Modular adjuvant-free pan-HLA-DR-immunotargeting subunit vaccine against SARS-CoV-2 elicits broad sarbecovirus-neutralizing antibody responses

A icle
Modula adju an - ee pan-HLA-DR-
immuno a ge ing subuni accine agains SARS-
CoV-2 elici s b oad sa beco i us-neu alizing
an ibody esponses
G aphical abs ac
Highligh s
dModula ITV design consis s o SARS-CoV-2 spike RBD used
o a pan-MHC class II mAb
dS uc u e o conse ed epi ope on HLA-DR o e s molecula
basis o b oad eac i i y
dAdju an - ee ITV immuniza ion elici s b oad neu alizing Ab
esponses in abbi s
dAdju an - ee ITV immuniza ion p o ec s e e s om SARS-
CoV-2 challenge
Au ho s
Aud ey Kassa djian, E ic Sun,
Jamie Sookhoo, ..., Shawn Babiuk,
B ian Ba be , Jean-Philippe Julien
Co espondence
jean-philippe.julie[email p o ec ed]
In b ie
Kassa djian e al. enginee and
cha ac e ize a modula accine sca old
o he deli e y o an igen o MHC class II
on an igen-p esen ing cells. This p o ein
accine induces b oad sa beco i us
neu alizing an ibody esponses and
p o ec s om SARS-CoV-2 i al
challenge independen ly o adju an co-
adminis a ion.
Kassa djian e al., 2023, Cell Repo s 42, 112391
Ap il 25, 2023 ª2023 The Au ho (s).
h ps://doi.o g/10.1016/j.cel ep.2023.112391 ll
A icle
Modula adju an - ee pan-HLA-DR-immuno a ge ing
subuni accine agains SARS-CoV-2 elici s
b oad sa beco i us-neu alizing an ibody esponses
Aud ey Kassa djian,
1,2
E ic Sun,
1,2
Jamie Sookhoo,
3,4
K i hika Mu hu aman,
1,5
Kayluz F ias Boligan,
6
Iga Kucha ska,
1
Edu ne Rujas,
1,7,8,9
A i Je ha,
1
Donald R. B anch,
6,10
Shawn Babiuk,
3,4
B ian Ba be ,
2
and Jean-Philippe Julien
1,2,5,11,
*
1
P og am in Molecula Medicine, The Hospi al o Sick Child en Resea ch Ins i u e, To on o, ON M5G 0A4, Canada
2
Depa men o Immunology, Uni e si y o To on o, To on o, ON M5S 1A8, Canada
3
Canadian Food Inspec ion Agency, Na ional Cen e o Fo eign Animal Disease, Winnipeg, MB R3E 3M4, Canada
4
Depa men o Immunology, Uni e si y o Mani oba, Winnipeg, MB R3E 0T5, Canada
5
Depa men o Biochemis y, Uni e si y o To on o, To on o, ON M5S 1A8, Canada
6
Canadian Blood Se ices, Keenan Resea ch Cen e, To on o, ON M5B 1W8, Canada
7
Ike basque, Basque Founda ion o Science, 48013 Bilbao, Spain
8
Pha macokine ic, Nano echnology and Gene The apy G oup, Facul y o Pha macy, Uni e si y o he Basque Coun y UPV/EHU, 01006
Vi o ia, Spain
9
Bioa aba, Mic obiology, In ec ious Disease, An imic obial Agen s, and Gene The apy, 01006 Vi o ia, Spain
10
Uni e si y o To on o, Depa men s o Medicine and Labo a o y Medicine and Pa hobiology, To on o, ON M5S 1A8, Canada
11
Lead con ac
*Co espondence: [email protected]
h ps://doi.o g/10.1016/j.cel ep.2023.112391
SUMMARY
Subuni accines ypically equi e co-adminis a ion wi h an adju an o elici p o ec i e immuni y, adding
de elopmen hu dles ha can impede apid pandemic esponses. To ci cum en he need o adju an in
a se e e acu e espi a o y synd ome co ona i us 2 (SARS-CoV-2) subuni accine, we enginee a he mo-
s able immuno a ge ing accine (ITV) ha le e ages he pan-HLA-DR monoclonal an ibody 44H10 o deli e
he i al spike p o ein ecep o -binding domain (RBD) o an igen-p esen ing cells. X- ay c ys allog aphy
shows ha 44H10 binds o a conse ed epi ope on HLA-DR, p o iding he basis o i s b oad HLA-DR eac-
i i y. Adju an - ee ITV immuniza ion in abbi s and e e s induces obus an i-RBD an ibody esponses ha
neu alize SARS-CoV-2 a ian s o conce n and p o ec ecipien s om SARS-CoV-2 challenge. We demon-
s a e ha he modula na u e o he ITV sca old wi h espec o helpe T cell epi opes and di e se RBD an-
igens acili a es b oad sa beco i us neu aliza ion. Ou indings suppo an i-HLA-DR immuno a ge ing as
an e ec i e means o induce s ong an ibody esponses o subuni an igens wi hou equi ing an adju an .
INTRODUCTION
Since he eme gence o se e e acu e espi a o y synd ome co o-
na i us 2 (SARS-CoV-2) in Decembe o 2019, join e o s by he
global scien i ic communi y ha e led o he de elopmen and
deploymen o accines a an unp eceden ed a e.
1,2
Cu en ly
licensed accines agains SARS-CoV-2 ha e coun e ed he
ad ance o he pandemic wi h conside able success; howe e ,
dispa i ies in global accina ion co e age, pe sis ence o ci cu-
la ing i us, and con inued i al e olu ion ha e highligh ed a
need o add ess he limi a ions o exis ing accine app oaches.
Agains pandemic pa hogens, accines mus exhibi high e icacy
and ideally p o ide du able immuni y and b oad p o ec ion
agains con inuously eme ging a ian s o conce n (VOCs). Mo e-
o e , accines wi h a o able p o iles o manu ac u ing and dis i-
bu ion would enable hei deploymen in coun ies wi h limi ed
in as uc u e o s o age and dis ibu ion, he eby inc easing ac-
cine accessibili y in u u e pandemic se ings.
3,4
Recombinan p o ein subuni accines can p o ide sa e and
e ec i e accina ion op ions o use ac oss di e se popula ions.
Agains apidly changing pa hogens, subuni -based app oaches
o e nume ous de elopmen al e iciencies ha can be le e aged
o pandemic esponses, including apid scalabili y, low de elop-
men and dis ibu ion cos s, and a educed eliance on sophis i-
ca ed cold-chain in as uc u e. Howe e , due o he limi ed
in insic immunogenici y o pu i ied p o ein an igens, subuni ac-
cine o mula ions usually equi e immunos imula o y agen s o
enhance he immune esponse.
5
The di e si y o a ailable adju-
an s, each wi h dis inc modes o ac ion, coupled wi h he unique
na u e o each an igen-adju an pai ing, poses conside able
de elopmen challenges o he ime-sensi i e deploymen o ad-
ju an ed subuni accines.
6,7
Mo eo e , cons ain s imposed by
he global supply and a ailabili y o widely used adju an s ha e
u he p ecluded he e icien de elopmen o subuni accines
o la ge-scale manu ac u ing.
8–10
To his end, al e na i e s a e-
gies o inc easing accine immunogenici y wi hou equisi e
Cell Repo s 42, 112391, Ap il 25, 2023 ª2023 The Au ho (s). 1
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
ll
OPEN ACCESS
pai ing o p o ein immunogens o ex insic adju an sys ems
emain a cen al pu sui in accine esea ch.
Ta ge ed an igen deli e y, also known as immuno a ge ing, is
one such s a egy p oposed o acili a e an igen up ake, p o-
cessing, and p esen a ion by an igen-p esen ing cells (APCs),
he eby enhancing accine-induced immune ac i a ion. P e i-
ous s udies ha e demons a ed he success o a ge ing APC e-
cep o s (such as majo his ocompa ibili y complex [MHC] class
II, C- ype lec in-like ecep o s, and umo nec osis ac o [TNF]
ecep o amily membe s) in enhancing immune esponses o
a ious ecombinan an igens ollowing accina ion.
11–14
One
pa icula MHC class II- a ge ing monoclonal an ibody (mAb),
44H10, has been success ully used in an immuno a ge ing
con ex o elici an igen-speci ic an ibody esponses o mul iple
accine candida es,
15–19
e en in he absence o adju an .
Though 44H10 was i s disco e ed as an an i-HLA-DR an ibody,
i is also c oss- eac i e wi h abbi and e e MHC class II mole-
cules,
16,20,21
enabling he in i o cha ac e iza ion o MHC class
II- a ge ing accine candida es in hese p e-clinical species.
P o iding a sou ce o e icien helpe T cell ac i a ion is ano he
independen s a egy o enhancing accine immunogenici y. In-
clusion o uni e sal helpe T cell epi opes can allow binding o
MHC class II molecules i espec i e o popula ion-le el allelic
a ia ion o p o ide e ec i e T cell help necessa y o op imal
an ibody esponses.
22–25
In his wo k, we epo he enginee ing, s uc u al cha ac e -
iza ion, and p e-clinical e alua ion o a he mos able adju an -
ee SARS-CoV-2 accine candida e ha le e ages he MHC
class II- a ge ing p ope ies o mAb 44H10 o elici p o ec i e
neu alizing an ibody esponses agains he SARS-CoV-2 ecep-
o -binding domain (RBD). We u he mo e explo e he ole o
syn he ic and na u ally de i ed T cell epi opes in enhancing ac-
cine immunogenici y. Las ly, we desc ibe he modula na u e o
he immuno a ge ing accine (ITV) immunoglobulin sca old ha
enables he inco po a ion o RBDs om mul iple sa beco i uses
on a single accine candida e o b oaden he elici ed neu alizing
esponse.
