ARTICLE OPEN
MTBVAC accina ion p o ec s hesus macaques agains
ae osol challenge wi h M. ube culosis and induces immune
signa u es analogous o hose obse ed in clinical s udies
And ew D. Whi e
1
✉, Lau a Sibley
1
, Cha lo e Sa as
1
, Alexand a Mo ison
1
, Jennie Gullick
1
, Simon Cla k
1
, Fe gus Gleeson
2
,
An hony McIn y e
2
, Cecilia Lindes am A lehamn
3
, Alessand o Se e
3
, F ancisco J. Salgue o
1
, Emma Rayne
1
, Es eban Rod iguez
4
,
Eugenia Puen es
4
, Dominick Laddy
5
, Ann Williams
1
, Mike Dennis
1
, Ca los Ma in
6
and Sally Sha pe
1
A single in ade mal accina ion wi h MTBVAC gi en o adul hesus macaques was well ole a ed and con e ed a significan
imp o emen in ou come ollowing ae osol exposu e o M. ube culosis compa ed o ha p o ided by a single BCG accina ion.
Vaccina ion wi h MTBVAC esul ed in a significan educ ion in M. ube culosis in ec ion-induced disease pa hology measu ed using
in i o medical imaging, in g oss pa hology lesion coun s and pa hology sco es eco ded a nec opsy, he equency and se e i y o
pulmona y g anulomas and he equency o eco e y o iable M. ube culosis om ex apulmona y issues ollowing challenge.
The immune p ofiles induced ollowing immunisa ion wi h MTBVAC eflec hose iden ified in human clinical ials o MTBVAC.
E alua ion o MTBVAC- and TB pep ide-pool-specific T-cell cy okine p oduc ion e ealed a p edominan ly Th1 esponse om poly-
(IFN-γ
+
TNF-α
+
IL2
+
) and mul i-(IFN-γ
+
TNF-α
+
) unc ional CD4 T cells, while only low le els o Th22, Th17 and cy okine-p oducing
CD8 T-cell popula ions we e de ec ed oge he wi h low-le el, bu significan , inc eases in CFP10-specific IFN-γsec e ing cells. In
his epo , we desc ibe conco dance be ween immune p ofiles measu ed in clinical ials and a macaque p e-clinical s udy
demons a ing significan ly imp o ed ou come a e M. ube culosis challenge as e idence o suppo he con inued de elopmen
o MTBVAC as an e ec i e p ophylac ic accine o TB accina ion campaigns.
npj Vaccines (2021)6:4 ; h ps://doi.o g/10.1038/s41541-020-00262-8
INTRODUCTION
Tube culosis (TB) is he leading cause o dea h o humans om a
single in ec ious agen wo ldwide. I is es ima ed ha 10 million
people ell ill wi h TB in 2018 and TB was esponsible o 1.5
million dea hs
1
. In o al, 1.7 billion people a e es ima ed o be
la en ly in ec ed wi h TB, and 10% o hese indi iduals a e a high
isk o elapsing wi h ac i e disease du ing hei li e ime. The
eme gence o mul id ug- esis an and ex ensi ely d ug- esis an
s ains o Mycobac e ium ube culosis (M. ube culosis), oge he
wi h he geog aphical o e lap be ween he HIV and TB epidemics,
mean ha he e is an u gen need o be e con ol o TB.
Vaccina ion is he mos e ec i e way o con ol any in ec ious
disease; howe e , he only accine cu en ly a ailable agains TB,
Mycobac e ium bo is bacillus Calme e-Gué in (BCG), whils
e ec i e agains se e e mani es a ions o in an ube culosis
2
,is
only pa ially e ec i e agains adul pulmona y TB
3
.
Non-human p ima e (NHP) models p o ide he mos ele an
p e-clinical models o human disease and play a c i ical ole in
accine de elopmen . Macaque models o M. ube culosis ha e
been es ablished
4–7
wi hin which BCG has been shown o con e
low-le el e ficacy agains in ec ious challenge
8
, hus p o iding an
a ena in which new accine egimens could demons a e supe io
e ficacy ela i e o bo h un accina ed o BCG- accina ed indi i-
duals. Immune signa u es ha associa e wi h an imp o ed
ou come o challenges ha e been iden ified in macaque
s udies
8–10
, and because o he simila i ies be ween he macaque
and human immune sys ems and he esponse o TB in ec ion
11
,i
is conside ed ha such immune signa u es would be ele an o
humans. Thus a i has no been possible o e i y his assump ion
o alida e pu a i e co ela es o p o ec ion because he e ha e
been insu ficien pa allels be ween TB accine clinical ials and
p e-clinical accine e ficacy s udies showing imp o ed ou come in
he non-human p ima e model o compa isons o p o ide
meaning ul da a.
MTBVAC is a li e-a enua ed s ain o M. ube culosis de i ed
om a clinical isola e belonging o mode n lineage 4, which is
known o ha e a wo ldwide pa e n o dis ibu ion. MTBVAC was
designed o s imula e specific hos immune esponses mimicking
na u al TB in ec ion wi hou causing disease h ough a ional
a enua ion by dele ion o he majo i ulence genes phoP and
adD26
12
. MTBVAC con ains all an igens p esen in M. ube culosis,
including hose con ained in he RD1 egion, ha a e absen om
BCG and ha e been associa ed wi h imp o ed p o ec ion in
animal models
13
. Since 2012, MTBVAC has been he only li e-
a enua ed M. ube culosis-based accine candida e in clinical
ials. The sa e y and immunogenici y o MTBVAC we e demon-
s a ed in adul s in a Phase Ia ial
14
, and in neona es by a Phase Ib
ial conduc ed in an endemic coun y, which showed ha
MTBVAC is as sa e as BCG and mo e immunogenic
15
. MTBVAC is
now in Phase IIa dose-defining ials in bo h adolescen s and
newbo ns in Sou h A ica (NCT02933281 and NCT03536117).
Fu he mo e, da a om p e-clinical es ing in small animal models
sugges ha MTBVAC accina ion is sa e, immunogenic and has
he po en ial o enhance p o ec ion agains expe imen al M.
ube culosis challenge ela i e o BCG
12,16,17
. E alua ion o he
1
Public Heal h England, Na ional In ec ion Se ice, Po on Down, Salisbu y SP4 0JG, UK.
2
The Chu chill Hospi al, Heading on, Ox o d, UK.
3
La Jolla Ins i u e o Alle gy and
Immunology, La Jolla, CA, USA.
4
Bio ab i, Pon e e d a, Spain.
5
Ae as, Rock ile, MD 20850, USA.
6
G upo de Gené ica de Micobac e ias, Depa amen o Mic obiología, Uni e sidad
de Za agoza, IIS-A agón, CIBERES, Za agoza, Spain. ✉email: [email p o ec ed]
www.na u e.com/npj accines
Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences
1234567890():,;
immunogenici y and e ficacy o MTBVAC in he macaque M.
ube culosis challenge model would p o ide da a o accele a e and
assis p oduc de elopmen pa hway decisions o mo e o e ficacy
ials.
This s udy aimed o de e mine he p o ec i e e ficacy agains
low-dose ae osol challenge wi h M. ube culosis con e ed by a
single in ade mal accina ion wi h MTBVAC o hesus macaques;
o cha ac e ise he immune esponse induced ollowing accina-
ion and o compa e he immune signa u es defined in macaques
wi h hose defined in MTBVAC immunised humans.
RESULTS
Vaccina ion
All animals in he s udy showed he weigh gain p ofiles expec ed
in no mal heal hy animals du ing he pe iod p io o challenge
and we e unpe u bed by accina ion (Supplemen a y Fig. 1A).
