science.sciencemag.org/cgi/content/full/science.abc6284/DC1

Supplementary Materials for

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Jingyou Yu*, Lisa H. Tostanoski*, Lauren Peter*, Noe B. Mercado*,

Katherine McMahan*, Shant H. Mahrokhian*, Joseph P. Nkolola*, Jinyan Liu*,

Zhenfeng Li*, Abishek Chandrashekar*, David R. Martinez, Carolin Loos,

Caroline Atyeo, Stephanie Fischinger, John S. Burke, Matthew D. Slein, Yuezhou Chen,

Adam Zuiani, Felipe J. N. Lelis, Meghan Travers, Shaghayegh Habibi, Laurent Pessaint,

Alex Van Ry, Kelvin Blade, Renita Brown, Anthony Cook, Brad Finneyfrock,

Alan Dodson, Elyse Teow, Jason Velasco, Roland Zahn, Frank Wegmann,

Esther A. Bondzie, Gabriel Dagotto, Makda S. Gebre, Xuan He, Catherine Jacob-Dolan,

Marinela Kirilova, Nicole Kordana, Zijin Lin, Lori F. Maxfield, Felix Nampanya,

Ramya Nityanandam, John D. Ventura, Huahua Wan, Yongfei Cai, Bing Chen,

Aaron G. Schmidt, Duane R. Wesemann, Ralph S. Baric, Galit Alter, Hanne Andersen,

Mark G. Lewis, Dan H. Barouch†

*These authors contributed equally to this work.

†Corresponding author. Email: dbarouch@bidmc.harvard.edu

Published 20 May 2020 on Science First Release

DOI: 10.1126/science.abc6284

This PDF file includes:

Materials and Methods

Figs. S1 to S13

References

Materials and Methods

Animals and study design. 35 outbred Indian-origin adult male and female rhesus

macaques (Macaca mulatta), 6-12 years old, were randomly allocated to groups. All animals

were housed at Bioqual, Inc. (Rockville, MD). Animals received DNA vaccines expressing S

(N=4), S.dCT (N=4), S.dTM (N=4), S1 (N=4), RBD (N=4), S.dTM.PP (N=5), and sham controls

(N=10). Animals received 5 mg DNA vaccines at week 0 and week 3. DNA vaccine were

administered by the intramuscular route with needle-and-syringe and without adjuvant. At week

6, all animals were challenged with 1.2x108 VP (1.1x104 PFU) SARS-CoV-2. Virus was

administered as 1 ml by the intranasal (IN) route (0.5 ml in each nare) and 1 ml by the

intratracheal (IT) route. All immunologic and virologic assays were performed blinded. All

animal studies were conducted in compliance with all relevant local, state, and federal

regulations and were approved by the Bioqual Institutional Animal Care and Use Committee

(IACUC).

Human samples. 27 de-identified human serum samples from SARS-CoV-2

convalescent individuals from Boston, MA were obtained from individuals at least 7 days after

documented recovery with negative nasal swab. All human studies were conducted in

compliance with all relevant local, state, and federal regulations and were approved by the

Partners Institutional Review Board (IRB).

DNA Vaccines. DNA vaccines were designed based on the SARS-CoV-2 spike (S)

protein sequence (Wuhan/WIV04/2019). Sequences were codon optimized and commercially

synthesized (Integrated DNA Technologies, NJ, USA). Six versions of S were produced (full

length S; deletion of cytoplasmic domain S.dCT; soluble ectodomain S.dTM; S1 domain with

foldon trimerization tag S1; receptor binding domain with foldon trimerization tag RBD; soluble

ectodomain with deletion of furin cleavage site, PP stabilizing mutations, and foldon

trimerization tag S.dTM.PP). Synthetic genes were cloned into the mammalian expression

plasmid pcDNA3.1+ (Invitrogen, CA, USA) and expanded with endotoxin-free gigaprep kits

(Machery-Nagel, Düren, Germany). All DNA vaccine sequences were confirmed by Sanger

DNA sequencing.

Western Blot. T-25 flasks seeded with 293T cells at 70-80% confluency were

transiently transfected with SARS-Cov-2 DNA expression plasmids (10µg DNA/construct) using

Lipofectamine 2000 (Invitrogen) and supernatants and cell lysates harvested 48 hours post-

transfection separately mixed with reducing sample buffer (Pierce), heated for 5 minutes at 95°C

and run on a precast 4-15% SDS-PAGE gel (Bio-Rad). Protein was transferred to a

polyvinylidene difluoride (PVDF) membrane using an iBlot dry blotting system (Invitrogen), and

membrane blocking performed overnight at 4°C in Dulbecco's phosphate-buffered saline T (D-

PBST) containing 0.2% Tween 20 (Sigma) (V/V) and 5% (W/V) non-fat milk powder.

Following overnight blocking, the PVDF membrane was incubated for 1 hour in 3% milk DPBS-

T containing a 1:10,000 dilution of polyclonal guinea pig anti-SARS antibody (BEI resources) or

a 1:10,000 dilution of polyclonal rabbit anti-SARS-CoV-2 RBD antibody (Sino Biological) for 1

hour. After this incubation, the PVDF membrane was washed five times with 5% milk DPBS-T

and subsequently incubated with 1:30,000 anti-guinea pig or anti-rabbit horseradish peroxidase

(HRP)-conjugated secondary antibody (Jackson Immunoresearch) in 3% milk DPBS-T. Finally,

the PVDF membrane was washed again five times with 5% milk DPBS-T, and developed using

an Amersham ECL Plus Western blotting detection system (GE Healthcare).

