of 39
1
Title:
Mosaic nanoparticles elicit cross
-
reactive immune responses to zoonotic coronaviruses in
mice
Authors:
Alexander A. Cohen
1
,
Priyanthi N.P. Gnanapragasam
1
, Yu E. Lee
1
,
Pauline R.
Hoffman
1
,
Susan Ou
1
,
Leesa M. Kakutani
1
,
Jennifer
R.
Keeffe
1
,
Hung
-
Jen Wu
2
, Mark Howarth
2
,
Anthony P. West
1
,
Christopher O. Barnes
1
,
Michel C. Nussenzweig
3
,
Pamela J. Bjorkman
1*
Affiliations:
1
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA
91125, USA
2
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
3
Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA
*Corresponding author: email:
bjorkman@caltech.edu
One sentence summary: Nanoparticle strategy for pan
-
sarbecovirus vaccine
Keywords: neutralizing antibodies, receptor
-
binding domain, sarbecoviruses, vaccine, zoonotic
coronaviruses
125
-
character summary for online ToC:
Immunizing with nanoparticles displaying diverse coronavirus RBDs elicits cross
-
reactive and
neutralizing antibody responses.
.
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2
Abstract
:
Protection against SARS
-
CoV
-
2 and SARS
-
related emergent zoonotic
coronaviruses is urgently
needed. W
e made homotypic nanoparticles displaying the receptor
-
binding domain (RBD) of
SARS
-
CoV
-
2 or co
-
displaying SARS
-
CoV
-
2 RBD along with RBDs from animal
betacoronaviruses that represent threats to humans (mosaic nanoparticle
s; 4
-
8 distinct RBDs).
Mice immunized with RBD
-
nanoparticles, but not soluble antigen, elicited cross
-
reactive binding
and neutralization responses. Mosaic
-
RBD
-
nanoparticles elicited antibodies with superior cross
-
reactive recognition of heterologous RBDs
compared to sera from immunizations with
homotypic SARS
-
CoV
-
2
RBD
-
nanoparticles or COVID
-
19 convalescent human plasmas.
Moreover, sera from mosaic
-
RBD
immunized mice neutralized heterologous pseudotyped
coronaviruses equivalently or better after priming th
an sera from homotypic SARS
-
CoV
-
2
RBD
-
nanoparticle immunizations, demonstrating
no
immunogenicity loss against particular
RBDs resulting from co
-
display. A single immunization with mosaic
-
RBD
-
nanoparticles
provides a potential strategy to simultaneously pr
otect against SARS
-
CoV
-
2 and emerging
zoonotic coronaviruses.
.
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3
Main Text:
SARS
-
CoV
-
2, a newly
-
emergent betacoronavirus, resulted in a
global
pandemic in 2020,
infecting millions and causing
the respiratory disease COVID
-
19
(
1, 2
)
.
Two other zoonotic
betacoronaviruses, SARS
-
CoV and MERS
-
CoV, also resulted in
outb
r
eaks
within the last 20
years
(
3
)
.
All three viruses
presumably
originated
in bats
(
4
)
,
with SARS
-
CoV and MERS
-
CoV
adapting
to inter
mediary
animal
hosts
before jumping to
humans.
S
ARS
-
like viruses circulate in
bats and s
erological surveillance of people living near caves where bats carry diverse
coronaviruses
demonstrates
dire
ct transmission of SARS
-
like viruses with pandemic potential
(
5
)
,
suggesting
a pan
-
coronavirus vaccine is needed to
protect against
future
outbreaks and
pandemics.
In particular,
t
he
bat
WIV1 and SHC014 strains are thought
to
represent an ongoing
threat to humans
(
6, 7
)
.
M
ost
current
SARS
-
CoV
-
2
vaccine candidates include
the spike trimer (S)
, the viral
protein
that
mediates
target
cell entry
after
one or more of its
receptor
-
binding domains (
RBDs
)
adopt
an “up”
position
to
bind
a
host receptor
(Fig. 1A)
. The RBDs of
human coronaviruses
SARS
-
CoV
-
2
,
SARS
-
CoV, HCoV
-
NL63, and related animal coronaviruses
(WIV1 and SCH014)
use
angiotensin
-
converting enzyme 2 (ACE2) as their host receptor
(
1, 8, 9
)
, while other
coronaviruses use receptors such as dipeptidyl peptidase 4
(
10
)
or sialic acids
(
11, 12
)
.