Body empe a u e, e y h ocy e sedimen a ion a e (ESR) and ed
cell haemoglobin concen a ion le el emained wi hin he no mal
ange o he species du ing he pe iod be ween accina ion and
challenge in all indi iduals (Supplemen a y Fig. 1B–D). Mild
indu a ion and e y hema occu ed a he si e o immunisa ion in
all he animals ha ecei ed an in ade mal accina ion wi h BCG
and in fi e o he eigh macaques ha ecei ed in ade mal
accina ion wi h MTBVAC. The skin eac ions induced we e
compa able in size and esol ed wi hin six weeks a e MTBVAC
accina ion and be ween six and ou een weeks a e BCG
(Supplemen a y Fig. 1E).
M. ube culosis exposu e and challenge ou come
Twen y-one weeks a e accina ion, he macaques we e exposed
o ae osols con aining an a e age o 27 CFU ( ange 14–30 CFU) M.
ube culosis E dman p o iding an es ima ed median e ained dose
in he lung o 4 CFU. To moni o disease de elopmen , CT scans
we e collec ed 3, 8, 12 and 16 weeks a e ae osol exposu e and
disease bu den e alua ed using a quan i a i e sco e sys em based
on he ex en and ea u es o he disease isible. Whils disease
bu den was compa able ac oss g oups h ee weeks a e
challenge, a week 8, significan ly imp o ed ou comes we e
obse ed in he wo accina ed g oups ela i e o he un acci-
na ed g oup (Fig. 1a–d) (MTBVAC g oup: o al CT sco e: P=0.0044,
lung CT sco e: P=0.0050, numbe o pulmona y nodules: P=
0.0008, pneumonia bu den: P=0.0014; BCG g oup: o al CT sco e:
P=0.0238, lung CT sco e: P=0.0286, numbe o pulmona y
nodules: P=0.0068, pneumonia bu den: P=0.0039). A week 12
(Fig. 1 –i), he disease bu den in he MTBVAC g oup emained
significan ly lowe han in bo h he un accina ed g oup ( o al CT
sco e: P=0.0208, lung CT sco e: P=0.0249, pneumonia bu den:
P=0.0182), and he BCG g oup ( o al CT sco e: P=0.0073, lung
CT sco e: P=0.0407, pneumonia bu den: P=0.0350). A end o
educed incidence o lymph node in ol emen , iden ified as
enla gemen and/o nec osis, was seen in he g oup ha ecei ed
MTBVAC (Fig. 1j) in compa ison o ha seen in he g oups ha
ecei ed BCG, o emained un accina ed.
Du ing he pos -M. ube culosis-exposu e pe iod, one o he
BCG- accina ed animals and h ee o he un accina ed animals
showed changes in beha iou and clinical pa ame e s (weigh
loss, anaemia o dyspnoea) consis en wi h p og ession o he
ube culosis-induced disease ha me humane endpoin c i e ia
and was eu hanized ahead o he planned end o he s udy. None
Fig. 1 S udy schedule and in i o CT imaging. a Diag am showing he week in which clinical examina ions (open ci cle), blood sample
collec ions (shaded ci cle), CT scan collec ion, he ae osol challenge wi h M. ube culosis and nec opsy (black ci cle) we e conduc ed ela i e o
accina ion. Disease bu den de elopmen quan ified om CT scans eflec s sco es de i ed o o al disease bu den (b), pulmona y disease
bu den (c), pneumonia (d) and he numbe o TB-induced nodules in he lung (e) om CT scans collec ed ea weeks 3, 8, 12 and 16 a e
challenge wi h M. ube culosis. Box plo s show g oup median alues +/−IQR wi h minimum and maximum alues indica ed by box whiske s.
Incidence o lymph node disease ac oss he 16-week s udy pe iod. Non-pa ame ic Mann–Whi ney U es s we e used o compa ison be ween
g oups wi h unadjus ed esul s epo ed as: *P≤0.05; **P ≤0.005.
A.D. Whi e e al.
2
npj Vaccines (2021) 4 Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences
1234567890():,;
o he animals in he MTBVAC g oup, o he emaining animals in
he BCG and un accina ed g oups, showed ad e se beha iou al o
p emo em clinical indica o s a he ime o e mina ion. The
p opo ion o , and ime a which, animals p og essed o mee p e-
defined humane endpoin c i e ia we e plo ed and accina ion
g oups compa ed using he log- ank es (Fig. 2a). This analysis
indica ed ends o imp o ed con ol o disease p og ession
du ing he fi s 16 weeks ollowing TB in ec ion in animals ha
had been accina ed wi h BCG, o MTBVAC, in compa ison o he
un accina ed g oup (MTBVAC s un accina ed: P=0.063, BCG s
un accina ed: P=0.23).
A he end o he s udy, a ange o app oaches we e applied o
measu e he le el o ube culosis-induced disease bu den.
Changes in body weigh , measu ed a nec opsy ela i e o he
peak weigh measu ed du ing he pos -challenge pe iod, we e
appa en in un accina ed animals and animals ha had been
emo ed om he s udy due o disease p og ession c i e ia (Fig.
2b). TB-associa ed, pa hological changes we e quan ified a
nec opsy using a g oss pa hology sco e sys em (Fig. 2c) and
e ealed ha g oss pa hology was significan ly educed in he
g oup ha ecei ed MTBVAC compa ed o macaques accina ed
wi h BCG ( o al pa hology sco e: P=0.005; lung pa hology sco e:
P=0.021; dissemina ed pa hology sco e: P=0.004), and un acci-
na ed macaques ( o al pa hology sco e: P=0.015; lung pa hology
sco e: P=0.003; dissemina ed pa hology sco e: P=0.003).
Pulmona y disease bu den measu ed by he manual coun ing
o bo h disc e e and coalesced lesions om se ial sec ions
e ealed significan ly ewe mac oscopic lesions in he lungs om
he MTBVAC- accina ed g oup compa ed o ei he he BCG-
accina ed g oup (P=0.04) o he un accina ed g oup (P=0.025)
(Fig. 2d). Mic oscopic g anulomas we e iden ified in H&E-s ained
sec ions p epa ed om ep esen a i e lung lobe and ex apul-
mona y issue samples, classified in o six s ages and coun ed.