Viral RNA assay. RT-PCR assays were utilized to monitor viral loads, essentially as

previously described (17). Briefly, RNA was extracted using a QIAcube HT (Qiagen,Germany)

and the Cador pathogen HT kit from bronchoalveolar lavage (BAL) supernatant and nasal

swabs. RNA was reverse transcribed using superscript VILO (Invitrogen) and ran in duplicate

using the QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems) according to

manufacturer’s specifications. Viral loads were calculated of viral RNA copies per mL or per

swab and the assay sensitivity was 50 copies. The target for amplification was the SARS-CoV2

N (nucleocapsid) gene. The primers and probes for the targets were:

2019-nCoV_N1-F :5’-GACCCCAAAATCAGCGAAAT-3’

2019-nCoV_N1-R: 5’-TCTGGTTACTGCCAGTTGAATCTG-3’

2019-nCoV_N1-P: 5’-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3’

Subgenomic mRNA assay. SARS-CoV-2 E gene subgenomic mRNA (sgmRNA) was

assessed by RT-PCR using an approach similar to previously described (18). To generate a

standard curve, the SARS-CoV-2 E gene sgmRNA was cloned into a pcDNA3.1 expression

plasmid; this insert was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit

(Cellscript) to obtain RNA for standards. Prior to RT-PCR, samples collected from challenged

animals or standards were reverse-transcribed using Superscript III VILO (Invitrogen) according

to the manufacturer’s instructions. A Taqman custom gene expression assay (ThermoFisher

Scientific) was designed using the sequences targeting the E gene sgmRNA (18). Reactions

were carried out on a QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems)

according to the manufacturer’s specifications. Standard curves were used to calculate sgmRNA

in copies per ml or per swab; the quantitative assay sensitivity was 50 copies per ml or per swab.

PFU assay. For plaque assays, confluent monolayers of Vero E6 cells were prepared in

6-well plates. Indicated samples collected from challenged animals were serially diluted, added

to wells, and incubated at 37oC for 1 hr. After incubation, 1.5 mL of 0.5% methylcellulose media

was added to each well and the plates were incubated at 37oC with 5% CO2 for 2 days. Plates

were fixed by adding 400 µL ice cold methanol per well and incubating at -20°C for 30 minutes.

After fixation, the methanol was discarded, and cell monolayers were stained with 600 µL per

well of 0.23% crystal violet for 30 minutes. After staining, the crystal violet was discarded, and

the plates were washed once with 600 µL water to visualize and count plaques.

ELISA. Briefly, 96-well plates were coated with 1µg/ml SARS-CoV-2 Spike (S) protein

(Sino Biological) in 1X DPBS and incubated at 4°C overnight. After incubation, plates were

washed once with wash buffer (0.05% Tween20 in 1 X DPBS) and blocked with 350 µL Casein

block/well for 2-3 hours at room temperature. After incubation, block solution was discarded and

plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in Casein block were

added to wells and plates were incubated for 1 hr at room temperature, prior to three further

washes and subsequent 1hr incubation with a 1:1000 dilution of anti-macaque IgG HRP (NIH

NHP Reagent Program) in the dark at room temperature. Plates were then washed three times

with wash buffer, and 100 µL of SeraCare KPL TMB SureBlue Start solution was added to each

well; plate development was halted by the addition of 100 µL SeraCare KPL TMB Stop solution

per well. The absorbance at 450nm was recorded using a VersaMax or Omega microplate reader.

ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an

absorbance > 0.2. Log10 endpoint titers are reported.

Pseudovirus neutralization assay. The SARS-CoV-2 pseudoviruses expressing a

luciferase reporter gene were generated in an approach similar to as described previously (10).

Briefly, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase

reporter plasmid pLenti-CMV Puro-Luc (Addgene), and Spike protein expressing pcDNA3.1-

SARS CoV-2 SΔCT were co-transfected into HEK293T cells with calcium phosphate. The

supernatants containing the pseudotype viruses were collected 48 hours post-transfection;

pseudotype viruses were purified by filtration with 0.45 µm filter. To determine the

neutralization activity of the antisera from vaccinated animals, HEK293T-hACE2 cells were

seeded in 96-well tissue culture plates at a density of 1.75 x 104 cells/well overnight. Two-fold

serial dilutions of heat inactivated serum samples were prepared and mixed with 50 µL of

pseudovirus. The mixture was incubated at 37oC for 1 hour before adding to HEK293T-hACE2

cells. Forty-eight hours after infection, cells were lysed in Steady-Glo Luciferase Assay

(Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were

defined as the sample dilution at which a 50% reduction in RLU was observed relative to the

average of the virus control wells.

Live virus neutralization assay. A full-length SARS-CoV-2 virus based on the Seattle

Washington isolate was designed to express luciferase and GFP and was recovered via reverse

genetics and described previously (14, 15). The virus was titered in Vero E6 USAMRID cells

to obtain a relative light units (RLU) signal of at least 10X the cell only control background.

Vero E6 USAMRID cells were plated at 20,000 cells per well the day prior in clear bottom

black walled 96-well plates. Neutralizing antibody serum samples were tested at a starting

dilution of 1:40 and were serially diluted 4-fold up to eight dilution spots. Antibody-virus

complexes were incubated at 37oC with 5% CO2 for 1 hour. Following incubation, growth

media was removed and virus-antibody dilution complexes were added to the cells in duplicate.