Consistent with its function in viral entry, S
is the primary target of neutralizing antibodies
(
13
-
22
)
,
with many
targeting
the
RBD
(
14
-
18, 21
-
26
)
.
M
ultivalent display of antigen enhances B
-
cell responses and can provide longer
-
lasting
immunity than mo
novalent antigens
(
27, 28
)
,
thus
protein
-
based vaccine candidates often
.
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4
involve a nanoparticle that enables antigen multimerization.
Many nanoparticle
s
and coupling
strategies have been explored for vaccine design
(
29
)
, with “plug and display” strateg
ies
being
especially useful
(
30, 31
)
. In
one
such
approach,
multiple copies of an engineered protein
domain called
SpyCatcher fused to subunits of a
virus
-
like particle
form spontaneous isopeptide
bonds to
purified antigen
s
tagged with a 13
-
residue SpyTag
(
29
-
32
)
. T
he SpyCatcher
-
SpyTag
system
was
used to prepare
multimerized
SARS
-
CoV
-
2 RBD
or
S trimer that
elicited
high titers
of neutralizing antibodies
(
33, 34
)
.
Although
promising
for protection against
SARS
-
CoV
-
2
,
coronav
irus reservoirs in bats suggest
future cross
-
species transmission
(
6
, 7, 35
)
,
necessitating
a
vaccine that protect
s
against emerging coronaviruses as well as SARS
-
CoV
-
2
.
Here we
prepared
SpyCatcher003
-
mi3
nanoparticles
(
31, 36
)
simultaneously displaying
SpyTagged
RBDs from
human and animal coronaviruses
to evaluate
whether
mosaic particles
can
elicit
cross
-
reactive
antibody responses
, as previously demonstrated for influenza head domain mosaic particles
(
37
)
.
W
e show that
mice immunized with
homotypic or mosaic nanoparticles
produce
d
broad
binding
and neutralizing
responses
,
in
cont
rast to
plasma
antibodies elicited in humans by SARS
-
CoV
-
2
infection
.
Moreover,
mosaic nanoparticles showed
enhanced
heterologous
binding and
neutralization properties
against human and bat
SARS
-
like betacoronaviruses (
sarbecoviruses
)
compared with
homotypic SARS
-
CoV
-
2 nanoparticles
.
We used a study of
sarbecovirus
RBD
receptor usage and
cell tropism
(
38
)
to guide our choice of
RBDs
for co
-
display on mosaic
particles
.
From 29 RB
Ds
that were classified into distinct clades
(
cl
ade
s
1, 2,
1/2,
and
3)
(
38
)
,
we identified
diverse
RBDs
from
SARS
-
CoV, WIV1
,
and
SHC014
(
clade 1
)
,
SARS
-
CoV
-
2
(c
lade
1/2)
,
Rs4081, Yunnan
20
11
(Yun11)
,
and Rf1
(
clade 2
),
and
B
M
48
-
31
(
clade 3
)
, of which SARS
-
CoV
-
2 and SARS
-
CoV are human coronaviruses and the
.
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rest are bat viruses originating in China
or Bulgaria (BM48
-
31)
.
We also
included
RBDs from
the
GX pangolin
clade 1/2
coronavirus
(
referred to here as
pang17
)
(
39
)
,
RaTG13
,
the
bat
clade
1/2 virus most closely
related to SARS
-
CoV
-
2
(
40
)
, RmYN02, a
clade 2
bat
virus
from C
hina
(
41
)
,
and BtKY72
, a Keny
an bat clade 3 virus
(
42
)
.
Mapping of the sequence conservation across
selected RBDs showed varying degrees of sequence identity (
68
-
95
%
), with highest sequence
variability
in
residues corresponding to the SARS
-
CoV
-
2
ACE2 receptor
-
binding motif (
Fig. 1A
-
D
; fig. S1).
