i uses
A icle
A Soluble Ve sion o Nipah Vi us Glycop o ein G
Deli e ed by Vaccinia Vi us MVA Ac i a es Speci ic
CD8 and CD4 T Cells in Mice
Geo gia Kalodimou 1,2, S enja Vei 1, Syl ia Jany 1, Ul ich Kalinke 3, Ch is ophe C. B ode 4,
Ge d Su e 1,2,* and Asisa Volz 1,2
1Ins i u e o In ec ious Diseases and Zoonoses, LMU Munich, 80539 Munich, Ge many;
geo gia.kalodimou@mic o. e med.uni-muenchen.de (G.K.); [email p o ec ed] (S.V.);
[email p o ec ed] (S.J.); asisa. olz@mic o. e med.uni-muenchen.de (A.V.)
2Ge man Cen e o In ec ion Resea ch (DZIF), pa ne si e Munich, 80539 Munich, Ge many
3Ins i u e o Expe imen al In ec ion Resea ch, TWINCORE, Cen e o Expe imen al and Clinical In ec ion
Resea ch, a join en u e be ween he Helmhol z Cen e o In ec ion Resea ch B aunschweig and he
Hanno e Medical School, 30625 Hanno e , Ge many; Kalinke.Ul ich@mh-hanno e .de
4Depa men o Mic obiology and Immunology, Uni o med Se ices Uni e si y o he Heal h Sciences,
Be hesda, MD 20814, USA; ch is ophe .b [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +49-89-2180-2514
Recei ed: 28 No embe 2019; Accep ed: 20 Decembe 2019; Published: 24 Decembe 2019


Abs ac :
Nipah i us (NiV) is an eme ging zoono ic i us ha is ansmi ed by ba s o humans and
o pigs, causing se e e espi a o y disease and o en a al encephali is. An ibodies di ec ed agains
he NiV-glycop o ein (G) p o ein a e known o play a majo ole in clea ing NiV in ec ion and in
p o iding accine-induced p o ec i e immuni y. Mo e ecen ly, T cells ha e been also shown o be
in ol ed in eco e y om NiV in ec ion. So a , ela i ely li le is known abou he ole o T cell
esponses and he an igenic a ge s o NiV-G ha a e ecognized by CD8 T cells. In his s udy, NiV-G
p o ein se ed as he a ge immunogen o ac i a e NiV-speci ic cellula immune esponses. Modi ied
Vaccinia i us Anka a (MVA), a sa e y- es ed s ain o accinia i us o p eclinical and clinical
accine esea ch, was used o he gene a ion o MVA–NiV-G candida e accines exp essing di e en
e sions o ecombinan NiV-G. O e lapping pep ides co e ing he en i e NiV-G p o ein we e used
o iden i y majo his ocompa ibili y complex class I/II- es ic ed T cell esponses in ype I in e e on
ecep o -de icien (IFNAR
−
/
−
) mice a e accina ion wi h he MVA–NiV-G candida e accines.
We ha e iden i ied an H2-b- es ic ed noname pep ide epi ope wi h CD8 T cell an igenici y and a
H2-b 15me wi h CD4 T cell an igenici y in he NiV-G p o ein. The iden i ica ion o his epi ope and
he a ailabili y o he MVA–NiV-G candida e accines will help o e alua e NiV-G-speci ic immune
esponses and he po en ial immune co ela es o accine-media ed p o ec ion in he app op ia e
mu ine models o NiV-G in ec ion. O no e, a soluble e sion o NiV-G was ad an ageous in ac i a ing
NiV-G-speci ic cellula immune esponses using hese pep ides.
Keywo ds: eme ging i uses; accina ion; MVA ec o accines; T cell esponses
1. In oduc ion
Nipah i us (NiV) is an eme ging zoono ic pa hogen o global conce n ha was anked ecen ly
by he Wo ld Heal h O ganiza ion (WHO) as a high-p io i y pa hogen. NiV is a nega i e-sense,
single-s anded RNA i us ha is a membe o he genus Henipa i us ( amily Pa amyxo i idae). NiV was
i s iden i ied du ing a la ge ou b eak a ec ing humans and pigs in Malaysia and Singapo e in 1999 [
1
].
F om 2001 onwa ds, seasonal ou b eaks a e obse ed almos annually in Bangladesh and spo adically
Vi uses 2020,12, 26; doi:10.3390/ 12010026 www.mdpi.com/jou nal/ i uses
Vi uses 2020,12, 26 2 o 24
in India [
2
]. Recen ou b eaks occu ed in Ke ala in May 2018 and June 2019, he i s ime NiV eme ged
in sou he n India [
2
]. Two s ains o NiV ha e been iden i ied, Malaysia and Bangladesh s ains, which
sha e 91.8% sequence homology [
3
]. NiV causes se e e espi a o y disease and encephali is [
4
–
6
], wi h
a e age case a ali y a es (CFR) o 40–75% [
7
]. Mo eo e , long- e m neu ological sequelae and e en
elapse o encephali is a e obse ed in many su i o s o in ec ions wi h bo h s ains o NiV [8–10].
The na u al ese oi o NiV and Hend a i us (HeV) a e he ui ba s o he genus P e opus,
which a e widely dis ibu ed in Asia, Aus alasia, and pa s o A ica [
11
,
12
]. Mo eo e , NiV has a
b oad species opism and can cause disease in a many animal species [
13
–
15
]. NiV can in ec humans
ia se e al ou es, which include he consump ion o ood con amina ed wi h ba sec e ions [
16
],
ansmission om ampli ica ion hos s such as pigs [
4
], and di ec human- o-human ansmission
be ween e y close con ac s [
17
]. Cu en ly he e a e no licensed ea men s o p e en a i e accines
a ailable o use in humans, which make he de elopmen o e ec i e p ophylac ic measu es impe a i e.
E idence o da e indica es ha he Henipa i us glycop o ein G is a highly p omising a ge o
i us-neu alizing an ibodies o coun e ac in ec ions wi h highly pa hogenic henipa i uses. The G
glycop o eins o NiV and HeV sha e 83% amino acid sequence homology and a e ype II ansmemb ane
p o eins[
18
]. Theglycop o einso bo h i uses bind o hehos cell ecep o seph in-B2and eph in-B3[
19
–
22
],
which a e highly conse ed ac oss many species [
23
]. To da e, he mos p omising he apeu ic app oach
agains Henipa i us in ec ion is he one based on he applica ion o he expe imen al human monoclonal
an ibody m102.4, which binds he eph in-B2 and eph in-B3 ecep o -binding si e on he glycop o eins o
HeV (HeV-G) and NiV (NiV-G) [
24
]. The p o ec i e e icacy o m102.4 has been success ully e alua ed
in di e en p eclinical models including e e s and A ican g een monkeys (AGM) [25–27].
Cu en ly, se e al accines deli e ing NiV-G ha e been shown o p o ec agains le hal challenge
in ec ions in p eclinical es ing. These candida e accines include ecombinan i uses de eloped using
accinia i us [
28
], cana ypox [
29
], esicula s oma i is i us (VSV) [
30
–
34
], abies i us [
35
], measles
i us [
36
], and adeno i us pla o ms [
37
,
38
]. To da e, he only licensed Henipa i us accine is he equine
accine agains HeV, Equi ac
®
HeV, which was app o ed o use in ho ses in Aus alia in 2012 [
39
].
This accine is a subuni accine, which comp ises he soluble o m o HeV-G (HeVsG) [
19
,
39
–
41
].
The subuni HeVsG accine has been shown o p o ec agains NiV in ca s [
42
], e e s [
43
], and non-human
p ima es [
44
], bu no in pigs [
45
]. The abili y o HeVsG o p o ec agains NiV in ec ion in some animal
models wa an s he e alua ion o a accine ha con ains a soluble o m o NiV glycop o ein G.