Virus-only controls and cell-only controls were included in each neutralization assay plate.

Following infection, plates were incubated at 37oC with 5% CO2 for 48 hours. After the 48 h

incubation, cells were lysed and luciferase activity was measured via Nano-Glo Luciferase

Assay System (Promega) according to the manufacturer specifications. SARS-CoV-2

neutralization titers were defined as the sample dilution at which a 50% reduction in RLU was

observed relative to the average of the virus control wells.

Systems serology. For the functional analysis of serum samples, bead-based assays were

used to quantify antibody-dependent cellular phagocytosis (ADCP), antibody-dependent

neutrophil phagocytosis (ADNP) and antibody-dependent complement deposition (ADCD), as

previously described (16). Protein antigens included receptor binding domain (RBD; courtesy

Aaron Schmidt, Ragon Institute and MassCPR), prefusion stabilized Spike ectodomain (S;

courtesy Bing Chen, Children’s Hospital and MassCPR), and nucleocapsid (N; Sino Biological).

Fluorescent streptavidin beads (Thermo Fisher) were coupled to biotinylated RBD, N and S and

incubated with diluted serum (ADCP and ADNP 1:100, ADCD 1:10). For ADCP, THPs were

added to the immune complexes and incubated for 16h at 37oC. For ADNP, primary neutrophils

were isolated using ammonium-chlor-ide potassium (ACK) lysis buffer from whole blood. After

1h incubation at 37oC, neutrophils were stained with an anti-CD66b PacBlue detection antibody

(Biolegend). For the ADCD assay, lyophilized guinea pig complement (Cedarlane) was

resuspended according to manufacturer’s instructions and diluted in gelatin veronal buffer with

calcium and magnesium (Boston BioProducts). Post incubation, C3 was detected with

Fluorescein-Conjugated Goat IgG Fraction to Guinea Pig Complement C3 (Mpbio). For

detection of antibody-dependent NK cell activity, an ELISA-based approach was used. Briefly,

plates were coated with 3 ug/mL of antigen (as mentioned above) and samples were added at a

1:50 dilution and incubated for 2h at 37oC. NK cells were isolated the day prior via RosetteSep

(Stem Cell Technologies) from healthy buffy coats and rested overnight in 1 ng/ml IL-15

(Stemcell). NK cells were incubated with immune complexes for 5h at 37oC with a staining

cocktail containing CD107a PE-Cy5 (BD), Golgi stop (BD) and Brefeldin A (BFA, Sigma

Aldrich). Post NK cell incubation, cells were fixed (Perm A, Life Tech) and stained for surface

markers with anti-CD16 APC-Cy7 (BD), anti-CD56 PE-Cy7 (BD) and anti-CD3 PacBlue (BD)

while fixing. Post permeabilization with Perm B (Life Tech), anti-IFN-gamma FITC (BD) and

anti-MIP-1β PE (BD) antibodies were used for intracellular staining. All assays were acquired

via flow cytometry with an iQue (Intellicyt) and an S-Lab robot (PAA). For ADCP, events were

gated on bead-positive cells, whereas neutrophils were defined as CD66b positive followed by

gating on bead-positive neutrophils. A phagocytosis score was calculated for ADCP and ADNP

as (percentage of bead-positive cells) x (MFI of bead-positive cells) divided by 10000. ADCD

was reported as MFI of C3 deposition. NK cells were defined as CD3-, CD16+ and CD56+. Data

were reported as percentage of cells positive for CD107a, MIP-1-alpha or IFN-gamma.

Both Pearson and Spearman correlations were used to explore linear and non-linear

relationships between antibody features and log10 peak sgmRNA copies/ml in BAL, respectively.

A Benjamini-Hochberg correction was used to correct for multiple comparisons. In addition,

Pearson correlations were used to test all pairwise correlations of antibody features. A principal

component analysis (PCA) was constructed using the R package ‘ropls’ to compare multivariate

profiles. To define the optimal features that correlate with protection, a partial least square

regression (PLSR) and random forest regression (RFR) using different sets of features were

compared. First, all isotypes/subclasses and Fc-receptor binding data were log10 transformed.

Candidate sets of features were obtained by exploring possible combinations with recursive

feature elimination and exhaustive comparison of all possible combinations of two features. The

PLSR was performed using the R package ‘ropls’, and the random forest was performed using

the R package ‘randomForest’. Each model (i.e. each set of features) was fitted for 10 repetitions

of 5-fold cross-validation. In each step of the feature elimination, for PLSR the feature which

had the lowest mean (across folds) variable importance of projection (VIP) score and for RFR

the feature with the lowest mean (across folds) importance measured as node impurity was

removed. For the best performing feature combinations and individual features, the models were

refitted using 100 repetitions of 5-fold cross-validation.

ELISPOT assay. ELISPOT plates were coated with mouse anti-human IFN-γ

monoclonal antibody from BD Pharmingen at a concentration of 5 µg/well overnight at 4°C.

Plates were washed with DPBS containing 0.25% Tween20, and blocked with R10 media (RPMI

with 11% FBS and 1.1% penicillin-streptomycin) for 1 h at 37°C. The Spike 1 and Spike 2

peptide pools contain 15 amino acid peptides overlapping by 11 amino acids that span the

protein sequence and reflect the N- and C- terminal halves of the protein, respectively. Spike 1

and Spike 2 peptide pools were prepared at a concentration of 2 µg/well, and 200,000 cells/well

were added. The peptides and cells were incubated for 18-24 h at 37°C. All steps following this

incubation were performed at room temperature. The plates were washed with coulter buffer and

incubated for 2 h with Rabbit polyclonal anti-human IFN-γ Biotin from U-Cytech (1 µg/mL).

