of 24
science.sciencemag.org/cgi/content/full/science.
abf6840
/DC1
Supplementary
Material
s for
Mosaic nanoparticles elicit cross
-reactive immune responses to zoonotic
coronaviruses in mice
Alexander A. Cohen, Priyanthi N. P. Gnanapragasam
, Yu E.
Lee
, Pauline R. Hoffman
,
Susan
Ou
, Leesa M.
Kakutani
, Jennifer R. Keeffe
, Hung-
Jen
Wu
, Mark
Howarth
,
Anthony P. West
, Christopher O. Barnes, Michel C.
Nussenzweig
, Pamela J.
Bjorkman*
*Corresponding author. Email:
bjorkman@caltech.edu
Published
12 January
2021 on
Science
First Release
DOI:
10.1126/science
.abf6840
This PDF file includes:
Materials and Methods
Figs. S1 to S5
References
Other supplementary material for this manuscript includes:
MDAR Reproducibility Checklist (PDF)
1
Materials and Methods
Phylogenetic tree.
A sequence alignment of coronavirus RBD
domains was made using Clustal
Omega
(
51
)
. A phylogenetic tree was
calculated from this amino acid alignment using PhyML
3.0
(
52
)
, and a figure of this tree was made
using PRESTO (
http://www
.
atgc
-
montpellier.fr/presto
).
Expression of RBD and S proteins
. Mammalian expression vectors encoding the RBDs of
SARS
-
CoV
-
2 (GenBank
MN985325.1; S protein residues 319
-
539) and SARS
-
CoV S
(GenBank AAP13441.1; residues 318
-
510) with an N
-
terminal human IL
-
2 or Mu phosphatase
signal peptide were previously described
(
47
)
. Expression vectors were constructed similarly for
RBDs from the following other sarbecovirus strains: RaTG13
-
CoV (GenBank QHR63300; S
protein residues 319
-
541
), SHC014
-
CoV (GenBank KC881005; residues 307
-
524), Rs4081
-
CoV
(GenBank KY417143; S protein residues 310
-
515), pangolin17
-
CoV (GenBank QIA48632;
residues 317
-
539), RmYN02
-
CoV (GSAID EPI_ISL_412977; residues 298
-
503), Rf1
-
CoV
(GenBank DQ412042; residues 310
-
515), W1V1
-
CoV (GenBank KF367457; residues 307
-
528),
Yun11
-
CoV (GenBank JX993988; residues 310
-
515), BM
-
4831
-
CoV (GenBank NC014470;
residues 310
-
530), BtkY72
-
CoV (GenBank KY352407; residues 309
-
530). Two versions of each
RBD expression vector were made: o
ne including a C
-
terminal hexahistidine tag (G
-
HHHHHH)
and SpyTag003 (RGVPHIVMVDAYKRYK)
(
43
)
(for coupling to SpyCatcher003
-
mi3) and one
with only a hexahistidine tag (for ELISAs). Biotinylated SARS
-
CoV
-
2 and Rs4081 RBDs were
produce
d by co
-
transfection of Avi/His
-
tagged RBD expression plasmids with an expression
plasmid encoding an ER
-
directed BirA enzyme (kind gift of Michael Anaya, Caltech). RBD
2
proteins were purified from transiently
-
transfected Expi293F cell (Gibco) supernatants
by nickel
affinity and size
-
exclusion chromatography
(
47
)
. Peak fractions corresponding to
RBDs were
identified by SDS
-
PAGE and then pooled and stored at 4 ̊C. A trimeric SARS
-
CoV
-
2
ectodomain with 6P stabilizing mutations
(
53
)
was expressed and purified as described
(
24
)
.
Correct folding of the soluble SARS
-
CoV
-
2 S trimer was verified by a 3.3 Å cryo
-
EM structure
of a neutralizing antibody complexed with the trimer preparation used for immunizations
(
24
)
.
To prepare fluorochrome
-
conjugated streptavidin
-
tetramerized RBDs, biotinylated SA
RS
-
2 and
Rs4081 RBDs were incubated with streptavidin
-
APC (eBioscience
TM
) and streptavidin
-
PE
(ThermoFisher), respectively, overnight at 4
o
C at a 1:1 molar ratio of RBD to streptavidin
subunit.
Preparation of human plasma IgGs
. Plasma samples collected f
rom COVID
-
19 convalescent
and healthy donors are described in
(
18
)
. Human IgGs were i
solated from heat
-
inactivated
plasma samples using 5
-
mL HiTrap MabSelect SuRe columns (GE Healthcare Life Sciences) as
described
(
24
)
.
Preparation of RBD
-
mi3 nanoparticles.
