferritin subunit into mammalian cells. We built on existingG

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unable to neutralize virus. This supports the notion that the
response to RBD may have largely focused on the nonneutralizing regions.
After a second dose of antigen, all antigens elicited
detectable neutralizing antibody titers (Figure 4E). Despite
the lack of detectable neutralizing signal in the RBD or SΔCGCN4 groups after a single dose (Figure 4C), these groups
exhibited titers roughly equivalent to those of the S-GCN4
group after a second dose (Figure 4E). Interestingly, mice
immunized with either S-Fer or SΔC-Fer had less variability inresponse than the other three groups (Figure 4E), suggestingthat multivalent presentation facilitates more consistentelicitation of neutralizing antibodies. Although all groups
elicited a neutralizing response after the second dose, the mice
immunized with SΔC-Fer had the highest overall neutralizing
titers and elicited signiﬁcantly higher neutralizing titers versus
both spike trimers tested (Figure 4E). Taken together, thesedata suggest that SΔC-Fer is the best-performing antigen outof those we tested here and could be a favorable candidate foruse in a subunit or nucleic acid vaccine against SARS-CoV-2.
Eﬀective global deployment of a SARS-CoV-2 vaccine will
depend on several logistical factors. The number of doses in an
immunization regimen required to achieve eﬃcacy will be ofcritical importance due to the number of doses required
worldwide and to the ability to access patients multiple times.
Therefore, assessing candidates that can achieve protection
after a single dose is highly important for implementing a
global vaccination strategy. Here, we have demonstrated that adose of either S-Fer or SΔC-Fer nanoparticles adjuvanted with
Quil-A and MPLA was suﬃcient to achieve neutralizing titers
greater than those in plasma from convalescent COVID-19
patients. Additionally, both S-Fer and SΔC-Fer form nanoparticles spontaneously following expression in mammaliancells without the need for a conjugation step, showcasing thepotential application of spike ferritin constructs to nucleic acidbased vaccine strategies. Taken together, our data indicate thatthe multivalent presentation of the SARS-CoV-2 spike is aneﬀective way to enhance the antibody response after a single
dose, providing key insight for the development of an eﬀective
and deployable vaccine to combat the COVID-19 pandemic.

work using ferritin nanoparticles to display other viral antigens
for vaccine development30,33−38 and designed nanoparticles
displaying the SARS-Cov-2 spike ectodomain. Importantly, thetwo ferritin-based antigens that we designed (S-Fer and SΔCFer) expressed comparably to spike trimers (S-GCN4 and
SΔC-GCN4) in mammalian cells (Figure S1), indicating thatfusing the spike to ferritin does not negatively impact protein
production. As glycosylation can inﬂuence proper folding ofviral antigens, it is also noteworthy that expression wasachieved in mammalian cells, which allows spike proteins to be
produced with native-like glycosylation.54
These constructs could also be important for the development of nucleic acid vaccines given the self-assembly of spike
ferritin nanoparticles following production in mammalian cells,
expression levels comparable to those of spike trimers, and theenhanced immune response after a single dose of antigen. We
administered equivalent doses of spike ferritin nanoparticle andtrimer and observed an enhanced response after single-doseand two-dose vaccine regimens with the SΔC-Fer (Figure
4C,E). Thus, administration of a nucleic acid vaccine encoding
a multimerized ferritin-based spike protein could potentially
elicit a heightened response versus constructs encoding spikealone, although we did not test that hypothesis here.
Following puriﬁcation of the spike-functionalized nanoparticles from Expi293F culture supernatant, we extensively
characterized their biophysical properties to ensure that the
spikes were properly folded and stable and could display
important epitopes of interest. We conﬁrmed the structure,homogeneity, and epitope conformations of spike-functionalized nanoparticles using cryo-EM (Figure 3A−C and FigureS3), SEC-MALS (Figure 2B and Figure S2), and BLImeasured binding to conformation-speciﬁc SARS-CoV-2
mAbs (Figure 3D).
Remarkably, mice immunized with a single dose of either SFer or SΔC-Fer elicited mean neutralizing antibody titers thatwere approximately 2-fold higher than mean titers observed inplasma from convalescent COVID-19 patients (Figure 4C).
Additionally, mice immunized with one dose of SΔC-Fer hadsigniﬁcantly higher neutralizing titers versus all nonferritingroups, whereas mice immunized with one dose of S-Fer hadsigniﬁcantly higher titers than the RBD and SΔC-GCN4 group(Figure 4C). These data clearly demonstrate that equivalentdoses of spike ferritin nanoparticles elicit a better neutralizing
response versus RBD or trimer alone in a single-dose setting
which is consistent with observations from other recent studies
investigating other multimerized SARS-CoV-2 vaccine platforms.31,32 A comparison of neutralizing antibody titers
observed in these other studies reveals that, despite the useof diﬀerent adjuvants, the spike ferritin nanoparticles we
present here elicit similar levels of neutralizing antibodies
following one or two doses.31,32
Mice immunized with the RBD exhibited no observable
serum neutralizing activity after a single dose (Figure 4C)
despite having RBD-speciﬁc antibody titers that were similar to
those in other groups (Figure 4B). This discordance suggeststhat RBD binding titers may not be a strong correlate ofneutralizing antibody responses in the context of vaccination,
particularly for candidates based on the RBD alone.52,79,80
Notably, the full-length spike ELISA binding titers fromanimals immunized with the RBD were signiﬁcantly lower thantiters from the other groups (Figure 4B), suggesting that
immunization with monomeric RBD elicits antibodies toepitopes that are occluded in full-length spike, rendering them■