RESULTS
ITV e ec i ely a ge s SARS-CoV-2 RBD an igen o MHC
class II
To enginee an ITV wi h desi able biochemical and immunolog-
ical p ope ies, we used he SARS-CoV-2 RBD o he hea y
chain C e minus o a chime ic 44H10 an ibody (c44H10) wi h
mouse a iable and human cons an egions (Figu e 1A). SDS-
PAGE analysis o pu i ied soluble RBD, c44H10 immunoglobulin
G (IgG), and ITV demons a ed he expec ed inc ease in he o e -
all size o he ecombinan ly exp essed ITV ela i e o c44H10
IgG unde bo h non- educing and educing condi ions (Fig-
u e 1B). Indeed, an upwa d shi o he c44H10 hea y-chain
band by 25 kDa in he ITV was obse ed, co esponding o
he RBD linkage o he ITV hea y chain. The hyd odynamic
adius o he ITV (9 nm) oughly co esponded o he sum o adii
measu ed o RBD and c44H10 (3.3 and 5.9 nm, espec i ely)
(Figu e 1C). Visualiza ion o pu i ied ITV by nega i e-s ain elec-
on mic oscopy u he con i med he inco po a ion o wo
RBDs pe ITV molecule, wi h conside able lexibili y be ween
he c44H10 IgG sca old and RBDs as con e ed by he
10-amino acid polypep ide linke joining hese domains
(Figu es 1D and S1).
ITV was ound o bind MHC class II on he human B lympho-
blas oid cell line BJAB in low cy ome y expe imen s (Figu e 1E),
and he s eng h o his in e ac ion was measu ed by biolaye
in e e ome y (BLI) using ecombinan HLA-DR, wi h an
appa en K
D
o 1.5 nM (Figu e 1F). In addi ion, he s uc u al
in eg i y o he RBD an igen in he con ex o he ITV was
con i med by low cy ome y using a panel o ou mAbs a ge -
ing dis inc con o ma ional epi opes on he SARS-CoV-2 RBD
(class I o IV)
26–31
(Figu e 1G). Toge he , hese da a demons a e
he abili y o he ITV o inco po a e he in ended biophysical and
unc ional p ope ies o i s cons i uen s, e aining bo h he s uc-
u al in eg i y o he RBD an igen and he MHC class II- a ge ing
abili y o he 44H10 an ibody.
c44H10 binds a conse ed si e on HLA-DR
A desi able cha ac e is ic o any accine is i s abili y o unc ion
e ec i ely ac oss he en i e a ge popula ion. As one o he mos
polymo phic se o alleles,
32
a ge ing o MHC class II ep esen s
a conside able challenge, and an e ec i e ITV a ge ing HLA-DR
would need o o e come he as majo i y o —i no all—HLA-DR
allelic a ia ion p esen wi hin he human popula ion. To e alua e
he obus ness o he 44H10 an ibody agains HLA-DR allelic
a ia ion, he binding o c44H10 was es ed agains a andom
se o 100 di e en dono pe iphe al blood mononuclea cell
(PBMC) samples om he G ea e To on o A ea. The c44H10
an ibody was eac i e agains 100% o he samples es ed (Fig-
u e 2A), sugges ing b oad eac i i y o he 44H10 speci ici y o-
wa d HLA-DR allelic a ian s in humans.
To de e mine he ex en o which 44H10 a ge s a uly mono-
mo phic epi ope on HLA-DR, as sugges ed by p e ious li e a u e
desc ibing eac i i y o DR-1,-2,-3,-4,-5, and -7pheno ypes,
20
we sol ed he c ys al s uc u e o he c44H10 Fab in complex
wi h he ex acellula HLA-DR ⍺/bhe e odime (HLA-
DRA*01:01, HLA-DRB1*04:01) by X- ay c ys allog aphy a a es-
olu ion o 3.1 A
˚(Figu e 2B; Table S1). S uc u al analysis o he
co-complex e ealed ha c44H10 binds away om he HLA-
DR pep ide-binding g oo e, a oiding egions o concen a ed
di e si y (Figu es 2B and S2A). Ins ead, an ibody-HLA-DR in e -
ac ions a e p edominan ly media ed by he p ac ically in a iable
HLA-DR ⍺chain, which con ibu es o e 70% o he in e ace
bu ied su ace a ea (BSA) (⍺chain BSA = 742 A
˚
2
; o al
BSA = 1,066 A
˚
2
) and media es 10 o he 13 in e molecula
hyd ogen bonds (Figu e 2C; Tables S2 and S3). Sequence align-
men o he IPD-MHC da abase indica ed ha mos HLA-DR es-
idues con ac ed by c44H10 a e conse ed ac oss majo -DRA
and -DRB1 allele g oups. Only h ee esidues a he ou mos pe-
iphe y o he an ibody-HLA in e ace (con ibu ing only 0.4%
o he in e ace o al BSA) displayed sligh sequence a iabili y
in o he HLA-DR allele g oups, namely ⍺-W168, b-F58, and
b-H60 (Figu es 2C and S2A–S2C). We assessed he impac o
his a iabili y on c44H10 an ibody binding by gene a ing a panel
o ecombinan HLA-DR mu an s subs i u ing hese esidues
wi h hei coun e pa s om o he HLA-DR alleles. BLI s udies e-
ealed ha subs i u ions a hese h ee posi ions minimally hin-
de ed he abili y o c44H10 o bind HLA-DR (Figu e 1D). Thus,
2Cell Repo s 42, 112391, Ap il 25, 2023
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he s uc u al cha ac e iza ion o he c44H10-HLA-DR in e ac-
ion in he con ex o comp ehensi e MHC class II gene ic
sequence da abases demons a es he abili y o he ITV o o e -
come ex eme HLA-DR polymo phisms in he human popula ion
by binding o a conse ed epi ope.
Unadju an ed ITV immuniza ion elici s obus
neu alizing an ibody esponses in abbi s
To e alua e he immunogenici y o he ITV and assess he
bene i o immuno a ge ing, abbi s we e immunized subcu a-
neously (s.c.) in a p ime-boos egimen wi h ei he 50 mg unad-
ju an ed ITV o an equimola dose o soluble RBD (sRBD)
(Figu e 3A). While unadju an ed sRBD immuniza ion did no
elici signi ican an ibody esponses, ITV immuniza ion induced
obus an i-RBD IgG i e s, pa icula ly a e a boos e dose a
day 35 pos -p iming (Figu e 3B). Ini ial assessmen o he unc-
ional quali y o he elici ed an ibody esponse om ITV-immu-
nized abbi s was ca ied ou using an ELISA-based su oga e
i us neu aliza ion (sVNT) assay, which e alua es he abili y
o se um an ibodies o dis up he RBD-ACE2 in e ac ion.
36,37
ITV immuniza ion elici ed RBD-neu alizing an ibody esponses
ha peaked a e he boos (day 49; ED
50
= 0.0064) and pe -
sis ed o e he expe imen al ime ame (Figu e 3C). These
da a we e co obo a ed by an o hogonal pseudo i us neu al-
iza ion (pVNT) assay (Figu e 3D), which measu es he abili y o
se um an ibodies o block he in ec ion o 293T cells exp essing
he human ACE2 (hACE2) ecep o by SARS-CoV-2 spike-
pseudo yped len i i us.
38
Rela i e o he sVNT, he pVNT assay
o e s inc eased sensi i i y h ough he de ec ion o an ibodies
ha neu alize ia mechanisms o he han s e ic hinde ance o
occlusion o he ACE2 binding si e.
27,39
Neu aliza ion i e s
de i ed om bo h assays we e no signi ican ly di e en (Fig-
u e 3E), sugges ing ha he neu alizing an ibody esponse eli-
ci ed by he ITV was la gely d i en by s e ic hinde ance o
occlusion o he ACE2 binding si e, as would be expec ed
om an RBD-based immunogen. In con as , no neu aliza ion
in he se a o abbi s immunized wi h sRBD was de ec ed by
ei he me hod. Collec i ely, hese da a demons a e he abili y
o unadju an ed ITV o elici neu alizing an ibody esponses in
immunized abbi s.
Figu e 1. Biophysical and unc ional cha ac e iza ion o he ITV agains SARS-CoV-2
(A) Schema ic ep esen a ion o he ITV agains SARS-CoV-2 c ea ed wi h BioRende .com.
(B) SDS-PAGE o pu i ied RBD, c44H10 IgG, and ITV unde non- educing (NR) and educing (R) condi ions.
(C) Dynamic ligh sca e ing p o iles o RBD, c44H10 IgG, and ITV.
(D) Rep esen a i e 2D class a e ages om nega i e-s ain elec on mic oscopy images o pu i ied ITV. The whi e scale ba on he op le panel co esponds o
10 nm. See also Figu e S1.
(E) Flow cy ome ic de ec ion o ITV binding o MHC class II exp essed on BJAB cells.
(F) Biolaye in e e ome y binding p o ile o pu i ied ITV o ecombinan HLA-DR, whe e black lines ep esen measu ed binding and o ange cu es co espond
he da a i ed o a 1:1 binding model (R
2
= 0.99).
(G) Binding o class I (REGN10933), II (COV2-2196), III (REGN10987), and IV (CR3022) mAbs a ge ing dis inc RBD con o ma ional epi opes o ITV measu ed by
low cy ome y.
Cell Repo s 42, 112391, Ap il 25, 2023 3
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Figu e 2. Molecula basis o MHC class II a ge ing by c44H10
(A) Flow cy ome ic de ec ion o c44H10 IgG binding o 100 dono PBMC samples om he G ea e To on o A ea. Da a a e ep esen ed as eac i i y o indi idual
PBMC samples + mean eac i i y in each g oup (ma ked by he ed lines) and analyzed by unpai ed es (****p < 0.0001).