The plates are washed a second time and incubated for 2 h with Streptavidin-alkaline

phosphatase antibody from Southern Biotechnology (1 µg/mL). The final wash was followed by

the addition of Nitor-blue Tetrazolium Chloride/5-bromo-4-chloro 3 ‘indolyl phosphate p-

toludine salt (NBT/BCIP chromagen) substrate solution for 7 minutes. The chromagen was

discarded and the plates were washed with water and dried in a dim place for 24 hours. Plates

were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.

Intracellular cytokine staining assay. 106 PBMCs/well were re-suspended in 100 µL of

R10 media supplemented with CD49d monoclonal antibody (1 µg/mL). Each sample was

assessed with mock (100 µL of R10 plus 0.5% DMSO; background control), Spike 1 and Spike 2

peptide pools (2 µg/mL), or 10 pg/mL phorbol myristate acetate (PMA) and 1 µg/mL ionomycin

(Sigma-Aldrich) (100µL; positive control) and incubated at 37°C for 1 h. After incubation, 0.25

µL of GolgiStop and 0.25 µL of GolgiPlug in 50 µL of R10 was added to each well and

incubated at 37°C for 8 h and then held at 4°C overnight. The next day, the cells were washed

twice with DPBS, stained with Near IR live/dead dye for 10 mins and then stained with

predetermined titers of mAbs against CD279 (clone EH12.1, BB700), CD38 (clone OKT10, PE),

CD28 (clone 28.2, PE CY5), CD4 (clone L200, BV510), CD45 (clone D058-1283, BUV615),

CD95 (clone DX2, BUV737), CD8 (clone SK1, BUV805), for 30 min. Cells were then washed

twice with 2% FBS/DPBS buffer and incubated for 15 min with 200µL of BD

CytoFix/CytoPerm Fixation/Permeabilization solution. Cells were washed twice with 1X Perm

Wash buffer (BD Perm/WashTM Buffer 10X in the CytoFix/CytoPerm Fixation/

Permeabilization kit diluted with MilliQ water and passed through 0.22µm filter) and stained

with intracellularly with mAbs against Ki67 (clone B56, FITC), CD69 (clone TP1.55.3, ECD),

IL10 (clone JES3-9D7, PE CY7), IL13 (clone JES10-5A2, BV421), TNF-α (clone Mab11,

BV650), IL4 (clone MP4-25D2, BV711), IFN-γ (clone B27; BUV395), IL2 (clone MQ1-17H12,

APC), CD3 (clone SP34.2, Alexa 700), for 30 min. Cells were washed twice with 1X Perm

Wash buffer and fixed with 250µL of freshly prepared 1.5% formaldehyde. Fixed cells were

transferred to 96-well round bottom plate and analyzed by BD FACSymphonyTM system.

Statistical analyses. Analysis of virologic and immunologic data was performed using

GraphPad Prism 8.4.2 (GraphPad Software). Comparison of data between groups was

performed using two-sided Mann-Whitney tests. Correlations were assessed by two-sided

Spearman rank-correlation tests. P-values of less than 0.05 were considered significant.

Supplemental Figure Legends

Figure S1. Correlation of pseudovirus and live virus NAb assays in vaccinated macaques.

Red line reflects the best-fit relationship between these variables. P and R values reflect two-

sided Spearman rank-correlation tests.

Figure S2. Viral RNA following SARS-CoV-2 challenge in sham controls in BAL and nasal

swabs. Red lines reflect median viral loads.

Figure S3. Viral RNA following SARS-CoV-2 challenge in vaccinated animals in BAL.

Red lines reflect median viral loads.

Figure S4. Viral RNA following SARS-CoV-2 challenge in vaccinated animals in nasal

swabs. Red lines reflect median viral loads.

Figure S5. Peak viral RNA and PFU titers following SARS-CoV-2 challenge in vaccinated

animals in BAL and nasal swabs. Red lines reflect median viral loads. P-values indicate two-

sided Mann-Whitney tests.

Figure S6. Correlations of log ELISA titers prior to challenge with log sgmRNA in BAL

and nasal swabs following challenge. Red lines reflect the best-fit relationship between these

variables. P and R values reflect two-sided Spearman rank-correlation tests.

Figure S7. Correlations of log ELISPOT responses prior to challenge with log sgmRNA in

BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between

these variables. P and R values reflect two-sided Spearman rank-correlation tests.

Figure S8. Correlations of log CD4+ ICS responses prior to challenge with log sgmRNA in

BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between

these variables. P and R values reflect two-sided Spearman rank-correlation tests.

Figure S9. Correlations of log CD8+ ICS responses prior to challenge with log sgmRNA

copies/ml in BAL and log sgmRNA copies/swab in nasal swabs following challenge. Red

lines reflect the best-fit relationship between these variables. P and R values reflect two-sided

Spearman rank-correlation tests.

Figure S10. Anamnestic ELISA responses following challenge. Responses on day 0 and day

14 following challenge are shown. Day 0 reflects study week 6. Red lines reflect median

responses.