SpyCatcher003
-
mi3 particles were prepared by
purification from BL21 (DE3)
-
RIPL
E coli
(Agilent) transformed with a pET28a
SpyCatcher003
-
mi3 gene (including an N
-
terminal 6x
-
His tag) as described
(
54
)
. Briefly, cell
pellets from transformed bacterial wer
e lysed with a cell disruptor in the presence of 2.0 mM
PMSF (Sigma). Lysates were spun at 21,000xg for 30 min, filtered with a 0.2 μm filter, and mi3
particles were isolated by Ni
-
NTA chromatography using a pre
-
packed HisTrap
TM
HP column
(GE Healthcare).
Eluted particles were concentrated using an Amicon Ultra 15 mL 30K
3
concentrator (MilliporeSigma) and purified by SEC using a HiLoad® 16/600 Superdex® 200
(GE Healthcare) column equilibrated with 25 mM Tris
-
HCl pH 8.0, 150 mM NaCl, 0.02% NaN
3
(TBS). SpyCatc
her
-
mi3 particles were stored at 4°C and used for conjugations for up to 1 month
after filtering with a 0.2 μm filter or spinning at 21,000xg for 10 min.
Purified SpyCatcher003
-
mi3 was incubated with a 3
-
fold molar excess (RBD to mi3 subunit) of
purified
SpyTagged RBD (either a single RBD for making homotypic SARS
-
CoV
-
2 RBD
particles or an equimolar mixture of four or eight RBDs for making mosaic particles) overnight
at room temperature in TBS. Conjugated mi3 particle were separated from free RBDs by SEC o
n
a Superose 6 10/300 column (GE Healthcare) equilibrated with PBS (20 mM sodium phosphate
pH 7.5, 150 mM NaCl). Fractions corresponding to conjugated mi3 particles were collected and
analyzed by SDS
-
PAGE. Concentrations of conjugated mi3 particles were de
termined using a
Bio
-
Rad Protein Assay.
Immunizations.
Animal procedures and experiments were performed according to protocols
approved by the IACUC. Experiments were done using 4
-
6 week old female Balb/c mice
(Charles River Laboratories), with 5 animals
each for cohorts immunized with soluble SARS
-
CoV
-
2 S or SpyCatcher003
-
mi3, and 10 animals each for remaining cohorts (Fig 3A).
Immunizations were carried out with intraperitoneal (ip) injections of either 5 μg of conjugated
RBD (calculated as the mass of
the RBD, assuming 100% efficiency of conjugation to
SpyCatcher003
-
mi3), 5 μg of soluble SARS
-
CoV
-
2 S, or 6 μg of unconjugated SpyCatcher003
-
mi3, in 100 μL of 50% v/v AddaVax
TM
adjuvant (Invivogen). Animals were boosted 4 weeks
after the prime with the same
quantity of antigen in adjuvant. Animals were bled every 2 weeks
4
via tail veins, and then euthanized 8 weeks after the prime (Day 56, 57) and bled through cardiac
puncture. Blood samples were allowed to clot at room temperature in MiniCollect® Serum and
P
lasma Tubes (Greiner), and serum was harvested, preserved in liquid nitrogen, and stored at
-
80 ̊C until use.
Sera for ELISAs were collected at Day 14 (Prime) and Day 42 (Boost). Sera for neutralization
assays were collected at Day 28 (Prime) and Day 56 (
Boost) (Fig. 3, fig. S3).
ELISAs.
10 μg/ml of a purified RBD (not SpyTagged) in 0.1 M NaHCO
3
pH 9.8 was coated
onto Nunc® MaxiSorp™ 384
-
well plates (Sigma) and stored overnight at 4
o
C. Plates were
washed with Tris
-
buffered saline with 0.1% Tween 20 (TBS
-
T
) after blocking with 3% bovine
serum albumin (BSA) in TBS
-
T for 1 hr at room temperature. Mouse serum was diluted 1:100
and then serially diluted by 4
-
fold with TBS
-
T/3% BSA and added to plates for 3 hr at room
temperature. A 1:50,000 dilution of secondar
y HRP
-
conjugated goat anti
-
mouse IgG (Abcam)
was added after washing for 1 hr at room temperature. Plates were developed using
SuperSignal™ ELISA Femto Maximum Sensitivity Substrate (ThermoFisher) and read at 425
nm. Curves were plotted and integrated to o
btain the area under the curve (AUC) using
Graphpad Prism 8.3 assuming a one
-
site binding model with a Hill coefficient (Fig. 3; fig. S3).
We also calculated EC
50
s and endpoint titers, which were determined using the dilution that was
at or below the mean
+ 2 x the standard deviation of the plate control (no primary serum added)
for ELISA binding data (fig. S3E,F). AUC calculations were used as they better capture changes
in maximum binding
(
55
)
. Statistical significance of titer differences between groups were
calculated using Tukey’s multiple comparison test using Graphpad Prism 8.3.