MATERIALS AND METHODSDNA Plasmid Construction and Propagation. All spike
constructs were cloned from a full-length spike expression
plasmid received from Dr. Florian Krammer.49 This constructcontains residues 1−1213 from the Wuhan-Hu-1 genome
sequence (GenBank MN9089473), followed by a thrombin
cleavage site, a T4 ﬁbritin trimerization domain, and a hexahistidine tag for puriﬁcation. This construct was cloned out ofthe parent vector and into an in-house pADD2 mammalian
expression vector using HiFi PCR (Takara) followed by InFusion (Takara) cloning with EcoRI/XhoI cut sites. This
construct was used to clone subsequent spike ferritin and spike
GCN4-pIQI trimer constructs. Full-length spike ferritin (S-Fer)and spikeΔCferritin (SΔC-Fer) were cloned by polymerase
chain reaction (PCR) ampliﬁcation of full-length spike(residues 1−1213) or spikeΔC (residues 1−1143) oﬀ theparent expression vector. This was followed by a stitching PCR
in which constructs were annealed to an amplicon containing
H. pylori ferritin (residues 5−168) originally generated as agene-block fragment from Integrated DNA Technologies(IDT). The spike and ferritin subunits were separated by a
SGG linker.33 Spike ferritin amplicons were inserted into theH

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Superfolder GFP (sfGFP) was tagged with either GCN4Avi-His (GCN4 tag domain amino acid sequence taken
from81) or His-tag only to quantify oﬀ-target tag antibody
responses. sfGFP-His and sf-GFP-GCN4-His encoded in
pET28a bacterial T7-expression vectors were transformed
into BL21 cells and induced at OD 0.6 with 1 mM isopropyl βD-1-thiogalactopyranoside (IPTG) for 3 h at 37 °C. Cells were
harvested by spinning at 7000g for 10 min, resuspended in 10
mM imidazole/1× PBS [pH 7.4], lysed by sonicating for 20 s
three times, and spun at 15 000g for 20 min following lysis.
sfGFP-His and sf-GFP-GCN4-His were then puriﬁed fromsupernatants using NiNTA resin as described below.
S-Fer, SΔC-Fer, and WT H. pylori ferritin nanoparticles
were isolated using anion-exchange chromatography followedby size-exclusion chromatography using an SRT SEC-1000(Sepax) column. Brieﬂy, Expi293F culture supernatants wereconcentrated using tangential ﬂow ﬁltration with a GE AKTA
Flux S with a 10 kDa molecular weight cutoﬀ (MWCO)
hollow ﬁber cartridge (UFP-10-E-4MA) and then buﬀerexchanged into 20 mM Tris [pH 8.0] via overnight dialysis at 4
°C using 100 kDa MWCO dialysis tubing. For some preps,
culture supernatant was dialyzed directly and not concentratedusing tangential ﬂow ﬁltration. Dialyzed culture supernatants
were ﬁltered through a 0.22 μm ﬁlter and loaded onto a 5 mL
HiTrapQ anion-exchange column (GE) equilibrated in 20 mM
Tris [pH 8.0] on a GE AKTA Pure system. Ferritin
nanoparticles were eluted with a sodium chloride (NaCl)
gradient, and the particle-containing fractions were identiﬁed
via Western blot with CR3022 for spike ferritins or SDS-PAGE
followed by staining with GelCode blue stain reagent(Thermo) for WT H. pylori ferritin. Fractions were pooled
and concentrated using a 100 kDa MWCO Amicon spin ﬁlter
and subsequently puriﬁed on a GE AKTA Pure system using
an SRT SEC-1000 SEC column equilibrated in 1× Dulbecco’s
phosphate-buﬀered saline (DPBS) (Gibco).
RBD, S-GCN4, SΔC-GCN4, sf-GFPHis, and sfGFP-GCN4His were puriﬁed with HisPur NiNTA resin (ThermoFisher).
Prior to puriﬁcation, resin was washed 3× with ∼10 column
volumes of wash buﬀer (10 mM imidazole/1× PBS [pH 7.4]).
Expi293F cell culture supernatants were diluted 1:1 with 10
mM imidazole/1× PBS [pH 7.4]; resin was added to diluted
cell supernatant and incubated at 4 °C. Resin/supernatantmixtures were agitated during binding using a stir-bar and amagnetic stir-plate. Resin/supernatant mixtures were added to
glass chromatography columns for gravity ﬂow puriﬁcation.
Resin was washed with 10 mM imidazole/1× PBS [pH 7.4],
and proteins were eluted with 250 mM imidazole/1× PBS.
NiNTA elutions were concentrated using Amicon spin
concentrators (10 kDa MWCO for RBD, sfGFP-His, and
sfGFP-GCN4-His and 100 kDa MWCO for spike trimers)
followed by size-exclusion chromatography. The RBD, sfGFPHis, and sfGFP-GCN4-His were puriﬁed using a GE Superdex
200 Increase 10/300 GL column, and the S-GCN4 and SΔCGCN4 proteins were puriﬁed using a GE Superose 6 Increase
10/300 GL column. Columns were pre-equilibrated in 1×
Dulbecco’s phosphate-buﬀered saline (DPBS) (Gibco) or 1×
PBS [pH 7.4].
Fractions were pooled based on A280 signals and/or SDSPAGE on 4−20% Mini-PROTEAN TGX protein gels stainedwith GelCode blue stain reagent (ThermoFisher). Samples for
immunizations were supplemented with 10% glycerol, ﬁlteredthrough a 0.22 μm ﬁlter, snap frozen, and stored at −20 °C
until use.

