1
Mosaic
sarbecovirus
vaccination
elicits
cross
-
reactive
responses
in
pre
-
immunized
animals
Alexander A. Cohen
1
,
1
3
, Jennifer R. Keeffe
1
,
1
3
,
Ariën
Schiepers
2
, Sandra E. Dross
3
,4
,
Allison J.
Greaney
5
,
Annie V. Rorick
1
,
Han
Gao
1
,
Priyanthi N.P. Gnanapragasam
1
,
Chengcheng Fan
1
,
Anthony P. West, Jr.
1
, Arlene I.
Ramsingh
6
,
Jesse H.
Erasmus
7
, Janice D.
Pata
8
,
Hiromi
Muramatsu
9
,
Norbert Pardi
9
, Paulo J.C. Lin
10
,
Scott Baxter
11
,
Rita Cruz
11
,
Martina Quintanar
-
Audelo
11,12
,
Ellis Robb
11
,
Cristina Serrano
-
Amatriain
11
,
Leonardo Magneschi
11
, I
an G
.
Fotheringham
11
, Deborah H. Fuller
3,4
, Gabriel D. Victora
2
,
Pamela J. Bjorkman
1,
14,*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
2
Laboratory of Lymphocyte Dynamics, The Rockefeller University, New York, NY,
10065,
USA
3
Department of Microbiology, University of Washington, Seattle, WA 98195, USA
4
National Primate Research Center, Seattle, WA 98121
, USA
5
Medical
Scientist Training Program, University of Washington, Seattle, WA 98195, USA.
6
Creative BioSolutions, LLC, Miami, FL,
33131
,
USA
7
HDT Bio, Seattle, WA,
98109,
USA
8
Wadsworth Center, New York State Department of Health and Department of
Biomedical
Sciences, University at Albany, Albany, NY, 12201, USA
9
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA,
19104,
USA
10
Acuitas Therapeutics, Vancouver, BC V6T 1Z3, Canada
11
Ingenza
Ltd, Roslin Innovation Centre, Charnock Bradley Building, Roslin, EH25 9RG, UK
12
Present address:
Centre for Inflammation Research
and
Institute of Regeneration and Repair
,
The University of Edinburgh
,
Edinburgh
,
EH16 4UU, UK
13
These authors contributed
equally.
14
Lead contact
*Correspondence:
bjorkman@caltech.edu
.
CC-BY-NC 4.0 International license
available under a
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;
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2
SUMMARY
Immunization with m
osaic
-
8b
[
60
-
mer
nanoparticles
presenting
8
SARS
-
like betacoronavirus
(
sarbecovirus
)
receptor
-
binding domains (RBDs
)
]
elicits
more broadly cross
-
reactive
antibod
i
es
than
homotypic
SARS
-
CoV
-
2 RBD
-
only
nanoparticles
and
protects against
sarbecoviruses
.
To
investigate
original antigenic sin
(OAS
)
effects
on mosaic
-
8b
efficacy
,
we
evaluated
effects of
prior
COVID
-
19
vaccinations
in non
-
human primates
and mice
on
sarbecovirus response
breadth
s elic
i
ted
by
mosaic
-
8
b
, admix
-
8b
(8 homotypics),
and homotypic
SARS
-
CoV
-
2
, finding greatest cross
-
reactivity
for
mosaic
-
8
b
.
As
demonstrated
by
molecular fate
-
mapping in which antibodies derived from specific
cohorts of B
cells
are
differentially detected
,
B
cells
primed by
WA1 spike
mRNA
-
LNP
dominated
antibody responses after
RBD
-
nanoparticle boosting
.
While
mosaic
-
8
b
-
and homotypic
-
nanoparticles
boosted
cross
-
reactive antibodies,
de novo
antibodies were
predominantly
induced
with
mosaic
-
8
b
boosting
,
and these were specific for variant RBDs with
increased
identity
to RBDs on
mosaic
-
8b
.
T
hese results
inform OAS
mechanisms and
support using
mosaic
-
8b
to protect
COVID
-
19
vaccinated/infected
humans
against
as
-
yet
-
unknown
SARS
-
CoV
-
2 variants and
animal
sarbecoviruses with
human
spillover potential
.
KEYWORDS
Antibody,
Macaque and
Mouse
M
odels,
Immune
I
mprinting,
Mosaic
-
8b
RBD
-
nanoparticle,
Original Antigenic Sin, Primary Addiction,
RBD,
S
arbecovirus,
SARS
-
CoV
-
2,
V
accin
ation
.