We chose 8
of
the
12
RBDs
for making
three types of mosaic
nano
particles
:
mosaic
-
4a
(
coupled to
SARS
-
2,
RaTG13
, SHC014, and Rs4081
RBDs)
, mosaic
-
4b
(
coupled to
pang17,
RmYN02, RF1, and WIV1
RBDs)
, a
nd mosaic
-
8
(coupled
to
all
eight RBDs
)
,
and
compare
d
them
with
homotypic mi3 particles constructed from SARS
-
CoV
-
2 RBD alone
(homotypic
SARS
-
2
)
.
RBDs from
SARS,
Yun11, BM
-
4831, and BtKY72
, which
were not
coupled to mosaic
particles
,
were
used to
evaluate
sera
for
cross
-
react
iv
e responses
.
SpyTag
003
-
RBDs were coupled
to
SpyCatcher003
-
mi3
(60 potential conjugation sites)
(
36, 43
)
to make ho
mo
t
ypic and
mosaic nanoparticles
(Fig 2A).
P
articles were purified
by
size exclusion
chromatography (SEC)
and analyzed by SDS
-
PAGE,
revealing
mono
disperse
SEC profiles and
nearly 100% conjugation
(Fig.
2B
,C
).
Representative
RBDs
were
conjugated to
SpyCatcher003
-
mi3
with similar or identical efficiencies
(
fig
. S2)
,
suggesting that
mosaic particles contain
ed
approximately equimolar mixtures of different
RBDs
.
We immunized
mice
with either
soluble SARS
-
CoV
-
2 spike trimer (SARS
-
2 S),
nano
particles
displaying only
SARS
-
2
RBD (homotypic
SARS
-
2
)
,
nano
particles
co
-
displaying RBDs (
mosaic
-
4a, mosaic
-
4b, mosaic
-
8
)
, or unconjugated nanoparticles
(mi3)
. I
gG
responses
were evaluated
.
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6
a
fter prime
or
boost
immunizations
(Fig. 3A)
by
ELISA
against
SARS
-
2 S (Fig. 3B) or
a panel
of
RBDs
(Fig.
3C
-
F;
fig. S3)
.
Sera from unconjugated nanoparticle
-
immunized animals (black in
Fig. 3
, fig. S3
) showed no responses above background.
A
nti
-
SARS
-
2
S trimer
and anti
-
SARS
-
2
RBD
serum responses were similar
(
Fig. 3B
,C
), demonstrating that
antibodies elicited against
RBDs
can access
their
epitopes on SARS
-
2 S trimer.
We also
conducted in vitro neutralization
assays using a pseudotyped virus assay that quantitat
ively correlates with authentic virus
neutralization
(
44
)
for strains known to infect
293T
ACE2
target cells (SARS
-
CoV
-
2, SARS, WIV1
and SHC104).
Neutralization and ELISA titers were s
ignificantly correlated (fig. S4
), thus
suggesting ELISAs are predictive of neutralization results
when pseudotyped neutralization
assays were not possible due to unknown
viral entry r
eceptor usage.
Mice immunized with soluble
SARS
-
2 S
trimer
(brown bars)
showed no binding
or
neutraliz
ation except for autologous
responses
against
SARS
-
2 after
boosting
(Fig.
3
C
-
F
).
By
contrast, sera
from RBD
-
nanoparticle
immunized animals
(red, green, yellow, and blue
bars
)
exhibited binding to all RBDs
(Fig. 3
C
-
F
;
fig. S
3A
)
and neutralization against all four strains
after boosting
(Fig.
3C
-
E
)
, consistent
with
increased immuno
genicities
of
multimerized
antigen
on nanoparticles versus soluble antigen
(
27, 28
)
.
Homotypic SARS
-
2 nano
particles, but not
soluble SARS
-
2 trimer, induced heterologous responses to zoonotic RBDs and neutralization of
heterolog
ous coronaviruses (Fig. 3D
-
F).
To address whether
co
-
display of SARS
-
2 RBD
along
with other RBDs
on
mosaic
-
4a
and
mosaic
-
8
versus
homotypic display of SARS
-
2 RBD
(
homotypic
SARS
-
2)
diminished anti
-
SARS
-
2 responses, we compared
SARS
-
2
specific
ELISA
and neutralization titers
for mosaic versus homotypic immunizations
(Fig. 3C)
:
there
were
no
significant difference
s
in IgG
anti
-
SARS
-
2
titers for animals immunized with homotypic
(red in
.