Figure S11. Anamnestic pseudovirus NAb responses following challenge. Responses on day

0 and day 14 following challenge are shown. Day 0 reflects study week 6. Red lines reflect

median responses.

Figure S12. Anamnestic live virus NAb responses following challenge. Responses on day 0

and day 14 following challenge are shown. Day 0 reflects study week 6. Red lines reflect

median responses.

Figure S13. Anamnestic ELISPOT responses following challenge. Responses on day 0 and

day 14 following challenge are shown. Day 0 reflects study week 6. Red lines reflect median

responses.

1.0 1.5 2.0 2.51.0

1.5

2.0

2.5

Log Pseudovirus NAb Titer

Log

Vir

us N

Ab

Tite

r P<0.0001R=0.8052

Figure S1

Days Following Challenge

Figure S2

Log

Vira

l RN

A C

opie

sBAL Nasal Swab

123456789

0 2 4 6 8 10 12 14

T576

5791

7137

7158

7223

T341

7148

7120

7121

7125

Med ian

123456789

0 2 4 6 8 10 12 14

T576

5791

7137

7158

7223

T341

7148

7120

7121

7125

Med ian

Sham Sham

Days Following ChallengeFigure S3

Log

Vira

l RN

A C

opie

s / m

lBAL

123456789

0 2 4 6 8 10 12 14

CG86

CF64

CF26

CC93

Med ian

123456789

0 2 4 6 8 10 12 14

BA76

9T7

6726

T783

Med ian

123456789

0 2 4 6 8 10 12 14

T784

T793

6416

6571

Med ian

S S.dCT S.dTM

123456789

0 2 4 6 8 10 12 14

T570

T572

5766

5796

Med ian

123456789

0 2 4 6 8 10 12 14

T573

T574

5764

38715

Med ian

123456789

0 2 4 6 8 10 12 14

T555

T560

T598

5771

5772

Med ian

S1 RBD S.dTM.PP

Days Following ChallengeFigure S4

Log

Vira

l RN

A C

opie

s / m

lNasal Swab

123456789

0 2 4 6 8 10 12 14

CG86

CF64

CF26

CC93

Med ian

123456789

0 2 4 6 8 10 12 14

BA76

9T7

6726

T783

Med ian

123456789

0 2 4 6 8 10 12 14

T784

T793

6416

6571

Med ian

S S.dCT S.dTM

123456789

0 2 4 6 8 10 12 14

T570

T572

5766

5796

Med ian

123456789

0 2 4 6 8 10 12 14

T573

T574

5764

38715

Med ian

123456789

0 2 4 6 8 10 12 14

T555

T560

T598

5771

5772

Med ian

S1 RBD S.dTM.PP

Figure S5

BAL Nasal Swab

Sha

m S

S.d

CT

S.d

TM S1

RB

D

S.d

TM.P

P

123456789

Log

Vir

al R

NA

Cop

ies

/ ml P=0.02

Sha

m S

S.d

CT

S.d

TM S1

RB

D

S.d

TM.P

P

123456789

Log

Vir

al R

NA

Cop

ies

/ Sw

ab P=0.04

Sham S

1

2

3

4

Log

PFU

/ S

wab

Nasal SwabP=0.04

Figure S6

BAL Nasal Swab

1.0 1.5 2.0 2.5 3.012345678

Log ELISA Titer

Log

sgm

RN

A C

opie

s / m

l

1.0 1.5 2.0 2.5 3.012345678

Log ELISA Titer

Log

sgm

RN

A C

opie

s / S

wabP=0.0041

R=-0.4733P=0.2712R=-0.2039

Figure S7

BAL Nasal Swab

1.0 1.5 2.0 2.5 3.012345678

Log ELISPOT SFC / 106 PBMC

Log

sgm

RN

A C

opie

s / m

l

1.0 1.5 2.0 2.5 3.012345678

Log ELISPOT SFC / 106 PBMC

Log

sgm

RN

A C

opie

s / S

wabP=0.9258

R=0.0196P=0.6037R=-0.1025

Figure S8

BAL Nasal Swab

-3.0 -2.5 -2.0 -1.5 -1.012345678

Log CD4 ICS

Log

sgm

RN

A C

opie

s / m

lP=0.4829R=-0.1383

-3.0 -2.5 -2.0 -1.5 -1.012345678

Log CD4 ICS

Log

sgm

RN

A C

opie

s / S

wab P=0.8855

R=0.0303

Figure S9

BAL Nasal Swab

-3.0 -2.5 -2.0 -1.5 -1.0 -0.512345678

Log CD8 ICS

Log

sgm

RN

A C

opie

s / m

l

-3.0 -2.5 -2.0 -1.5 -1.0 -0.512345678

Log CD8 ICS

Log

sgm

RN

A C

opie

s / S

wabP=0.4655

R=-0.1438P=0.8176R=-0.0485

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000

100000EL

ISA

Tite

r

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000

100000

ELIS

A Ti

ter

Day 0 Day 14

Figure S10

Day 0 Day 14

Sha

m S

S.d

CT

S.d

TM S1

RB

D

S.d

TM.P

P

10

100

1000

10000P

seud

ovir

us N

Ab

Tite

r

Sha

m S

S.d

CT

S.d

TM S1

RB

D

S.d

TM.P

P

10

100

1000

10000

Pse

udov

irus

NA

b Ti

ter

Figure S11

Day 0 Day 14

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000

100000Vi

rus

NA

b Ti

ter

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000

100000

Viru

s N

Ab

Tite

rFigure S12

Day 0 Day 14

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000SF