The ole o an ibodies in p o ec i e immuniza ion agains NiV in ec ion has been widely epo ed,
howe e he e is limi ed in o ma ion on NiV-speci ic T-cell-media ed immuni y. Consequen ly, i is o
in e es o be e unde s and he ole o T cells in accine-induced p o ec ion agains NiV. This, in u n,
will aide in he de elopmen o new and imp o ed accine candida es.
In his s udy, we in es iga ed ecombinan Modi ied Vaccinia i us Anka a (MVA) o deli e y o
NiV-G an igens [
46
]. We cons uc ed wo MVA–NiV-G accine candida es o exp ess ull-leng h NiV-G
o he soluble o m NiVsG. The candida e accines we e gene ically s able and e icien ly eplica ed in
p ima y chicken emb yo ib oblas s, a cell cul u e sys em used o manu ac u ing o MVA accines.
Impo an ly, accina ion o mice lacking he in e e on alpha/be a ecep o (IFNAR
−
/
−
) elici ed eadily
de ec able NiV-G-speci ic CD8 and CD4 T cells. We iden i ied a po en ial H2-b- es ic ed epi ope
in he NiV-G ha s imula ed an igen-speci ic CD8 T cells and a po en ial H2-IAb- es ic ed epi ope
ha s imula ed an igen-speci ic CD4 T cells. In e es ingly, in compa ison wi h ull-leng h NiV-G, he
soluble an igen NiVsG induced signi ican ly s onge epi ope-speci ic T cell esponses. Ou wo k will
be ele an o u u e s udies cha ac e izing NiV-G-speci ic T cell esponses in NiV p eclinical models.
2. Ma e ials and Me hods
2.1. Mice
IFNAR
−
/
−
mice [
47
] backc ossed mo e han 20- old on he C57BL/6 backg ound (we e b ed
unde speci ied-pa hogen- ee (SPF) condi ions, housed in isola ed cage uni s (IsoCage, Tecniplas ,
Vi uses 2020,12, 26 3 o 24
Hohenpeißenbe g, Ge many) and had access o ood and wa e ad libi um. All expe imen s we e
app o ed by he Go e nmen o Uppe Ba a ia, Munich Ge many and we e pe o med in compliance
wi h he Ge man Animal Wel a e Ac (55.2Ve -2532.Ve _02-17-93, 09.01.2017).
2.2. Cells
P ima y chicken emb yo ib oblas s (CEF) we e isola ed om 10-day-old SPF chicken
emb yos (VALO, Cuxha en, Ge many) and g own in Minimum Essen ial Medium (MEM)
(Sigma-Ald ich, Tau ki chen, Ge many) supplemen ed wi h 10% hea -inac i a ed e al bo ine se um
(FBS) (Sigma-Ald ich), 1% Penicillin–S ep omycin (Sigma-Ald ich), and 1% MEM nonessen ial
amino acid solu ion (Sigma-Ald ich). Human HeLa cells (ATCC CCL-2) we e main ained in
MEM supplemen ed wi h 10% hea -inac i a ed FBS (Sigma-Ald ich) and he abo e an ibio ics.
DF-1 cells (ATCC CRL-12203) we e g own in VLE Dulbecco’s Modi ied Eagle’s Medium (DMEM)
(Me ck, Da ms ad , Ge many) supplemen ed wi h 10% hea -inac i a ed FBS (Sigma-Ald ich), 1%
Penicillin–S ep omycin (Sigma-Ald ich), 1% MEM nonessen ial amino acid solu ion (Sigma-Ald ich),
and 1% HEPES solu ion (Sigma-Ald ich). Cells we e main ained a 37
◦
C in a humidi ied 5%
CO2a mosphe e.
2.3. Plasmid Cons uc ions
The cDNA ha encoded o he en i e 602 amino acid sequence o he NiV-G p o ein (Nipah
i us isola e UMMC1, GenBank accession numbe AY029767.1) was modi ied in silico by in oducing
silen codon al e a ions o emo e e mina ion signals o accinia i us ea ly ansc ip ion (TTTTTNT)
and G/C nucleo ide uns. Fo cons uc ion o he soluble o m o NiV-G p o ein (NiVsG), he
cy oplasmic and ansmemb ane domains we e dele ed and an in e nal leade sequence and amino
acid linke sequences we e added as desc ibed p e iously [
40
,
48
]. Fo accinia i us (VACV)-speci ic
ansc ip ional egula ion, we placed he NiVsG gene sequences unde con ol o he syn he ic VACV
ea ly/la e p omo e (PmH5) [
49
] and used he s ong na u al ea ly VACV p omo e P g [
50
–
52
] o
exp ession o NiV-G sequences. The cDNAs encoding o NiV-G o NiVsG including he clea age si es
o he es ic ion endonucleases Xhol and Apal we e gene a ed by DNA syn hesis (GeneWiz, Leipzig,
Ge many). cDNA sequences we e cloned in o he MVA ans e plasmid pLW-73 [
53
], which di ec s
inse ion o he e ologous sequences o a si e be ween he open eading ames (ORF) o he essen ial
i al genes, MVA069R and MVA070L.
2.4. Gene a ion o Recombinan Vi uses
Recombinan MVA i uses exp essing he NiVsG and NiV-G p o eins we e gene a ed as desc ibed
p e iously [
54
,
55
]. To summa ize, CEF cells a 80–90% con luence we e in ec ed wi h non ecombinan
MVA (clonal isola e MVA F6) a a mul iplici y o in ec ion (MOI) o 0.05 and ans ec ed wi h ec o
plasmid DNA con aining NiVsG o NiV-G gene sequences using X- emeGENE
™
HP DNA T ans ec ion
Reagen (Roche Diagnos ics, Penzbe g, Ge many). A e 48 h incuba ion, cells we e ha es ed, and
he ecombinan i uses MVA–NiVsG and MVA–NiV-G we e clonally isola ed by sc eening o
co-exp ession o he luo escen p o ein ma ke GFP and plaque passaging se e al imes. Resul an
ec o i us isola es we e quali y con olled using s anda d p o ocols [
54
]. Polyme ase chain eac ion
(PCR) analysis o genomic i al DNA se ed o con i m he gene ic iden i y and genomic s abili y o
he MVA ec o i uses. The eplica i e capaci y o he ecombinan MVA–NiV i uses compa ed wi h
non ecombinan MVA was es ed by mul i-s ep g ow h expe imen s in CEF and human HeLa cells.
To gene a e accine p epa a ions, ecombinan MVA–NiV i uses we e ampli ied in CEF, pu i ied by
ul acen i uga ion h ough 36% suc ose cushions, and econs i u ed in 10 mM T is-HCl bu e , pH 9.0,
o make s ock p epa a ions [54].
