nature nanotechnology
https://doi.org/10.1038/s41565-024-01655-9
Artic�e
Proactive vaccination using multiviral
Quartet Nanocages to elicit broad anti-
coronavirus responses
In the format provided by the
authors and unedited
* = fully conserved
: = strongly similar
. = weakly similar
Rs4081
RVSPTHEVVRFPNITNRCPFDKVFNASRFPNVYAWERTKISDCVADYTVLYNS
-
TSFSTFKCYGVSPSKLIDLCFTSVYADTFL
RmYN02
RILPSTEVVRFPNITNFCPFDKVFNATRFPNVYAWQRTKISDCIADYTVLYNS
-
TSFSTFKCYGVSPSKLIDLCFTSVYADTFL
Rf1
RVSPVTEVVRFPNITNLCPFDKVFNATRFPSVYAWERTKISDCVADYTVFYNS
-
TSFSTFNCYGVSPSKLIDLCFTSVYADTFL
BM
-
4831
RVTPTTEVVRFPNITQLCPFNEVFNITSFPSVYAWERMRITNCVADYSVLYNSSASFSTFQCYGVSPTKLNDLCFSSVYADYFV
BtKY72
RVSPSTEVVRFPNITNLCPFGQVFNASNFPSVYAWERLRISDCVADYAVLYNSSSSFSTFKCYGVSPTKLNDLCFSSVYADYFV
pang17
RVQPTISIVRFPNITNLCPFGEVFNASKFASVYAWNRKRISNCVADYSVLYNS
-
TSFSTFKCYGVSPTKLNDLCFTNVYADSFV
SARS2
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNS
-
ASFSTFKCYGVSPTKLNDLCFTNVYADSFV
RaTG13
RVQPTDSIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNS
-
TSFSTFKCYGVSPTKLNDLCFTNVYADSFV
SHC014
RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNS
-
TSFSTFKCYGVSATKLNDLCFSNVYADSFV
SARS1
RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNS
-
TFFSTFKCYGVSATKLNDLCFSNVYADSFV
WIV1
RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNS
-
TSFSTFKCYGVSATKLNDLCFSNVYADSFV
*: * .::******: ***..*** : * .****:* :*::*:***:*:*** : ****:***** :** ****:.**** *:
Rs4081
IRSSEVRQVAPGETGVIADYNYKLPDDFTGCVIAWNTAKQDQG
-----
QYYYRSSRKTKLKPFERDLTSDE
-------------
RmYN02
IRFSEVRQIAPGETGVIADYNYKLPDDFTGCVLAWNTAQQDIG
-----
SYFYRSHRAVKLKPFERDLSSDE
-------------
Rf1
IRFSEVRQVAPGQTGVIADYNYKLPDDFTGCVIAWNTAKQDVG
-----
SYFYRSHRSSKLKPFERDLSSEE
-------------
BM
-
4831
VKGDDVRQIAPAQTGVIADYNYKLPDDFTGCVIAWNTNSLDS
--
SNE
--
FFYRRFRHGKIKPYGRDLSNVLFNPSGGTCSA
-
EG
BtKY72
VKGDDVRQIAPAQTGVIADYNYKLPDDFTGCVLAWNTNSVDSKSGNN
--
FYYRLFRHGKIKPYERDISNVLYNSAGGTCSSISQ
pang17
VKGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNSVKQDALTGGNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVG
SARS2
IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG
RaTG13
ITGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSKHIDAKEGGNFNYLYRLFRKANLKPFERDISTEIYQAGSKPCNGQTG
SHC014
VKGDDVRQIAPGQTGVIADYNYKLPDDFLGCVLAWNTNSKDSSTSGNYNYLYRWVRRSKLNPYERDLSNDIYSPGGQSCSA
-
VG
SARS1
VKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTP
-
PA
WIV1
VKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTP
-
PA
: .:***:**.:** **********:* ***:***: * : ** * ::.*: **::.