G anulomas we e mos p e alen in he pulmona y issues om
he un accina ed g oup and significan ly less abundan in he
g oup ha ecei ed MTBVAC (P=0.02) (Fig. 2e). Compa ison o
No accine
BCG
MTBVAC
0
5
10
15
20
200
400
600
nuoc noiseL
)de adilosnoc + e e csiD(
*
*
No accine
BCG
MTBVAC
0
50
100
150
Sco e
*
**
To al sco e
No accine
BCG
MTBVAC
**
*
Lung sco e
No accine
BCG
MTBVAC
**
*
To al Dissemina ed
051015
0
25
50
75
100
Weeks
Pe cen di
sease p og ession
No accine
BCG
MTBVAC
No accine s BCG: 0.23
s MTBVAC: 0.06
BCG s MTBVAC: 0.32
p- alue (unadjus ed)
Con ols
BCG
MTBVAC
0
10
20
30
40
Numbe o samples
LLOD
>LLOD
**
*
a
0255075100
MTBVAC
BCG
No accine
S age I
S age II
S age III
S age IV
S age V
S age VI
Summed g anuloma coun
No accine
BCG
MTBVAC
0
50
100
150
200
300
400
500
Summed g anuloma coun
*
No accin e
BCG
MTBVAC
-30
-20
-10
0
10
%change om peak pos challenge
No accine
BCG
MTBVAC
No accine
BCG
MTBVAC
0
2
4
6
Log
10
CFU
*
LALN Ex a-pulmona y
bc
de
hi
g
02505007501000
MTBVAC
BCG
No accine
S age I
S age II
S age III
S age IV
S age V
S age VI
Summed g anuloma coun
***
***
***
***
Fig. 2 Tube culosis-induced disease bu den. a Kaplan–Meie plo showing he de elopmen o p og essi e disease o a le el ha me
humane endpoin c i e ia in accina ed and un accina ed macaques a e challenge wi h M. ube culosis. Unadjus ed P alues om log- ank
compa isons a e shown. bChange in body weigh exp essed as a pe cen age o he peak weigh measu ed du ing he pos -challenge s udy
pe iod. cTo al, pulmona y and dissemina ed (spleen, li e , kidneys) ube culosis-induced disease bu den measu ed using a g oss pa hology
sco e sys em. dThe numbe o mac oscopic lesions in he lungs ollowing se ial sec ioning. eTo al numbe o g anulomas (s age I–VI
combined) iden ified in he lung om ep esen a i e H&E-s ained sec ions. To al numbe o g anulomas (s age I–VI combined) iden ified in
he lung om ep esen a i e H&E-s ained sec ions whe e s acked ba s indica e he numbe o g anulomas a each s age I–VI and he
combined o al wi hin each expe imen al g oup. gTo al numbe o g anulomas (s age I–VI combined) iden ified in he ex apulmona y issues
(spleen, li e , kidneys) om ep esen a i e H&E-s ained sec ions. hThe p opo ion o issue samples cul u ed om which M. ube culosis was
isola ed (>LLOD: CFU alue g ea e han he lowe limi o de ec ion o he assay; LLOD: alue eco ded as he lowe limi o de ec ion o he
assay). iBac e ial bu den de e mined in lung-associa ed lymph nodes (LALN) and ex apulmona y issues. The colou and symbol coding pe
indi idual is consis en h oughou and be ween figu es. Upside down iangula symbols indica e animals in which disease p og essed o
mee humane endpoin c i e ia. Non-pa ame ic Mann–Whi ney U es s we e used o g oup-wise compa isons o pa hology sco es,
g anuloma/lesion coun s and iable CFU coun s eco e ed om issues, wi h unadjus ed esul s epo ed as: *P≤0.05; **P ≤0.01.Χ
2
es s we e
used o compa e he p opo ion o issues wi h iable M. ube culosis CFU coun s abo e o below he LLOD o he assay be ween accina ion
g oups and Coch an–A mi age me hod Χ
2
es s o compa e g anuloma s age sco es be ween g oups, unadjus ed esul s a e epo ed as *P≤
0.05; **P ≤0.01; ***P ≤0.001.
A.D. Whi e e al.
3
Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences npj Vaccines (2021) 4
he combined numbe o each ype o g anuloma iden ified in he
sec ions examined om pulmona y (Fig. 2 ) and ex apulmona y
(Fig. 2g) issues e ealed g anulomas o all s ages we e obse ed
in all g oups, bu he numbe and dis ibu ion a ied amongs
g oups. Type V and ype VI g anulomas, ep esen ing mo e
ad anced lesion de elopmen , we e mos nume ous in he
un accina ed g oup making up app oxima ely hal o he
g anulomas obse ed in issue sec ions; by con as , ype V and
ype VI g anulomas made up a smalle p opo ion o he
g anulomas obse ed in he g oup ha ecei ed MTBVAC. The
numbe o acid- as bacilli (AFB) associa ed wi h each g anuloma
inc eased alongside he de elopmen o g anulomas. AFB we e
no obse ed wi hin ype I o II g anulomas collec ed om any o
he macaques and only a low numbe s in g anulomas o ype III,
IV and V. The majo i y o AFB we e ound wi hin he nec o ic co es
o ype VI g anulomas. AFB we e mos p e alen in g anulomas
om he un accina ed g oup and less abundan in hose om
accina ed g oups, wi h he ewes coun ed in hose om he
MTBVAC- accina ed g oup (Supplemen a y Table 1).
The spleen, kidneys, li e and lung-associa ed lymph nodes
(LALN) we e sampled o he p esence o iable M. ube culosis
pos -mo em. Bac e ia we e cul u ed om significan ly ewe o
he samples collec ed om he macaques gi en MTBVAC han
om hose collec ed om he BCG- accina ed g oup (P=0.011)
o he un accina ed con ol g oup (P=0.003) (Fig. 2h). While he
bac e ial bu den measu ed in he LALN was simila ac oss all he
s udy g oups, he bu den measu ed in he ex apulmona y issues
collec ed om MTBVAC- accina ed (P=0.0337) and he BCG-
accina ed animals was educed compa ed o ha measu ed in
issues om un accina ed animals (Fig. 2i).
Immune esponses ollowing accina ion and in ec ion
The mycobac e ium-specific IFN-γ esponse induced by accina-
ion and M. ube culosis challenge was measu ed using an ex i o
ELISpo assay applied a 2-week in e als h ough he s udy (Fig.
3a). In he pe iod be o e he challenge, PPD-specific IFN-γspo
o ming uni (SFU) equencies significan ly inc eased abo e he
h eshold in all macaques ha ecei ed accina ions wi h BCG o
MTBVAC (BCG: P=0.0002; MTBVAC: P=0.0002). The peak PPD-
specific esponse occu ed ou weeks a e accina ion in he
MTBVAC g oup and 2 weeks la e in he BCG- accina ed g oup a
week 6. Twen y weeks a e accina ion and 1 week be o e
challenge wi h M. ube culosis, PPD-specific IFN-γSFU equencies
measu ed in he accina ed g oups we e significan ly highe han
he le els measu ed in he un accina ed g oup (BCG: P=0.0006;
MTBVAC: P=0.0019). CFP10-specific IFN-γSFU equencies
exceeding he assay h eshold be ween accina ion and challenge
we e only de ec ed in he MTBVAC g oup and consequen ly, he
esponse was significan ly highe han hose in he BCG-
accina ed (P=0.0011) and he un accina ed g oups (P=
0.0033), wi h he la ges esponses seen be ween 6 and 10 weeks
(Fig. 3b). Du ing he same pe iod be ween accina ion and
challenge, ESAT6-specific SFU equencies did no inc ease abo e
backg ound le els in any o he es g oups. Following exposu e o
M. ube culosis, educed equencies o PPD, CFP10 and ESAT6-
specific SFU we e seen in he MTBVAC and BCG- accina ed g oups
ela i e o he un accina ed g oup, wi h he di e ences eaching
significance o PPD and ESAT6 in he MTBVAC- accina ed g oup
(PPD: P=0.0030; ESAT6: P=0.0030) and o PPD and CFP10 in he
BCG- accina ed g oup (PPD: P=0.0104; CFP10: P=0.0070). Fou -
een weeks a e he challenge, he MTBVAC g oup showed
significan ly lowe le els o PPD-, CFP10- and ESAT6-specific SFU
in he pe iphe y han hose eco ded in he BCG- accina ed g oup
(PPD:P=0.0379; CFP10:P=0.0148; ESAT6: P=0.0148) which
could be in e p e ed as be e con ol o he M. ube culosis
in ec ion.
Mul ipa ame e flow cy ome y assays we e applied o explo e
he pheno ype and unc ional p ofile o he cellula immune
esponse. Applica ion o in acellula cy okine s aining o measu e
an igen-specific cy okine p oduc ion e ealed inc eased equen-
cies o mul i unc ional CD4 T-cells p oducing IFN-γ, IL-2 and TNF-α
(poly unc ional), o IFN-γand TNF-α, in animals ha ecei ed BCG
o MTBVAC, wi h he equency o cy okine-p oducing cells
eaching significance abo e p e- accina ion le els om ou
weeks a e accina ion (Fig. 4a, b and Supplemen a y Fig. 2A,
B). Cy okine-p oducing CD4 T-cell popula ions emained
unchanged in he un accina ed g oup consis en wi h he lack
o expe imen al in e en ion applied o his g oup a his s age o
he expe imen (Fig. 4c and Supplemen a y Fig. 2C). Compa ison
o mul i unc ional T-cell equencies be ween he accina ion
g oups indica ed ha significan ly highe equencies o CD4 T-
cells p oducing IFN-γ, IL-2 and TNF-α(Fig. 4d); o IFN-γand TNF-α
(compa a i e plo no shown) simul aneously we e p esen in he
BCG- and MTBVAC- accina ed g oups ela i e o he un accina ed
animals a weeks 4, 8, 12 and 18. Changes in he equency o
an igen-specific CD4 T-cells p oducing IL-17 o IL-22 we e no
de ec ed ollowing accina ion and di e ences we e no appa en
be ween he accina ion g oups (Fig. 4e, ), indica ing ha bo h
BCG and MTBVAC accina ion induced a p edominan ly Th1 CD4
T-cell esponse. Vaccina ion-induced cy okine-p oducing CD8 T-
cell popula ions we e de ec ed a a low equency ela i e o he
CD4 T-cell subse s and gene ally did no inc ease significan ly
abo e p e- accina ion le els (Supplemen a y Figs. 3 and 4).