(legend con inued on nex page)
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Inclusion o T cell epi opes in ITV design enhances RBD-
speci ic an ibody esponses
The inco po a ion o uni e sally immunogenic T cell epi opes
(TCEs) in he ITV c ea es he po en ial o enhance immune e-
sponses elici ed by his accine candida e by bypassing popula-
ion-le el MHC class II allelic a ia ion o p o ide an e icien
sou ce o T cell help. To explo e he ole o TCEs in augmen ing
ITV immunogenici y, TpD ( e anus and diph he ia oxoid epi-
opes sepa a ed by a ca hepsin clea age si e)
24
o PADRE
(pan HLA-DR-binding epi ope)
22,23
pep ide sequences we e
used o he ligh -chain C e minus o he ITV by a sho GGS
linke , and he induced an ibody esponse elici ed in abbi s
immunized s.c. wi h unadju an ed ITV, ITV-TpD, o ITV-PADRE
was compa ed. Inco po a ion o ei he T helpe pep ide in ITVs
nea ly doubled he RBD-speci ic an ibody esponse ela i e o
he ITV wi hou any TCEs (Figu e 4A) and u he enhanced ac-
cine-induced se um neu aliza ion as measu ed by bo h sVNT
and pVNT (Figu e 4B). The addi ion o TCEs enhanced peak
(day 49) se a neu aliza ion po ency mo e han 5- old (ED
50
=
0.0013) as measu ed by pVNT, wi h peak an ibody esponses
oughly equi alen o 100 mg/mL o he highly neu alizing he a-
peu ic mAb REGN10987 (IC
50
= 0.04 nM).
31
To compa a i ely
assess he impac o ou e o accine adminis a ion on ITV
immunogenici y, abbi s we e also immunized in amuscula ly
(i.m.) wi h ITV-TpD. Consis en wi h p e ious indings,
40,41
g ea e an i-RBD an ibody esponses we e elici ed in i.m.-immu-
nized abbi s compa ed wi h hose immunized s.c. (Figu e S3A),
and hese esponses we e also accompanied by g ea e neu al-
iza ion po ency measu ed by bo h pVNT and sVNT (Figu es S3B
and S3C). Collec i ely, hese da a suppo he inco po a ion
o enginee ed TCEs and i.m. deli e y as a s a egy o
enhance an igen-speci ic an ibody esponses in he con ex o
immuno a ge ing.
P e-exis ing immuni y o TD enhances neu alizing
an ibody i e s elici ed by ITV-TpD
Gi en i con ains TCEs de i ed om e anus and diph he ia (TD)
oxoids, he TpD pep ide p o ides an oppo uni y o enhance he
(B) 3.1 A
˚c ys al s uc u e o he c44H10 Fab in complex wi h HA pep ide-bound HLA-DR (HLA-DRA*01:01 [da k blue], HLA-DRB1*04:01 [ligh blue]). HC, hea y
chain; KC, kappa chain.
(C) Bu ied su ace a ea (BSA) con ibu ion o HLA-DR esidues con ac ed by c44H10 as de e mined by PDBePISA in e ace analysis.
33
Residues in ol ed in
hyd ogen bonds (H) and disul ide b idges (S) a e indica ed by black do ed lines. Below is he HLA-DR aand bchain sequence di e si y o con ac ed esidues
om he IPD-IMGT/HLA da abase in Weblogo ep esen a ion,
34,35
whe e esidues om he c ys allized allele a e black and al e na i e esidues om o he alleles
a e g ay. The h ee esidues in he HLA-DR epi ope a ge ed by c44H10 ha display sligh sequence di e si y (a-W168, b-F58, and b-H60) a e indica ed by boxes
and colo ed in o ange. See also Figu e S2.
(D) BLI binding o c44H10 o HLA-DR mu an s wi h subs i u ions in key pe iphe ally con ac ed esidues. Black lines ep esen measu ed binding and o ange
cu es co espond o he da a i ed o a 1:1 binding model (R
2
< 0.98).
Figu e 3. Adju an - ee ITV immuniza ion in abbi s induces obus and neu alizing an i-RBD esponses
(A) Schedule o abbi immuniza ions and sampling (n = 5 abbi s/g oup). Schema ic c ea ed wi h BioRende .com.
(B) Quan i ica ion o an i-RBD IgG i e s elici ed by unadju an ed ITV o sRBD immuniza ion in abbi s. Da a a e ep esen ed as he mean ±SEM and analyzed by
Mann-Whi ney es (*p < 0.05; ** <0.01; ***p < 0.001).
(C and D) Se um neu aliza ion po ency agains wild- ype SARS-CoV-2 (WIV04/2019) a selec ed ime poin s measu ed by sVNT (C) and pVNT (D). Da a a e
ep esen ed as he mean ±SEM o a leas wo expe imen al eplica es assaying se a pooled om abbi s wi hin each g oup.
(E) Compa ison o se um neu aliza ion i e s de e mined by sVNT and pVNT a selec ed ime poin s, whe e neu aliza ion i e co esponds o he se um dilu ion a
which 50% inhibi ion o neu aliza ion is measu ed. Da a a e ep esen ed as he mean ±SEM o indi idual expe imen al eplica es assaying se a pooled om
abbi s wi hin each g oup and analyzed by a Mann-Whi ney es .
Cell Repo s 42, 112391, Ap il 25, 2023 5
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esponse o o eign an igens deli e ed on an ITV ia i s po en ial
o ecall helpe T cell esponses in popula ions p e iously immu-
nized agains TD.
24
To assess whe he p e-exis ing an i-TD im-
muni y could enhance he immunogenici y o TpD-con aining
ITVs, abbi s we e i s p e-immunized wi h ei he he licensed
Sano i Td Adso bed accine (Td) o PBS and subsequen ly
immunized i.m. wi h ei he ITV o ITV-TpD (Figu e 4C). Among
Td p e-immunized abbi s, signi ican ly highe an i-RBD IgG i-
e s we e obse ed in abbi s immunized wi h ITV-TpD
compa ed wi h hose immunized wi h ITV alone (Figu es 4D
and 4E). In e es ingly, an ibody esponses om bo h g oups
immunized wi h ITV-TpD also displayed highe a idi y han hose
immunized wi h ITV, sugges ing imp o ed quali y o he an ibody
esponse and p esumably e lec ing he a o able con ibu ion o
TpD o ge minal cen e dynamics and a ini y ma u a ion (Fig-
u e S3D). Fu he mo e, among abbi s immunized wi h ITV-
TpD, he highes induc ion o an igen-speci ic an ibodies
occu ed in hose p e iously immunized wi h Td accine, sug-
ges ing ha immuni y es ablished by Td p e-immuniza ion
con ibu ed o he enhancemen o ITV-TpD immunogenici y.
Addi ionally, Td-p e-immunized abbi s showed enhanced
neu aliza ion po ency ela i e o con ol abbi s, which was
pa icula ly appa en a e a single dose o ITV-TpD, whe e
highe se um an ibody i e s we e accompanied by a 3- old in-
c ease in neu aliza ion po ency as measu ed by sVNT
(Figu e 4F).
As an eme ging lead, we e alua ed he s abili y o pu i ied ITV-
TpD s o ed a h ee di e en empe a u es (20C, 4C, and
40C) o up o 3 weeks. ITV-TpD emained monodispe se and
e ained RBD s uc u al in eg i y and binding o MHC class II
Figu e 4. ITV-induced an i-RBD esponses a e enhanced by enginee ed T cell epi opes and p e-exis ing immuni y
(A) An i-RBD IgG i e s elici ed by unadju an ed ITV, ITV-TpD, o ITV-PADRE in immunized abbi s measu ed by ELISA (n = 5–10 abbi s/g oup). Da a a e
ep esen ed as he mean ±SEM o indi idual abbi measu emen s wi hin each g oup.
(B) Neu aliza ion po ency o accine-induced an ibody esponses measu ed by sVNT and pVNT, benchma ked o a p e-immunized se um sample o which
100 mg/mL o mAb REGN10987 was added. Da a a e ep esen ed as he mean ±SEM o a leas wo (sVNT) o h ee (pVNT) expe imen al eplica es.
(C) Immuniza ion schedule o e alua e he ole o p e-exis ing an i- e anus and diph he ia (TD) oxoid immuni y in enhancing ITV-TpD immunogenici y. Schema ic
c ea ed wi h BioRende .com.
(D–F) Quan i ica ion (D and E) and neu aliza ion po ency (F) o an i-RBD IgG i e s elici ed in abbi s a e one (D and F) o wo (E) doses o ITV/ITV-TpD. sVNT da a
a e ep esen ed as he mean ±SEM o expe imen al duplica es assaying se a pooled om abbi s wi hin each g oup and we e analyzed by K uskal Wallis
ollowed by Dunn’s mul iple compa isons es (*p < 0.05; ** <0.01).
See also Figu e S3.
6Cell Repo s 42, 112391, Ap il 25, 2023
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e en unde high he mal s ess o p olonged pe iods o ime
(Figu e S4), demons a ing he high he mos abili y o his mole-
cule as buil on he Ig sca old. Toge he , hese da a emphasize
he con ibu ion o TCEs o accine immunogenici y and he
obus ness o he IgG sca old in p o ein enginee ing and accine
de elopmen .
Adju an - ee ITV-TpD immuniza ion p o ec s e e s
om SARS-CoV-2 challenge
To assess he abili y o ITV-TpD immuniza ion o p o ec om
SARS-CoV-2 challenge, e e s we e immunized i.m. wi h
50 mg unadju an ed ITV-TpD o an equimola dose o Alum-ad-
ju an ed sRBD (sRBD-Alum) in a p ime-boos egimen and
we e subsequen ly challenged in anasally wi h 10
6
PFU li e
SARS-CoV-2 i us (Wuhan-Hu-1) a 2 weeks pos -boos (Fig-
u e 5A). An i-RBD an ibody i e s elici ed by immuniza ion we e
signi ican ly g ea e in e e s immunized wi h unadju an ed
ITV-TpD han sRBD-Alum, especially a e one dose o accine
(Figu e 5B). Co espondingly, se a om he unadju an ed ITV-
TpD g oup displayed g ea e i us neu aliza ion po ency han
ha o he sRBD-Alum g oup, as measu ed by plaque educ ion
neu aliza ion assay (Figu e 5C).