CC-BY-NC 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2024.
;
https://doi.org/10.1101/2024.02.08.576722
doi:
bioRxiv preprint
3
Introduction
Spillover of animal SARS
-
like betacoronaviruses (sarbecoviruses) resulted in
two health crises in
the past 20 years:
the SARS
-
CoV
(SARS
-
1)
epidemic in the early 2000s and the
recent
COVID
-
19 pandemic caused by SARS
-
CoV
-
2
(SARS
-
2)
.
C
ross
-
species transmission
of
a new
sarbecovirus
that is easily spread between humans
from
reservoirs in bats or other animals
could
result in another pandemic
.
1
-
3
In addition,
COVID
-
19 continues to be a
health concern due to
long
COVID complications
4
as well as
SARS
-
2
variants of concern (VOCs)
that
show
apparent
ly
increased transmissibility and/or resistance to neutralizing antibodies
(Abs)
elicited by infection
or vaccination
.
5
-
8
For example,
in Omicron VOCs,
substitutions in the SARS
-
2 spike protein
receptor
-
binding domain
s
(RBD), the major target of neutralizing
Abs
and detectable cross
-
variant
neutralization
,
9,10
have
re
duced
the
efficacies
of vaccines and therapeutic monoclonal
Abs
(mAbs)
.
8,11
One
strategy to
increase protective responses to SARS
-
2 VOCs
involves
novel variant
vaccine boosters
.
12
Since
repeated
updating
of COVID vaccines
is impractical and expensive
, a
more optimal strategy would be a
vaccine that
does
not require
changing
to
protect against
both
emerging
sarbe
coviruses
and SARS
-
2 VOCs
.
To develop
a
vaccine
that
w
ould
protect
against unknown
sarbecoviruses and new SARS
-
2
variants, w
e
used
a
n
approach involving simultaneous display of
eight different
sarbecovirus
RBDs
arranged randomly
on protein
-
based
60
-
mer
nanoparticles
(mosaic
-
8
b
RBD
-
nanoparticles)
(Figure 1A)
and evaluated Ab responses
against RBDs representing
both matched
(RBD on the
nanoparticle
)
and mismatched
(RBD not on the nanoparticle)
viruses
.
13
We found that mosaic
-
8
b
nanoparticles
showed enhanced heterologous binding, neutralization, and protection from
sarbecovirus challenges compared with homotypic (SARS
-
2 RBD
only) nanoparticles in animal
models
.
13,14
For example
, mosaic
-
8b immunizations
showed protection in
K18
-
hACE2 transgenic
mice,
a stringent model of coronavirus infection
,
15
from
both SARS
-
2 and
a
mismatched
SARS
-
1
challenge, whereas
homotypic
SARS
-
2 immunized mice were protected only from SARS
-
2
challenge
.
14
To explain
the
increased cross
-
reactivity of Abs elicited by mosaic
-
8b
, we
hypothesized that
B
cells with B
cell receptors (BCRs) that can crosslink
using both of their antigen
-
binding
Fab
arms
between adjacent
non
-
identical
RBDs
to allow recognition
of
conserved epitopes would be
preferentially
stimulated
to produce cross
-
reactive Abs
,
as compared with B
cells presenting
BCRs that bind to
variable
epitopes
, which could rarely, if ever, crosslink between
non
-
identical
RBDs arranged randomly on a nanoparticle
14
(Figure S1A).
By contrast, homotypic RBD
-
nanoparticles
presenting identical RBDs
are
predicted to
bind
BCRs against immunodominant
strain
-
specific epitopes presented on
adjacent identical
RBDs
.
E
pitope mapping of polyclonal
antisera elicited by mosaic
-
8b
versus homotypic SARS
-
2 RBD
-
nanoparticles using
deep
mutational scanning (DMS)
16
provided evidence supporting this model:
Abs
from
mosaic
-
8b
antisera
primarily targeted
more conserved class 4 and class 1/4
RBD
epitopes
(Figure 1B)
(RBD
epitope nomenclature from ref.
17
)
that show less variability in
sarbecoviruses and
SARS
-
2
VOCs
because they contact other portions of the spike trimer
,
14
whereas
homotypic
antiserum
Abs
primarily targeted
variable
class 1 and 2 RBD epitope regions
that
are
more accessible and are
not involved in
contacts with non
-
RBD portions of spike
.