Vi uses 2020,12, 26 4 o 24
2.5. Wes e n Blo Analysis o Recombinan P o eins
CEF and HeLa cells we e in ec ed wi h ecombinan MVA–NiVsG and MVA–NiV-G i uses a
a MOI o 5. Cells in ec ed wi h MVA (MOI 5) and inocula ion medium alone (mock) we e used as
con ols. Cell lysa es we e p epa ed o cul u e supe na an s we e collec ed a a ious ime poin s a e
in ec ion and s o ed a
−
80
◦
C. Cell-associa ed and sec e ed p o eins we e esol ed by sodium dodecyl
sul a e (SDS)-polyac ylamide (10%) gel elec opho esis (SDS-PAGE), and p o eins we e ans e ed
on o ni ocellulose memb anes by we elec oblo ing. To in es iga e he glycosyla ion pa e n, p o eins
we e p e ea ed using he P o ein Deglycosyla ion Mix II ki (New England Biolabs, Ipswich, MA,
USA) acco ding o he manu ac u e ’s ins uc ions p io o SDS PAGE. Memb anes we e blocked wi h
blocking bu e , which consis ed o PBS con aining 5% non- a d ied milk powde (Ca l Ro h, Ka ls uhe,
Ge many) and 0.1% / Tween 20 (Sigma-Ald ich) o one hou a oom empe a u e. Memb anes
we e hen p obed wi h he p ima y an ibody (polyclonal mouse an i-NiV-G (1:2000) o polyclonal
abbi an i-NiV-G (1:5000)) dilu ed in blocking bu e o e nigh a 4
◦
C. Memb anes we e washed h ee
imes wi h PBS con aining 0.1% Tween 20 (PBS/T) and p obed wi h goa an i-mouse IgG conjuga ed
o ho se adish pe oxidase (HRP) (1:5000; Agilen Dako, Glos up, Denma k) o goa an i- abbi IgG
HRP (1:5000; Cell Signaling Technology, Leiden, The Ne he lands). Memb anes we e washed again
wi h PBS/T and we e de eloped using Supe Signal
®
Wes Du a Ex ended Du a ion subs a e (The mo
Fishe Scien i ic, Planegg, Gemany). Blo s we e isualized using a Mic oChemi 4.2 image (DNR
Bio-Imaging Sys ems, Ne e Yamin, Is ael).
2.6. Immuno luo escence
Con luen monolaye s o HeLa cells we e in ec ed wi h ecombinan MVA–NiVsG o MVA–NiV-G
i uses a MOI 0.05. Con ols included MVA (MOI 0.05) and inocula ion medium alone (mock).
A e in ec ion, cells we e incuba ed o 16 h in a 37
◦
C incuba o . Cells we e ixed wi h 4%
pa a o maldehyde (PFA) (Sigma-Ald ich) and on some occasions, pe meabilized wi h 0.5% T i on
X-100 (Sigma-Ald ich). Cells we e blocked in blocking bu e con aining 1% bo ine se um albumin
(BSA) (Sigma-Ald ich). Cells we e s ained wi h polyclonal abbi an i-NiV-G dilu ed 1:10,000 in PBS
con aining 0.5% BSA (PBS/BSA). Cells we e washed wi h PBS and s ained wi h he seconda y an ibody
goa an i-mouse IgG Alexa Fluo (AF) 568 (1:1000; The mo Fishe Scien i ic) dilu ed in PBS/BSA bu e .
Fluo escence was isualized using he Keyence BZ-X710 mic oscope (Keyence, Osaka, Japan).
2.7. Immuniza ion Expe imen s in Mice
Mice we e immunized wi h 10
8
PFU in 50
µ
L accine bu e (10 mM T is and 140 mM NaCl,
pH 7.4) o ecombinan MVA–NiVsG, ecombinan MVA–NiV-G o MVA o accine bu e as a mock
accine con ol ia he in ape i oneal o in amuscula ou es. Mice ecei ed ei he one (p ime) o wo
(p ime-boos ) immuniza ions o e a 21 day in e al. Fo T cell analysis, mice we e eu hanized 8 days
a e immuniza ion. Blood was collec ed on days 0, 18, and 31. Coagula ed blood was cen i uged a
1300
×
g o 5 min in MiniCollec ials (G eine Bio-One, Alphen aan den Rijn, The Ne he lands) in
o de o sepa a e se um, which was subsequen ly s o ed a −20 ◦C un il u he use.
2.8. Quan i ica ion o To al An igen-Speci ic IgG An ibodies
An igen-speci ic IgG esponses induced by immuniza ion wi h he accine candida es we e
analyzed by enzyme-linked immunoso ben assay (ELISA) using pu i ied soluble ecombinan NiV
glycop o ein G exp essed in mammalian HEK293 cells. Fla bo om 96-well ELISA pla es (Nunc
™
MaxiSo p
™
Pla es, The mo Scien i ic) we e coa ed wi h 50 ng/well ecombinan p o ein (100
µ
L olume)
and incuba ed o e nigh a 4
◦
C. Pla es we e washed h ee imes wi h 200
µ
L/well PBS/T. Pla es we e
blocked wi h blocking bu e con aining 1% bo ine se um albumin (Sigma-Ald ich) and 0.15 M suc ose
(Sigma-Ald ich) in PBS o 1 h a 37
◦
C. Pla es we e hen washed wi h PBS/T as desc ibed abo e.
Se a we e se ially dilu ed in PBS con aining 1% BSA (PBS/BSA) h ee- old down he pla e, s a ing a
Vi uses 2020,12, 26 5 o 24
a dilu ion o 1:100 (100
µ
L olume/well) and incuba ed o 1 h a 37
◦
C. A e washing, pla es we e
incuba ed wi h 100
µ
L/well goa an i-mouse IgG conjuga ed HRP (1:2000; Agilen Dako, Denma k)
dilu ed in PBS/BSA o 1 h a 37
◦
C. Pla es we e hen washed wi h PBS/T as desc ibed ea lie . Then,
100
µ
L/well 3,3
0
, 5,5
0
-Te ame hylbenzidine (TMB) Liquid Subs a e Sys em o ELISA (Sigma-Ald ich)
was added, and pla es we e incuba ed un il a colo change was obse ed. The eac ion was s opped by
he addi ion o 100
µ
L/well S op Reagen o TMB Subs a e (450 nm, Sigma-Ald ich). The abso bance
was measu ed on an ELISA pla e eade a 450 nm wi h a 620 nm e e ence wa eleng h. To al IgG
i e s we e calcula ed om he in lec ion poin o he i a ion cu e as loga i hms o he ecip ocal.
2.9. Gene a ion o Syn he ic Pep ides, Design o Pep ide Pools, and Pep ide P edic ion
The p o ein sequence o NiV-G was ob ained om he UniP o da abase (ID: Q9IH62). Using an
in silico app oach, we iden i ied 130 indi idual syn he ic pep ides ha spanned he ex e nal domain
NiV-G p o ein, s a ing om he hi d amino acid o he N- e minal side o he C e minus (amino
acids 73–602). Ou pep ide lib a y consis ed o 15me pep ides ha o e lapped by 11me . All pep ides
we e syn hesized by The mo Fishe Scien i ic as c ude ma e ial (<50% pu i y) on a 1–4 mg scale.
The wo-dimensional pep ide pool ma ix sys em was used o sc eening [
56
,
57
]. B ie ly, pep ides we e
o ganized in o wo-dimensional ma ix pep ide pools (H1–H10 and V1–V11) con aining 11–13 pep ides.
Fo mapping o po en ial CD8 T cell epi opes in posi i e 15me pep ides, e e y possible sequence o
pep ides 8–11me in leng h was de e mined. Theo e ical pep ides we e hen analyzed o binding o
he mouse majo his ocompa ibili y complex (MHC) class I allele H2-b using he SYFPEITHI da abase.
The pep ides o each amino acid sequence leng h we e syn hesized and es ed. Fo iden i ica ion o
CD4 T cell epi opes, MHC class II binding p edic ions we e pe o med on 15me pep ides ound wi hin
posi i e pep ide pools iden i ied by IFN-
γ
Enzyme-Linked Immunospo assay (ELISPOT). Using he
MHC Class II Binding, T Cell Epi ope P edic ion esou ce o he Immune Epi ope Da abase (IEDB,
h ps://www.iedb.o g/), pep ides es ic ed o mouse MHC class II allele H2-IAb we e analyzed using
“IEDB Recommended” p edic ion me hod [
58
]. The mos p omising candida es we e hen chosen o
u he expe imen al epi ope p edic ion s udies. All pep ides we e dissol ed o a concen a ion o
2 mg/mL in PBS, aliquo ed, and s o ed a −20 ◦C un il use.