C /

106 P

BM

C

Sham

S

S.dC

T

S.dT

M S1

RB

D

S.dT

M.P

P10

100

1000

10000

SFC

/ 10

6 PB

MC

Figure S13

References and Notes

1. F. Wu, S. Zhao, B. Yu, Y.-M. Chen, W. Wang, Z.-G. Song, Y. Hu, Z.-W. Tao, J.-H. Tian, Y.-

Y. Pei, M.-L. Yuan, Y.-L. Zhang, F.-H. Dai, Y. Liu, Q.-M. Wang, J.-J. Zheng, L. Xu, E.

C. Holmes, Y.-Z. Zhang, A new coronavirus associated with human respiratory disease in

China. Nature 579, 265–269 (2020). doi:10.1038/s41586-020-2008-3 Medline

2. P. Zhou, X.-L. Yang, X.-G. Wang, B. Hu, L. Zhang, W. Zhang, H.-R. Si, Y. Zhu, B. Li, C.-L.

Huang, H.-D. Chen, J. Chen, Y. Luo, H. Guo, R.-D. Jiang, M.-Q. Liu, Y. Chen, X.-R.

Shen, X. Wang, X.-S. Zheng, K. Zhao, Q.-J. Chen, F. Deng, L.-L. Liu, B. Yan, F.-X.

Zhan, Y.-Y. Wang, G.-F. Xiao, Z.-L. Shi, A pneumonia outbreak associated with a new

coronavirus of probable bat origin. Nature 579, 270–273 (2020). doi:10.1038/s41586-

020-2012-7 Medline

3. M. L. Holshue, C. DeBolt, S. Lindquist, K. H. Lofy, J. Wiesman, H. Bruce, C. Spitters, K.

Ericson, S. Wilkerson, A. Tural, G. Diaz, A. Cohn, L. Fox, A. Patel, S. I. Gerber, L. Kim,

S. Tong, X. Lu, S. Lindstrom, M. A. Pallansch, W. C. Weldon, H. M. Biggs, T. M.

Uyeki, S. K. Pillai; Washington State 2019-nCoV Case Investigation Team, First Case of

2019 Novel Coronavirus in the United States. N. Engl. J. Med. 382, 929–936 (2020).

doi:10.1056/NEJMoa2001191 Medline

4. Q. Li, X. Guan, P. Wu, X. Wang, L. Zhou, Y. Tong, R. Ren, K. S. M. Leung, E. H. Y. Lau, J.

Y. Wong, X. Xing, N. Xiang, Y. Wu, C. Li, Q. Chen, D. Li, T. Liu, J. Zhao, M. Liu, W.

Tu, C. Chen, L. Jin, R. Yang, Q. Wang, S. Zhou, R. Wang, H. Liu, Y. Luo, Y. Liu, G.

Shao, H. Li, Z. Tao, Y. Yang, Z. Deng, B. Liu, Z. Ma, Y. Zhang, G. Shi, T. T. Y. Lam, J.

T. Wu, G. F. Gao, B. J. Cowling, B. Yang, G. M. Leung, Z. Feng, Early Transmission

Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N. Engl. J. Med.

382, 1199–1207 (2020). doi:10.1056/NEJMoa2001316 Medline

5. N. Zhu, D. Zhang, W. Wang, X. Li, B. Yang, J. Song, X. Zhao, B. Huang, W. Shi, R. Lu, P.

Niu, F. Zhan, X. Ma, D. Wang, W. Xu, G. Wu, G. F. Gao, W. Tan; China Novel

Coronavirus Investigating and Research Team, A Novel Coronavirus from Patients with

Pneumonia in China, 2019. N. Engl. J. Med. 382, 727–733 (2020).

doi:10.1056/NEJMoa2001017 Medline

6. N. Chen, M. Zhou, X. Dong, J. Qu, F. Gong, Y. Han, Y. Qiu, J. Wang, Y. Liu, Y. Wei, J. Xia,

T. Yu, X. Zhang, L. Zhang, Epidemiological and clinical characteristics of 99 cases of

2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 395,

507–513 (2020). doi:10.1016/S0140-6736(20)30211-7 Medline

7. C. Huang, Y. Wang, X. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, X. Gu, Z. Cheng,

T. Yu, J. Xia, Y. Wei, W. Wu, X. Xie, W. Yin, H. Li, M. Liu, Y. Xiao, H. Gao, L. Guo, J.

Xie, G. Wang, R. Jiang, Z. Gao, Q. Jin, J. Wang, B. Cao, Clinical features of patients

infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020).

doi:10.1016/S0140-6736(20)30183-5 Medline

8. J. F. Chan, S. Yuan, K.-H. Kok, K. K.-W. To, H. Chu, J. Yang, F. Xing, J. Liu, C. C.-Y. Yip,

R. W.-S. Poon, H.-W. Tsoi, S. K.-F. Lo, K.-H. Chan, V. K.-M. Poon, W.-M. Chan, J. D.

Ip, J.-P. Cai, V. C.-C. Cheng, H. Chen, C. K.-M. Hui, K.-Y. Yuen, A familial cluster of

pneumonia associated with the 2019 novel coronavirus indicating person-to-person

transmission: A study of a family cluster. Lancet 395, 514–523 (2020).

doi:10.1016/S0140-6736(20)30154-9 Medline

9. A. Chandrashekar, J. Liu, A. J. Martinot, K. McMahan, N. B. Mercado, L. Peter, L. H.

Tostanoski, J. Yu, Z. Maliga, M. Nekorchuk, K. Busman-Sahay, M. Terry, L. M. Wrijil,

S. Ducat, D. R. Martinez, C. Atyeo, S. Fischinger, J. S. Burke, M. D. Slein, L. Pessaint,

A. Van Ry, J. Greenhouse, T. Taylor, K. Blade, A. Cook, B. Finneyfrock, R. Brown, E.

Teow, J. Velasco, R. Zahn, F. Wegmann, P. Abbink, E. A. Bondzie, G. Dagotto, M. S.