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Fig. 3
C
)
versus
mosaic nanoparticles
(green
and blue
in Fig. 3
C
)
.
Thus there was no advantage
of im
munization with a homotypic RBD
-
nanoparticle versus a mosaic
-
nanoparticle
that included
SARS
-
2
RBD
in terms of the magnitude of immune responses
against SARS
-
2.
W
e
next
compared serum responses
against
matched RBDs (RBDs present on
an
injected
nanoparticle; gray horizontal
shading
) versus
mismatch
ed RBDs (RBDs not p
resent on injected
nanoparticle;
r
ed horizontal
shading
)
(Fig. 3; fig. S3)
.
Although
SARS
-
2 RBD was not presented
on mosaic
-
4b,
antibody titers elicited by mosaic
-
4b
immunization
(yellow) were not significantly
different than tit
ers elicited by
matched nanoparticle immunizations
(
homotypic SARS
-
2
(red)
,
mosaic
-
4a (green), and mosaic
-
8 (blue))
, and sera from boosted mosaic
-
4b
immunized mice
neutralized SARS
-
2 pseudovirus (Fig.
3C
).
In other matched versus
mismatch
ed comparisons,
sera showed binding and neutralization
of
SHC014 and WIV1 regardless of whether these RBDs
were included on the injected nanoparticle (Fig. 3D)
, underscoring sharing of common epitopes
among RBDs (Fig. 1A)
.
Demonstrating advantages of mos
aic versus homotypic SARS
-
2 nanoparticles, sera from
mosaic
-
8
immunized mice bound SHC014 and WIV1 RBDs significantly better after the prime
than sera from homotypic SARS
-
2
immunized mice and retained better binding to SHC014
RBD after boosting (Fig. 3D).
Thus
the
potentia
l increased avidity of the
homotypic SARS
-
2
nanoparticle
displaying only one type of RBD
over
the
mosaic
-
8
nanoparticle
s
did not confer
increased breadth.
Moreover,
mosaic
-
8
immunized and boosted
sera
were
7
-
44
fold
more potent
than sera from homotypic SARS
-
2
immunized animals
in neutralizing SHC014 and WIV1
(Fig.
3D
).
Neutralization of the SHC014 and WIV1 pseudoviruses by mosaic
-
8 sera suggests
that
.
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combining RBDs on a mosaic nanoparticle does not diminish the immune
response against a
particular RBD
, also suggested by ELISA
binding
of sera to
Rs4081 and RaTG13 (fig.
S3
A
,B
).
To
further
address whether RBD
-
nanoparticles elicit
ed
antibodies
that recognized
totally
mis
matched strains
and SARS
-
CoV
-
2 RBD mutants
, we evaluated
sera for
binding
to
SARS,
Yun11, BM
-
4831, and BtKY72
RBDs
(Fig. 3
E,F
)
, SARS
-
2 RBD mutants (fig. S3
C
),
MERS
-
CoV RBD (fig. S3D),
and
for neutralization
in SARS
pseudovirus
assays
(Fig. 3E)
.
W
e
found no
reductions in
SARS
-
2
RBD
b
inding as a result of
mutations (
Y453F
,
the “Danish mink variant”
(
45
)
or a Q493K/Q498Y/P499T triple mutant
(
46
)
)
(fig. S3
C
),
no binding of any elicited sera to
MERS
-
CoV
RBD (fig. S3D), and
higher and more cross
-
reactive antibody responses for mosaic
immunizations compared with homotypic SARS
-
2 immunizations: e
.g., mosaic
-
8
primed and
boosted animals showed significantly higher titers against SARS RBD than
sera from
homotypic
SARS
-
2
immunized mice (Fig.
3E
).
After the prime, sera from the homotypic SARS
-
2
immunized animals
did not neutralize
SARS, whereas the mosaic
-
4b and mo
saic
-
8 sera were
neutralizing (Fig. 3E), perhaps fa
cilitated by
these nanoparticles including
WIV1 RBD, which is
related by 95%
amino acid
identity to SARS RBD (Fig. 1D
). After boosting, SARS
-
2 and
mosaic
-
4a sera were also neutra
lizing, although titers wer
e ~4
-
fold lower than for mosaic
-
8
immunized animals (Fig. 3E).