2.10. T Cell Analysis
2.10.1. Enzyme-Linked Immunospo assay (ELISPOT)
T cell analysis by ELISPOT was pe o med as desc ibed p e iously [
55
]. B ie ly, spleens
we e collec ed om mice 8 days a e he inal immuniza ion. Single-cell suspensions we e
p epa ed by easing spleens h ough a 70
µ
m cell s aine (Falcon
®
Co ning, Co ning, NY,
USA). Red blood cells (RBC) we e emo ed using Red Cell Lysis Bu e (Sigma-Ald ich), and
cells we e washed and esuspended in RPMI-10, which consis ed o RPMI-1640 (Sigma-Ald ich)
con aining 10% hea -inac i a ed FBS (Sigma-Ald ich) and 1% Penicillin–S ep omycin (Sigma-Ald ich).
Fo expe imen s ha equi ed CD4 and CD8 T cell pu i ica ion, splenocy es we e incuba ed wi h
mouse CD4 and CD8 Mic oBeads (Mil enyi Bio ec, Be gisch Gladbach, Ge many) and p ocessed by
nega i e selec ion using he Quad oMACS Sepa a o (Mil enyi Bio ec).
IFN-
γ
-p oducing cells we e measu ed by IFN-
γ
ELISPOT assay using he Mouse IFN-
γ
ELISpo PLUS ki (Mab ech, S ockholm, Sweden) as desc ibed in he manu ac u e ’s p o ocol.
In summa y, 2
×
10
5
splenocy es we e seeded on o 96-well la bo om pla es (100
µ
L/well) (Sa s ed ,
Nümb ech , Ge many), and 100
µ
L/well pep ide pools, subpools, o indi idual pep ides we e added
(each pep ide dilu ed o 2
µ
g/mL in RPMI-10). A e mixing, he splenocy e/pep ide mix u es we e
ans e ed on o pla es p ecoa ed wi h IFN-
γ
de ec ion an ibody and incuba ed o 48 h a 37
◦
C.
Nons imula ed cells we e used as a mock con ol, and he posi i e con ols cul u es we e ea ed
wi h pho bol my is a e ace a e (PMA) and ionomycin (bo h om Sigma-Ald ich) o accinia i us
(VACV)-speci ic CD8 T cell epi ope, B8R
20–27
(TSYKFESV) [
59
]. A e incuba ing, pla es we e p ocessed

Vi uses 2020,12, 26 6 o 24
as desc ibed in he ki manu ac u e ’s p o ocol (Mab ech). Spo s we e coun ed and analyzed using he
Au oma ed ELISPOT pla e eade (A. EL. VIS Eli.Scan and A. EL. VIS ELISPOT Analysis So wa e,
Hanno e , Ge many).
2.10.2. In acellula Cy okine S aining and Flow Cy ome y
Splenocy es we e dilu ed o 1
×
10
7
cells/mL in RPMI-10, and 100
µ
L/well (1
×
10
6
cells) was
added on o a 96-well U-bo om pla e. Then, 100
µ
L/well pep ide dilu ed o 16
µ
g/mL in RPMI-10 was
added o gi e a inal pep ide concen a ion o 8
µ
g/mL. The VACV CD8 T cell epi ope B8R
20–27
( inal
concen a ion o 8
µ
g/mL) was used as a posi i e con ol along wi h PMA (10 ng/mL) plus ionomycin
(500 ng/mL). PBS dilu ed in RPMI-10 was used as a mock s imula ed con ol. A e pla ing, cells we e
incuba ed o 2 h a 37
◦
C. Then, 20
µ
L/well 10x B e eldin A, a Golgi inhibi o ha was p epa ed by
dilu ing 1000X B e eldin A (Biolegend, San Diego, CA, USA) in RMPI-10, was added o gi e a inal
dilu ion o 1x B e eldin A. Cells we e hen incuba ed o an addi ional 4 h a 37
◦
C. A e incuba ing,
pla es we e cen i uged (500 g o 3 min), and he supe na an was emo ed. Cells we e washed
once wi h 200
µ
L/well FACS bu e (MACSQuan Running Bu e wi h 2% FBS). Ex acellula s aining
was pe o med wi h he ollowing an ibodies dilu ed in FACS bu e : an i-mouse CD3 phycoe i h in
(PE)-Cy7 (clone 17A2, 1:100, Biolegend), an i-mouse CD4 B illian Viole 421 (clone GK1.5, 1:600,
Biolegend), an i-mouse CD8
α
Alexa Fluo 488 (clone 53-6.8, 1:300, Biolegend), and pu i ied CD16/CD32
(Fc block; clone 93, 1:500, Biolegend). Cells we e s ained in 50
µ
L/well o 30 min on ice in he da k, hen
washed once wi h 200
µ
L/well FACS bu e and hen wice wi h 200
µ
L/well PBS. Cells we e hen s ained
wi h 100
µ
L/well o he ixable dead cell iabili y dye Zombie Aqua (1:800, Biolegend) dilu ed in PBS
o 30 min on ice in he da k. Cells we e washed wice wi h 200
µ
L/well PBS and ixed wi h 100
µ
L/well
o Fixa ion Bu e (Biolegend) o 20 min a oom empe a u e in he da k. Cells we e washed wice
wi h 200
µ
L/well PBS and esuspended in 200
µ
L/well FACS bu e . Pla es we e s o ed o e nigh a
4
◦
C, p o ec ed om ligh . The ollowing day, cells we e pe meabilized by washing h ee imes wi h
200
µ
L/well 1
×
Pe m Wash bu e , which con ained In acellula S aining Pe meabiliza ion Wash Bu e
(10
×
) (Biolegend) dilu ed o 1
×
wi h dis illed wa e . Cells we e s ained in acellula ly in 100
µ
L/well o
an i-mouse IFN-
γ
(clone XMG1.2, 1:200, Biolegend) plus TNF-
α
(clone MP6-XT22, 1:200, Biolegend)
dilu ed in 1X Pe m Wash bu e o 30 min a oom empe a u e, p o ec ed om ligh . Cells we e hen
washed h ee imes wi h 200
µ
L/well 1X Pe m Wash and hen esuspended in 300
µ
L/well FACS bu e .
Samples we e il e ed h ough 50
µ
m nylon mesh (Se a P y L d., Hun ingwood, NSW, Aus alia)
in o 5 mL ound bo om FACS ubes (Sa s ed ). Single-colo con ols we e p epa ed o each FACS
analysis using OneComp eBeads
™
Compensa ion Beads (eBioscience, The mo Fishe Scien i ic) o
luo opho e-conjuga ed an ibodies and cells o he iabili y dye Zombie Aqua. Da a acquisi ion was
pe o med by MACSQuan VYB Flow Analyse (Mil enyi Bio ec), and da a was analyzed using FlowJo
(FlowJo LLC, BD Li e Sciences, Ashland, OR, USA).
2.11. S a is ical Analysis
Da a we e analyzed using G aphPad P ism e sion 5.0 (G aphPad So wa e Inc., San Diego,
CA, USA) and we e exp essed as mean
±
s anda d e o o he mean (SEM). S a is ical analysis was
pe o med using he unpai ed, wo- ailed - es o compa e wo g oups and one-way ANOVA o
compa e h ee o mo e g oups. The h eshold o s a is ical signi icance was p<0.05.
