Sa e y and immunogenici y o he no el H4:IC31 ube culosis accine
candida e in BCG- accina ed adul s: Two phase I dose escala ion ials
Ma ia No by
a,1
, Timo Vesika i
b,1
, La s Lindq is
a
, Ma kus Maeu e
c
, Raija Ahmed
c
, Shahnaz Mahda i a
c
,
Sean Benne
d
, J. B uce McClain
d
, Ba ba a M. Shephe d
d
, Dane Li
d
, Da id A. Hokey
d
, Ing id K omann
e
,
Sø en T. Ho
e
, Pe e Ande sen
e
, Ad ië e W. de Visse
, Simone A. Joos en
, Tom H.M. O enho
,
Jan Ande sson
a,g
, Susanna B ighen i
g,
⇑
a
Di ision o In ec ious Diseases, Ka olinska Uni e si y Hospi al, S ockholm, Sweden
b
Vaccine Resea ch Cen e , Uni e si y o Tampe e, Tampe e, Finland
c
TIM, Depa men o Labo a o y Medicine and CAST, Ka olinska Ins i u e , S ockholm, Sweden
d
Ae as, Rock ille, MD, USA
e
S a ens Se um Ins i u , Copenhagen, Denma k
Depa men o In ec ious Diseases, Leiden Uni e si y Medical Cen e , Leiden, The Ne he lands
g
Cen e o In ec ious Medicine (CIM), Ka olinska Ins i u e , S ockholm, Sweden
a icle in o
A icle his o y:
Recei ed 2 July 2016
Recei ed in e ised o m 28 Decembe 2016
Accep ed 20 Janua y 2017
A ailable online 17 Feb ua y 2017
Keywo ds:
Tube culosis
Vaccine
Human
Clinical ial
Sa e y
Immuni y
abs ac
Backg ound: No el accine s a egies a e equi ed o p o ide p o ec i e immuni y in ube culosis (TB)
and p e en de elopmen o ac i e disease. We in es iga ed he sa e y and immunogenici y o a no el
TB accine candida e, H4:IC31 (AERAS-404) ha is composed o a usion p o ein o M. ube culosis an i-
gens Ag85B and TB10.4 combined wi h an IC31
Ò
adju an .
Me hods: BCG- accina ed heal hy subjec s we e immunized wi h a ious an igen (5, 15, 50, 150
l
g) and
adju an (0, 100, 500 nmol) doses o he H4:IC31 accine (n = 106) o placebo (n = 18) in wo andomized,
double-blind, placebo-con olled phase I s udies conduc ed in a low TB endemic se ing in Sweden and
Finland. The subjec s we e ollowed o ad e se e en s and CD4
+
T cell esponses.
Resul s: H4:IC31 accina ion was well ole a ed wi h a sa e y p o ile consis ing o mos ly mild o mod-
e a e sel -limi ed injec ion si e pain, myalgia, a h algia, e e and pos - accina ion in lamma o y eac-
ion a he sc eening ube culin skin es injec ion si e. The H4:IC31 accine elici ed an igen-speci ic
CD4
+
T cell p oli e a ion and cy okine p oduc ion ha pe sis ed 18 weeks a e he las accina ion.
CD4
+
T cell expansion, IFN-
c
p oduc ion and mul i unc ional CD4
+
Th1 esponses we e mos p ominen
a e wo doses o H4:IC31 con aining 5, 15, o 50
l
g o H4 in combina ion wi h he 500 nmol IC31 adju-
an dose.
Conclusions: The no el TB accine candida e, H4:IC31, demons a ed an accep able sa e y p o ile and was
immunogenic, capable o igge ing mul i unc ional CD4
+
T cell esponses in p e iously BCG- accina ed
heal hy indi iduals. These dose-escala ion ials p o ided e idence ha he op imal an igen-adju an
dose combina ions a e 5, 15, o 50
l
g o H4 and 500 nmol o IC31.
Conclusions: T ial egis a ion: ClinicalT ials.go , NCT02066428 and NCT02074956.
Ó2017 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
WHO has decla ed ube culosis (TB) as a global heal h eme -
gency and despi e socioeconomic imp o emen , TB con inues o
cause a conside able numbe o dea hs. A p e en i e TB accine
could educe he sp ead and se e i y o TB disease signi ican ly.