5
Neutralization assays
. SARS
-
CoV
-
2, SARS, WIV1, and SHC014
pseudoviruses based on HIV
lentiviral particles were prepared as described
(
18, 56
)
using genes encoding S protein sequences
lacking C
-
terminal residues in the cytoplasmic tail: 21 amino acid deletions for SARS
-
CoV
-
2,
WIV1, and SHC014 and a 19 amino acid deletion for SARS
-
CoV. IC
50
values derived from
this
pseudotyped neutralization assay method were shown to quantitatively correlate with results
from neutralization assays using authentic SARS
-
CoV
-
2 virus
(
44
)
. For pseudovirus
neutralization assays, four
-
fold seriall
y diluted sera from immunized mice were incubated with a
pseudotyped virus for 1 hour at 37 ̊C. After incubation with 293T
ACE2
target cells for 48 hours at
37 ̊C, cells were washed twice with phosphate
-
buffered saline (PBS) and lysed with Luciferase
Cell Cul
ture Lysis 5x reagent (Promega). NanoLuc Luciferase activity in lysates was measured
using the Nano
-
Glo Luciferase Assay System (Promega). Relative luminescence units (RLUs)
were normalized to values derived from cells infected with pseudotyped virus in th
e absence of
serum. Half
-
maximal inhibitory dilutions (ID
50
values) were determined using 4
-
parameter
nonlinear regression in AntibodyDatabase
(
57
)
. Statistical significance of titer differences
between groups were calculated using Tukey’s multiple comparison test of ID
50
s converted to
log
10
scale using Graphpad Prism 8.3.
Statistical Analysis.
Comparisons between groups for ELISAs and neutralization assays were
calculated with one
-
way analysis of variance (ANOVA) using Tukey’s post hoc test in Prism 9.0
(Graphpad). For correlation anal
ysis between ELISA and neutralization titers, significance (p),
Spearman coefficients (r
s
), and linear plots were calculated using Prism 9.0 (Graphpad).
Differences were considered significant when p values were less than 0.05. Exact p values are in
6
releva
nt figure near each corresponding line, with asterisks denoting level of significance (*
denotes 0.01<p<0.05, ** denotes 0.001<p<0.01, *** denotes 0.0001<p<0.001, and **** denotes
p<0.0001).
Flow cytometry
. B
-
cell analysis using flow cytometry was carried out as described
(
54
)
. Briefly,
single
-
cell suspensions were prepared from mouse spleens using mechanical dissociation, and
red blood cells were removed using ACK lysing buffer (Gibco). The white blood
cell preparation
was enriched for IgG+ B
-
cells using the negative selection protocol in a mouse memory B
-
cell
isolation kit (Miltenyi). The following commercial reagents were used to stain enriched
splenocytes: CD4
-
APC
-
eFluor 780 (clone: RM4
-
5), F4/80
-
APC
-
eFluor 780 (clone: BM8),
CD8a
-
APC
-
eFluor 780 (clone: 53
-
6.7), Ly
-
6G
-
APC
-
eFluor 780 (clone: RB6
-
8C5), IgM
-
APC
-
eFluor 780 (clone: II/41) (Thermo Fisher Scientific), CD19
-
FITC (clone: 6D5) (Biolegend),
IgG1 BV421 (clone: X40) and IgG2 BV421 (clone: R19
-
15) (
BD Bioscience). SARS
-
2 RBD
-
APC and Rs4081 RBD
-
PE for used to identify antigen
-
specific B
-
cells. Cell viability was
analyzed with Fixable Viability Stain 700 (BD Bioscience). Stained cells were analyzed with a
SY3200 Cell Sorter (Sony) configured to detect
6 fluorochromes. 2,000,000 events were
collected per sample and analyzed via FlowJo software (TreeStar).
7
8
Fig. S1.
Alignment of RBD sequences used for making mosaic particles. Sequences shown are for the
RBDs of SARS
-
CoV
-
2 (SARS
-
2, GenBank: MN985325.
1), RaTG13 (QHR63300), SHC014
(RsSHC014, KC881005), Rs4081 (KY417143), PCoV_GX
-
P5L (pang17) (QIA48632),
RmYN02 (GSAID EPI_ISL_412977), Rf1 (DQ412042), WIV1 (KF367457), SARS
-
CoV
(AAP13441.1), Yun11 (Cp/Yunnan2011, JX993988), BM
-
4831 (BM48
-
31/BGR/2008,
NC014
470), and BtKY72 (KY352407). SARS
-
2 RBD residues that interact directly with ACE2
(
58
)
are indicated by an asterisk. We note that antibody neutralization by direct binding of
ACE2
-
binding residues does not represent the only mechanism of neu
tralization for ACE2
-
tropic
viruses. This has been shown for monoclonal human antibodies derived from COVID
-
19
patients: some neutralizing antibodies do not directly interact with the ACE2
-
binding site on
RBD (for example, class 3 anti
-
SARS
-
CoV
-
2 neutraliz
ing antibodies
(
24
)
). Red shad
ing
indicates strains known to use ACE2 as a receptor.