Gebre, X. He, C. Jacob-Dolan, N. Kordana, Z. Li, M. A. Lifton, S. H. Mahrokhian, L. F.

Maxfield, R. Nityanandam, J. P. Nkolola, A. G. Schmidt, A. D. Miller, R. S. Baric, G.

Alter, P. K. Sorger, J. D. Estes, H. Andersen, M. G. Lewis, D. H. Barouch, SARS-CoV-2

infection protects against rechallenge in rhesus macaques. Science 369, 812–817 (2020).

doi:10.1126/science.abc4776 Medline

10. Z. Y. Yang, W. P. Kong, Y. Huang, A. Roberts, B. R. Murphy, K. Subbarao, G. J. Nabel, A

DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice.

Nature 428, 561–564 (2004). doi:10.1038/nature02463 Medline

11. R. N. Kirchdoerfer, C. A. Cottrell, N. Wang, J. Pallesen, H. M. Yassine, H. L. Turner, K. S.

Corbett, B. S. Graham, J. S. McLellan, A. B. Ward, Pre-fusion structure of a human

coronavirus spike protein. Nature 531, 118–121 (2016). doi:10.1038/nature17200

Medline

12. J. Pallesen, N. Wang, K. S. Corbett, D. Wrapp, R. N. Kirchdoerfer, H. L. Turner, C. A.

Cottrell, M. M. Becker, L. Wang, W. Shi, W.-P. Kong, E. L. Andres, A. N. Kettenbach,

M. R. Denison, J. D. Chappell, B. S. Graham, A. B. Ward, J. S. McLellan,

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike

antigen. Proc. Natl. Acad. Sci. U.S.A. 114, E7348–E7357 (2017).

doi:10.1073/pnas.1707304114 Medline

13. D. Wrapp, N. Wang, K. S. Corbett, J. A. Goldsmith, C.-L. Hsieh, O. Abiona, B. S. Graham,

J. S. McLellan, Cryo-EM structure of the 2019-nCoV spike in the prefusion

conformation. Science 367, 1260–1263 (2020). doi:10.1126/science.abb2507 Medline

14. T. Scobey, B. L. Yount, A. C. Sims, E. F. Donaldson, S. S. Agnihothram, V. D. Menachery,

R. L. Graham, J. Swanstrom, P. F. Bove, J. D. Kim, S. Grego, S. H. Randell, R. S. Baric,

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory

syndrome coronavirus. Proc. Natl. Acad. Sci. U.S.A. 110, 16157–16162 (2013).

doi:10.1073/pnas.1311542110 Medline

15. B. Yount, K. M. Curtis, E. A. Fritz, L. E. Hensley, P. B. Jahrling, E. Prentice, M. R. Denison,

T. W. Geisbert, R. S. Baric, Reverse genetics with a full-length infectious cDNA of

severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. U.S.A. 100, 12995–

13000 (2003). doi:10.1073/pnas.1735582100 Medline

16. A. W. Chung, M. P. Kumar, K. B. Arnold, W. H. Yu, M. K. Schoen, L. J. Dunphy, T. J.

Suscovich, N. Frahm, C. Linde, A. E. Mahan, M. Hoffner, H. Streeck, M. E. Ackerman,

M. J. McElrath, H. Schuitemaker, M. G. Pau, L. R. Baden, J. H. Kim, N. L. Michael, D.

H. Barouch, D. A. Lauffenburger, G. Alter, Dissecting Polyclonal Vaccine-Induced

Humoral Immunity against HIV Using Systems Serology. Cell 163, 988–998 (2015).

doi:10.1016/j.cell.2015.10.027 Medline

17. P. Abbink, N. B. Mercado, J. P. Nkolola, R. L. Peterson, H. Tuyishime, K. McMahan, E. T.

Moseley, E. N. Borducchi, A. Chandrashekar, E. A. Bondzie, A. Agarwal, A. J. Belli, K.

A. Reimann, B. F. Keele, R. Geleziunas, M. G. Lewis, D. H. Barouch, Lack of

therapeutic efficacy of an antibody to α4β7 in SIVmac251-infected rhesus macaques.

Science 365, 1029–1033 (2019). doi:10.1126/science.aaw8562 Medline

18. R. Wölfel, V. M. Corman, W. Guggemos, M. Seilmaier, S. Zange, M. A. Müller, D.

Niemeyer, T. C. Jones, P. Vollmar, C. Rothe, M. Hoelscher, T. Bleicker, S. Brünink, J.