pADD2 mammalian expression vector via In-Fusion using
EcoRI/XhoI cut sites. The spike trimer constructs were cloned
similarly and fused to the GCN4-pIQI46 domain instead offerritin. The spikeΔC trimer included residues 1−1137
followed by a glycine residue and then the GCN4-pIQI
domain. The full-length spike trimer included residues 1−1213
followed by a GGGGS linker and then the GCN4-pIQI
domain. Both trimer constructs contained a hexa-histidine tagfor NiNTA puriﬁcation.
The SARS-CoV-2 RBD construct was kindly provided by
Dr. Florian Krammer.49 This construct contains the native
signal peptide (residues 1−14) followed by residues 319−541
from the SARS-CoV-2 Wuhan-Hu-1 genome sequence(GenBank MN908947.3) and a hexa-histidine tag at the Cterminus for puriﬁcation. This expression plasmid (pCAGGS)
contains a CMV promoter for protein expression in
mammalian cells.
The variable heavy chain (HC) and variable light chain(LC) sequences for SARS-CoV-2 reactive mAbs, CR3022 (HCGenBank DQ168569, LC Genbank DQ168570), CB6 (HCGenBank MT470197, LC GenBank MT470196), and COVA2-15 (HC GenBank MT599861, LC GenBank MT599945)
were codon optimized for human expression using the IDT
Codon Optimization Tool and ordered as gene-block fragments from IDT. Fragments were PCR ampliﬁed and inserted
into linearized CMV/R expression vectors containing theheavy chain or light chain Fc sequence from VRC01 using InFusion.
Soluble human ACE2 with an Fc tag was constructed by
PCR amplifying ACE2 (residues 1−615) from Addgene
plasmid 1786 (a kind gift from Dr. Jesse Bloom) and fusing
it to a human Fc domain from VRC01, separated by a TEVGSGG linker using a stitching PCR step. ACE2-Fc wasinserted into the pADD2 mammalian expression vector via InFusion using EcoRI/XhoI cut sites.
After all cloned plasmids were sequence conﬁrmed using
Sanger sequencing, plasmids were transformed into Stellar
Cells (Takara) and grown overnight in 2xYT/carbenicillin
cultures, with the exception of the CMV/R mAb plasmids
which were grown in 2xYT/kanamycin cultures. Plasmids wereprepared for mammalian cell transfection using Machery Nagel
Maxi Prep columns. Eluted DNA was ﬁltered in a biosafety
hood using a 0.22 μm ﬁlter prior to transfection.
Expression and Puriﬁcation of SARS-CoV-2 Antigens,mAbs, and Soluble ACE2. All antigens for immunization,
mAbs, and soluble human ACE2-Fc were expressed andpuriﬁed from Expi293F cells. Expi293F cells were cultured
using 66% FreeStyle 293 Expression/33% Expi293 Expression
medium (ThermoFisher) and grown in polycarbonate baﬄedshaking ﬂasks at 37 °C and 8% CO2 while shaking at 120 rpm.
Cells were transfected at a density of approximately (3−4) ×
106 cells/mL. Transfection mixtures were made by adding 568
μg of maxi-prepped DNA to 113 mL of culture medium (perliter of transfected cells) followed by addition of 1.48 mL of
FectoPro (Polyplus). For mAbs, cells were transfected with a
1:1 ratio of HC:LC plasmid DNA. Mixtures were incubated atroom temperature for 10 min and then added to cells. Cells
were immediately boosted with D-glucose (4 g/L ﬁnal
concentration) and 2-propylpentanoic (valproic) acid (3 mM
ﬁnal concentration). Cells were harvested 3−5 days posttransfection via centrifugation at 7000g for 15 min. Culture
supernatants were ﬁltered with a 0.22 μm ﬁlter.
I