Rs4081
-
NGVRTLSTYDFYPNVPIEYQATRVVVLSFELLNAPATVCGPKLSTALVKNQCVNF
RmYN02
-
NGVRTLSTYDFNPNVPLDYQATRVVVLSFELLNAPATVCGPKLSTQLVKNRCVNF
Rf1
-
NGVRTLSTYDFNQNVPLEYQATRVVVLSFELLNAPATVCGPKLSTSLVKNQCVNF
BM
-
4831
LNCYKPLASYGFTQSSGIGFQPYRVVVLSFELLNAPATVCGPKQSTELVKNKCVNF
BtKY72
LGCYEPLKSYGFTPTVGVGYQPYRVVVLSFELLNAPATVCGPKKSTELVKNKCVNF
pang17
LNCYYPLERYGFHPTTGVNYQPFRVVVLSFELLNGPATVCGPKLSTTLVKDKCVNF
SARS2
FNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
RaTG13
LNCYYPLYRYGFYPTDGVGHQPYRVVVLSFELLNAPATVCGPKKSTNLVKNKCVNF
SHC014
PNCYNPLRPYGFFTTAGVGHQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF
SARS1
LNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF
WIV1
FNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF
. * *.* . : .* **********:.******** ** *:*::****
320
340
360
380
400
420
440
460
480
500
520
540
Supplementary Fig. 1. Sarbecovirus RBD sequence alignment
. Amino acid sequence
alignment of sarbecovirus RBDs used in this study, numbered according to Spike protein
of
SARS2 Wuhan variant.
A
Fully Conserved
Strongly Similar
Weakly Similar
Non
-
conserved
Sarbecovirus RBD Protein Identity
Map of Residue Conservation
B
180
°
90
°
Supplementary Fig. 2. RBD residue conservation
. (
A
) Heat map of percent amino acid
identity between sarbecovirus RBDs used in this study
. (
B
) Conservation of residues between
sarbecoviruses used in the study, as mapped onto the SARS2 Wuhan RBD crystal structure
(PDB ID: 6ZER). Multiple orientations of the same RBD are shown, represented as the van der
Waals surface.
2
3
1b
1a
Clade
A
SpySwitch
-
mediated purification
B
RBD Quartet
Alternate RBD Quartet
C
D
Quartet [SARS1]
-
250
-
13
0
-
-
10
0
-
-
7
0
-
-
5
5
-
-
35
-
-
25
-
-
FT
6
5
4
3
2
1
1
2
Wash
Elution
FT
6
5
4
3
2
1
1
2
Wash
Elution
-
250
-
13
0
-
-
10
0
-
-
7
0
-
-
5
5
-
-
35
-
-
-
250
-
13
0
-
-
10
0
-
-
7
0
-
-
5
5
-
-
35
-
-
25
-
-
FT
6
5
4
3
2
1
W
Elution
SN
Cell supernatant containing
SpyTagged
Quartet
Resin
Resin
SpySwitch resin
captures SpyTag
-
fusion at neutral pH
SpyTag
-
fusion
elutes at weakly
acidic pH
SpySwitch
SpySwitch
Purified Quartet
Supplementary Fig. 3. SpySwitch purification of RBD quartets
. (
A
) Schematic of
SpySwitch affinity purification. SpyTag genetically fused to the Quartet has a non
-
covalent
interaction with SpySwitch at a neutral pH, before eluting at a weakly acidic pH through charge
-
charge repulsion. This system was used to purify (
B
) RBD Quartet, (
C
) Alternate RBD Quartet,
and (
D
) RBD Quartet with SARS1 in place of SARS2. The supernatant (SN), flowthrough (FT),
wash (W) and elution fractions were
analyzed
by SDS
-
PAGE with Coomassie staining. A
representative gel from two independent purifications. Molecular weight markers are in kDa.
Quartet
Alt.
Quartet
SARS1
Quartet
Supplementary Fig. 4. Size
-
exclusion chromatography of Quartets.
Quartets were run on
an S200
Sephacryl
column in PBS pH 7.4 after purification by SpySwitch, with gel filtration
standards shown. Absorbance is measured in
mAU
. Absorbance peak size differs in line with
the different loaded concentrations.