14
The
se results, combined with
animal
experiments showing
promising cross
-
reactive binding and neutralization data for
elicited
polyclonal antisera
13,14
and
mAbs
,
18
suggested that mosaic
-
8b
RBD
-
nanoparticles
represent a
promising vaccine strategy to protect against
current and future SARS
-
2 VOCs as well as
additional
animal sarbecoviruses
that could spill
over into humans.
The m
osaic
-
8b
immunized animals
in our previous studies
were naïve
with respect to SARS
-
2
exposure
. By contrast, the
majority of
humans receiving a mosaic
-
8b
vaccine
would have
already
.
CC-BY-NC 4.0 International license
available under a
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The copyright holder for this preprint
this version posted February 9, 2024.
;
https://doi.org/10.1101/2024.02.08.576722
doi:
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4
been
infected with SARS
-
2, vaccinated, or both.
An important question, therefore, is whether
mosaic
-
8b
immunization would
elicit
broad
ly
cross
-
reactive
recognition
of
conserved
sarbecovirus
RBD epitopes
in
animals that
were pre
-
vaccinated with
spike
-
based
COVID
-
19 vaccines
;
e.g.,
mRNA
-
lipid
nanoparticle
(LNP) vaccines
.
Specifically, we aimed to understand the role of i
mmune
imprinting,
also known
as original antigenic sin
(OAS)
,
as it relates to eliciting breadth in non
-
naïve individuals. Immune imprinting
was
initially
postulated
to
explain why
influenza virus
infections with
new
and
distinct viral strains
resulted in
preferential
boosting of
Ab
responses
against epitopes shared with the original strain
.
19,20
Recently,
experiments in
Ab
fate
-
mapping
(
Igk
Tag
) mice
demonstrated
that
,
upon repeated immunization, serum Abs
continue to derive
from
B
cells
that were initially
activated
in the primary response.
21
Abs induced
de novo
in subsequent
responses were suppressed,
consistent
with OAS.
This
reliance
on
primary cohort B
cells
(primary addiction)
decreased with antigenic distance, resulting in partial alleviation of OAS upon
boosting with BA.1
s
pike
mRNA
-
LNP
after
single
-
dose
WA1 mRNA priming.
21
T
o
i
nvestigate
basic mechanisms in
B
cell activation in
animals with previous antigenic exposure and to
inform
future
mosaic
-
8b
immunizations
in humans with
previous SARS
-
2 exposure, we
designed
experiments to compare
immune responses to RBD
-
nanoparticles and
additional
COVID
-
19
immunizations
in pre
-
vaccinated anima
ls.
Here, we
evaluate
d
immune responses
to mosaic
-
8b
,
mosaic
-
7
(mosaic
-
8b without a SARS
-
2
RBD)
,
homotypic SARS
-
2
, and
admix
-
8b
(mixture of
8 homotypic
nanoparticles)
(Figure 1A)
in
non
-
human primates (NHPs) and in mice that were
previously vaccinated with
DNA, mRNA,
a
self
-
amplifying RNA (repRNA
)
,
or
adenovirus
-
vectored
COVID
-
19
vaccine
s
(Table S1)
.
As
previously observed in SARS
-
2 naïve animals,
13,14
we found that
m
osaic
-
8b immunization
i
n three
independent
pre
-
vaccinated animal models
elicit
ed
broadly cross
-
reactive binding and
neutralizing Ab responses
against
viral strains
that were both
matched
and mismatched
.
As
also
observed in naïve animals, we
found
increased cross
-
reactivity of Abs elicited by
mosaic
-
8b
compared with homotypic SARS
-
2 RBD
-
nanoparticles
in pre
-
vaccinated animals
,
and also
demonstrated
increased cross
-
reactivity for mosaic
-
8b versus
admix
-
8b
sera
in mice
that were
pre
-
vaccinated with
a Pfizer
-
like
mRNA
-
LNP
.
Molecular fate
-
mapping
21
studies in
mice
showed
that
de novo
Abs
were
predominantly
induced
in pre
-
vaccinated mice only
with mosaic
-
8b boosting,
and
that these Abs
were specific for variant RBDs with increased identity to RBDs on mosaic
-
8b.
