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: [email protected]
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
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