2
0
15
0
-
5
A
280
(mAU)
-
Void
-
158 kDa
-
44 kDa
-
17 kDa
-
1.3 kDa
-
Void
-
158 kDa
-
44 kDa
-
17 kDa
-
1.3 kDa
Elution Volume (mL)
2
0
100
40
6
0
80
12
0
Elution Volume (mL)
2
0
100
40
6
0
80
12
0
Elution Volume (mL)
2
0
100
40
6
0
80
12
0
Elution Volume (mL)
2
0
100
40
6
0
80
12
0
20
0
40
1
0
3
0
0
12
0
80
4
0
Elution Volume (mL)
2
0
100
40
6
0
80
12
0
0
0
0
4
0
0
80
20
60
-
Void
-
158 kDa
-
44 kDa
-
17 kDa
-
1.3 kDa
-
Void
-
158 kDa
-
44 kDa
-
17 kDa
-
1.3 kDa
-
Void
-
158 kDa
-
44 kDa
-
17 kDa
-
1.3 kDa
Quartet
-
SpyTag
SpyTag
-
Quartet
Alternate Quartet
Quartet[SARS1]
Kraken Quartet
A
B
C
D
E
5
10
2
0
15
0
-
5
5
10
A
280
(mAU)
A
280
(mAU)
A
280
(mAU)
A
280
(mAU)
A
Post
-
Prime RBD ELISAs
0.0
0.5
1.0
1.5
2.0
2.5
IgG
titer
AUC
SARS1
0.0
0.5
1.0
1.5
2.0
2.5
IgG
titer
AUC
SARS2 Wuhan
B
Uncoupled
RBD
Uncoupled
Quartet
Homotypic
Nanocage
Quartet
Nanocage
0.0
1.0
2.0
3.0
IgG
titer
AUC
IgG
titer
AUC
IgG
titer
AUC
Post
-
Boost SARS2 Spike ELISAs
Wuhan
Beta
Delta
0.0
1.0
2.0
3.0
0.0
1.0
2.0
3.0
Supplementary Fig. 5. Further Breadth of Immune Response from Immunization with
Quartet Nanocages
. Binding data for serum IgG antibodies presented as area under the curve
of a serial sera dilution. Sera samples are from mice immunized with uncoupled SARS2 Wuhan
RBD (orange), Uncoupled Quartet (yellow), SARS2 Wuhan RBD coupled to SpyCatcher003
-
mi3 (Homotypic Nanocage, green), and Quartet Nanocage (blue) as outlined in Fig. 2. Solid
gray
rectangles under samples indicate the ELISA is against a component of that vaccine
(matched), while striped rectangles indicate the ELISA is against an antigen absent in that
vaccine (mismatched). Each dot represents serum from one animal. The mean is denoted by a
bar, shown
±
1
s.d.
, n = 6. Significance was calculated with an ANOVA test using Tukey’s post
hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; other comparisons were non
-
significant. Graphs
demonstrate the binding of (
A
) post
-
prime sera to RBDs and (
B
) post
-
boost sera to SARS2
variant Spike proteins.
Mismatched
Matched
Post
-
Boost
SARS2 Wuhan
Post
-
Boost SHC014
Post
-
Boost Rs4081
Post
-
Boost RaTG13
Post
-
Boost SARS1
Post
-
Boost BM
-
4831
Uncoupled
RBD
Uncoupled
Quartet
Homotypic
Nanocage
Quartet
Nanocage
Mismatched
Matched
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
10
0
Midpoint Titer
10
1
10
2
10
3
10
4
Supplementary Fig. 6. Midpoint Titers from Immunization with Quartet Nanocages.
Binding
data for serum IgG antibodies presented as midpoint titers, calculated from curve of a serial
dilution of serum. Sera samples are from mice immunized with uncoupled SARS2 Wuhan RBD
(orange), Uncoupled Quartet (yellow), SARS2 Wuhan RBD coupled to SpyCatcher003
-
mi3
(Homotypic Nanocage, green), and Quartet Nanocage (blue) as outlined in Fig. 2. Solid
gray
rectangles under samples indicate the ELISA is against a component of that vaccine (matched),
while striped rectangles indicate the ELISA is against an antigen absent in that vaccine
(mismatched). Each dot represents serum from one animal. The mean is denoted by a bar,
shown + 1
s.d.
, n = 6. Significance was calculated with an ANOVA test, followed by Tukey’s
multiple comparison post hoc test of ID
50
values converted to log
10
scale. * p < 0.05, ** p < 0.01,
*** p < 0.001; other comparisons were non
-
significant.
Dashed horizontal lines represent the limit
of detection.
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Dilution
Post
-
Boost SARS2
Wuhan
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Post
-
Boost SHC014
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Post
-
Boost Rs4081
Dilution
Dilution
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Dilution
Post
-
Boost RaTG13
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Post
-
Boost SARS1
A
405
0
0.5
1.0
1.5
2.0
10
2
10
3
10
4
10
5
10
6
10
7
Post
-
Boost BM
-
4831
Dilution
Dilution
Supplementary Fig. 7. Serum binding curves
from immunization
with Quartet
Nanocages.
ELISA binding data are presented for a serial dilution of sera from mice
immunized with uncoupled SARS2 Wuhan RBD (orange), Uncoupled Quartet (yellow), SARS2
Wuhan RBD coupled to SpyCatcher003
-
mi3 (Homotypic Nanocage, green), and Quartet
Nanocage (blue) as outlined in Fig. 2. The mean absorbance (duplicate measurements for
n=6 serum samples) for each immunization condition at each dilution is plotted. A curve is fit
and plotted for each immunization condition.