3. Resul s
3.1. Gene a ion and Cha ac e isa ion o MVA Vec o Vaccines Deli e ing NiV-G An igens
To gene a e he ecombinan MVA i uses deli e ing NiV-G an igens, we used he gene om NiV
Malaysia (isola e UMMC1, GenBank accession numbe AY029767.1) and gene a ed codon-op imized
gene sequences encoding a ull-leng h glycop o ein G (NiV-G) o a soluble ex e nal domain G
p o ein (NiVsG). These syn he ic gene sequences we e placed unde he ansc ip ional con ol o he
Vi uses 2020,12, 26 7 o 24
accinia i us-speci ic p omo e s P g o PmH5 and in oduced in o he MVA genome by homologous
ecombina ion a ge ing he in e genic si e be ween he essen ial MVA genes 069 and 070L. The clonal
isola ion o he ecombinan i uses MVA–NiVsG and MVA–NiV-G was acili a ed by he co-p oduc ion
o he g een luo escen epo e p o ein (GFP), as p e iously desc ibed [
54
]. The inal ecombinan
i uses con aining he NiV-G gene sequences (MVA–NiV-G and MVA–NiVsG) we e ob ained a e
emo al o he GFP epo e gene by in agenomic homologous ecombina ion (Figu e S1A, ma ke gene
dele ion). To e i y he iden i y o he desi ed modi ica ion, we pe o med he s anda d quali y con ol
expe imen s as desc ibed p e iously [
54
]. PCR analysis o i al genomic DNA con i med he p ope
inse ion o he ecombinan gene sequences a he a ge si e in he genome o MVA (Figu e S1B–E)
and he gene ic cha ac e is ics and s abili y o he ecombinan MVA i uses. We assessed he g ow h
beha io o he ecombinan i uses MVA–NiVsG and MVA–NiV-G in mul i-s ep g ow h analyses
in human HeLa cells and p ima y CEF, which a e ou inely used o ampli ica ion o ecombinan
MVA in accine manu ac u ing (Figu e S1F). MVA–NiVsG and MVA–NiV-G e icien ly eplica ed
in CEF and demons a ed an inc ease o in ec i i y i e s ha was compa able o ha ob ained wi h
non ecombinan MVA. Howe e , MVA–NiV-G and MVA–NiVsG did no p oduc i ely g ow in human
HeLa cells, con i ming ha hey had e ained he cha ac e is ic eplica ion de iciency o MVA in cells
o mammalian o igin. These indings co obo a ed he expec ed MVA pheno ype and con i med ha
he ecombinan i uses could be handled unde labo a o y condi ions o biosa e y le el 1. O no e, we
o iginally gene a ed ano he ecombinan MVA i us using he syn he ic ea ly–la e p omo e PmH5
o ansc ip ional con ol o ecombinan gene exp ession and p oduc ion o he ull-leng h NiV-G
p o ein. Howe e , upon g ow h es ing, his candida e i us ailed o each le els o in ec ious p ogeny
in CEF o be eligible o la ge-scale ampli ica ion as needed o accine p oduc ion p ocesses.
3.2. Cha ac e isa ion o Recombinan NiV-G P o eins
Ou accine candida es, MVA–NiVsG and MVA–NiV-G, should p oduce ecombinan NiV
glycop o ein G in i s ull-leng h o m (NiV-G) and, in pa allel, he soluble o m (NiVsG). NiV-G is a 602
amino acid long p o ein consis ing o an N- e minal in e nal domain, a ansmemb ane domain, and a
C- e minal ex e nal domain (Figu e 1A). Fo NiVsG p o ein, an in e nal leade sequence and h ee
amino acid linke s ha e eplaced he in e nal and ansmemb ane domains (Figu e 1A), as desc ibed
p e iously [
19
,
48
]. We assessed he co ec exp ession and s udied he cellula localiza ion o he
NiV-G and NiVsG by immuno luo escence mic oscopy o MVA–NiV-in ec ed cells immunolabeled
wi h an i-NiV-G an ibody, ollowed by a luo escen ly labelled seconda y an ibody. Cell nuclei we e
s ained wi h DAPI (300 nM). As an icipa ed, we obse ed di e en pa e ns o g een luo escence,
wi h a ying cellula localiza ions depending on he MVA–NiV cons uc . G een luo escence, speci ic
o NiV-G, was iden i ied in pe meabilized and nonpe meabilized cells in ec ed wi h MVA–NiV-G
(Figu e 1B), bu no in MVA-in ec ed o mock con ol cells. This da a con i med ha he ecombinan
ull-leng h NiV-G p o ein encoded by MVA–NiV-G was indeed ancho ed on he cell su ace. In con as ,
he NiV-G-speci ic s aining in cells in ec ed wi h MVA–NiVsG i us appea ed o be p edominan ly
loca ed wi hin he cells and was eadily de ec ed a e pe meabiliza ion. As an icipa ed, we ailed
o de ec NiVsG in conside able amoun s wi hou pe meabiliza ion, con i ming ha NiVsG was no
exp essed on he cell su ace (Figu e 1B).
To u he in es iga e he syn hesis o NiV-G p o eins a e in ec ion wi h MVA–NiVsG and
MVA–NiV-G, espec i ely, o al p o eins om in ec ed CEF and HeLa cell cul u es we e analyzed
by Wes e n blo using a NiV-G-speci ic an ibody. To al cell lysa es o cul u e supe na an s ob ained
om CEF and HeLa cul u es in ec ed wi h ecombinan MVA i us we e sepa a ed by SDS-PAGE and
immunoblo ed. We speci ically de ec ed a p o ein wi h an es ima ed molecula mass o app oxima ely
72–75 kDa in lysa es om CEF cells and HeLa cells in ec ed ei he wi h MVA–NiV-G o MVA–NiVsG
(Figu e 1C). In he cell lysa es, he glycop o ein was i s de ec able a 4 h pos -in ec ion. The amoun
o p o ein inc eased o e ime, esul ing in a p ominen band a 16 h pos -in ec ion, which was
main ained h oughou he 72 h ime cou se. To moni o he elease o ecombinan NiV-G p o ein
Vi uses 2020,12, 26 8 o 24
om MVA–NiVsG in ec ed cells, we also analyzed he supe na an s o in ec ed CEF and HeLa cell
cul u es. A p o ein wi h a molecula mass o app oxima ely 72–75 kDa sized band was obse ed in
supe na an s om MVA–NiVsG-in ec ed cells (Figu e 1C), indica ing ha in ec ed cells we e sec e ing
he soluble NiV-G p o ein. Sec e ed NiV-G p o ein was i s obse ed a 16 h pos -in ec ion in he
supe na an s o MVA–NiVsG-in ec ed cul u es and became especially p ominen by 24 h pos -in ec ion.
The le els o sec e ed NiV-G seemed o decline by 72 h pos -in ec ion. We did no howe e de ec
NiV-G p o ein in supe na an s o MVA–NiV-G-in ec ed cells.
Vi uses 2020, 12, x FOR PEER REVIEW 8 o 25
Figu e 1. Analysis o ecombinan Nipah i us ull-leng h glycop o ein G (NiV-G) p o ein p oduced
by cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. (A) Schema ic diag am o ecombinan NiV
ull-leng h glycop o ein G (NiV-G) and NiV soluble glycop o ein G (NiVsG). Colo ed ec angles
ep esen indi idual p o ein domains, and b acke ed ex displays he s a and end o amino acid
sequences o each domain. (B) Immuno luo escence s aining o cells in ec ed wi h MVA–NiVsG and
MVA–NiV-G. HeLa cells we e in ec ed a MOI 0.05 wi h he abo e i uses o 16 h. MVA and mock
Figu e 1.
Analysis o ecombinan Nipah i us ull-leng h glycop o ein G (NiV-G) p o ein p oduced by
cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. (
A
) Schema ic diag am o ecombinan NiV ull-leng h
Vi uses 2020,12, 26 9 o 24
glycop o ein G (NiV-G) and NiV soluble glycop o ein G (NiVsG). Colo ed ec angles ep esen
indi idual p o ein domains, and b acke ed ex displays he s a and end o amino acid sequences o
each domain. (
B
) Immuno luo escence s aining o cells in ec ed wi h MVA–NiVsG and MVA–NiV-G.