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weights were determined using ASTRA 7.3.2 (Wyatt
Technologies).
Cryo-EM Data Acquisition. Samples were diluted to a
ﬁnal concentration of ∼0.4 mg/mL for both the S-Fer and
SΔC-Fer particles after puriﬁcation. The samples (3 μL) wereapplied onto glow-discharged 200-mesh R2/1 Quantifoil grids
coated with continuous carbon. The grids were blotted for 2 s
and rapidly cryocooled in liquid ethane using a Vitrobot MarkIV instrument (Thermo Fisher Scientiﬁc) at 4 °C and 100%
humidity. Samples were screened using a Talos Arctica
cryoelectron microscope (Thermo Fisher Scientiﬁc) operated
at 200 kV. The spikeΔC ferritin sample was imaged in a Titan
Krios cryoelectron microscope (Thermo Fisher Scientiﬁc)
operated at 300 kV with GIF energy ﬁlter (Gatan) at amagniﬁcation of 130 000× (corresponding to a calibrated
sampling of 1.06 Å per pixel). Micrographs were recorded withEPU 2.6 (Thermo Fisher Scientiﬁc) with a Gatan K2 Summit
direct electron detector; each image was composed of 30
individual frames with an exposure time of 6 s and an exposure
rate of 7.8 electrons per second per Å2. A total of 3684 movie
stacks was collected.
Single-Particle Image Processing and 3D Reconstruction. All movie stacks were ﬁrst imported into Relion 3.0.682
for image processing. Motion-correction was performed with
MotionCor2 1.3.2,83 and the contrast transfer function wasdetermined with CTFFIND4 4.1.13.84 All particles were
autopicked using the NeuralNet option in EMAN2 2.31,85
yielding 152 734 particles from 3540 selected micrographs.
Then, particle coordinates were imported into Relion 3.0.6,
where poor 2D class averages were removed through several
rounds of 2D classiﬁcation. The initial model was built in
cryoSPARC86 using the ab initio reconstruction option withoctahedral symmetry applied. The ﬁnal 3D reﬁnement was
performed using 62 837 particles with or without octahedral
symmetry applied; a 13.5 Å map and a 23.6 Å map wereobtained, respectively. Resolution for the ﬁnal maps wasestimated with the 0.143 criterion of the Fourier shellcorrelation curve. A Gaussian low-pass ﬁlter was applied tothe ﬁnal 3D maps displayed in the UCSF Chimera 1.13.1
software package.87
BLI of mAbs Binding to SARS-CoV-2 Puriﬁed
Antigens. BLI was performed on an OctetRed 96 system(ForteBio). Antigens and mAbs were diluted in Octet buﬀer(0.5% bovine serum albumin, 0.02% Tween, 1× Dulbecco’s
phosphate-buﬀered saline (DPBS) (Gibco)) and plated in 96well ﬂat-bottom black plates (Greiner). Tips were preequilibrated in Octet buﬀer and regenerated in 100 mM
glycine [pH 1.5] prior to binding antigens. Anti-Human Fc
sensor tips (Forte) were dipped into 200 nM mAb and thensubmerged into wells containing 100 nM (protomer ormonomer concentration) of each antigen. The ﬁnal concentration of each antigen was as follows: 100 nM RBD monomer,
33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Ferand SΔC-Fer nanoparticle. Background subtraction was
performed using an mAb-loaded sensor tip submerged into awell containing buﬀer only.
ELISA with Puriﬁed mAbs and Mouse Serum. ELISA of
SARS-CoV-2 antigens was performed by coating antigens on
MaxiSorp 96-well plates (ThermoFisher) at 2 μg/mL in 1×
PBS [pH 7.4] overnight at 4 °C. Mouse serum ELISAs were
performed using RBD or full-length spike ectodomain with a
T4 ﬁbritin (foldon) trimerization domain, described in detailin ref 49. ELISAs to quantify oﬀ-target antibody responses