Uncoupled
RBD
Uncoupled
Quartet
Homotypic
Nanocage
Quartet
Nanocage
Supplementary Fig. 8. Schematic of Different Quartets
. Genetic organization of (
A
) SpyTag
-
Quartet, (
B
) Alternate RBD Quartet, (
C
) Quartet [SARS1], and (
D
) Kraken Quartet (with SARS2
Omicron XBB.1.5)
.
These schematics indicate the virus origin of each RBD, predicted N
-
linked
glycosylation sites, tag location, and nucleotide number.
500
1,000
1,500
2,000
2,500
SHC014
RBD
Rs4081
RBD
RaTG13
RBD
SARS2 Wuhan
RBD
Glycosylation
Signal Peptide
SpyTag
1
Nucleotide
2,760
500
1,000
1,500
2,000
2,500
pang17
RBD
RmYN02
RBD
Rf1
RBD
WIV1
RBD
Glycosylation
Signal Peptide
SpyTag
1
Nucleotide
2,787
500
1,000
1,500
2,000
2,500
SHC014
RBD
Rs4081
RBD
RaTG13
RBD
SARS1
RBD
Glycosylation
Signal Peptide
SpyTag
1
Nucleotide
2,829
A
C
B
SpyTag
-
Quartet
Alternate Quartet
Quartet [SARS1]
500
1,000
1,500
2,000
2,500
SHC014
RBD
Rs4081
RBD
RaTG13
RBD
SARS2 XBB.1.5
RBD
Glycosylation
Signal Peptide
SpyTag
1
Nucleotide
2,760
D
Kraken Quartet
A
Negative Stain Transmission Electron Microscopy
Nanocage
Mosaic
-
8
Quartet
Nanocage
Nanocage
Quartet
Nanocage
Mosaic
-
8
0
10
20
30
40
0
10
20
30
40
Relative Frequency (%)
0
10
20
30
0
10
20
30
40
Relative Frequency (%)
40
0
10
20
30
0
10
20
30
40
Relative Frequency (%)
40
50
Diameter (nm)
Diameter (nm)
Diameter (nm)
Diameter
(nm)
Nanocage
28.5
±
2.2
Mosaic
-
8
27.9
±
1.7
Quartet
Nanocage
28.5
±
2.3
Supplementary Fig. 9. Negative stain
TEM of
nanocage immunogens.
(
A
) Representative
negative stain TEM
images of
uncoupled Nanocage, Mosaic
-
8 nanoparticles, and Quartet
Nanocages. Scale bar is 20 nm. (
B
) Size distribution of nanoparticles measured by TEM with
2 nm bin size (n = 75). (
C
)
Table of the size
distribution of
different nanoparticles: mean
±
1
s.d
.
(n = 75).
B
C
Homotypic nanocage
Mosaic
-
4
Mosaic
-
8
Quartet Nanocage
Dual Quartet Nanocage
Uncoupled RBD quartet
Supplementary Fig. 10.
Breadth of antibody induction by Quartet and Mosaic
Immuno
gens.
(
A
) Summary of timeline for this set of immunizations with 0.02 nmol antigen
per dose.
(B
-
C)
ELISA for serum IgG from mice immunized with the indicated immunogen.
Each dot represents serum from one animal. (
B
) Post
-
prime response to SARS2 or SARS1.
(
C
) Post
-
boost response to SpyCatcher003
-
mi3 or SpyTag
-
MBP.
The mean is denoted by a
bar, with error bars
±
1
s.d.
, n = 6. Significance was calculated with an ANOVA test using
Tukey’s post hoc test. No significance test was performed for SpyTag
-
MBP responses. * p <
0.05, ** p < 0.01, *** p < 0.001; other comparisons were non
-
significant.
Mismatched
Matched
B
Post
-
Prime SARS2 Wuhan
C
Post
-
Boost SpyTag
-
MBP
0.0
0.2
0.4
0.6
0.8
1.0
IgG
titer
AUC
Post
-
Prime SARS1
Prime
Day 0
Post
-
Prime
Bleed
Day 13
Boost
Day 14
Post
-
Boost
Bleed
Day 34
A
0.0
0.2
0.4
0.6
0.8
1.0
IgG
titer
AUC
Post
-
Boost SpyCatcher003
-
mi3
IgG
titer
AUC
5.0
4.0
3.0
1.0
0.0
2.0
IgG
titer
AUC
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0