Measu emen s o i al i e s om nasal washes by qRT-PCR in
he 10 days ollowing challenge e ealed a ma ked educ ion in
SARS-CoV-2 i al i e s in bo h he unadju an ed ITV-TpD- and
sRBD-Alum-immunized g oups compa ed wi h he con ol PBS
g oup (Figu e 5D). These da a we e addi ionally con i med by
he quan i ica ion o i us isola ed om nasal washes o chal-
lenged animals, wi h lowe i al i e s measu ed in e e s immu-
nized wi h unadju an ed ITV-TpD and sRBD-Alum compa ed
wi h he con ol g oup (Figu e 5E). Pos -challenge moni o ing
o symp oms con i med he es ablishmen o SARS-CoV-2 in ec-
ion in con ol e e s, which de eloped signs o disease a ound
day 7–10 pos -challenge (Figu e 5F). While clinical signs o dis-
ease we e obse ed in some animals o he sRBD-Alum g oup,
all e e s in he unadju an ed ITV-TpD g oup emained heal hy
and esponsi e h oughou he du a ion o he s udy. Toge he ,
hese da a suppo he abili y o unadju an ed ITV-TpD o p o-
ec e e s om SARS-CoV-2-associa ed disease and elici
obus neu alizing an i-RBD esponses a le els su passing
Alum-adju an ed RBD.
Modula ITV design allows inclusion o mul iple RBDs o
achie e b oad sa beco i us neu aliza ion
To add ess he need o nex -gene a ion COVID-19 accines o
b oadly coun e con inuously eme ging VOCs, we assessed
he abili y o ITVs o simul aneously ca y RBD an igens om
wo dis inc sa beco i uses. Speci ically, he SARS-CoV-2 and
SARS-CoV-1 spike p o ein RBDs we e, espec i ely, used o
he c44H10 IgG hea y and ligh chains (Figu e 6A). Wi h consid-
e a ion o ou esul s on he impo ance o TCEs o ITV immuno-
genici y, TpD was also used o he C e minus o he SARS-CoV-
1 RBD on he ITV ligh chain. Bian igenic ITV-TpD mig a ion in
SDS-PAGE unde educing condi ions e ealed hea y- and
ligh -chain bands a 75 and 50 kDa, espec i ely, consis en
wi h he usion o an RBD o each an ibody chain (Figu e 6B). A
panel o non-c oss- eac i e mAbs was used in low cy ome y
expe imen s o assess he p ope olding o bo h RBD an igens
on he ITV-TpD. SARS-CoV-1 RBD-speci ic mAbs m396
and 80R
42,43
only displayed binding o bian igenic ITV-TpD,
whe eas SARS-CoV-2 RBD-speci ic mAbs REGN10987 and
REGN10933
31
bound o bo h mono- and bian igenic ITV-TpD
molecules (Figu e 6C), con i ming he abili y o he ITV pla o m
o success ully co-display di e se and con o ma ionally in ac
RBD an igens.
Rabbi s immunized wi h bian igenic ITV-TpD elici ed signi i-
can ly highe an i-SARS-CoV-1 RBD esponses han hose
immunized wi h mono-an igenic ITV-TpD (Figu e 6D). Impo -
an ly, he enhancemen in an i-SARS-CoV-1 esponses elici ed
by bian igenic ITV-TpD did no comp omise he de elopmen o
an i-SARS-CoV-2 an ibody esponses, which we e equi alen in
bo h mono- and bian igenic g oups. In e es ingly, mono-an i-
genic ITV-TpD elici ed a measu able an i-SARS-CoV-1 RBD
esponse despi e no ha ing SARS-CoV-1 RBD on he immu-
nogen, sugges ing he elici a ion o an ibodies c oss- eac i e
be ween bo h SARS-CoV-1 and SARS-CoV-2 RBDs. Roughly
equi alen le els o an i-SARS-CoV-1 and SARS-CoV-2 an i-
bodies we e elici ed by bian igenic ITV-TpD immuniza ion, sug-
ges ing ha bo h si es o RBD inco po a ion on he c44H10
IgG sca old (hea y chain and/o ligh chain) we e equally e ec-
i e o an igen immunogenici y. Bo h immunogens elici ed
obus neu alizing an ibody esponses agains wild- ype
SARS-CoV-2 and e ained neu aliza ion agains a wide ange
o VOCs, including he highly di e gen Omic on BA.1 and
BA.5 a ian s (Figu es 6E and S5B). In con as , signi ican ly
highe neu aliza ion agains SARS-CoV-1 was con e ed by
he inco po a ion o he SARS-CoV-1 RBD on he bian igenic
ITV-TpD (Figu es 6E and S5A). Collec i ely, hese da a suppo
he modula na u e o he ITV sca old as an e ec i e adju an -
ee accine pla o m o he deli e y and induc ion o obus
an ibody esponses agains di e se RBD an igens.
DISCUSSION
Immuno a ge ing as an app oach o accine design has p e i-
ously been explo ed in he con ex o di e en an igens and
a ge molecules on he su ace o a ious APC subse s.
13,44,45
Ta ge ing o MHC class II has been he mos widely e alua ed,
wi h se e al s udies ha ing epo ed he elici a ion o an igen-
speci ic humo al esponses enhanced ela i e o adminis a ion
o an igen alone.
12,14,46
In addi ion, an igen unlinked o bu co-
adminis e ed wi h MHC class II- a ge ing mAb ails o induce sig-
ni ican IgG an ibody esponses o he an igen.
11,15–17,47,48
In
mice, an igen linked o an iso ype-ma ched an ibody o i ele an
speci ici y does no elici an igen-speci ic an ibody esponses,
and ma ching he speci ici y o he a ge ing an ibody o he e-
cipien ’s MHC haplo ype was demons a ed o be essen ial o
esponsi eness,
11,49
a ibu ing he obse ed e ec o he a -
ge ing p ope y o he sca old an ibody used.
The ITV desc ibed he ein builds upon hese p inciples, elici ing
obus neu alizing an ibody esponses agains he SARS-CoV-2
RBD e en in he absence o adju an . In e es ingly, a ecen
epo desc ibed a accine candida e a ge ing he SARS-CoV-
2 spike p o ein RBD o MHC class II by use o an alpaca-de i ed
nanobody (VHH
MHCII
).
12
Howe e , he elici ed obus humo al
and cellula immuni y agains SARS-CoV-2 and i s a ian s
Cell Repo s 42, 112391, Ap il 25, 2023 7
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equi ed co-adminis a ion o poly(dI-dC) wi h an i-CD40. The
po en ial o an igen immuno a ge ing o a COVID-19 accine
has also been explo ed in he con ex o o he APC ecep o s.
In one ins ance, a accine a ge ing he SARS-CoV-2 spike p o-
ein RBD o CD40 molecules exp essed on APCs con e ed p o-
ec ion agains u he COVID-19 in ec ion in con alescen
macaques when adminis e ed wi hou adju an .
13
Fu he mo e,
in anasal unadju an ed spike p o ein boos ing in mice p e i-
Figu e 5. An ibody esponses elici ed by
ITV-TpD immuniza ion in e e s p o ec
agains SARS-CoV-2 challenge
(A) Schedule o e e immuniza ion, sampling, and
challenge (n = 6 e e s/g oup). Schema ic c ea ed
wi h BioRende .com.
(B) An i-RBD endpoin i e s elici ed by ITV-TpD o
sRBD-Alum immuniza ion p io o challenge as
measu ed by ELISA. As e isks indica e signi ican
di e ences be ween ITV-TpD and sRBD-Alum
g oups. Da a analyzed by K uskal-Wallis ollowed
by Dunn’s mul iple compa isons es (*p < 0.05;
**p < 0.01).
(C) Se um neu aliza ion i e s o li e SARS-CoV-2
i us measu ed by plaque educ ion neu aliza ion
assay a e one (D33) o wo (D47) accine doses.
Da a analyzed by Mann-Whi ney es (**p < 0.01).
(D and E) qRT-PCR de ec ion o i al RNA (D) and
i us isola ion o quan i y iable i us (E) om pos -
challenge nasal washes collec ed om e e s.
As e isks indica e signi ican di e ences be ween
ITV-TpD and PBS g oups. Da a analyzed by
K uskal-Wallis ollowed by Dunn’s mul iple com-
pa isons es (*p < 0.05; **p < 0.01).
(F) De elopmen o symp oms in e e s in he
14 days pos -challenge as assessed by a blind
obse e .
Fo all panels, da a a e ep esen ed as he mean ±
SEM o indi idual e e measu emen s wi hin
each g oup.
ously immunized wi h mRNA-LNP ac-
cine was ecen ly shown o be highly
e ec i e in gene a ing mucosal humo al
immune esponses.
50
Thus, he ad an-
ages o de eloping ITVs a e appa en
o he immuniza ion o bo h an igen-
expe ienced and an igen-nai e popula-
ions, emphasizing he po en ial o be
used in bo h p iming and boos ing sce-
na ios. A sys emic head- o-head com-
pa ison o a ious ITV modali ies a ge -
ing di e en APC ecep o s emains o
be explo ed o de e mine whe he one e-
cep o su passes he o he s o he elici-
a ion o neu alizing an ibody esponses
o whe he he simul aneous a ge ing o
mul iple ecep o s could syne gis ically
enhance his esponse.
Though i is one o he bes -cha ac e -
ized APC ecep o s o immuno a ge ing,
he ex eme polymo phism o he MHC
locus ep esen s a subs an ial challenge o he de elopmen
o an ITV ha is b oadly eac i e in he human popula ion.