Simila ly, po en ial accina ion-induced changes in he equency
o inna e lymphoid cells we e explo ed by quan ifica ion o
mucosal-associa ed in a ian T cells (MAIT’s) and na u al kille (NK)
cell subse s. Howe e , MAIT cell equencies de ec ed in BCG,
MTBVAC and un accina ed g oups emained a low equency
(g oup medians below 1% o he o al T-cell popula ion) ollowing
accina ion (Supplemen a y Fig. 5A), and NK cell popula ions
(iden ified by he pa e n o CD56 and CD16 exp ession) did no
di e significan ly a any ime poin in compa ison o p e-
accina ion le els o be ween he accina ion g oups (Supple-
men a y Fig. 5B). Fu he mo e, cy okine p oduc ion om he NK
cell popula ion as a whole ( ega dless o CD56 and CD16
exp ession) was assessed (Supplemen a y Fig. 5C–H), bu sig-
nifican di e ences we e no de ec ed in he o al equency o NK
cells p oducing IFN-γ, IL-2, TNF-α, IL-17 o IL-22 ollowing MTBVAC
o BCG accina ion in compa ison o p e- accina ion le els.
An igen-specific cy okine p oduc ion by γδ T-cell popula ions
was ound o p ima ily consis o TNF-αand IL-17 p oducing cells,
wi h highe equencies de ec ed in esponse o s imula ion wi h
MTBVAC a he han pooled M. ube culosis-de i ed pep ides
(Supplemen a y Figs 6 and 7). T ends o a accine-induced
inc ease in he equency o cy okine-p oducing γδ T-cell
popula ions we e appa en in bo h he BCG and MTBVAC-
accina ed g oups; howe e , hese esponses ailed o each
significance abo e p e- accina ion le els (Supplemen a y Figs 6
and 7). Compa ison o IL-17-p oducing γδ T-cell equencies
be ween g oups a each ime poin ollowing accina ion
(Supplemen a y Fig. 6) indica ed a end o inc eased p oduc ion
o IL-17 a weeks 4 and 8 ollowing MTBVAC accina ion, whe eas
by weeks 12 and 18 g ea e equencies we e measu ed in he
BCG- accina ed g oup. Despi e hese ends, he di e ences
measu ed be ween he accina ion g oups did no each s a is ical
significance. Ne e heless, as a la ge p opo ion o he γδ T-cell
popula ion p oduced an igen-specific IL-17 ollowing accina ion,
which was in con as o he low equency o IL-17-p oducing
CD4 and CD8 T cells, his indica es a po en ially impo an ole o
he γδ T-cell subse in he p oduc ion o his impo an
immunomodula o y cy okine.
A.D. Whi e e al.
4
npj Vaccines (2021) 4 Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences
DISCUSSION
In ade mal accina ion wi h MTBVAC was well ole a ed by adul
hesus macaques and con e ed a significan imp o emen in
ou come ollowing ae osol exposu e o M. ube culosis compa ed
o ha p o ided by BCG accina ion. S a is ically significan
di e ences in disease bu den de elopmen we e demons a ed
be ween g oups measu ed using a sco ing sys em applied o CT
scan images. This app oach e ealed he disease bu den in he
MTBVAC g oup o be significan ly educed compa ed o ha in
he un accina ed con ol g oup 8 weeks a e challenge and
significan ly educed in compa ison o he BCG- accina ed g oup
by week 12. This finding was eflec ed in he esul s o he
analyses applied a he end o he s udy schedule, 16 weeks a e
challenge, when measu es based on he assessmen o mac o-
scopic and mic oscopic pa hology and bac e ial bu den e ified
an imp o ed ou come a e challenge in he MTBVAC- accina ed
g oup ela i e o he BCG- accina ed and he un accina ed
g oups. Fu he mo e, a e he challenge, highe le els o CFP10
and ESAT6-specific IFN-γ-sec e ing cells we e de ec ed in he
un accina ed ela i e o he accina ed g oups wi h he lowes
le els p esen in he MTBVAC g oup. The associa ion be ween
inc eased equencies o CFP10 and ESAT6-specific esponses
pos -challenge wi h he poo e pos -challenge ou come is
consis en wi h o he epo s and sugges s ha he equency o
esponses o hese diagnos ic an igens p o ides a bioma ke
eflec i e o disease p og ession and an igen load. The hesus
macaque p o ides a s ingen model, in which only a ew accines
ha e con e ed significan imp o emen s in ou come a e M.
ube culosis challenge
8–10,18–20
and only one o he new TB accine
has a o ded significan ly supe io p o ec ion compa ed o
in ade mally deli e ed BCG
19
. Taken oge he , hese da a suppo
he con inued de elopmen o MTBVAC as a mo e e ec i e
p ophylac ic accine o TB accina ion campaigns.
The immune p ofiles induced ollowing immunisa ion wi h
MTBVAC eflec hose iden ified in human clinical ials
14,15,17
.
E alua ion o MTBVAC- and TB pep ide-pool-specific T-cell
cy okine p oduc ion e ealed a p edominan ly Th1 esponse
p ofile om poly- (IFN-γ
+
TNF-α
+
IL2
+
) and mul i- (IFN-γ
+
TNF-α
+
)
unc ional CD4 T cells, while only low le els o Th22, Th17 and
cy okine-p oducing CD8 T-cell popula ions we e de ec ed. As in
clinical ials, hese p ofiles we e simila in p ofile and scale o
hose iden ified a e BCG accina ion; al hough di e ences in
an igen ecogni ion we e de ec ed ha eflec p o ec i e immu-
nological signa u es epo ed om small animal s udies
17
, which
No accine
BCG
MTBVAC
0
500
1000
1500
2000
2500
IF N yELISP O T CFP10 AUC
**
**
-10-5 0 5 101520253035
0
1000
2000
3000
4000
6000
SFU pe million cells
M. b
MTBVAC
-10-5 0 5 101520253035
0
1000
2000
3000
4000
6000
M. b
BCG
-10-5 0 5 101520253035
0
1000
2000
3000
4000
6000
M. b
-10 -5 0 5 10 15 20 25 30 35
0
100
200
300
400
500
2000
4000
SFUpe million cells
M. bMTBVAC
-10-5 0 5 101520253035
0
100
200
300
400
500
2000
4000
M. bBCG
-10-5 0 5 101520253035
0
100
200
300
400
500
2000
4000
M. b
-10-5 0 5 101520253035
0
100
200
300
400
500
2000
4000
Week
SFU pe million cells
M. bMTBVAC
-10-5 0 5 101520253035
0
100
200
300
400
500
2000
4000
Week
M. bBCG
-10-5 0 5 101520253035
0
100
200
300
400
500
2000
4000
Week
M. b
MTBVAC g oup BCG g oup No accine g oup
a.
PPD
ESAT-6
CFP-10
b.