Results from these
studies
contribute to increased understanding of
basic immune phenomena
involving
B
cell
responses
under
potential OAS
conditions
. In addition,
they
support the use of
a
mosaic
-
8b RBD nanoparticle
vaccine in
humans with
previous
SARS
-
2
exposure
to pro
tect
against
future
SARS
-
2 variants
, and
also of critical importance
,
to prevent another pandemic caused by
spillover of
an
animal
sarbecovirus
with
the potential for human
-
to
-
human
transmission.
RESULTS
We used the SpyCatcher
-
SpyTag system
22,23
to covalently attach RBDs with C
-
terminal SpyTag
sequences to a 60
-
mer nanoparticle (SpyCatcher
-
mi3)
24
to make RBD
-
nanoparticles
designated
as either
mosaic
-
8
b
(each nanoparticle presenting the SARS
-
2 Beta RBD
and
seven other
sarbecovirus RBDs attached
randomly
to
60 sites)
,
14
mosaic
-
7 (
mosaic
-
8b without the SARS
-
2
Beta RBD
)
,
homotypic
SARS
-
2
(each nanoparticle presenting 60 copies of SARS
-
2 Beta RBD
)
,
14
or
admix
-
8b (
mixture of
8 homotypic RBD
-
nanoparticles corresponding to the RBDs in mosaic
-
8b)
(
Figure
1
; Figure S1
)
.
We used
RBD
-
nanoparticle
doses
based upon
RBD molarity
; thus,
animal cohorts
received
a consistent
RBD dose
but
homotypic SARS
-
2 RBD
-
nanoparticles have
8 times as many SARS
-
2 Beta RBDs as either mosaic
-
8b or admix
-
8b.
For each
immunogen
evaluated
in
a
pre
-
vaccinated animal model, we
assessed serum Ab binding
to
spike antigens
(
different sarbecovirus
RBDs
or
WA1 spike)
by ELISA
and
using
an
in vitro
.
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The copyright holder for this preprint
this version posted February 9, 2024.
;
https://doi.org/10.1101/2024.02.08.576722
doi:
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5
neutralization assay.
Of potential relevance to
results in
humans, both binding and neutralizing
Ab levels correlate with vaccine efficacy.
25
We present ELISA and neutralization data in the form
of radar plots
(
mean binding E
D
50
or
mean
neutralization ID
50
for
each
cohort
shown as
contours
across multiple antigens
simultaneously
) to convey
trends across
multiple
antigens
.
To evaluate
statistical significance
of differences between immunized cohorts
, w
e also present binding and
neutralization data as histograms of the mean of mean titers
across all antigens.
Mosaic
-
8b
immunizations
in previously
-
vaccinated NHPs elicits cross
-
reactive
Ab
responses
To address
the immunogenicity of a mosaic
-
8
b
immunization in a pre
-
vaccinated
animal model
,
we
immunized
14
NHPs that
had
previously
received
nucleic acid
-
based
SARS
-
2 vaccines.
T
he
se
animals
received
four doses of DNA or repRNA vaccines
between weeks
-
64
and
-
30
as
described in the methods
,
Figure 2A
, and
Table S1
.
26
-
28
Eight
weeks before
immunization
, we re
-
stratified the animals into three groups (n
=
4
-
5)
, each of which
was
similar in mean and range of
weigh
ts
, age
s
, sex, and Ab neutralization titers
against SARS
-
2 D614G (Figure S2A)
, without
considering
vaccine history
.
The
mixed
immune history in these
groups of
pre
-
vaccinated NHPs
is r
epresentative of
a
complex
immune history in
people
who
have been vaccinated and/or
infected multiple times
. The
NHPs were
immunized
at week 0
with two doses (8 weeks apart) of
either mosaic
-
8b or homotypic SARS
-
2 RBD
-
nanoparticles or with
a
bivalent repRNA
-
LION
28
vaccine encoding
the WA
-
1
and
the
Omicron BA.1
s
pike
proteins
, which mimicked
immunogens
used in commercial bivalent mRNA
-
LNP vaccines
(Figure 2A).
Serum was then sampled
every
two
weeks for 22 weeks
after the
first nanoparticle or bivalent repRNA boost
to
measure the peak
and subsequent
contraction
of
serum binding and neutraliz
ing Ab
responses
(Figure 2A)
.