HeLa cells we e in ec ed a MOI 0.05 wi h he abo e i uses o 16 h. MVA and mock in ec ed HeLa
cells we e used as con ols. Fixed pe meabilized and ixed nonpe meabilized cells we e immunos ained
wi h abbi polyclonal an ibody o NiV-G and he seconda y an ibody an i- abbi Alexa Fluo 488.
Nuclei we e s ained wi h DAPI solu ion. Panel shows ep esen a i e pic u es o ixed/pe meabilized
and ixed/nonpe meabilized in ec ed HeLa cells a 40
×
magni ica ion. (
C
) Wes e n blo analysis o
ecombinan NiV-G p o eins p oduced by chicken emb yo ib oblas s (CEF) and HeLa cells in ec ed
wi h MVA–NiVsG and MVA–NiV-G. Lysa es and cul u e supe na an s we e collec ed om cell
cul u es in ec ed a MOI 5 wi h he abo e i uses, wild- ype MVA o nonin ec ed con ols (mock).
Samples we e collec ed a indica ed hou s pos -in ec ion. Cell lysa es and p o eins we e es ed by
immunoblo ing using a NiV-G-speci ic polyclonal mouse an ibody. P o ein bands co esponding o
he expec ed molecula weigh s o ecombinan NiV-G and NiVsG p o ein (~65
−
70 kDa) a e indica ed.
(
D
) Wes e n Blo analysis o ecombinan p o eins p oduced by DF-1 cells in ec ed wi h MVA–NiVsG
and MVA–NiV-G a MOI 5 o 36 h. MVA and mock in ec ed cells we e used as con ols. Cell lysa es
and cul u e supe na an s we e incuba ed wi h (+) o wi hou (
−
) enzymes o deglycosyla e p o eins,
analyzed by SDS-PAGE, and immunoblo ed wi h a abbi polyclonal an ibody o NiV-G. Be a-ac in
was used as a loading con ol o lysa es. Solid black a ow ep esen s glycosyla ed ecombinan NiV-G
and NiVsG p o ein (~65–70 kDa), and dashed black a ow ep esen s deglycosyla ed ecombinan
NiV-G and NiVsG p o ein (~58 kDa). Blue solid a ow ep esen s be a-ac in (~40 kDa). MVA: modi ied
Vaccinia i us Anka a.
Since he NiVsG gene encoding sequences we e modi ied o esul in he sec e ed soluble e sion
(sG), we pe o med addi ional Wes e n blo expe imen s o de e mine whe he he p o ein s ill
main ained glycosyla ion si es compa able o wild- ype NiV-G. Lysa es and supe na an s ob ained
om cul u es o DF-1 cells in ec ed wi h MVA–NiVsG we e ea ed wi h enzymes ha deglycosyla e
p o eins and analyzed by wes e n blo ing. Enzyme ea men esul ed in a educ ion in he molecula
mass o ecombinan NiVsG p o ein om 70–75 kDa o 58–60 kDa (Figu e 1D), ma ching he expec ed
size o unmodi ied G p o ein. This sugges ed ha ecombinan NiVsG has e ained he no mal
glycosyla ion pa e n o wild- ype NiV-G.
3.3. An ibody Responses in Vaccina ed IFNAR−/−Mice
To assess he immunogenici y o he ecombinan MVA–NiV-G/NiVsG candida e accines, we
accina ed IFNAR
−
/
−
mice wi h 10
8
PFU ia he in amuscula ou e a days 0 and 21. Se um samples
we e es ed o NiV-G-binding IgG an ibodies by ELISA 18 days a e he i s immuniza ion (P ime)
and 10 days a e he second immuniza ion (P ime-Boos ) (Figu e 2). E en a single applica ion o
he MVA–NiV-G accines induced abundan le els o NiV-G-speci ic IgG an ibodies in he mice.
A e boos e immuniza ion, all accina ed animals p oduced e en highe le els o ci cula ing
NiV-G-speci ic an ibodies, wi h he an ibody i e s inc easing by app oxima ely en- old.
Vi uses 2020,12, 26 16 o 24
3.4.4. Iden i ica ion o Po en ial H2-IAb-Res ic ed CD4 T Cell Epi opes o NiV-G
Ini ially, we also analyzed pu i ied CD4 T cell cul u es om MVA–NiVsG immunized mice o
hei abili y o s imula e IFN-
γ
by ELISPOT using a wo-dimensional pooled-pep ide ma ix sys em
(Figu e 3A, Sec ion 3.4.1). In con as o CD8 T cell-en iched splenocy es, he IFN-
γ
SFC signals in
hese cul u es we e lowe (Figu es 3B and 6A). In o de o de e mine de ini i ely which pep ide pools
we e abo e backg ound le els, we selec ed a cu o alue o 20 IFN-
γ
SFC/10
6
cells (Figu e 6A, g ey
line). Mean IFN-
γ
SFC alues abo e backg ound we e obse ed in 8 ou o he 21 pools (pools H3, H4,
V2, V3, V5, V6, V9, and V11) (Figu e 6A).
Vi uses 2020, 12, x FOR PEER REVIEW 18 o 25
Figu e 6. Ac i a ion o an igen-speci ic CD4 T cells a e immuniza ion wi h he ecombinan MVA
candida e accines exp essing NiV-G o NiV-sG. G oups o IFNAR−/− mice (n = 2–7) we e immunized
wice wi h MVA–NiVsG, MVA–NiV-G, MVA, o saline (mock) ia he i.p. o i.m. ou e o e a 21 day
pe iod. Eigh days a e he inal immuniza ion, CD4 T cell-en iched splenocy es o o al splenocy es
we e es imula ed and measu ed by IFN-γ ELISPOT assay o IFN-γ in acellula cy okine s aining
(ICS) plus FACS analysis. (A) Sc eening o H2-IAb- es ic ed T cell epi opes o NiV-G in CD4 T cell-
en iched splenocy es ob ained om i.p. immunized mice. T cell esponses we e measu ed by IFN-γ
ELISPOT assay. G aph shows IFN-γ SFC o CD4 T cell-en iched splenocy es s imula ed wi h pep ide
Figu e 6.
Ac i a ion o an igen-speci ic CD4 T cells a e immuniza ion wi h he ecombinan MVA
candida e accines exp essing NiV-G o NiV-sG. G oups o IFNAR
−
/
−
mice (n=2–7) we e immunized

Vi uses 2020,12, 26 17 o 24
wice wi h MVA–NiVsG, MVA–NiV-G, MVA, o saline (mock) ia he i.p. o i.m. ou e o e a
21 day pe iod. Eigh days a e he inal immuniza ion, CD4 T cell-en iched splenocy es o o al
splenocy es we e es imula ed and measu ed by IFN-
γ
ELISPOT assay o IFN-
γ
in acellula cy okine
s aining (ICS) plus FACS analysis. (
A
) Sc eening o H2-IAb- es ic ed T cell epi opes o NiV-G
in CD4 T cell-en iched splenocy es ob ained om i.p. immunized mice. T cell esponses we e
measu ed by IFN-
γ
ELISPOT assay. G aph shows IFN-
γ
SFC o CD4 T cell-en iched splenocy es
s imula ed wi h pep ide pools H1–H10 and V1–V11. The g ey dashed line ep esen s he g oup mean
cu o alue (20 IFN-
γ
SFC/10
6
cells) o iden i ying posi i e pep ide pools. (
B,C
) Iden i ica ion o
H2-IAb- es ic ed candida e epi opes o NiV-G by IFN-
γ
ICS and FACS analysis. To al splenocy es
om i.m. immunized mice we e es imula ed wi h wo p omising candida e H2-IAb- es ic ed 15me
pep ides, #49 (LFMTNVWTPPNPNTV) and #50 (NVWTPPNPNTVYHCS), iden i ied by pep ide pool
sc eening and in silico H2-IAb-binding p edic ions using he IEDB da abase. (
B
) Rep esen a i e low
cy ome y do plo s showing IFN-
γ
p oduc ion in he splenic CD4 T cell compa men . (
C
) F equency
and absolu e numbe (pe 10
6
splenocy es) o IFN-
γ
+CD4 T cells. Dashed line on g aphs ep esen
he cu o o de ini i ely posi i e samples. Di e ences be ween indi idual g oups we e analyzed
by one-way ANOVA and Tukey pos -hoc es . As e isks ep esen s a is ically signi ican di e ences
be ween wo g oups o a speci ic pep ide. * p<0.05, ** p<0.01.