mAbs and ACE2-hFc were puriﬁed using Protein A agarose
resin (Pierce). Filtered cell culture supernatant was diluted 1:1
with 1× PBS [pH 7.4] and added to Protein A resin which was
prewashed with ∼10 column volumes of 1× PBS [pH 7.4].
Resin/supernatant mixtures were batch bound at 4 °C.
Proteins were eluted with 100 mM glycine [pH 2.8], andelutions were neutralized via addition of 1/10th volume 1 M
Tris [pH 8.0].
Western Blot Analysis of Expi293F Culture Supernatants. Expi293F culture supernatants were collected 3 days
after transfection, harvested via spinning at 7000g for 15 min,and ﬁltered through a 0.22 μm ﬁlter. Samples were diluted in
SDS-PAGE Laemmli loading buﬀer (Bio-Rad), boiled at 95°C, and run on a 4−20% Mini-PROTEAN TGX protein gel(Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were
blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20)
and then washed with PBST. In-house-made primary antibodies CR3022, COVA2-15, and CB6 (approximate concentrations 0.8−1.3 mg/mL) were added at a 1:10 000 dilution in
PBST. Blots were washed with PBST, and secondary rabbit
antihuman IgG H&L HRP (abcam ab6759) was added at
1:10 000 in PBST. Blots were developed using Pierce ECL
substrate and imaged using a GE Amersham imager 600.
Dot Blot Analysis of Expi293F Culture Supernatants.
Puriﬁed proteins were diluted into conditioned expression
medium (medium harvested 3 days post-mock-transfection) toa ﬁnal concentration of 0.1 mg/mL and serially diluted 3-foldusing conditioned medium as diluent. Dilution series weremade in two independent replicates. Each concentration (2μL) was dotted onto a nitrocellulose membrane to produce a
standard curve. Expi293F culture supernatants from spike
antigen expressions were harvested 3 days post-transfection via
centrifugation at 7000g for 15 min and ﬁltered through a 0.22
μm ﬁlter. Supernatants were spotted on the same blot as thestandard curve. Blots were left to dry for 20 min in a fume
hood and then blocked in 5% milk/PBST for 10 min at room
temperature. CR3022 (4 μg) was added to blocking solution(0.4 μg/mL ﬁnal concentration) and incubated for 1 h at room
temperature. Blots were washed 16 times with 9 mL of PBST.
Secondary antibody was added at 1:10 000 (abcam ab6759,
rabbit antihuman IgG H&L HRP) in 5% milk/PBST andincubated for 1 h at room temperature. Blots were washed 16
times with 9 mL of PBST, developed using Pierce ECL
Western blotting substrate, and imaged using a GE Amersham
imager 600. Replicate protein expressions (n = 5) were
performed and included in the analysis. Dots were quantiﬁedusing the gel analysis protocol in Fiji (ImageJ), and curves
were ﬁtted using a linear regression in GraphPad Prism 8.4.1.
Statistical analysis on expression data presented in Figure S1B
was assessed using an ordinary one-way ANOVA with Tukey’s
multiple comparisons tests using GraphPad Prism 8.4.1. * = p≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.
SEC-MALS of SARS-CoV-2 Antigens. SEC-MALS was
performed on an Agilent 1260 Inﬁnity II HPLC with Wyatt
detectors for light scattering (miniDAWN) and refractive
index (Optilab). Puriﬁed antigen (1−10 μg) was loaded onto a
Superdex 200 Increase 3.2/200 (RBD) or onto an SRT SEC1000 4.6 × 300 mm (spike proteins) column equilibrated in
1× PBS [pH 7.4] or 1× Dulbecco’s phosphate-buﬀered saline(DPBS) (Gibco). Columns were ﬂowed at a rate of 0.15 mL/
min (S200) or 0.35 mL/min (SRT SEC-1000). MolecularJ