We performed
ELISAs against a panel of RBD and
s
pike
protein
s
to assess serum Ab binding
titers and pseudovirus assays against available viral strains
to
determine Ab neutralizing titers
(Figure 2; Figure S2B
-
E)
. Responses at week 0
(immediately prior to
nanoparticle or
repRNA
-
LION
immunizations)
show
ed
equivalent
titers
across
the
three groups
(Figure S2A)
. The mosaic
-
8b and homotypic SARS
-
2
nanoparticle
-
immunized
NHPs
elicited
significantly
high
er
lev
el
s
of
binding (Figure 2B,C; Figure S2B,C) and neutralization (Figure 2D,E; Figure S2D,E) than the
bivalent repRNA
-
LI
ON immunization after week 0
.
Notably,
RBD
-
nanoparticle
immunizations
in
pre
-
immunized
NHPs elicited
high overall
ELISA and neutralization titers
against
a diverse array
of
both matched and mismatched
sarbecovirus
es (Figure 2C,E; Figure S2C,E)
.
As generally
observed for vaccines,
29
-
31
we found that elicited
Ab binding responses
contracted
after both RBD
-
nanoparticle and bivalent
rep
RNA
immunizations
(5
-
8 fold
from
w
eek 2 to
w
eek 8
)
and after
boosting (~10 fold
from
w
eek 10 to
w
eek 22
)
. Contractions in binding Ab levels
largely
correlated
with drops in neutralizing Ab titers (Figure
2B,D; Figure S2B,D
).
Mean serum binding
titer
s for all RBD variants evaluated by ELISA were significantly higher at
5
of
6
time points evaluated after priming pre
-
vaccinated NHPs for
mosaic
-
8b
compared with
homotypic SARS
-
2 samples (Figure 2C; Figure S2C).
In addition, at
weeks 2, 4, and 8
(after only
a single nanoparticle immunization), mosaic
-
8b elicited
significantly higher neutralization titers
compared to
immunization with
homotypic SARS
-
2 (Figure 2E; Figure S2E). At every time point,
either mean binding titers, mean neutralization titers, or both, were significantly higher for mosaic
-
8b than
for
homotypic samples
across
divergent sarbecoviruses, a
required
result for potentially
broad protection since both Ab binding titer and neutralization correlate with vaccine efficacy in
licensed SARS
-
2 vaccines.
31
In summary,
critical for its potential use in non
-
naïve humans,
mosaic
-
8b elicited cross
-
reactive Ab responses even in a background of four prior COVID
-
19
vaccinations
, and
mosaic
-
8b
immunizations
consistently
elicited a broader
Ab
response than
.
CC-BY-NC 4.0 International license
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The copyright holder for this preprint
this version posted February 9, 2024.
;
https://doi.org/10.1101/2024.02.08.576722
doi:
bioRxiv preprint
6
homotypic SARS
-
2 and bivalent
repRNA
immunizations in
the pre
-
vaccinated NHP model
(Figure
2; Figure S2).
Mosaic
-
8b immunizations
outperform genetically
-
encoded vaccine and
non
-
mosaic
RBD
-
nanoparticle immunizations
in pre
-
vaccinated mice
T
o
generalize our findings in pre
-
vaccinated NHPs
to a different animal model
and
to
other
vaccine modalities
, we
conducted
experiments in mice
that had been
pre
-
immunized with
genetically
-
encoded vaccines
,
either
an
mRNA
-
LNP
formulation equivalent to
Pfizer
-
BioNTech
’s
WA1 spike
-
encoding
BNT162b2
vaccine
(Figure
3
; Figure S
3
; Table S1
)
or
with
an adenovirus
-
vectored vaccine
(
research
-
grade
ChAdOx1 nCoV
-
19
produced by
the Viral Vector Core Facility
,
Oxford University
)
also encoding the WA1 spike
(Figure
4
; Figure S
4
; Table S1
)
.
For mouse
experiments
, we
could increase
the number of
animals per cohort
and
also
add
new
cohort
s
(admix
-
8b
and mosaic
-
7
)
to
comparison
s
of
mosaic
-
8b, homotypic SARS
-
2, and
mRNA
-
LNP
or
viral vector
immunizations after
pre
-
vaccinations
(
Figure
3A; Figure
4A
)
.
Pre
-
vaccinated mice were bled
prior to
immunizations
at day 0
and
after
immunizations
at
days
28 and 56
.
As in the
NHP experiments, we
performed
ELISAs against a panel of RBD and
s
pike
protein
s
and pseudovirus
assays against available viral strains (Figure
3
B
-
E
; Figure 4
B
-
E
)
.