He e, ou molecula cha ac e iza ion o mAb 44H10 builds on
p io li e a u e es ablishing i as a pan-HLA-DR mAb
20
and p o-
ides impo an insigh s in o he abili y o 44H10 o o e come
HLA-DR di e si y h ough a ge ing a highly conse ed epi ope,
u he suppo ing he use o his an ibody o immuno a ge ing
ac oss di e se human popula ions. In ac , hese molecula
8Cell Repo s 42, 112391, Ap il 25, 2023
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT o RESOURCE SOURCE IDENTIFIER
An ibodies
Alexa Fluo 488 A iniPu e Goa An i-Human IgG,
Fcg agmen speci ic
Jackson ImmunoResea ch Ca #109-545-098; RRID: AB_2337840
Goa An i-Rabbi IgG H&L (HRP) Abcam Ca #ab97051; RRID: AB_10679369
Goa An i-Fe e IgG H&L (HRP) Abcam Ca #ab112770
RRID: AB_10862402
Bac e ial and i us s ains
SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) Vaccine and In ec ious
Disease O ganiza ion (VIDO)
N/A
Biological samples
Human PBMCs Canadian Blood Se ices (CBS) N/A
Chemicals, pep ides, and ecombinan p o eins
SARS-CoV-2 spike p o ein RBD Flo ian K amme , Icahn School
o Medicine a Moun Sinai
N/A
SARS-CoV Spike/RBD P o ein (RBD, His Tag) Sino Biological Ca #40150-V08B2
Human ACE2 (hACE2) Rujas e al.
70
N/A
c44H10 IgG This pape N/A
c44H10 Fab This pape N/A
ITV This pape N/A
ITV-TpD This pape N/A
ITV-PADRE This pape N/A
ITV-TpD (Bi-An igenic) This pape N/A
HLA-DR (A*01:01 / B1*04:01) This pape N/A
HLA-DR (A*01:01+W168R / B1*04:01) This pape N/A
HLA-DR (A*01:01 / B1*04:01+F31I/H33N) This pape N/A
HLA-DR (A*01:01 / B1*04:01+F31V/H33N) This pape N/A
HLA-DR (A*01:01+W168R / B1*04:01+F31I/H33N) This pape N/A
HLA-DR (A*01:01+W168R / B1*04:01+F31V/H33N) This pape N/A
SARS-CoV-2 Spike p o ein (RBD, A i & His Tag)-HRP Gensc ip Ca #Z03594
GIBCO
TM
F eeS yle
TM
293 Exp ession Medium The mo Fishe Scien i ic Ca #12338026
Fec oPRO DNA T ans ec ion Reagen VWR Ca #10118-444
BioT ans ec ion eagen Bioland Scien i ic Ca #B01-01
b i eli e plus Repo e Gene Assay Sys em Pe kin Elme Ca #6066769
Td ADSORBED Sano i Pas eu , VaccineShoppeCanada Ca #482229
Alhyd ogel(alum) adju an 2% In i ogen Ca # ac-alu-50
Alexa Fluo
TM
488 C
5
Maleimide The mo Fishe Scien i ic Ca #A10254
T iPu e
TM
Isola ion Reagen Sigma Ald ich Ca #11667157001
U anyl Fo ma e The mo Fishe Scien i ic Ca #50-189-8757
C i ical comme cial assays
Oc e P o ein A (P oA) Biosenso s Sa o ius Ca #18-5010
Oc e An i-Pen a-HIS (HIS1K) Biosenso s Sa o ius Ca #18-5120
4X TaqMan Fas Vi us one s ep RT-PCR ki The mo Fishe Scien i ic Ca # 4444434
gBlock In eg a ed DNA Technologies Cus om
(Con inued on nex page)
Cell Repo s 42, 112391, Ap il 25, 2023 15
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Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
Deposi ed da a
C ys al s uc u e o c44H10 Fab-MHC class II complex This pape PDB: 8EUQ
Expe imen al models: Cell lines
F eeS yle
TM
293-F Cells The mo Fishe Scien i ic Ca #R79007
Expi293F GnTI
-/-
Cells The mo Fishe Scien i ic Ca #A39240
BJAB Cells DSMZ Ca #ACC757
HEK293T Cells ATCC Ca #CRL-3216
HEK293T-ACE2 Cells BEI Resou ces Ca #NR52511
Ve o E6 Cells ATCC Ca #CRL-1586
Expe imen al models: O ganisms/s ains
New Zealand Whi e Rabbi s Ceda lane Labo a o ies N/A
Eu opean Fe e s Ma shall Bio esou ces N/A
Recombinan DNA
pCAGGS-SARS-CoV-2-RBD_His Flo ian K amme , Icahn School
o Medicine a Moun Sinai
N/A
pcDNA3.4-hACE2_His Rujas e al.
70
N/A
pcDNA3.4-ITV-HC-CoV2RBD This pape N/A
pcDNA3.4-ITV-KC This pape N/A
pcDNA3.4-ITV-KC-TpD This pape N/A
pcDNA3.4-ITV-KC-PADRE This pape N/A
pcDNA3.4-ITV-KC-CoV1RBD-TpD This pape N/A
pcDNA3.4-HLA-DR-A*01:01_His This pape N/A
pcDNA3.4-HLA-DR-A*01:01-W168R_His This pape N/A
pcDNA3.4-HLA-DR-B*04:01_His This pape N/A
pcDNA3.4-HLA-DR-B*04:01- F31I/H33N_His This pape N/A
pcDNA3.4-HLA-DR-B*04:01- F31V/H33N_His This pape N/A
pcDNA3.4-REGN10933-IgG-HC Hansen e al.
31
N/A
pcDNA3.4-REGN10933-KC Hansen e al.
31
N/A
pcDNA3.4-REGN10987-IgG-HC Hansen e al.
31
N/A
pcDNA3.4-REGN10987-KC Hansen e al.
31
N/A
pcDNA3.4-COV2-2196-IgG-HC Zos e al.
30
N/A
pcDNA3.4-COV2-2196-KC Zos e al.
30
N/A
pcDNA3.4-CR3022-IgG-HC Yuan e al.
29
N/A
pcDNA3.4-CR3022-KC Yuan e al.
29
N/A
pcDNA3.4-m396-IgG-HC P abaka an e al.
42
N/A
pcDNA3.4-m396-KC P abaka an e al.
42
N/A
pcDNA3.4-80R-IgG-HC Hwang e al.
43
N/A
pcDNA3.4-80R-KC Hwang e al.
43
N/A
pCAGGS-SARS-CoV-2-Wuhan-Hu-1
Spike Glycop o ein Gene
BEI Resou ces Ca #NR52310
Len i i al backbone wi h Luc2;
ZsG een inse
BEI Resou ces Ca #NR52516
Helpe plasmid wi h Ta 1b inse BEI Resou ces Ca #NR52518
Helpe plasmid wi h Gag; pol inse BEI Resou ces Ca #NR52517
Helpe plasmid wi h Re 1b inse BEI Resou ces Ca #NR52519
SARS-CoV-2-Wuhan-Hu-1 Spike
Glycop o ein Gene, Be a a ian
Da id Ho, Columbia Uni e si y N/A
SARS-CoV-2-Wuhan-Hu-1 Spike
Glycop o ein Gene, Gamma a ian
Da id Ho, Columbia Uni e si y N/A
(Con inued on nex page)
16 Cell Repo s 42, 112391, Ap il 25, 2023
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RESOURCE AVAILABILITY
Lead con ac
Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he Lead Con ac , Jean-
Philippe Julien ([email p o ec ed]).
Ma e ials a ailabili y
All unique and s able eagen s gene a ed in his s udy a e a ailable ia he lead con ac upon a easonable eques .
Da a and code a ailabili y
dThe c ys al s uc u e has been deposi ed o he P o ein Da a Bank and is publicly a ailable as o he da e o publica ion. Acces-
sion numbe s a e lis ed in he key esou ces able.
dThis pape does no epo o iginal code.
dAny addi ional in o ma ion equi ed o eanalyze he da a epo ed in his pape is a ailable om he lead con ac upon eques .
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mammalian cell lines and cul u e condi ions
Female mammalian cells (F eeS yle
TM
293-F cells, The mo Fishe Scien i ic; HEK 293S, GnT I
-/-
cells, ATCC) we e cul u ed in sus-
pension in GIBCO
TM
F eeS yle
TM
293 Exp ession Medium (The mo Fishe Scien i ic) a 37C in a Mul i on P o Shake (In o s HT)
wi h 70% humidi y, 8% CO
2
and o a ing a 130 pm. B lymphoblas oid BJAB cells (DSMZ)
77
we e g own in RPMI medium supple-
men ed wi h 10% e al bo ine se um (FBS) and 1% penicillin/s ep omycin (all om The mo Scien i ic) in a 37C, 5% CO
2
incuba o .
Fo pseudo i us neu aliza ion assays, HEK293T cells (ATCC) and HEK293T-ACE2 cells (BEI NR52511) we e cul u ed in DMEM me-
dia supplemen ed wi h 10% hea -inac i a ed FBS (Gibco), 2.5% HEPES (Gibco) and 0.5% gen amicin (The moFishe ) in a 37C, 5%
CO
2
incuba o . Ve o E6 cells (ATCC) we e main ained in a humidi ied incuba o a 37C, 5% CO
2
in DMEM (Co ning) supplemen ed
wi h 10% FBS.
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
SARS-CoV-2-Wuhan-Hu-1 Spike
Glycop o ein Gene, Del a a ian
Da id Ho, Columbia Uni e si y N/A
SARS-CoV-2-Wuhan-Hu-1 Spike
Glycop o ein Gene, Omic on BA.1 a ian
Dennis Bu on, Sc ipps Resea ch Ins i u e N/A
SARS-CoV-2-Wuhan-Hu-1 Spike
Glycop o ein Gene, Omic on BA.5 a ian
GISAID Accession #EPI_ISL_11542604
So wa e and algo i hms
Relion Sche es
71
h ps:// elion. ead hedocs.io/en/
elease-3.1/Ins alla ion.h ml
FlowJo FlowJo, LLC h ps://www. lowjo.com/solu ions/
lowjo/downloads
SBG id Mo in a al.