Fig. 3 Immune esponse o accina ion and challenge. The equency o M. ube culosis an igen-specific IFN-γ-sec e ing cells induced
ollowing BCG accina ion and challenge measu ed by ELISpo (a). Top ow: PPD-specific esponse p ofiles, middle ow: ESAT6-specific
esponse p ofiles, bo om ow: CFP10-specific esponse p ofiles. Le : esponse p ofiles in he MTBVAC- accina ed g oup; middle: esponse
p ofiles in he BCG- accina ed g oup; igh : esponse p ofiles in he un accina ed g oup. Vaccina ion wi h MTBVAC, o BCG indica ed by he
do ed line, and ae osol exposu e o M. ube culosis indica ed by he dashed line a week 21. Compa ison o he CFP10-specific IFN-γ esponse
measu ed by ELISpo induced ollowing accina ion wi h BCG o MTBVAC de e mined by analysis o he a ea unde he esponse cu e
be ween week 2 and 20 a e accina ion wi h g oup compa ison made using Mann–Whi ney be ween g oups **P≤0.005 (b). All line g aphs
show indi idual esponse (ci cula symbol) and G oup median esponse (solid line). The colou and symbol coding pe indi idual is consis en
h oughou and be ween figu es. Upside down iangula symbols indica e animals in which disease p og essed o mee humane endpoin
c i e ia. The assay h eshold (mean o p e- accina ion alues plus 1.5× s anda d de ia ion) is indica ed by a ho izon al do ed line (PPD:
119 spo o ming uni s (SFU) pe million cells; ESAT6: 40 SFU pe million cells; CFP10: 28 SFU pe million cells).
A.D. Whi e e al.
5
Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences npj Vaccines (2021) 4

included he de ec ion o low-le el CFP10-specific IFN-γ esponses
ollowing MTBVAC, bu no BCG, accina ion. This is analogous o
he findings o Aguilo e al.
17
, who ha e demons a ed a link
be ween MTBVAC-induced CFP10 and ESAT6-specific IFN-γsec e-
ion and imp o ed p o ec ion agains M. ube culosis challenge in
he C3H/HeNRj mouse s ain, an imp o emen ha was nega ed
when mice we e accina ed wi h he ESAT6 and CFP10 abla ed
MTBVAC-ΔE6C10 cons uc . Low-le el, bu significan , inc eases in
CFP10-specific IFN-γ-sec e ing cells ha e also been de ec ed in
MTBVAC- accina ed human olun ee s; al hough, in con as o
mu ine s udies, ESAT6-specific cy okine p oduc ion did no
inc ease significan ly in his popula ion
14,17
. This p ofile o an igen
specifici y was mi o ed di ec ly by he cellula immune esponses
measu ed in macaques whe e significan inc eases in CFP10-, bu
no ESAT6-, specific IFN-γsec e ing cells we e de ec ed ollowing
MTBVAC accina ion. This no only p o ides u he e idence ha
a low-le el CFP10-specific immune esponse is a key ea u e o he
MTBVAC-induced cell-media ed esponse bu is also a alida ion
ha he immunogenici y p ofiles measu ed in macaques a e
ep esen a i e o immune signa u es obse ed in humans. This
conco dance be ween immune p ofiles measu ed in clinical ials
and a macaque p e-clinical assessmen o a no el TB accine
candida e demons a ing significan ly imp o ed ou come a e M.
ube culosis challenge is a p omising indica ion ha he imp o ed
p o ec ion p o ided by MTBVAC accina ion o macaques and
mice will ansla e o he human popula ion.
METHODS
Expe imen al animals
Twen y- ou male and emale hesus macaques (Macaca mula a) o Indian
o igin aged be ween 4.2 and 5.2 yea s o age we e sou ced om an
es ablished, cha ac e ised, closed UK b eeding colony. Compa ible social
g oups we e housed in acco dance wi h Home O fice (UK)
21
and NC3Rs
guidelines
22
in cages wi h high-le el obse a ion balconies, ex ensi e
en i onmen al s imula ion and p o ided wi h a wide ange o die a y
en ichmen
23
. Animal p ocedu es and s udy designs we e app o ed by he
Es ablishmen o Animal Wel a e and E hical Re iew Commi ee and
au ho ised unde a UK Home O fice p ojec licence. P io o challenge wi h
M. ube culosis macaques we e housed in cages app oxima ely 2.5-m high
by 4-m long by 2-m deep, cons uc ed wi h high-le el obse a ion
balconies and wi h a floo o deep li e o allow o aging. Following he
challenge, animals we e ans e ed o banks o cages placed in di ec ional
ai flow con ainmen sys ems ha allowed g oup housing and en i on-
men al con ol whils p o iding a con inuous, s anda dised inwa d flow o
ully condi ioned esh ai iden ical o all g oups. Addi ional en i onmen al
en ichmen was a o ded by he p o ision o oys, swings, eeding puzzles
and DVDs o isual s imula ion. In addi ion o s anda d old-wo ld p ima e
pelle s, he die was supplemen ed wi h a selec ion o esh ege ables and
ui . Fo each p ocedu e, seda ion was applied by in amuscula injec ion
wi h ke amine hyd ochlo ide (10 mg/kg) (Ke ase , Fo Dodge Animal
Heal h L d, Sou hamp on, UK). None o he animals had been used
p e iously o expe imen al p ocedu es, and each socially compa ible
g oup was andomly assigned o s udy ea men . P io o s udy
en olmen , p e ious exposu e o mycobac e ial an igens was assessed
using an IFN-γELISPOT (MabTech, Nacka. Sweden) o de ec esponses o
Fig. 4 M. ube culosis pep ide-specific CD4 T-cell cy okine sec e ion p ofiles measu ed by whole blood in acellula cy okine s aining. Box
plo s show he accina ion g oup median equency o CD4 T-cells p oducing combina ions o he cy okines IFN-γ, IL-2 and TNF-α(a–d); IL-17
(e) o IL-22 ( )+/−IQR wi h minimum and maximum alues indica ed by box whiske s. Cy okine p oduc ion was measu ed a weeks p io o
(−1), and ollowing (4, 8, 12 and 18) BCG o MTBVAC accina ion. Significan di e ences be ween p e- and pos - accina ion alues (Wilcoxon
signed- ank) and be ween g oups (Mann–Whi ney U es ) a e indica ed by ba s and as e isks: *P≤0.05, **P≤0.01, ***P≤0.001 (all P alues a e
unadjus ed o mul iple compa isons). F equencies measu ed in indi idual animals a e ep esen ed by do s.
A.D. Whi e e al.
6
npj Vaccines (2021) 4 Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences
ube culin-PPD (SSI, Copenhagen, Denma k), and pooled 15-me pep ides
o ESAT6 and CFP10 (Pep ide P o ein Resea ch LTD, Fa eham, U.K.).
Vaccina ion
The accina ion schedule ela i e o he ae osol challenge is shown in Fig.
1a. The sample size o he expe imen was de e mined using o al
pa hology sco e as he p ima y e ficacy eadou and was powe ed o
de ec a nine-poin educ ion (a bi a y uni s) wi h a powe o 80% and an
αo 0.05. Eigh o he macaques we e immunised by in ade mal (ID)
injec ion in he uppe le a m wi h 100 μl o BCG accine, Danish s ain
1331 (1s WHO e e ence eagen , NIBSC, UK), he second g oup o eigh
macaques ecei ed an ID accina ion wi h 100 μl o MTBVAC (Bio ab i,
Spain) and he emaining eigh animals we e le un ea ed as a nega i e
con ol g oup. The BCG and MTBVAC accines we e p epa ed o
in ade mal adminis a ion acco ding o he manu ac u e ’s ins uc ions
o adminis a ion o humans. One ml o Sau ons diluen was added o a
ial o BCG accine o gi e a suspension o BCG a an es ima ed
concen a ion o be ween 2 and 8 × 10
6
CFU/ml and 1 ml o s e ile wa e
was added o MTBVAC o gi e a concen a ion o 3–17 × 10
6
CFU. Due o
di e ences in accine diluen solu ions, i was no possible o deli e a
mock accina ion o animals in he un accina ed con ol g oup.