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were performed by coating either WT H. pylori ferritin
particles, sfGFP-GCN4-Avi-His (GCN4-Avi-His sequencetaken from ref 81), or sfGFP-His. After coating, plates were
washed 3× with PBST and blocked overnight at 4 °C with
ChonBlock Blocking/Dilution ELISA buﬀer (Chondrex).
ChonBlock was removed manually, and plates were washed
3× with PBST. Mouse serum samples, puriﬁed mAbs, andACE2-Fc were serially diluted in diluent buﬀer (1× PBS, 0.5%
bovine serum albumin, 2% ﬁltered fetal bovine serum, 0.2%
bovine gamma globulins (Sigma), 5 mM EDTA, 0.1% Tween)
starting at 1:50 serum dilution or 10 μg/mL and then added tocoated plates for 1 h at room temperature. Plates were washed
3× with PBST. For mouse serum ELISAs, HRP goat
antimouse (BioLegend 405306) was added at a 1:10 000
dilution in diluent buﬀer for 1 h at room temperature.
Isotyping experiments to quantify isotypes of RBD-speciﬁc
antibodies were performed by adding a 1:10 000 dilution ofeither HRP antimouse IgG1 (abcam ab97240), HRP
antimouse IgG2a heavy chain (abcam ab97245), HRP
antimouse IgG2b heavy chain (abcam ab97250), HRP
antimouse IgA alpha chain (abcam ab97235), or HRP
antimouse IgM mu chain (abcam ab97230). For puriﬁed
mAbs and ACE2-Fc, Direct-Blot HRP antihuman IgG1 Fc
antibody (Biolegend 410604) was added at a 1:10 000 dilution
in diluent buﬀer for 1 h at room temperature.
ACE2 blocking assays were performed by coating RBD as
described for other antigens, incubating serially diluted mouseserum for 1 h at room temperature, and then adding ACE2-Fc
at a ﬁnal concentration of 1 μg/mL to wells for 1 h at room
temperature. ACE2 binding was measured with Direct-Blot
HRP antihuman IgG1 Fc at a 1:10 000 dilution, and blocking
was quantiﬁed as the decrease in observed ACE2 binding as afunction of serum dilution.
After incubation with secondary antibody, ELISA plates
were washed 6× with PBST. Plates were developed for 6 minusing One-Step Turbo TMB substrate (Pierce) and werequenched with 2 M sulfuric acid. Absorbance at 450 nm wasdetermined using a BioTek plate reader. Background wasdetermined using an average of wells containing no serum,mAb, or ACE2. This value was then subtracted from all wellson the plate. Background subtracted values were then imported
into GraphPad Prism 8.4.1 and ﬁtted with a three-parameter
nonlinear regression to obtain EC50 or apparent KD values.
For ACE2 blocking assays, A450 values from ACE2 only
wells and background wells (no serum, no ACE2) wereaveraged and set at either 0% ACE2 blocking (ACE2 only) or
100% ACE2 blocking (background). A450 values for each
serum concentration were then normalized in GraphPad Prism
8.4.1 using these values. Percent ACE2 blocking at either 1:50
or 1:500 serum dilution can be found in Figure S6 for both day
21 and day 28 immunization time points.
Mouse Immunizations. Balb/c female mice (6−8 weeksold) were procured from The Jackson Laboratory. All mice
were maintained at Stanford University according to the Public
Health Service Policy for “Humane Care and Use ofLaboratory Animals” following a protocol approved by
Stanford University Administrative Panel on Laboratory
Animal Care (APLAC-33709). Mice were immunized via
subcutaneous injection of 10 μg of antigen with the exceptionof SΔC-Fer dosing and longevity studies in which animals were
immunized with either 0.1, 1, 10, or 20 μg as speciﬁed in
Figure S10. All antigen doses were formulated with 10 μg of
Quil-A (InVivogen) and 10 μg of monophosphoryl lipid A

Research Article(InVivogen) diluted in 1× Dulbecco’s phosphate-buﬀeredsaline (DPBS) (Gibco) in a total volume of 100 μL per
injection. Mice were immunized at day 0 and day 21. Serumwas collected at days 0, 21, and 28 and processed using
Sarstedt serum collection tubes. Day 0 serum was analyzed forboth ELISA binding and lentiviral neutralization and showedno evidence of binding or neutralizing activity (data notshown).
Collection of Plasma from Convalescent COVID-19
Patients. Convalescent COVID-19 plasma (CCP) donor
samples were obtained from residual ethylenediaminetetraacetic acid (EDTA) specimens at the time of collection,aliquoted, and stored at −80 °C. CCP samples were obtained
from donors who had a clinical diagnosis of COVID-19 andeither a positive RT-PCR or SARS-CoV-2 antibody test. On
the day of donation, donors were required to be healthy andasymptomatic for at least 4 weeks. The date of collection
ranged from 4 to 10 weeks after symptom resolution.
SARS-CoV-2 Pseudotyped Lentivirus Production and
Viral Neutralization Assays. SARS-CoV-2 spike-pseudotyped lentivirus was produced in HEK293T cells via calcium
phosphate transfection. Six million cells were seeded in D10
medium (DMEM + additives: 10% fetal bovine serum, Lglutamate, penicillin, streptomycin, and 10 mM HEPES) in 10
cm plates 1 day prior to transfection. A ﬁve-plasmid system47
was used for viral production: the lentiviral packaging vector(pHAGE_Luc2_IRES_ZsGreen), the SARS-CoV-2 spike, andlentiviral helper plasmids (HDM-Hgpm2, HDM-Tat1b, andpRC-CMV_Rev1b). The spike vector contained the full-length
wild-type spike sequence from the Wuhan-Hu-1 strain of
SARS-CoV-2 (GenBank NC_045512). Plasmids were added
to ﬁlter-sterilized water as follows: 10 μg of pHAGE_Luc2_IRS_ZsGreen, 3.4 μg of SARS-CoV-2 spike, 2.2 μg of
HDM-Hgpm2, 2.2 μg of HDM-Tat1b, and 2.2 μg of pRCCMV_Rev1b in a ﬁnal volume of 500 μL. HEPES-buﬀered
Saline (2×, pH 7.0) was added dropwise to this mixture to a
ﬁnal volume of 1 mL. To form transfection complexes, 100 μL
of 2.5 M CaCl2 was added dropwise while the solution was
gently agitated. Transfection reactions were incubated for 20
min at room temperature and then added dropwise to plated
cells. Medium was removed ∼24 h post-transfection andreplaced with fresh D10 medium. Virus-containing culture
supernatants were harvested ∼72 h post-transfection via
centrifugation at 300g for 5 min and ﬁltered through a 0.45
μm ﬁlter. Viral stocks were aliquoted and stored at −80 °C
until use.
For viral neutralization assays, ACE2/HeLa48 cells wereplated in white-walled clear-bottom 96-well plates at 5000
cells/well 1 day prior to infection or at 2500 cells/well 2 days
prior to infection. Mouse serum was centrifuged at 2000g for
15 min, heat inactivated for 30 min at 56 °C, and diluted in
D10 medium. Virus was diluted in D10 medium, supplemented with Polybrene, and then added to inhibitor dilutions.
Polybrene was present at a ﬁnal concentration of 5 μg/mL in
diluted samples. Puriﬁed mAbs were ﬁltered through a 0.22 μm
ﬁlter prior to neutralization assays. Virus/inhibitor dilutions
were incubated at 37 °C for 1 h. After incubation, medium wasremoved from cells, replaced with an equivalent volume ofinhibitor/virus dilutions, and incubated at 37 °C for ∼48 h.
Cells were lysed by adding BriteLite assay readout solution(PerkinElmer), and luminescence values were measured with a
BioTek plate reader. Each plate was normalized by averaging
RLUs from wells with cells only (0% infectivity) and virus onlyK