72
h ps://sbg id.o g/so wa e/
i les/sbg id-ins alle
Phenix Adams e al.
73
h p://www.phenix-online.o g/
XDS Kabsch
74
h ps://xds.m .mpg.de/h ml_doc/
downloading.h ml
Coo Emsley e al.
75
h ps://www2.m c-lmb.cam.ac.uk/
pe sonal/pemsley/coo /
PRISM G aphpad G aphPad So wa e, LLC h ps://www.g aphpad.com/
scien i ic-so wa e/p ism/
O he
PDBePisa se e K issinel and Hen ick
33
h ps://www.ebi.ac.uk/pdbe/pisa/
IPD-IMGT/HLA da abase Robinson e al.
34
h ps://www.ebi.ac.uk/ipd/img /hla/
Homemade ca bon g ids Boo h e al.
76
N/A
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Rabbi s
New Zealand Whi e Rabbi s we e b ed in e nally and housed indi idually o in pai s a Ceda lane Labo a o ies’ egis e ed acili y (Bu -
ling on, On a io, Canada). Immuniza ion s udies we e conduc ed on emale abbi s aged app oxima ely 3 mon hs. All p ocedu es
we e conduc ed in acco dance wi h Ceda lane’s Animal Ca e Commi ee and s anda d ope a ing p o ocols we e e iewed and
app o ed by he acili y’s go e ning body.
Fe e s
Eu opean e e s (Mus ela u o) we e o de ed om Ma shall Fa ms (New Yo k) and anspo ed o he Canadian Science Cen e o
Human and Animal Heal h (CSCHAH) in a clima e-con olled ehicle. Each expe imen al g oup was composed o 3 male and 3 emale
e e s aged app oxima ely 6 mon hs. On a i al, he animals we e housed in caging uni s in he BSL3 Ag acili y a he Canadian Food
Inspec ion Agency – Na ional Cen e o Fo eign Animal Disease (CFIA-NCFAD). Animals we e acclima ized o 7 days p io o he
s a o he expe imen and we e checked daily by ained animal ca e s a . Animals we e obse ed du ing acclima ion, con ales-
cence and all s ages o in ec ion. Fe e expe imen s we e conduc ed unde he app o al o he CSCHAH Animal Ca e Commi ee
which ollows he guidelines o he Canadian Council on Animal Ca e.
Human PBMCs
PBMC samples we e om heal hy dono s p o ided as EDTA whole blood by he Canadian Blood Se ices. All dono s p o ided
in o med consen o use o dona ions o esea ch pu poses p io o dona ion.
METHOD DETAILS
Plasmid design and syn hesis
DNA plasmids o he exp ession o all p o eins desc ibed in his wo k we e designed in he pcDNA3.4 TOPO mammalian exp ession
ec o and op imized o Homo sapiens exp ession a GeneA (In i ogen). These cons uc s we e maxip epped using Pu eLink
HiPu e Plasmid Maxip ep Ki s (In i ogen).
Exp ession and pu i ica ion o ecombinan ITVs and an i-RBD mAbs
F eeS yle 293-F cells we e spli o a densi y o 0.8 x 10
6
cells/mL a leas one hou be o e ans ec ion. Cells we e ans ec ed using
Fec oPRO Reagen (Polyplus) ollowing manu ac u e ins uc ions a a 1:1 DNA o Fec oPRO a io. 90 mg o plasmid DNA was used
o ans ec ion (2:1 a io o hea y and ligh chain DNA plasmids) o e e y 200 mL o cell cul u e. T ans ec ed cells we e incuba ed in a
37C, 5% CO
2
shaking incuba o o 5 o 7 days o allow o he exp ession and pai ing o hea y and ligh chain gene p oduc s. T ans-
ec ed cell cul u e supe na an s we e collec ed and il e ed h ough 0.22 mM S e i op il e s (Millipo e Sigma) be o e loading on o p o-
ein A a ini y columns using he A
¨KTA s a p o ein pu i ica ion sys em (Cy i a Li e Sciences). Following loading, samples we e
washed wi h 1X phospha e-bu e ed saline (PBS) hen elu ed wi h 100 mM glycine, pH 2.2 and immedia ely neu alized wi h 1 M
T is, pH 9.0. Elu ion ac ions we e concen a ed using Amicon 30K Ul a-0.5 mL Cen i ugal Fil e s (Millipo e Sigma) and bu e -
exchanged in o PBS wi h PD-10 desal ing columns (Cy i a). All pu i ied p o eins we e alida ed o in eg i y and pu i y ia sodium
dodecyl sul a e polyac ylamide gel elec opho esis (SDS-PAGE) and s o ed a -80C un il use.
Exp ession and pu i ica ion o ecombinan c44H10 Fab
c44H10 Fab hea y and ligh chain plasmids we e ans ec ed in o F eeS yle 293-F cells as desc ibed abo e. Recombinan c44H10
Fab was pu i ied by KappaSelec a ini y ch oma og aphy wi h 100 mM glycine, pH 2.2 elu ion and immedia e 1 M T is, pH 9.0
neu aliza ion, ollowed by MonoS ion exchange ch oma og aphy using 20 mM NaOAc, pH 5.6 ±1M KCl, and size exclusion ch o-
ma og aphy on a Supe dex 200 Inc ease 10/300 GL in 20 mM T is, pH 8.0, 150 mM NaCl (TBS) (Cy i a).
Exp ession and pu i ica ion o ecombinan SARS-CoV-2 RBD and hACE2
F eeS yle 293-F cells we e ans ec ed as desc ibed abo e wi h 50 mg o plasmid DNA encoding he SARS-CoV-2 RBD o human
ACE2 (hACE2) pe 200 mL o cell cul u e. Recombinan p o eins we e pu i ied by a ini y ch oma og aphy ia a HisT ap Ni-NTA col-
umn (Cy i a) and elu ed using 20 mM T is pH 8.0, 500 mM imidazole bu e . Subsequen size exclusion ch oma og aphy was pe -
o med in 20 mM T is, pH 8.0, 150 mM NaCl on a Supe dex 200 Inc ease column (Cy i a).
Design, exp ession and pu i ica ion o ecombinan MHC Class II
The ex acellula domains o MHC Class II (DRA*01:01/DRB1*04:01) aand bchains we e designed in pcDNA3.4 TOPO, wi h bo h
chains con aining C- e minal Tobacco E ch Vi us (TEV)-clea able Fos/Jun zippe s o p omo e dime iza ion and 6xHis ags o pu i-
ica ion pu poses.
78
The MHC Class II molecule was exp essed wi h he In luenza Hemagglu inin (HA) pep ide co alen ly a ached ia
a lexible linke o he N e minus o he bchain o p omo e p ope olding o he a/bdime . HLA-DR aand bchain plasmids we e co-
ans ec ed in a 1:1 a io (50 mg o al pe 200 mL o cell cul u e) in o HEK 293S (GnT I
-/-
) cells. Recombinan MHC Class II was pu i ied
by a ini y ch oma og aphy ia a HisT ap Ni-NTA column (Cy i a) and elu ed using 20 mM T is, pH 8.0, 500 mM imidazole. Subse-
quen size exclusion ch oma og aphy was pe o med in TBS, pH 8.0 on a Supe dex 200 Inc ease 10/300 GL (Cy i a). Pu i ied
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MHC Class II was subjec ed o o e nigh ea men wi h EndoH and TEV p o eases a a ios o 5:1 and 20:1, espec i ely, o degly-
cosyla ion and clea age o he Fos/Jun zippe . A second ound o a ini y and size exclusion pu i ica ions was pe o med on clea ed
MHC Class II be o e complexa ion wi h c44H10 Fab o c ys alliza ion ials.
Dynamic Ligh Sca e ing o RBD, c44H10 IgG and ITV
Dynamic ligh sca e ing (DLS) analysis was pe o med using a DynaP o Pla e Reade III (Wya Technology). 20 mL o each p o ein a
1 mg/mL we e added o a 384-well black, clea bo om pla e (Co ning) and measu ed a a ixed empe a u e o 25 C wi h a du a ion
o 5 s pe ead. Pa icle hyd odynamic adii (Rh) and polydispe si y (% Pd) we e ob ained om he accumula ion o en eads om
duplica e samples using he Dynamics so wa e (Wya Technology).
Nega i e-s ain elec on mic oscopy
Pu i ied ITV p o ein was dilu ed o 20 mg/mL, applied on o homemade ca bon ilm-coa ed g ids
76
(p e iously glow-discha ged in ai
o 15 s) and s ained wi h 2% u anyl o ma e. G ids we e imaged wi h a Hi achi HT7800 TEM ope a ing a 120 kV, wi h a calib a ed
pixel size o 1.83 A
˚/pix. Pa icle selec ion, ex ac ion and 2D classi ica ion we e pe o med wi h Relion 3.1.
71
Fluo escen labeling o an i-RBD mAbs o low cy ome y
Pu i ied an i-RBD mAbs we e dilu ed o 100 mM in PBS and incuba ed in a 10- old mola excess o TCEP a oom empe a u e o
30 min. Alexa Fluo 488 (AF488) C
5
Maleimide dye (In i ogen) was added o each eac ion a a concen a ion o 10 mM. Samples
we e incuba ed o e nigh a 4C p o ec ed om ligh . F ee, unconjuga ed dye was washed ou o solu ion using PBS and Amicon
30K Ul a-0.5 mL Cen i ugal Fil e s (Millipo e Sigma). The concen a ion o labeled p o ein was assessed by Nanod op measu emen
a 280 nm.