In the
WA1
mRNA
-
LNP
pre
-
vaccination
experiments, r
esponses
at
day
0
(
after all animals
had
received
the same course of
mRNA
-
LNP
vaccines
)
show
ed
significant
differences in
binding
titers
elicited
by the pre
-
vaccination
s
across the cohorts (Figure
S
3
B
-
C
,
G
-
H
), possibly because of
differences
i
n
initial
vaccine responses or in
A
b
contraction
. T
hus
,
we appli
ed
baseline
corrections to account
for
different
mean responses
at day 0
in each of the four groups
(see Methods).
Neutralization
potenc
ies
at day 0
were
similar for all
cohorts
and therefore
were
not
baseline
-
corrected
(Figure
S
3D
-
E)
.
Baseline corrections were not required for
the
ChAdOx1
pre
-
va
ccination experiments
(Figure S4)
.
mRNA
-
LNP pre
-
vaccinated mice
.
In the WA1
Pfizer
-
like
mRNA
-
LNP
pre
-
vaccinated animals
(Figure
3
A
; Table S1
)
,
the
increase in
Ab binding
titers
was
higher
at
day
s
28 and 56
for mosaic
-
8b than
for
the other cohorts when evaluated
against
sarbecovirus RBDs
(
especially
notable
for
day
56
)
(Figure
3
B)
.
When
the mean of mean
changes
from day 0
across all strains
was
calculated
,
mosaic
-
8b consistently induced significantly higher
titers than the other coho
rts
(Figure
3
C). In
general,
while
RBD
-
nanoparticle
immunization
s
boosted Ab binding against RBDs
,
immunization with
an additional dose of
WA1 mRNA
-
LNP
did not
appreciably boost
A
b
responses
, especially at
day
56
(Figure
3
C).
When comparing
responses to
different
protein
nanoparticles
,
mosaic
-
8b
immunizations i
nduced
significantly higher
increases in
mean binding
titers
than
either admix
-
8b or homotypic SARS
-
2
immunizations.
However, m
ean neutralization
titers
did not show
as pronounced
d
ifferences
between cohorts
(Figure 3D)
,
although
comparisons for the mean of mean titers at day 56 after RBD nanoparticle
immunization
rose to
significance for the mosaic
-
8b/admix
-
8b
(p=0.0
21)
and mosaic
-
8b/mRNA
-
LNP
(p=0.035)
comparisons
(Figure 3E)
.
Also, m
ean
neutralization titers for mosaic
-
8b were higher than titers
for
admix
-
8b or homotypic SARS
-
2 against some
viral strains
(
e.g., SARS
-
1
,
SHC014
, and WIV1
)
(
Figure 3D)
.
To evaluate
whether
a mosaic RBD
-
nanoparticle without
a
SARS
-
2 RBD
would
also
overcome
potential OAS effects, we compared
Ab responses
raised by
mosaic
-
7
(
include
s
all mosaic
-
8b
RBDs except for SARS
-
2
;
Figure
1A)
,
to mosaic
-
8b
(Figure
3
F
-
J
)
. Mice were
pre
-
vaccinated with
two doses of
WA1 mRNA
-
LNP and
one
dose of
WA1/BA.
5
bivalent mRNA
-
LNP
and then
immunized with either
two doses of
an RBD
-
nanoparticle (
mosaic
-
8b or mosaic
-
7
)
or
an additional
dose of
WA1
/BA.5
mRNA
-
LNP
, which
was
included
to
mimic
a more
up
-
to
-
date immune history
.
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;
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7
in the human population
)
(Figure 3F).
We found
consistently
greater
increases
in mean Ab binding
titers
at
day
28 and
day
56
with respect to day 0
for
mosaic
-
7
compared with
mosaic
-
8b
immunized
animals
across
all RBDs
(Figure 3G)
,
which
were
reflected
as
significantly
higher
m
ean
fold changes
in
Ab binding titers
for mosaic
-
7
(Figure 3H)
.
Although
increase
s
in binding
titer
s
with respect to
d
ay 0 values
were higher
for mosaic
-
7
than
for mosaic
-
8b
,
differences in
the
mean of
mean
titer
s
for the corresponding non
-
baseline corrected data
were
only marginal
between the two groups
at day 28,
dropping to
no significance
at day 56 (Figure S3
K
-
N)
,
suggesting that immunization by these RBD
-
nanoparticles had elicited a maximum binding
anti
-
RBD
Ab
response
. In addition, there were no neutralization differences
for
mosaic
-
8b and mosaic
-
7 antisera
across strains (Figure 3I) or significant differences between mean neutralization titers
(Figure 3J)
,
as also observed for
the
non
-
baseline
corrected
me
an binding
titers
.