Schneider, R. Ehmann, K. Zwirglmaier, C. Drosten, C. Wendtner, Virological assessment

of hospitalized patients with COVID-2019. Nature 581, 465–469 (2020).

doi:10.1038/s41586-020-2196-x Medline

19. Q. Gao, L. Bao, H. Mao, L. Wang, K. Xu, M. Yang, Y. Li, L. Zhu, N. Wang, Z. Lv, H. Gao,

X. Ge, B. Kan, Y. Hu, J. Liu, F. Cai, D. Jiang, Y. Yin, C. Qin, J. Li, X. Gong, X. Lou, W.

Shi, D. Wu, H. Zhang, L. Zhu, W. Deng, Y. Li, J. Lu, C. Li, X. Wang, W. Yin, Y. Zhang,

C. Qin, Development of an inactivated vaccine candidate for SARS-CoV-2. Science 369,

77–81 (2020). doi:10.1126/science.abc1932 Medline

20. B. L. Corey, J. R. Mascola, A. S. Fauci, F. S. Collins, A strategic approach to COVID-19

vaccine R&D. Science 368, 948–950 (2020). doi:10.1126/science.abc5312 Medline

21. R. Liu, J. Wang, Y. Shao, X. Wang, H. Zhang, L. Shuai, J. Ge, Z. Wen, Z. Bu, A

recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell

responses in rhesus monkeys after single dose immunization. Antiviral Res. 150, 30–38

(2018). doi:10.1016/j.antiviral.2017.12.007 Medline

22. K. Muthumani, D. Falzarano, E. L. Reuschel, C. Tingey, S. Flingai, D. O. Villarreal, M.

Wise, A. Patel, A. Izmirly, A. Aljuaid, A. M. Seliga, G. Soule, M. Morrow, K. A.

Kraynyak, A. S. Khan, D. P. Scott, F. Feldmann, R. LaCasse, K. Meade-White, A.

Okumura, K. E. Ugen, N. Y. Sardesai, J. J. Kim, G. Kobinger, H. Feldmann, D. B.

Weiner, A synthetic consensus anti–spike protein DNA vaccine induces protective

immunity against Middle East respiratory syndrome coronavirus in nonhuman primates.

Sci. Transl. Med. 7, 301ra132 (2015). doi:10.1126/scitranslmed.aac7462 Medline

23. G. P. Kobinger, J. M. Figueredo, T. Rowe, Y. Zhi, G. Gao, J. C. Sanmiguel, P. Bell, N. A.

Wivel, L. A. Zitzow, D. B. Flieder, R. J. Hogan, J. M. Wilson, Adenovirus-based vaccine

prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates

robust immune responses in macaques. Vaccine 25, 5220–5231 (2007).

doi:10.1016/j.vaccine.2007.04.065 Medline

24. J. Zhou, W. Wang, Q. Zhong, W. Hou, Z. Yang, S.-Y. Xiao, R. Zhu, Z. Tang, Y. Wang, Q.

Xian, H. Tang, L. Wen, Immunogenicity, safety, and protective efficacy of an inactivated

SARS-associated coronavirus vaccine in rhesus monkeys. Vaccine 23, 3202–3209

(2005). doi:10.1016/j.vaccine.2004.11.075 Medline

25. J. E. Martin, M. K. Louder, L. A. Holman, I. J. Gordon, M. E. Enama, B. D. Larkin, C. A.

Andrews, L. Vogel, R. A. Koup, M. Roederer, R. T. Bailer, P. L. Gomez, M. Nason, J. R.

Mascola, G. J. Nabel, B. S. Graham; VRC 301 Study Team, A SARS DNA vaccine

induces neutralizing antibody and cellular immune responses in healthy adults in a Phase

I clinical trial. Vaccine 26, 6338–6343 (2008). doi:10.1016/j.vaccine.2008.09.026

Medline

26. P. Abbink, R. A. Larocca, K. Visitsunthorn, M. Boyd, R. A. De La Barrera, G. D.

Gromowski, M. Kirilova, R. Peterson, Z. Li, O. Nanayakkara, R. Nityanandam, N. B.

Mercado, E. N. Borducchi, A. Chandrashekar, D. Jetton, S. Mojta, P. Gandhi, J. LeSuer,

S. Khatiwada, M. G. Lewis, K. Modjarrad, R. G. Jarman, K. H. Eckels, S. J. Thomas, N.

L. Michael, D. H. Barouch, Durability and correlates of vaccine protection against Zika

virus in rhesus monkeys. Sci. Transl. Med. 9, eaao4163 (2017).

doi:10.1126/scitranslmed.aao4163 Medline

27. C. T. Tseng, E. Sbrana, N. Iwata-Yoshikawa, P. C. Newman, T. Garron, R. L. Atmar, C. J.

Peters, R. B. Couch, Immunization with SARS coronavirus vaccines leads to pulmonary

immunopathology on challenge with the SARS virus. PLOS ONE 7, e35421 (2012).

doi:10.1371/journal.pone.0035421 Medline

28. L. Liu, Q. Wei, Q. Lin, J. Fang, H. Wang, H. Kwok, H. Tang, K. Nishiura, J. Peng, Z. Tan,

T. Wu, K.-W. Cheung, K.-H. Chan, X. Alvarez, C. Qin, A. Lackner, S. Perlman, K.-Y.

Yuen, Z. Chen, Anti-spike IgG causes severe acute lung injury by skewing macrophage

responses during acute SARS-CoV infection. JCI Insight 4, e123158 (2019).

doi:10.1172/jci.insight.123158 Medline

29. B. S. Graham, Rapid COVID-19 vaccine development. Science 368, 945–946 (2020).

doi:10.1126/science.abb8923 Medline