Flow cy ome y o con i ma ion o MHC class II- a ge ing and RBD s uc u al in eg i y
BJAB cells we e collec ed in a conical ube and cen i uged a 300 g o 5 min. Cell pelle s we e esuspended in s aining bu e (PBS,
2% FBS, 0.05% NaN
3
)a 1x10
6
cells/mL, and 200 ml o he cell suspension was dispensed in o he wells o a polys y ene, V-bo om
96-well pla e (G eine Bio-One) o s aining. Cells we e cen i uged a 300 g o 5 min, and hen incuba ed in Fc Block (BD Biosci-
ences) o 10 min a oom empe a u e. Pu i ied ITV was hen added o he cells a 10 mg/mL and le o incuba e o 1 h a 4C.
Fo he con i ma ion o ITV binding o MHC Class II, cells we e washed wice, hen s ained wi h AF488 A iniPu e Goa An i-
Human IgG, Fcg agmen speci ic (1:1,000, Jackson ImmunoResea ch) o 30 min a 4C. Fo he p obing o RBD s uc u al in eg i y,
AF488 p e-labeled an i-RBD mAbs (REGN10987, REGN10933, m396, 80R) we e used as seconda y a 10 mg/mL in lieu o an i-IgG
seconda y. A e wo addi ional washes, cells we e esuspended in p opidium iodide (1:100, The mo Scien i ic) o he exclusion o
dead cells and deb is. An iso ype-ma ched an ibody se ed as a nega i e con ol. Samples we e acqui ed on BD LSR II o LSR Fo -
essa cell analyze s using he BD FACSDi a
TM
So wa e, and u he analyzed using he FlowJo
TM
So wa e.
Flow cy ome y o 44H10 binding o human PBMCs
Pe iphe al blood mononuclea cells (PBMC) cells we e isola ed by densi y cen i uga ion using Ficoll-Hypaque solu ion (GE Heal h-
ca e). PBMC we e esuspended in s aining bu e (PBS, 2% FCS, 1mM EDTA) and Fc ecep o s we e blocked wi h Human T uS ain
FcX
TM
(Biolegend) acco ding o he manu ac u e ’s ins uc ions. PBMCs we e hen s ained o 1 h a 4C wi h 50 mL o 0.1 mg/mL
AF488 p e-labeled c44H10 IgG. AF488-conjuga ed Human IgG1 was used as iso ype con ol. Samples we e acqui ed on a Sony
SP6800 Spec al Analyze and p ocessed using he FlowJo
TM
So wa e.
Biolaye in e e ome y o measu emen o ecombinan MHC class II-c44H10/ITV binding
Real- ime analysis o binding kine ics was measu ed using he Oc e RED96 BLI sys em (Sa o ius). Baseline, associa ion, and disso-
cia ion s eps we e conduc ed a 25C o 180 s in kine ics bu e (PBS, pH 7.4, 0.01% BSA, 0.002% Tween). Recombinan MHC Class
II was loaded on o Pen a-His Biosenso s (Fo e
´Bio) a 10 mg/mL un il a h eshold esponse o 0.7 nm. Associa ion e en s we e
measu ed by dipping loaded biosenso s in o wells con aining a wo- old se ial dilu ion o c44H10 IgG o ITV a a 250 nM s a ing con-
cen a ion. Dissocia ion was measu ed by ans e o biosenso s back in o bu e -con aining wells. Biosenso s we e egene a ed in
10 mM glycine, pH 1.5. Kine ics da a we e analyzed using he Fo e
´Bio Oc e Da a Analysis so wa e 9.0.0.6, and cu es we e i ed o
a 1:1 binding model o calcula ion o K
D
,K
on
and K
o
.
Biolaye in e e ome y o measu emen o ITV binding o hACE2
Pu i ied ITV was loaded on o P o ein A biosenso s (Fo e
´Bio) a 10 mg/mL un il a h eshold esponse o 0.7 nm, and associa ion e en s
we e measu ed by dipping loaded biosenso s in o wells con aining a wo- old se ial dilu ion o hACE2 a a 500 nM s a ing concen-
a ion. Dissocia ion was measu ed by ans e o biosenso s back in o bu e -con aining wells. Biosenso s we e egene a ed in
100 mM glycine, pH 2.2. Kine ics da a we e analyzed using he Fo e
´Bio Oc e Da a Analysis so wa e 9.0.0.6, and cu es we e i ed
o a 1:1 binding model o calcula ion o K
D
,K
on
and K
o
.
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Co-c ys alliza ion and s uc u e de e mina ion o he c44H10 Fab-MHC class II complex
c44H10 Fab was mixed wi h ecombinan MHC Class II in a 1.5-mola excess, and excess Fab was pu i ied away ia size exclusion
ch oma og aphy (Supe dex 200 Inc ease 10/300 GL, Cy i a). The p o ein complex was concen a ed o 8 mg/mL and mixed wi h a
mo he liquo o 1.7 M ammonium sul a e, 15% glyce ol, 0.085 M HEPES, 1.7% PEG400, as well as c ys al seeds p e iously ob ained
in a condi ion o 2 M ammonium sul a e and 0.1 M bis- is pH 5.5, in a a io o 2:1:3 (p o ein:seed:mo he liquo ). C ys als appea ed
a e 120 days and g ew s eadily un il day 160, a which ime hey we e c yop o ec ed in 15% ( / ) e hylene glycol be o e being
lash- ozen in liquid ni ogen. Da a we e collec ed a he 23-ID-D beamline a he A gonne Na ional Labo a o y Ad anced Pho on
Sou ce. Da ase s we e p ocessed, me ged and scaled using XDS and Xp ep.
74
The s uc u e was de e mined by molecula eplace-
men using Phase .
79
Re inemen o he s uc u e was pe o med using phenix. e ine
73
and Coo .
75
Access o all so wa e was sup-
po ed h ough SBG id.
72
Sequences o known HLA-DRA and HLA-DRB1 alleles we e aligned using he IPD-IMGT/HLA da abase
sequence alignmen ool,
34
and in e ac ions we e analyzed using he PDBePisa se e .
33
Endo oxin measu emen s and emo al o in i o s udies
Endo oxin le els in RBD, ITV, ITV-TpD o ITV-PADRE p o ein samples we e measu ed using he EndoSa e Nexgen-PTS Sys em
(Cha les Ri e ). The h eshold o samples sui able o immuniza ion was < 5 EU/mL. When equi ed, endo oxin emo al was pe -
o med using a ToxinE ase endo oxin emo al ki (GenSc ip ) pe he manu ac u e ’s ins uc ions and e es ed un il endo oxin le els
measu ed we e below he h eshold alue.
Rabbi immuniza ions
Female New Zealand whi e abbi s housed a Ceda lane Labo a o ies we e immunized a day 0 ia SQ o IM injec ion wi h un-
adju an ed RBD, ITV, ITV-TpD o ITV-PADRE, ollowed by a boos a day 35. Fo he s udy assessing he ole o exis ing an i-TD
immuni y in ITV-TpD immunogenici y, abbi s we e in amuscula ly immunized 28 days be o e he ITV/ITV-TpD p ime wi h ¼ o
he human dose o Td Adso bed accine (Sano iª), o PBS. Fo se um p epa a ion, blood was collec ed in o ed op acu aine
ubes and incuba ed a 3-4 h a oom empe a u e o allow o clo ing. The ubes we e cen i uged a 4 C a 3000 pm o
20 min, and supe na an s we e pou ed o in o app op ia e ubes and s o ed a -20C be o e shipping on d y ice.
ELISA measu emen o an i-RBD i e s elici ed in abbi s
Immulon 4 HBX ELISA pla es (The mo Scien i ic) we e coa ed o e nigh a 4C wi h 100 ng/well SARS-CoV-2 spike RBD (p o-
duced in-house) o SARS-CoV-1 spike RBD (Sino Biological). All subsequen s eps we e conduc ed a oom empe a u e.
Pla es we e washed h ee imes wi h PBS-T (PBS, 0.1% Tween), hen incuba ed wi h blocking bu e (PBS-T, 3% non- a
milk) o 1 h. The blocking solu ion was disca ded and 100 mL o abbi se a p e-dilu ed in diluen bu e (PBS-T, 1% milk)
and s anda d ( abbi an i-SARS-CoV-2 spike RBD polyclonal an ibody, Ceda lane) we e added o he ELISA pla es. A e a
2 h incuba ion, pla es we e washed h ee imes wi h PBS-T and incuba ed wi h Goa An i-Rabbi IgG H&L (HRP) seconda y
an ibody (1:10,000, Abcam) o 1 h. Pla es we e once mo e washed h ee imes, hen de eloped using a TMB Subs a e Re-
agen Se (BD) ollowing manu ac u e ins uc ions; eac ions we e s opped a 5 min by he addi ion o 2 N HCl. Abso bance
eadings a 450 nm we e acqui ed using a Syne gy Neo2 Mul i-Mode Assay Mic opla e Reade (Bio ek Ins umen s). Da a
we e plo ed in P ism 9.3.1 (G aphPad) and an ibody concen a ion was ex apola ed om abso bance using ou -pa ame e
logis ic (4PL) eg ession o log- ans o med alues.
ELISA measu emen o an ibody a idi y index
ELISAs we e pe o med as desc ibed abo e, wi h an added 15 min incuba ion wi h 1.5 M NaSCN ollowing he p ima y incuba ion
wi h abbi se um. A idi y index was de ined as he pe cen age o an ibodies in se um ha emain bound o he RBD-coa ed pla e
a e chao ope ea men , and was calcula ed using he ollowing o mula:
A idi y Index =Ti e wi h 1:5M NaSCN ea men
Ti e wi hou NaSCN ea men 3100%
Su oga e i us neu aliza ion assay (sVNT)
Immulon 4 HBX ELISA pla es (The mo Scien i ic) we e coa ed o e nigh a 4C wi h 200 ng/well ecombinan hACE2, ollowed by
blocking wi h 3% BSA in PBS-T o 1 h a oom empe a u e. To simula e i al neu aliza ion, a 1:500 dilu ion o RBD-HRP
(GenSc ip ) was p e-incuba ed wi h se ially dilu ed se um samples o 1 h a 37C, subsequen ly added o blocked pla es, and u he
incuba ed o 1 h a oom empe a u e. Pla es we e washed h ee imes wi h PBS-T p io o colo ime ic de elopmen wi h TMB o
15 min. Abso bance da a a 450 nm we e con e ed o % inhibi ion using he ollowing o mula:
%Inhibi ion =1ðODsample ODminÞ
ðODmax ODminÞ3100%
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Pseudo i us p oduc ion
Pseudo i us p oduc ion was conduc ed as p e iously desc ibed.