In o de o iden i y po en ial CD4 T cell epi opes o H2-IAb, we pe o med a compu a ional
analysis o he 15me pep ides in he i e mos posi i e pep ide pools measu ed by ELISPOT assay
(pools H3, H4, V3, V6, and V9). Using MHC II binding p edic ions ob ained om he IEDB online
esou ce, we ound wo p omising pep ides. These pep ides we e #49 (LFMTNVWTPPNPNTV) and
#50 (NVWTPPNPNTVYHCS) (Table 2). Nex , we used IFN-
γ
ICS o iden i y di ec ly an igen-speci ic
CD4 T cells a e s imula ion wi h hese pep ides. Fo his, splenocy es om mice ha had been
accina ed wi h MVA–NiV-G o MVA–NiVsG we e s imula ed wi h pep ides #49 and #50 and analyzed
by low cy ome y. A small popula ion o pep ide-speci ic IFN-
γ
+CD4 T cells was obse ed in he
MVA–NiVsG and MVA–NiV-G g oups (Figu e 6B). Due o low equencies o IFN-
γ
-p oducing cells,
we chose a cu o alue o 0.1% o di e en ia e be ween posi i e signals and backg ound. O e all, CD4
T cells om he MVA–NiVsG g oup had a equency o IFN-
γ
+abo e he cu o ela i e o MVA–NiV-G
g oup (Figu e 6C). The mean pe cen age o IFN-
γ
+CD4 T cells o he wo pep ides was 0.09–0.17%
and 0.04–0.07% o MVA–NiVsG and MVA–NiV-G, espec i ely. Mo eo e , he equency and absolu e
numbe o IFN-
γ
-p oducing CD4 T cells in pep ide #50 s imula ed cul u es was signi ican ly highe
in he MVA–NiVsG g oup when compa ed wi h he MVA–NiV-G g oup (mean =290
±
65 and
140 ±46 cells/106
splenocy es espec i ely). Fo pep ide #49, MVA–NiVsG accina ed mice showed
a signi ican ly ele a ed equency o IFN-
γ
+CD4 T cells only. In conclusion, ou da a indica e ha
pep ide #50 (NVWTPPNPNTVYHCS) is a p omising H2-IAb- es ic ed CD4 T cell epi ope candida e
o NiV-G. The alignmen o he pep ide o he ull amino acid sequence o wild- ype NiV-G is shown in
Figu e S3.
4. Discussion
The con inuous h ea o suddenly eme ging NiV ou b eaks, pa icula ly in Bangladesh and India,
demons a e he need o coun e measu e app oaches eady o use in an immedia e public heal h
esponse. A p esen , he e a e no licensed NiV accines o use in humans a ailable. The exis ence o
a NiV candida e accine should signi ican ly educe he isk o in ec ion and ansmission o he i us
in he case o an ou b eak scena io. The e a e some expe imen al NiV accines ha ha e al eady been
es ed in di e en p eclinical animal models. The majo ocus o hese app oaches was o e alua e
he immunogenici y and e icacy in he con ex o NiV challenge in ec ion. In hose s udies, e icacy
has been mos ly associa ed wi h he gene a ion o NiV-speci ic an ibodies, and immune moni o ing is
mainly elying on he de ec ion o i us-neu alizing an ibodies [
31
,
60
,
61
]. Howe e , he e is ela i ely
li le known abou he induc ion and he ele ance o NiV-speci ic cellula immune esponses. In ha
con ex , he a ailabili y o app op ia e ools o in es iga e he ole o T cells in NiV-speci ic immuni y
is an impo an p e equisi e in he de elopmen o new accines and he apeu ics. Thus, i will be
Vi uses 2020,12, 26 18 o 24
indispensable o moni o in animal models he con ibu ion o i us-speci ic T cells o p o ec i e
immuni y bu also o po en ial NiV an igen-speci ic immune pa hology.
He e, we iden i ied a majo his ocompa ibili y complex (MHC) haplo ype H2-b- es ic ed pep ide
epi ope in he NiV-G p o ein by s imula ing T cells om MVA
−
NiVsG accina ed IFNAR
−
/
−
mice
wi h a wo-dimensional (2D) ma ix pool o o e lapping pep ides. IFNAR
−
/
−
mice ha e been al eady
es ablished as a aluable p eclinical animal model o NiV in ec ion wi h a LD
50
o 8
×
10
3
p u a e
in ape i oneal challenge in ec ion [
15
]. Mo eo e , in p e ious s udies, we ha e al eady success ully
demons a ed ha in e e on ype I ecep o knockou mice (IFNAR
−
/
−
) [
47
] can be eadily used as
animal models o s udy he immunogenici y and p o ec i e capaci y o MVA immuniza ion [
62
,
63
].
He e, we wished o speci ically assess he abili y o MVA-deli e ed NiV-G an igen o induce he
ac i a ion o cellula immune esponses in mice. In gene al, he en elope G p o ein is known as he
well-conse ed a achmen glycop o ein o bo h HeV and NiV. In a p e ious s udy, a ecombinan
adeno-associa ed i us accine exp essing a ull-leng h NiV-G p o ein p o ec ed hams e s in an NiV
in ec ion model [
38
]. In ano he app oach, a soluble e sion o HeV-G has been enginee ed and showed
an e en mo e ad an ageous e icacy when es ed in di e en p eclinical animal models [
42
,
43
,
64
].
Using HeVsG, a monoclonal an ibody m102.4 was de i ed and has al eady been success ully es ed as
a he apeu ic app oach in humans. To u he enhance Henipa i us G p o ein-induced immunogenici y,
we also designed and es ed a soluble e sion o he NiV-G p o ein (NiVsG) simila o he HeVsG
an igen used o he gene a ion o m102.4. To compa a i ely e alua e he immunogenici y o he NiVsG
p o ein, we also gene a ed an MVA exp essing ull-leng h G. The ecombinan i uses MVA–NiV-G
and MVA–NiVsG p oduced s able amoun s o NiV-G an igen upon
in i o
in ec ion o human cells,
indica ing he unimpai ed exp ession o he a ge gene unde ansc ip ional con ol o he syn he ic
accinia i us-speci ic ea ly–la e p omo e PmH5 o he s ong na u al ea ly p omo e P g . In he
case o MVA–NiVsG, emo al o he ansmemb ane domain and cy oplasmic ail esul ed in he
sec e ion o he NiV-G om MVA-in ec ed cells and accumula ion also in he supe na an o cell
cul u es, as demons a ed in Wes e n blo analysis and immunos aining. A simila esul was ob ained
wi h HeVsG, as exp essed by con en ional ecombinan VACV [
41
]. In con as , he ull-leng h
MVA-p oduced NiV-G p o ein was no eleased in he supe na an , indica ing he s able p esen a ion
on he cell su ace h ough he ansmemb ane domains. Ano he impo an aspec o p ope p o ein
exp ession, olding and con o ma ional s abili y is in luenced by he N-glycans. Recen s udies indica ed
ha NiV-G N-glycans educe usion e iciency because emo al o some N-glycans caused cell–cell
hype usogenici y and inc eased i al en y [
65
]. Glycosidase ea men o ull-leng h MVA-p oduced
NiV-G esul ed in a polypep ide o 58 kDa, co esponding o he molecula mass p edic ed om he
NiV G gene encoding sequences. The glycosidase ea men o he NiVsG also indica ed he p esence
o all he N-glycans si es wi hin he soluble e sion o he glycop o ein. A i s
in i o
e alua ion in
mice e ealed ha ea men wi h he MVA–NiV-G and MVA–NiVsG candida e accines esul ed in
he induc ion o simila le els o G-binding se um an ibodies, con i ming he immunogenici y o bo h
an igen e sions [
66
]. In ha con ex , he p esence o binding an ibodies seems o play a subs an ial
ole in he blocking o NiV en y, since he mechanism o NiV neu aliza ion is complex and in ol es
mo e an igenic si es han hose equi ed o simple ecep o binding [
67
]. Howe e , mo e ecen s udies
in di e en animal models sugges ha cellula immune esponses a e also in ol ed in media ing
p o ec ion agains NiV in ec ion [
38
,
68
]. This obse a ion is u he suppo ed by s udies in a pig
model o NiV in ec ion showing subs an ial ac i a ion o CD8 T cells a e o al in ec ion wi h NiV [
45
].