https://dx.doi.org/10.1021/acscentsci.0c01405
ACS Cent. Sci. XXXX, XXX, XXX−XXXACS Central Science■

http://pubs.acs.org/journal/acscii(100% infectivity). Normalized values were ﬁtted with a threeparameter nonlinear regression inhibitor curve in GraphPad
Prism 8.4.1 to obtain IC50 values. Fits for all serum
neutralization assays were constrained to have a value of 0%
at the bottom of the ﬁt. The limit of quantitation for this assay
is approximately 1:100 serum dilution. Serum samples thatfailed to neutralize or that neutralized at levels higher than
1:100 were set at the limit of quantitation for statisticalanalyses. Plots in Figure 4B−E are shown starting at thereciprocal serum dilution of the limit of quantitation (102).
Statistical Analyses. All normalization, cure-ﬁtting, andstatistical analysis described below were performed using
GraphPad Prism 8.4.1.
For ELISA, binding titers of RBD and spike were
determined for each mouse by performing 8-point serial
serum dilutions started at 1:50 with 10-fold dilutions induplicate, with replicates performed on separate days.
Background subtracted replicates for each mouse werecompiled and ﬁtted with a three-parameter nonlinear
regression activation curve to obtain an EC50 value for each
serum binding from each animal to each antigen. Data werethen compiled per group for each time point. Statisticalcomparisons between groups were performed using a Kruskal−
Wallis ANOVA followed by Dunn’s multiple comparisons test.
Signiﬁcance for comparisons between groups is indicated in
Figure 4B,D: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001,**** = p ≤ 0.0001. Compiled data from Dunn’s multiple
comparisons tests between groups for ELISA titers can befound in Table S2.
For lentiviral neutralization assays, serum from each animal
at each time point was assessed in duplicate with a 6-point
serial dilution (starting at 1:50 with 5-fold dilutions). Eachassay was performed again in a separate experimental replicate.
Each dilution series was normalized to 0% and 100% infectivity
using the average RLU values of wells with cells only and withvirus only, respectively, from each plate. Four normalized
curves for each animal at each time point were compiled toobtain an IC50 value. Statistical comparisons between groups
were performed using a Kruskal−Wallis ANOVA followed by
Dunn’s multiple comparisons test. Signiﬁcance for comparisons between groups is indicated in Figure 4C,E: * = p ≤ 0.05,** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Compiled
data from Dunn’s multiple comparisons tests between groups
for neutralization titers can be found in Table S3.
Safety Statement. No unexpected or unusually high safety
hazards were encountered.
Data Availability. The cryo-EM map of the SΔC-Fer
nanoparticle has been deposited in the wwPDB OneDep
System under EMD accession code EMD-23220.