70
B ie ly, 293T cells we e co- ans ec ed wi h a len i i al backbone
encoding he luci e ase epo e gene (BEI NR52516), a plasmid exp essing he SARS-CoV-2 Spike (BEI NR52310) and plasmids en-
coding he HIV s uc u al and egula o y p o eins Ta (BEI NR52518), Gag-pol (BEI NR52517) and Re (BEI NR52519). Co- ans ec ion
o he i e plasmids was pe o med using BioT eagen (Bioland Scien i ics) ollowing manu ac u e ins uc ions. 24 h pos - ans ec-
ion a 37C, he media was supplemen ed wi h 5 mM sodium bu y a e (NaB) and he cells we e incuba ed o an addi ional 24 h a
30C p io o pseudo i us (PsV) ha es ing. Be a (B.1.351), Gamma (P.1), and Del a (B.1.617.2) SARS-CoV-2 PsV a ian s we e
gene a ed by subs i u ing he wild- ype spike plasmid wi h he espec i e VOC spike using plasmids kindly p o ided by Da id Ho
(Columbia). Omic on (BA.1) a ian PsV was gene a ed using a spike plasmid kindly p o ided by Dennis Bu on (The Sc ipps
Resea ch Ins i u e), and Omic on (BA.5) a ian PsV was gene a ed using a spike plasmid syn hesized a GeneA (In i ogen).
PsV we e ha es ed, il e ed h ough 0.45 mm s e ile il e s, and concen a ed using 100 K Amicon il e s (Millipo e Sigma).
Pseudo i us neu aliza ion assay (pVNT)
Neu aliza ion assays we e pe o med using 293T-ACE2 cells (BEI NR52511) as p e iously desc ibed
38
wi h ew modi ica ions.
70
B ie ly,
abbi se a we e inac i a ed o 30 min a 56C. Se ial dilu ions o he inac i a ed se a we e incuba ed o 1 h a 37Cwi hSARS-CoV-2
PsV and subsequen ly added o 293T-ACE2 cells (BEI NR52511) seeded in Poly-L-lysine (Sigma-Ald ich) coa ed pla es 24 h p io o he
expe imen . A inal concen a ion o 5 mg/ml polyb ene (Sigma-Ald ich) was added o he PsV-se a mix u es. A e 48 h o incuba ion a
37C, neu aliza ion was moni o ed by adding 50 ml o B i eli e plus eagen (Pe kinElme ) o 50 ml o cells o 2 min. Supe na an s we e
ans e ed o a 96-well whi e pla e (Sigma-Ald ich) o measu e luminescence in ela i e ligh uni s (RLUs) using a Syne gy Neo2 Mul i-
Mode Assay Mic opla e Reade (Bio ek Ins umen s). Abso bance da a we e con e ed o % inhibi ion using he same o mula as used
in he sVNT. Plo ed da a a e he a e age o a leas h ee eplica e measu emen s. Da a we e i ed using 4PL eg ession cons ained a
op = 100% and bo om = 0% in G aphPad P ism 9.3.1 o de e mina ion o neu aliza ion i e s.
Fe e immuniza ions and challenge
Fe e s we e immunized in amuscula ly wi h 50 mg o unadju an ed ITV-TpD o an RBD-equimola dose o Alum-adju an ed RBD on
day 0 and day 35. Un accina ed e e s we e immunized wi h an equal olume o s e ile PBS. Each g oup o six animals consis ed o
h ee males and h ee emales. SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) (VIDO) was g own and main ained in Ve oE6
cells a CFIA-NCFAD Le el 3 (Zoono ic). Fe e s we e challenged on day 49 wi h 10
6
p u SARS-CoV-2 (Wuhan-Hu-1) in anasally
(50 mL pe nos il, 100 mL o al). Blood samples we e collec ed in BD se um-sepa a o mic o aine ubes (BD 365967) excep on
day 14 pos -challenge, whe e blood was collec ed in 5 mL BD acu aine se um-sepa a o ubes (BD 367989) upon eu hanasia.
Se um sepa a ed om blood and nasal washes pe o med using 1 mL PBS we e s o ed in 2 mL sc ew cap mic o ubes (Sa s ed )
a -80C un il use.
ELISA measu emen o an i-RBD i e s elici ed in e e s
96-well la bo om pla es(Nunc) (The mo Scien i ic) we e coa ed wi h 50 ng/well o RBD in 0.05 M ca bona e-bica bona e bu e (Sigma-
Ald ich) o e nigh a 4C. Pla es we e washed 5 imeswi h0.01 M PBS-T and blocked wi h1X Casein Blocking Bu e (Sigma-Ald ich) o
1 h shaking a 37C. Pla es we e washed and dilu ions o se um samples we e p epa ed in casein blocking bu e and incuba ed o 1 h
shaking a 37C. Pla es we e washed and incuba ed wi h goa an i- e e IgG-HRP (1:10,000, Abcam) o 1 h shaking a 37C. Pla es we e
washed and de eloped wi h TMB (The moFishe Scien i ic) ollowing manu ac u e ins uc ions; eac ions we e s opped by he addi ion
o S op Solu ion 0.16 M sulphu ic acid (The moFishe Scien i ic). Abso bance o he pla es was ead a 450 nm.
Plaque educ ion neu aliza ion i e (PRNT) assay
Se um samples we e hea -inac i a ed a 56C o 30 min. Two- old se ial dilu ions o inac i a ed se a we e incuba ed wi h 100 p u o
SARS-CoV-2 i us o 1 h a 37C. Each i us-se um mix u e was hen added o wells o > 90% con luen Ve o E6 cells in a 48-well
o ma , incuba ed o 1 h a 37Cin5%CO
2
, hen o e laid wi h 500 mL o 2% ca boxyme hyl cellulose (Sigma) in supplemen ed
DMEM (Co ning) pe well. Pla es we e incuba ed a 37C o 72 h, ixed wi h 10% bu e ed o malin and s ained wi h 0.5% c ys al
iole . Se um dilu ions esul ing in > 70% educ ion o plaque coun s compa ed o i us-only con ols we e conside ed posi i e
o i us neu aliza ion.
RNA ex ac ion om nasal washes
To al RNA ex ac ion om nasal washes was conduc ed using he MagMAX CORE Nucleic Acid Pu i ica ion Ki (The moFishe ) on he
The mo Scien i ic King ishe bench op au oma ed ex ac ion ins umen , using T iPu e Isola ion eagen (Sigma Ald ich) in a 1:9 /
a io ins ead o he ki -supplied Lysis Solu ion. 650 mL inac i a ed sample, 30 mL binding beads and 350 mL binding bu e spiked wi h
A mou ed RNA-En e o i us (ARM-ENTERO, Asu agen) we e hen used o RNA ex ac ion in 96 deep-well pla es. Ex ac ed RNA was
eco e ed in 30 mL elu ion bu e . The spiked en e o i al a mou ed RNA was used as an exogenous RNA ex ac ion con ol.
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RT-qPCR o measu emen o i al i e s in nasal washes
RNA ex ac ed om nasal washes was es ed o he p esence o SARS-CoV-2 RNA by an E gene RT-qPCR ha de ec s a b oad
ange o human and ba co ona i uses.
80
Fo he de ec ion o SARS-CoV-2 RNA by RT-qPCR, 4X TaqMan Fas Vi us one s ep
RT-PCR ki (Li eTech) was used acco ding o manu ac u e ’s ecommenda ions. Fo each RT-qPCR eac ion, 0.4 mM o E gene o -
wa d and e e se p ime s, 0.2 mM o ARM-ENTERO o wa d and e e se p ime s and 0.2 mM o bo h p obes we e used. RT-qPCR
uns we e pe o med using a 7500 Fas Real-Time PCR sys em (Applied Biosys ems) using he ollowing cycle condi ions: 50C o
5 min, 95C o 20 s, hen 40 cycles o 95C o 3 s ollowed by 60C o 30 s. RT-qPCR semi-quan i a i e esul s we e calcula ed
based on a gBlock (In eg a ed DNA Technologies) s anda d cu e o SARS-CoV-2 E gene.
Vi us isola ion om nasal washes
To i a e li e SARS-CoV-2 i us in he nasal washes o he e e s, wo- old se ial dilu ions o nasal washes we e added o wells
o > 90% con luen Ve o E6 cells in a 48-well o ma , incuba ed o 1 h a 37Cin5%CO
2
, hen o e laid wi h 500 mL o 2% ca box-
yme hyl cellulose (Sigma) in supplemen ed DMEM (Co ning) pe well. Pla es we e incuba ed a 37C o 72 h, ixed wi h 10% bu e ed
o malin and s ained wi h 0.5% c ys al iole . Plaques we e coun ed and he endpoin dilu ion was aken as he ela i e li e- i us i es
in he nasal washes.
QUANTIFICATION AND STATISTICAL ANALYSIS
All da a and s a is ical analyses we e pe o med using P ism 9.3.1 (G aphPad). Whe e e applicable, he no mali y o he da a was
de e mined by Shapi o-Wilk es o selec he mos app op ia e s a is ical es . Fo compa ison o wo g oups, S uden ’s- es
(no mal dis ibu ion) o Mann-Whi ney (abno mal dis ibu ion) we e pe o med. Fo compa ison o mo e han wo g oups, one-way
ANOVA (no mal dis ibu ion) o K uskal-Wallis ollowed by Dunn’s mul iple compa isons es (abno mal dis ibu ion) we e pe o med.
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