In line wi h hese p eclinical da a, humans su i ing NiV in ec ion [
2
] showed signi ican le els o
p oli e a ing (Ki-67+) CD8 T cells, indica ing he p esence o acu e e ec o cells. These da a emphasize
ha in addi ion o he humo al immune esponses, T cells could be associa ed wi h eco e y om NiV
in ec ion. In a mo e ecen s udy, S oh and cowo ke s con i med he ac i a ion o NiV-speci ic CD8 T
cells in mice a e accina ion wi h NiV-like pa icles. These da a u he highligh ha T cells may play
a c i ical ole in NiV in ec ion [
68
]. Ano he hypo hesis is ha NiV-speci ic T cells could be in ol ed in
po en ial immunopa hologies. In his con ex , da a om in ec ions wi h o he neu o opic i uses,
Vi uses 2020,12, 26 19 o 24
o example, Wes Nile i us, demons a ed ha an igen-speci ic T cells can open he blood b ain
ba ie and con ibu e o i us in ec ions o he b ain [
69
–
73
]. Thus, o allow o mo e de ailed s udies
cha ac e izing T cells in NiV-associa ed immuni y o pa hogenesis, i is essen ial o iden i y NiV-G
pep ide epi opes allowing o he speci ic MHC- es ic ed an igen p esen a ion and he ac i a ion o
NiV-speci ic T cells. We iden i ied a noname epi ope NiV-G-9.3
75–83
(RSTDNQAVI). Analysis o his
pep ide sequence showed ha NiV-G 9.3
75–83
could be unc ionally conse ed in Hend a i us, bu no
Ceda i us (ano he ecognized Henipa i us), G an igens (Figu e S3). S uc u al and unc ional analyses
e eal p omiscuous and species-speci ic use o eph in ecep o s by Ceda i us [
74
]. This po en ial
epi ope will suppo a mo e de ailed expe imen al cha ac e iza ion o T cells induced by NiV in ec ion
in he mouse model and hei con ibu ion o pa hogenesis and p o ec ion. In his s udy, we ound
ha MVA–NiVsG accina ion induced signi ican ly highe amoun s o NiV-G epi ope-speci ic CD8 T
cells compa ed wi h he MVA–NiV-G candida e accine. This was a somewha su p ising obse a ion
as he immuniza ions wi h bo h an igens had elici ed e y compa able le els o G-speci ic an ibodies.
I is emp ing o specula e ha NiVsG, as a soluble an igen, can igge enhanced T cell esponses
because i is a ailable o wo di e en pa hways o an igen p esen a ion. On he one hand, NiVsG
as in acellula ly syn he ized an igen is endogenously p ocessed and p esen ed ia MHC-I on he
cell su ace o ac i a e CD8 T cells. In addi ion, he NiVsG is sec e ed in high amoun s om he
MVA–NiVsG-in ec ed cells, and such ex acellula an igen can e icien ly uel he c oss-p esen a ion
pa hway and he eby induce ele a ed CD8 and CD4 T cell immune esponses [
75
,
76
]. In e es ingly, he
MVA–NiVsG candida e accine also p o ed o apidly induce NiV-G epi ope-speci ic CD8 T cells a e
single-dose applica ion. These da a a e o ele ance, since he epidemiology o he mo e ecen NiV
ou b eaks demons a ed ha a po en ial accine candida e should apidly p o ec . Impo an ly, an
H2b- es ic ed epi ope has been iden i ied in IFNAR
−
/
−
mice, which se e as an es ablished small
animal model o NiV in ec ion [
15
]. In addi ion, we showed he induc ion o NiV-G-speci ic CD4
T cells upon p ime-boos immuniza ion in he IFNAR
−
/
−
wi h he MVA–NiV accines and using
pep ides o
in i o
s imula ion, as iden i ied by using MHC II binding p edic ions ob ained om he
IEDB online esou ce (www.iedb.o g). This da a goes well along wi h he hypo hesis ha CD4 T cell
esponses a e also signi ican ly ele a ed upon in ec ion [
77
]. Again, MVA–NiVsG accina ion esul s
in mo e e icien ac i a ion o CD4 T cell esponses. Fu he expe imen s will be needed o cha ac e ize
he con ibu ion o NiV G-speci ic CD4 T cells o NiV in ec ion in mo e de ail. Taken oge he , ou
indings showed he ac i a ion o NiV-G-speci ic T cells in IFNAR
−
/
−
mice ollowing accina ion
wi h MVA-based candida e accines. We con i med he iden i ica ion o po en ial H2-b- es ic ed
NiV-G CD8 and CD4 T cell pep ide epi opes. In his s udy we also demons a ed ha an MVA–NiVsG
candida e accine may ha e supe io immunogenici y, esul ing in NiV-speci ic an ibodies and T cells
in IFNAR
−
/
−
mice. These da a emphasize he p omise o u u e s udies in his animal model u he
e alua ing he ole o NiV-speci ic T cells ac i a ed by he G-9.3
75–83
and G-50
269–283
pep ide epi opes,
in bo h accine-induced p o ec ion and po en ial con ibu ion o NiV-induced pa hologies.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/1999-4915/12/1/26/s1,
Figu e S1: Gene a ion and cha ac e iza ion o ecombinan MVA–NiVsG and MVA–NiV-G, Figu e S2: IFN-
γ
p oduc ion by CD8 and CD4 T cells s imula ed by he NiV-G pep ide 9.3, Figu e S3: Amino acid alignmen o
H2-b- and H2-IAb- es ic ed pep ides, Figu e S4: Quali y o ac i a ed CD8 T cells om mice immunized wi h
ecombinan MVA exp essing NiV-G.
Au ho Con ibu ions:
G.K., S.V., C.C.B., G.S. and A.V. concei ed and designed he expe imen s; G.K., S.V., S.J.
and A.V. pe o med he expe imen s; G.K., S.V., U.K., C.C.B., G.S. and A.V. analyzed he da a; G.K., G.S. and A.V.
w o e he pape . All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This wo k was suppo ed by he Fede al Minis y o Educa ion and Resea ch (BMBF), g an numbe
DZIF TTU 01.805.
Acknowledgmen s:
U sula Klos e meie , Pa izia Bone , Johannes Dö ing and Axel G oß p o ided aluable
help wi h he animal s udies.
Vi uses 2020,12, 26 20 o 24
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he
s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o
publish he esul s.
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