■

Research Article

AUTHOR INFORMATION

Corresponding AuthorPeter S. Kim − Department of Biochemistry & Stanford
ChEM-H, Stanford University, Stanford, California 94305,
United States; Chan Zuckerberg Biohub, San Francisco,California 94158, United States; orcid.org/0000-00016503-4541; Email: kimpeter@stanford.edu
Authors

Abigail E. Powell − Department of Biochemistry & Stanford
ChEM-H, Stanford University, Stanford, California 94305,
United States; orcid.org/0000-0001-6408-9495
Kaiming Zhang − Department of Bioengineering & James H.
Clark Center, Stanford University, Stanford, California
94305, United States; orcid.org/0000-0003-0414-4776
Mrinmoy Sanyal − Department of Biochemistry & Stanford
ChEM-H, Stanford University, Stanford, California 94305,
United States
Shaogeng Tang − Department of Biochemistry & Stanford
ChEM-H, Stanford University, Stanford, California 94305,
United States
Payton A. Weidenbacher − Department of Biochemistry &Stanford ChEM-H and Department of Chemistry, Stanford
University, Stanford, California 94305, United States
Shanshan Li − Department of Bioengineering & James H.
Clark Center, Stanford University, Stanford, California
94305, United States
Tho D. Pham − Department of Pathology, Stanford
University, Stanford, California 94305, United States;
Stanford Blood Center, Palo Alto, California 94304, UnitedStatesJohn E. Pak − Chan Zuckerberg Biohub, San Francisco,California 94158, United States
Wah Chiu − Department of Bioengineering & James H. Clark
Center, Stanford University, Stanford, California 94305,
United States; Chan Zuckerberg Biohub, San Francisco,California 94158, United States; Division of CryoEM and
Bioimaging, SSRL, SLAC National Accelerator Laboratory,
Menlo Park, California 94025, United States; orcid.org/
0000-0002-8910-3078
Complete contact information is available at:https://pubs.acs.org/10.1021/acscentsci.0c01405
Author Contributions

A.E.P., P.A.W., S.T., M.S., and P.S.K. designed experiments.
A.E.P. and S.T. puriﬁed antigens for immunization. M.S.
performed mouse work. P.A.W. performed protein quantitation experiments and analysis. A.E.P. performed antigen
characterization, serum ELISAs and neutralizations, and data
analysis. K.Z. performed cryo-EM sample preparation, datacollection, and image processing. S.L. assisted with image
processing. K.Z., S.L., and W.C. analyzed the cryo-EM data.
T.D.P. collected convalescent plasma samples. J.E.P. assistedwith protein characterization methods and optimization. A.E.P.
and P.S.K. wrote the manuscript. All authors assisted withediting and approved the ﬁnal version of the manuscript.

ASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acscentsci.0c01405.

Notes

The authors declare the following competing ﬁnancial
interest(s): A.E.P., P.A.W., and P.S.K. are named as inventors
on a provisional patent application applied for by Stanford
University and the Chan Zuckerberg Biohub on immunogenic
coronavirus fusion proteins and related methods.

Additional data and ﬁgures including expression levels,
size-exclusion chromatography, cryo-EM, ELISA results,
assay validation, and oﬀ-target antibody responses(PDF)
L

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ACKNOWLEDGMENTS
We thank Dr. Jesse Bloom, Kate Crawford, Dr. Dennis Burton,and Dr. Deli Huang for sharing the plasmids, cells, andinvaluable advice for implementation of the spike-pseudotyped
lentiviral neutralization assay. We thank Dr. Florian Krammer
and Fatima Amanat for providing the SARS-CoV-2 RBD and
FL 2P spike plasmids for protein production. We thankNielson Weng and Dr. Nicholas Tierney for advice on
statistical analyses. We thank Dr. Duo Xu for designing andproviding the structural representations of spike and ferritin in
Figure 1. We thank Dr. Corey Liu and the Stanford ChEM-H
Macromolecular Knowledge Center for kindly relocating the
GE Amersham 600 imager to our lab space during the
pandemic shutdown for us to rapidly begin this work. We
thank Drs. Corey Hecksel and Patrick Mitchell for expert
maintenance of Stanford-SLAC Cryo-EM Center and theSLAC National Accelerator Laboratory for supporting thesestudies during the pandemic shutdown. We thank members of
the Kim Lab for fruitful discussions and insight on project
design as well as helpful comments on the manuscript. We
acknowledge BioRender for the images used in Figure 2A. The
CMV/R expression vectors were received from the NIH AIDS
Reagent Program. This work was supported by the Stanford
Maternal and Child Health Research Institute postdoctoral
fellowship (to A.E.P.), the Damon Runyon Cancer Research
Foundation Merck Fellowship (DRG-2301-17 to S.T.),
National Institutes of Health grants (P41GM103832,
R01AI148382, P01AI120943, S10OD02160 to W.C.), Chan
Zuckerberg Biohub (to W.C. and P.S.K.), the Virginia and D.
K. Ludwig Fund for Cancer Research (to P.S.K.), and theFrank Quattrone and Denise Foderaro Family Research Fund(to P.S.K.).

■

Research Article

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