Vaccina ions we e adminis e ed wi hin 1 h o accine econs i u ion. Pla e
cul u e o esidual accine confi med he iabili y o he BCG and MTBVAC
accines, and he mean a e age adminis e ed dose o each accine was
calcula ed o be 1.2 × 10
6
(SD 3.4 × 10
4
) CFU and 8.2 × 10
5
(SD 3.9 × 10
4
)
CFU o BCG and MTBVAC espec i ely. The injec ion si es we e moni o ed
o local eac ions a e accina ion wi h BCG o MTBVAC such ha
eac ions we e measu ed and assessed.
M. ube culosis challenge s ain
TheE dmanK01s ock(HPA-Sep 2011)used o hechallengewas
p epa ed om s ocks o he M. ube culosis E dman s ain K01 (BEI
Resou ces). A s ock suspension was ini ially p epa ed om a 5-ml
bac e ial s a e cul u e o iginally gene a ed om colonies g own on
Middleb ook 7H11 supplemen ed wi h oleic acid, albumin, dex ose and
ca alase (OADC) selec i e aga (BioMe ieux, UK). A liquid ba ch cul u e
was hen g own o loga i hmic g ow h phase in 7H9 medium (Sigma-
Ald ich, UK) supplemen ed wi h 0.05% ( / ) Tween 80 (Sigma-Ald ich,
UK). Aliquo s we e s o ed a −80 °C. The concen a ion o colony-
o ming uni s (CFU)/ml in he s ock suspension was de e mined om
hawed aliquo s by enume a ion o CFU cul u ed on Middleb ook 7H11
OADC selec i e aga .
Ae osol exposu e
Twen y-one weeks a e accina ion, all animals we e challenged by
exposu e o ae osols o M. ube culosis, as p e iously desc ibed
24,25
. Mono-
dispe sed bac e ia in pa icles we e gene a ed using a h ee-je Collison
nebulise (BGI) and, in conjunc ion wi h a modified Hende son appa a us
26
,
deli e ed o he na es o each seda ed p ima e ia a modified e e ina y
anaes he ic mask. Challenge was pe o med on seda ed animals placed
wi hin a ‘head-ou ’, ple hysmog aphy chambe (Buxco, Wilming on, No h
Ca olina, USA) o enable he ae osol o be deli e ed simul aneously wi h
he measu emen o espi a ion a e and espi ed olume. The calcula ions
o de i e he p esen ed dose (PD) ( he numbe o o ganisms ha he
animals inhale) and he e ained dose ( he numbe o o ganisms assumed
o be e ained in he lung) ha e been desc ibed p e iously
27,28
. A nebulise
concen a ion was selec ed o esul in a e ained dose o app oxima ely
fi e iable CFU.
Clinical p ocedu es
Full clinical examina ions we e conduc ed a 2-week in e als h oughou
he s udy. Macaques we e seda ed, ho acic adiog aphs we e aken, body
weigh and empe a u e measu ed and blood samples collec ed. Red
blood cell (RBC) haemoglobin le els we e measu ed using a HaemaCue
haemoglobinome e (Haemacue L d, D onfield, UK), and ESR was
de e mined using he Sediplas sys em (Gues Medical, Edenb idge, UK).
Obse ed beha iou was moni o ed o con a-indica o s. The ime o
nec opsy, i p io o he end o he planned s udy pe iod, was de e mined
by expe ienced p ima ology s a and based on a combina ion o he
ollowing ad e se indica o s: dep ession o wi hd awn beha iou , abno -
mal espi a ion (dyspnoea), loss o 20% o peak pos -challenge body
weigh , ESR le els ele a ed abo e no mal (>20 mm), haemoglobin le el
below no mal limi s (<100 g/dL), inc eased empe a u e (>41 °C) and
abno mal findings on he ho acic adiog aphs.
Compu ed omog aphy (CT) imaging
CT scans we e collec ed om seda ed animals using a 16-slice Ligh speed
CT scanne (Gene al Elec ic Heal hca e, Milwaukee, WI, USA) 3, 8, 12 and
16 weeks a e ae osol exposu e o M. ube culosis, as desc ibed
p e iously
24
. To acili a e ull examina ion o lesions and lymph nodes,
Niopam 300 (B acco, Milan, I aly), a non-ionic, iodina ed con as medium,
was adminis e ed in a enously (IV) a 2 ml/kg body weigh . Scans we e
e alua ed by an expe ho acic adiologis blinded o he animal’s
ea men and clinical s a us, o he numbe and dis ibu ion ac oss lung
lobes o pulmona y lesions and he p esence o disease ea u es, such as
nodula conglome a ion, ca i a ion, consolida ion (an indica o o al eola
pneumonia), a ‘ ee-in–bud’pa e n (an indica o o b onchocen ic
pneumonia) and lobula collapse. The ai ways we e e alua ed o he
occu ence o wall hickening and he p esence o b onchocele. The lymph
nodes we e assessed o enla gemen and he p esence o nec osis.
Ex apulmona y issues, including li e , kidneys and spleen we e examined
o he p esence o single o mul iple oci o disease, ca i a ion o nec osis.
The disease bu den a ibu able o in ec ion wi h M. ube culosis was
sco ed using a ela i e sco ing sys em based on he numbe o lesions
p esen in lungs, spleen, li e , kidney and lymph nodes, and he p esence
and ex en o TB-induced s uc u al abno mali ies as desc ibed p e-
iously
23
. The sco es o consolida ion and ee-in-bud we e summed o
p o ide a pneumonia sco e. The sco es a ibu ed o each issue (lung lobe,
o gan, lymph node) we e summed o gi e he o al CT sco e o p o ide a
measu e o pulmona y and ex apulmona y disease bu den.
In e e on-gamma (IFN-γ) ELISpo
Pe iphe al blood mononuclea cells (PBMC) we e isola ed om hepa in
an i-coagula ed blood using s anda d me hods. An IFN-γELISpo assay was
used o quan i y he numbe mycobac e ia-specific IFN-γ-p oducing T cells
in PBMCs using a human/simian IFN-γki (MabTech, Nacka. Sweden), as
desc ibed p e iously
24
. In b ie , 2 × 10
5
PBMCs we e cul u ed wi h 10 μg/ml
PPD (SSI, Copenhagen, Denma k) o pools o o e lapping 15-me pep ides
spanning CFP10 o ESAT6 (Pep ide P o ein Resea ch L d, Wickham, UK) in
duplica e, o wi hou an igen, in quad uplica e, and incuba ed o 18 h.
Pho bol 12-my is a e (Sigma-Ald ich Do se , UK) (100 ng/ml) and ionomy-
cin (CN Biosciences, No ingham, UK) (1 μg/ml) we e used as a posi i e
con ol. A e cul u e, spo s we e de eloped acco ding o he manu ac-
u e ’s ins uc ions. Pla es we e scanned, and spo s enume a ed using a
CTL Immunospo S6 eade and so wa e. De e mina ions om eplica e
es s we e a e aged, and he da a we e analysed by sub ac ing he mean
numbe o spo s in he medium-only con ol wells om he mean coun s
o spo s in wells wi h an igen, o pep ide pools, o de i e an an igen-
specific spo coun . This alue was mul iplied by a ac o o fi e and
epo ed as IFN-γSFU equency pe million PBMCs. A minimum h eshold
le el o ac i i y was calcula ed o each mycobac e ial an igen by analysis
o he dis ibu ion o SFU equencies quan ified p io o mycobac e ial
accina ion o challenge (baseline SFU). Th eshold alues o 119 SFU
(mean +1.5 SD) o PPD-; 28 SFU (mean +2.0 SD) o CFP10-; and 40 SFU
(mean +2.0 SD) o ESAT6-specific SFU we e selec ed o encompass he
95 h pe cen ile o SFU equencies measu ed a baseline. Only esponses
exceeding hese h esholds we e conside ed as posi i e. An igen-specific
IFN-γSFU p ofiles we e plo ed using G aphpad 7.0 (G aphpad Inc, USA)
and used o calcula e a ea unde he cu e (AUC) alues o compa ison o
accina ion g oup median AUC by Mann–Whi ney U es .