B
oth mosaic
-
8b
and
mosaic
-
7 were significantly
better than
an additional dose of
WA1/
BA.5
(Figure S3K
-
N).
ChAdOx1
pre
-
vaccinated mice
.
We also
examined the responses to mosaic
-
8b, homotypic
SARS
-
2,
and
admix
-
8b
in
ChAdOx1
pre
-
vaccinated animals (Figure 4
; Figure S4
; Table S1
).
In
these experiments, we
compared
RBD
-
nanoparticle immunization with
immunization
of
an
addit
ional
dose
of ChAdOx
1
or
of
WA1 mRNA
-
LNP
(Figure 4A)
.
Overall
Ab
binding
responses
across
RBDs
were
generally
higher
for mosaic
-
8b and admix
-
8b than for homotypic SARS
-
2
, and
the three nanoparticle
immunizations produced higher binding titers than
an
additional
immunization with
WA1 mRNA
-
LNP
or
ChAdOx
1
(Figure 4B)
,
as
reflected in some cases with
significan
t
differences in mean ELISA binding titers (Figure 4
C
).
Neutralization
titers against
SARS
-
1
were higher for
mosaic
-
8b
and
admix
-
8b
than
for
homotypic SARS
-
2
at day 56
(Figure
4D)
, but differences in mean of mean neutralization titers
were not significant across cohorts
(Figure 4E).
Overall, mosaic
-
8b and admix
-
8b boosting elicited the broadest binding and
neutralization responses.
To
determine
which
RBD
epitopes
were
targeted
in
pre
-
vaccinated and then immunized mice
,
we
performed
DMS
16
using yeast display libraries derived from WA1 and XBB1.5 RBDs
for
ChAdOx1
pre
-
vaccinated mic
e
that were immunized with either mosaic
-
8b,
admix
-
8b
, homotypic SARS
-
2,
or WA1 mRNA
-
LNP
(Figure
4A;
Figure 5)
.
To aid in interpreting epitope mapping of polyclonal
antisera
, w
e
first
conducted
control
experiments
by
compar
ing
DMS
of individual mAbs
with
known epitopes
(class 1, 2, 3,
4, and 1/
4; Figure 1B)
to
DMS
using different mixtures of
the same
mAbs
(Figure S5).
As previously shown
for mapping of mAb epitopes,
32,33
DMS
revealed the
expected
escape
profiles
matching
the
epitopes
of the control mAbs (Figure S5).
However
, when
mAbs were mixed in various ratios,
DMS signals were obscured
, with
little
or
no
highlighting of
epitopes
in the equimolar
mix
and only partial highlighting when
the mixtures contained either
more class 3
,
class
1/4
,
and
class
4 mAbs or
more
class 1
/
class 2 mAbs (Figure S5).
Thus,
a
polyclonal
serum
including
anti
-
RBD
Abs
that
are
e
qually distributed across all classes
(i.e., a
“polyclass” antiserum)
will likely
display low
DMS
escape
fractions
across all RBD residues
,
whereas a polyclonal serum response that is
more
focused
on
particular epitope(s)
will
show
more
defined
escape peaks
.
We next
analyzed
d
ifferences in epitope targeting of Abs elicited by mosaic
-
8b
,
admix
-
8b
,
homotypic
SARS
-
2
,
and
WA1 mRNA
-
LNP
in ChAdOx1
pre
-
vaccinated mice
(Figure 5)
, keeping
in mind
(
i
)
the possibility of inducing polyclass Abs
,
and
(
i
i
)
that
DMS results
can depend on the
particular RBD library used in an experiment. F
or example, class 1 and class 2 anti
-
RBD Abs
from WA1 mRNA
-
LNP vaccinated animals
do not
bind to XBB1.5,
which would
emphasiz
e
detection of
responses to
the
more conserved class 3 and class 4
epitopes
.
We found that both
homotypic SARS
-
2 and WA1 mRNA
-
LNP
immunizations
resulted in
relatively
defined
class 1
, 2, and 3
DMS profiles
against
the
WA1
library
(Figure
5A
-
B)
and
defined class 3
.
CC-BY-NC 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2024.
;
https://doi.org/10.1101/2024.02.08.576722
doi:
bioRxiv preprint