ELISA titers against other mismatched RBDs (Yun11, BM
-
4831,
BtKY72) were significantly higher for sera collected after mosaic
-
8 priming compared to sera
from homotypic SARS
-
2 priming, and heigh
tened binding was retained after boosting (Fig. 3F).
Thus mosaic nanoparticles, particularly mosaic
-
8, induce higher antibody titers against
mismatch
ed RBDs than homotypic SARS
-
2 nanoparticles, again favoring the co
-
display
approach for inducing broader anti
-
coronavirus responses
, especially
after a
single
prime
.
.
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We
investigated the potential for cross
-
reactive recognition using
flow cytometry to ask
whether
B
-
c
ell receptors on IgG+ splenic B
-
cells from
RBD
-
nanoparticle
boosted animals could
simultaneously recognize
RBDs from
SARS
-
2 and Rs4081 (
r
elated by 70% sequence identity
)
(
Fig. 1D
;
fig. S5
)
.
Whereas control animals were negative, all other
groups showed B
-
cells that
recognized SARS
-
2 and Rs4081 RBDs simultaneously, suggesting the existence of antibodies
that cross
-
react with both RBDs (fig. S5E).
To
compare antibodies elicited by
RBD
-
nano
particle immunization
to
antibodies
elicited by
SARS
-
CoV
-
2 infection
, we repeated ELISAs against
the RBD panel
using IgGs from
COVID
-
19
plasma donors
(
47
)
(Fig. 4
)
.
Most
of the convalescent
plasmas
showed detectable binding
to
SARS
-
2
RBD
(Fig. 4A)
.
However,
binding
to
other
sarbecovirus RBDs (
RaTG13
, SHC014,
WIV1, Rs4081 and BM
-
4831)
was significantly
weaker than binding to SARS 2
RBD
, with
many
human
plasma
IgG
s
showing
no binding above background
(Fig. 4B
-
G
)
.
In addition,
although conva
lescent plasma IgGs
neutralized
SARS
-
CoV
-
2 pseudoviruses
, they showed
weak
or
no neutralization of SARS, SHC014, or WIV1 pseudoviruses
(Fig. 4H)
. These results are
consistent with little to no cross
-
reactive recognition of RBDs from zoonotic coronavirus strains
resulting from SARS
-
CoV
-
2 infection in humans.
In conclusion, we
confirm
ed
that
multimerization of RBDs on nanoparticles enhance
s
immunogenicity compared wit
h soluble antigen
(
33, 48
)
and further showed that
homotypic
SARS
-
2 nanoparticle immunization produced IgG responses that bound
zoonotic
RBDs
and
neutralized heterologous coronaviruses
after boosting
. By c
ontrast
,
soluble SARS
-
2 S
.
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immunization and natural infection w
ith SARS
-
CoV
-
2
resulted in
weak or no
heterologous
responses
in plasmas
.
C
o
-
display of
SARS
-
2 RBD along with
diverse RBDs on
mosaic
nano
particles
showed
no disadvantage
s
for eliciting neutralizing
antibod
i
es against SARS
-
CoV
-
2
compared with homotypic SARS
-
2 nanoparticles
, suggesting
mosaic nanoparticles as a candidate
vaccine to protect against COVID
-
19
.
Furthermore
,
compared with homotypic SARS
-
2 RBD
particles,
the
mosai
c
co
-
display
strategy
demonstrated
advantages for eliciting
neutralizing
antibodies
against
zoonotic sarbecoviruses
, thus potentially
also
providing protection against
emerging coronaviruses with
human
spillover potential
.
,
Neutralization
of matched and
mismatch
ed strains was observed after
mosaic
priming
, suggesting a single injection of a mosaic
-
RBD
nano
particle might be sufficient in a vaccine.
Since COVID
-
19 convalescent plasmas
showed little to no recognition of coronavirus
RBD
s other
than SARS
-
CoV
-
2,
COVD
-
19
induced immunity
in humans
may not protect
against another emergent coronavirus
.
However,
the
mosaic nanoparticle
s
described here
could be
used as described or
easily adapted to present
RBDs from
newly
-
discovered
zoonotic coronaviruses
.
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