Whole blood in acellula cy okine s aining (WB ICS) assay
In i o s imula ion. The me hod used o s imula e blood cells o ICS was
closely aligned wi h p o ocols applied in MTBVAC clinical ials conduc ed
by he Sou h A ican Tube culosis Vaccine Ini ia i e (SATVI)
15
. In b ie , WB
ICS was pe o med using 450 μl o sodium hepa in (Sigma-Ald ich, UK)
an i-coagula ed blood incuba ed o a o al o 12 h wi h 0.25 μg/ml o an i-
CD28 and an i-CD49d co-s imula o y an ibodies (bo h om BD Biosciences,
UK) and 1 μg/ml o selec ed M. ube culosis pep ides (TB Mega-pool
p o ided by C. Lindes am-A lehamn, La Jolla Ins i u e o Alle gy and
Immunology, USA), 1 × 10
6
CFU/ml o MTBVAC (Bio ab i, Spain), 5 μg/ml o
s aphylococcus en e o oxin B (SEB) as a posi i e con ol (Sigma-Ald ich,
UK), o s e ile phospha e bu e saline (PBS) as a nega i e con ol (Se e n
Bio ech, UK). An igenic s imula ion was ini ia ed wi hin 2 h o blood sample
collec ion and p oceeded o 7 h in a 37 °C wa e ba h be o e 50 μl o
A.D. Whi e e al.
7
Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences npj Vaccines (2021) 4
plasma was emo ed om each sample and ozen a −80 °C as a esou ce
o u u e in e oga ion o sec e ed bioma ke s. The p o ein anspo
inhibi o B e eldin-A (Sigma-Ald ich, UK) was added o he mix u e a a
final concen a ion o 10 μg/ml, and he samples e u ned o incuba ion
condi ions o a u he 5 h. Following he comple ion o he incuba ion
pe iod, EDTA solu ion (Sigma-Ald ich, UK) was added a a concen a ion o
2 mM o de ach adhe en cells. A e 15 min, he sample was dilu ed 1:10 in
FACSlyse solu ion (BD Biosciences, UK) and incuba ed o 15 min o emo e
ed blood cell con amina ion. Finally, he cellula ac ion o he sample
was sepa a ed by cen i uga ion and esuspended in 1 ml o c yosolu ion
consis ing o 30% oe al cal se um (Lab ech,UK), 20% DMSO and 50%
RPMI cul u e medium (bo h om Sigma-Ald ich, UK), be o e eezing unde
a con olled cooling a e and c yop ese a ion in liquid ni ogen
apou phase.
Samples we e hawed in ba ches o flow cy ome y analysis such ha
all he samples om an indi idual animal we e s ained wi h an ibodies and
analysed on he same occasion. The c yop ese ed ma e ial was apidly
hawed a 37 °C and washed wice by en old dilu ion in RPMI +1U/ml
DNase (Sigma-Ald ich, UK) ollowed by cen i uga ion. Cells we e
pe meabilised using BD cy ofix/cy ope m eagen , washed and esus-
pended in 50 μl pe m wash bu e (BD Biosciences, UK) o su ace and
in acellula an ibody s aining. The cell su ace and in acellula s aining
mix u e we e p epa ed in B illian S aining bu e (BD Biosciences, UK) and
consis ed o he ollowing fluo och ome-conjuga ed an ibodies: an i-CD3
AF700; an i-CD4 Pe CP-Cy5.5; an i-CD161 Pe-Cy7; an i-IFN-γFITC; an i-TNF-
αBUV395 (all om BD Biosciences, UK); an i-CD8 APC-Fi e750; an i-Vα7.2
PE-Dazzle 594; an i-CD16 BV785; an i-γδ-TCR BV421; an i-IL-17 BV711; an i-
CD56 BV605 (all om Biolegend, UK); an i-IL-22 APC (The mo Fishe
Scien ific, UK) and an i-IL-2 PE (Mil enyi Bio ech, UK). BD Compbeads (BD
Biosciences, UK) we e labelled wi h he abo e fluo och omes o use as
compensa ion con ols. Following an ibody labelling, cells and beads we e
washed by cen i uga ion and fixed in 4% pa a o maldehyde solu ion
(Sigma-Ald ich, Gillingham, UK) p io o flow cy ome ic acquisi ion.
Flow cy ome ic acquisi ion and analysis. Flow cy ome y da a was
acqui ed using a fi e-lase LSRII Fo essa ins umen (BD Biosciences,
Ox o d, UK) and da a we e analysed using FlowJo ( e sion 10.5.3 BD
Biosciences, UK) (Supplemen a y Fig. 8). Cell agg ega es and deb is we e
excluded om he analysis using o wa d sca e -heigh (FSC-H) e sus
o wa d sca e -a ea (FSC-A), ollowed by side sca e -heigh (SSC-H) e sus
side sca e -a ea (SSC-A) plo s. Cy okine-p oducing lymphocy e popula-
ions we e hen iden ified using a FSC-H e sus SSC-A do plo , ollowed by
sequen ial ga ing h ough CD3
+
, ollowed by CD4
+
, CD8
+
o γδ-TCR
+
ga es o iden ifica ion o he majo T-cell popula ions, o CD3
−
, CD8
+
,
CD56
+
and/o CD16
+
NK cells, be o e indi idual cy okine ga es o iden i y
IFN-γ, IL-2, IL-17, IL-22 and TNF-αp oducing popula ions. Poly unc ional
T cells we e iden ified using Boolean ga ing combina ions o indi idual
cy okine-p oducing CD4, CD8 o γδ T cells. The so wa e package PESTLE
( e sion 1.8) was used o backg ound sub ac ion o ob ain an igen-
specific cy okine esponses, and G aphpad ( e sion 8.0.1) was used o
gene a e g aphical ep esen a ions o flow cy ome y da a (G aphpad
So wa e Inc, USA).
Nec opsy
The nec opsies we e conduc ed ei he when he disease p og essed o
mee he c i e ia se as a humane endpoin o 16 o 18 weeks a e M.
ube culosis ae osol challenge. Animals we e anaes he ised, and clinical
da a collec ed. Blood samples we e aken p io o eu hanasia by
in aca diac injec ion o a le hal dose o anaes he ic (Dolele hal, Vé oquinol
UK L d, 140 mg/kg). A pos -mo em examina ion was pe o med immedi-
a ely and g oss pa hological changes we e sco ed using an es ablished
sys em based on he numbe and ex en o lesions p esen in he lungs,
spleen, li e , kidney and lymph nodes, as desc ibed p e iously
29
. Samples
o spleen, li e , kidneys and acheob onchial, inguinal and axilla y lymph
nodes we e emo ed and sampled o quan i a i e bac e iology. The lung,
oge he wi h he hea and a ached acheob onchial and associa ed
lymph nodes, we e emo ed in ac . The lymph nodes we e measu ed and
examined o lesions. These we e fixed by in a- acheal in usion wi h 10%
neu al bu e ed o malin (NBF) using a sy inge and 13CH Nela on ca he e
(J.A.K. Ma ke ing, Yo k, UK). The ca he e ip was inse ed in o each
b onchus in u n ia he achea; he lungs we e in used un il hey we e
expanded o a size conside ed o be no mal inspi a o y dimensions, and
he achea liga ed o e ain he fluid. The in used lung was imme sed in
10% NBF. In addi ion, samples o kidneys, li e , spleen, and sub cla icula ,
hepa ic, inguinal and axilla y lymph nodes we e fixed in 10% NBF.
Pa hology s udies
G oss examina ion ollowing fixa ion. The lung lobes we e sliced se ially
(~5-mm in e als). Disc e e and coalesced lesions we e coun ed and
eco ded, and he dimensions o he la e we e measu ed. A issue slice
con aining ob ious lesions (when p esen ) was chosen om each lung
lobe. Whe e g oss lesions we e no isible, a sample was aken om a p e-
defined ana omical loca ion om each lobe o es ablish consis ency
be ween animals. Sec ions o LALN ( hose associa ed wi h he dis al
achea and bi u ca ion) and om ex a- ho acic o gans desc ibed
p e iously, we e also fixed in o malin.
His opa hological examina ion. All samples we e p ocessed o pa a fin
wax blocks using s anda d p ocedu es, sec ioned a ~4 μm, s ained wi h
haema oxylin and eosin (H&E) and Ziehl-Neelsen (ZN) s ains. Slides we e
scanned digi ally using ‘3D His ech’slide scanne and p op ie a y
‘Case iewe ’so wa e used o cap u e, s o e and anno a e digi al images.
All g oss and his opa hological examina ions we e ca ied ou by a
qualified e e ina y pa hologis blinded o he ea men g oup. Each slide
was e alua ed o he p esence o ube culous lesions and AFBs. The TB-
associa ed lesions we e eco ded using he sco ing sys em desc ibed by
Rayne e al.
30
. B iefly, six di e en ypes o g anulomas we e iden ified.
Types one (I), wo (II) and h ee (III) we e conside ed as “uno ganised”
lesions, while ypes ou (IV), fi e (V) and six (VI) we e desc ibed as
“o ganised”g anulomas. Type I was small, di use oci o mac ophages and
lymphocy es wi h sca e ed neu ophils and eosinophils, lacking clea ly
defined ma gins, infil a e al eola walls and ex end in o al eoli. Type II
lesions we e composed o simila cell ypes as ype I bu we e la ge in size
and o ming a mo e defined ci cumsc ibed ocus o g anuloma ous
inflamma ion, equen ly ci cula and wi h a iably dema ca ed bo de .
Type III was as ype II bu wi h ocal nec osis p esen , cha ac e ised by
nuclea pyknosis and ka yo hexis wi h he loss o cellula a chi ec u e.
Type IV was cha ac e is ically ci cumsc ibed g anulomas consis ing
p ima ily o mac ophages admixed wi h neu ophils and o he leucocy es,
wi h e idence o a ew pe iphe al lymphocy es. Type V was o ganised
lesions exhibi ing nec o ic oci wi h degene a ed neu ophils and ype VI
we e classic, la gely well-dema ca ed, g anulomas wi h cen al caseous
nec osis and a a iable im o lymphocy es. Fo each issue sec ion, he
o al a ea o g oss disease was calcula ed and he o al numbe o
g anulomas o each ype we e coun ed and eco ded. Mo eo e , he o al
numbe o AFBs we e coun ed o each g anuloma in he ZN-s ained
sec ions om he lung and LALN.
Bac e iology
The spleen, kidneys, li e and acheob onchial lymph nodes we e sampled
o he p esence o iable M. ube culosis pos -mo em, as desc ibed
p e iously
20
. Weighed issue samples we e homogenised in 2 ml o s e ile
wa e , hen ei he se ially dilu ed in s e ile wa e p io o being pla ed, o
pla ed di ec ly on o Middleb ook 7H11 OADC selec i e aga . Pla es we e
incuba ed o h ee weeks a 37 °C and esul an M. ube culosis colonies
coun ed. Mean CFU coun s om eplica e issue samples we e used o
g aphical p esen a ion. The lowe limi o de ec ion o cul u e on pla es
was fi e CFUs pe mL o homogena e.
S a is ical analyses
G aphPad P ism e sion 7.0 (G aphPad So wa e Inc, La Jolla, Cali o nia,
USA) was used o all g aphical da a ep esen a ions and s a is ical
analyses, hese included: compa ison o an igen-specific IFN-γSFU
equencies measu ed using he ex i o ELISpo assay, equencies o
specific cell subse s and mul i unc ional cell popula ions measu ed by flow
cy ome y, di e ences in quali a i e pa hology sco es and his opa hology
g anuloma se e i y sco es, quan ifica ion o iable M. ube culosis in
ex apulmona y issues and compa isons o clinical pa ame e s be ween
accina ion g oups, using non-pa ame ic Wilcoxon signed- ank o
Mann–Whi ney U es s. Chi-squa ed (Χ
2
es s we e used o compa e
di e ences be ween he accina ion g oups in he p opo ion o
bac e iology issue samples whe e iable M. ube culosis CFU le els we e
below he lowe limi o de ec ion o he assay and Coch an–A mi age
me hod Χ
2
es s o he compa ison o he equency o g anuloma se e i y
da a. Disease p og ession be ween g oups was compa ed using log- ank
su i al analysis. Whe e applicable, all s a is ical analyses we e conduc ed
A.D. Whi e e al.
8
npj Vaccines (2021) 4 Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences
using wo- ailed es s and P alues a e unadjus ed o mul iple
compa isons.
Repo ing summa y
Fu he in o ma ion on esea ch design is a ailable in he Na u e Resea ch
Repo ing Summa y linked o his a icle.
DATA AVAILABILITY
The da ase s gene a ed and analysed in his s udy a e a ailable om he
co esponding au ho s upon easonable eques . Likewise, bioma e ials a chi ed
om his s udy may be sha ed o u he esea ch.
Recei ed: 22 July 2020; Accep ed: 13 No embe 2020;
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ACKNOWLEDGEMENTS
This wo k was suppo ed by he Depa men o Heal h, UK and a g an om Ae as.
The iews exp essed in his publica ion a e hose o he au ho s and no necessa ily
hose o he Depa men o Heal h. We hank he s a o he Biological In es iga ions
G oup a PHE Po on and he PHE macaque colonies o assis ance in conduc ing
s udies, Lau a Hun e o his ology suppo , Faye Lanni and Neil McCloud o
bac e iology and ae obiology suppo , Helen Mea ns om SATVI o ad ice aligning
macaque immunological me hods wi h p o ocols applied in MTBVAC clinical ials.
AUTHOR CONTRIBUTIONS
A.D.W. con ibu ed o he design o he wo k, da a acquisi ion, analysis and
in e p e a ion. L.S. con ibu ed o da a acquisi ion, analysis and in e p e a ion. C.S.
con ibu ed o da a acquisi ion, analysis and in e p e a ion. A.M. con ibu ed o da a
acquisi ion, analysis and in e p e a ion. J.G. con ibu ed o da a acquisi ion, analysis
and in e p e a ion. F.G. con ibu ed o da a acquisi ion, analysis and in e p e a ion.
A.M. con ibu ed o da a acquisi ion, analysis and in e p e a ion. S.C. con ibu ed o
da a acquisi ion, analysis and in e p e a ion. C.L.A. con ibu ed o da a acquisi ion.
A.S. con ibu ed o da a acquisi ion. F.J.S. con ibu ed o da a acquisi ion, analysis and
in e p e a ion. Emma Rayne con ibu ed o da a acquisi ion, analysis and
in e p e a ion. Es eban Rod iguez con ibu ed o he design and e iew o he wo k.
E.P. con ibu ed o he design and e iew o he wo k. D.L. con ibu ed o he design
o he wo k. A.W. con ibu ed o he design o he wo k and da a analysis and
in e p e a ion. M.D. con ibu ed o he concep ion and design o he wo k and da a
acquisi ion, analysis and in e p e a ion. C.M. con ibu ed o he design o he wo k
and da a in e p e a ion. S.S. con ibu ed o he concep ion and design o he wo k,
da a acquisi ion, analysis and in e p e a ion.
COMPETING INTERESTS
E.P., E.R. and C.M. a e co-in en o s on a pa en on MTBVAC held by he Uni e si y o
Za agoza and Bio ab i.
ADDITIONAL INFORMATION
Supplemen a y in o ma ion is a ailable o his pape a h ps://doi.o g/10.1038/
s41541-020-00262-8.
Co espondence and eques s o ma e ials should be add essed o A.D.W.
Rep in s and pe mission in o ma ion is a ailable a h p://www.na u e.com/
ep in s
A.D. Whi e e al.
9
Published in pa ne ship wi h he Sealy Ins i u e o Vaccine Sciences npj Vaccines (2021) 4