The bacillus Calme e-Gué in (BCG) accine p o ides incomple e
p o ec ion agains pulmona y TB and a boos wi h BCG does no
consis en ly p o ide addi ional p o ec ion [1,2]. Howe e , new-
bo n BCG accine p e en ion is s ill a majo global s a egy o
h p://dx.doi.o g/10.1016/j. accine.2017.01.055
0264-410X/Ó2017 The Au ho s. Published by Else ie L d.
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Abb e ia ions: TB, ube culosis; M b, Mycobac e ium ube culosis; BCG, bacillus
Calme e-Gué in; MDR, mul id ug- esis an ; TST, ube culin skin es ; ICS, in a-
cellula cy okine s aining; FASCIA, Flow-cy ome ic Assay o Speci ic Cell-media ed
Immune- esponse in Ac i a ed whole blood; AE, ad e se e en s; INR, in e na ional
no malized a io; MHC, majo his ocompa ibili y complex.
⇑
Co esponding au ho a : Ka olinska Ins i u e , Cen e o In ec ious Medicine
(CIM), Depa men o Medicine Huddinge, Ka olinska Uni e si y Hospi al Huddinge,
141 86 S ockholm, Sweden.
E-mail add ess: [email p o ec ed] (S. B ighen i).
1
MN and TV con ibu ed equally o his manusc ip .
Vaccine 35 (2017) 1652–1661
Con en s lis s a ailable a ScienceDi ec
Vaccine
jou nal homepage: www.else ie .com/loca e/ accine
he con ol o TB, which unde lines he impo ance o de eloping
an imp o ed TB accine ha would mo e e ec i ely boos BCG.
H4:IC31 (AERAS-404) is an in es iga ional accine ha is com-
posed o wo ac i e componen s: he H4 an igen which is a usion
p o ein c ea ed om wo Mycobac e ium ube culosis (M b)-
an igens, an igen 85B (Ag85B) and TB10.4, and an immunological
adju an called IC31
Ò
. The a ionale o he H4:IC31 accine is ha
p esen a ion o M b-speci ic an igens in his se ing could augmen
T cell immuni y induced by BCG and hus imp o e p o ec ion
agains TB. Ag85B is a 30 kDa mycolyl ans e ase p o ein [3,4] ha
has p e iously been demons a ed o induce subs an ial p o ec i e
immuni y agains ae osol challenge wi h he highly i ulen M b
E dman s ain in guinea pigs [5]. TB10.4 is one o h ee membe s
o he e y simila ea ly sec e o y an igenic a ge (ESAT)-6 g oup
o p o eins ound in M b cul u e supe na an s [6]. TB10.4 has been
shown o induce la ge and b oade immune esponses in T cells
isola ed om TB pa ien s compa ed o BCG- accina ed and non-
accina ed dono s [7]. Immuniza ion o mice wi h a usion p o ein
o TB10.4 and Ag85B has been shown o induce a signi ican syne -
gis ic p o ec i e e ec agains subsequen ae osol challenge wi h
M b [8]. Simila ly, Ag85B and ESAT-6 (H1 an igen) induced po en
and long-li ed e ec o T cell esponses in naï e olun ee s when
adminis a ed in he p esence o he IC31 adju an [9,10]. The p o-
p ie a y IC31 adju an (Valne a, Vienna, Aus ia) is a combina ion
o a leucine- ich pep ide, KLK, and a syn he ic oligonucleo ide,
ODN1a. KLK enhances he up ake o an igens in o an igen-
p esen ing cells and he e o e imp o es he immune esponse o
pep ide an igens. ODN1a is a syn he ic bac e ial DNA analogue ha
esembles a CpG mo i ha will p omo e a Th1 esponse and he
p oduc ion o IFN-
c
and IL-2, which a e conside ed o play essen-
ial oles in p o ec ion agains TB [11]. In addi ion, he IC31 adju-
an enhances he p oduc ion o bo h IFN-
c
and humo al
esponses ia he TLR9 signaling pa hway [12,13].
I has been demons a ed in animal s udies ha H4:IC31 boos s
BCG immuni y and p o ides g ea e p o ec ion agains de elop-
men o TB disease han BCG alone [14–16]. The e o e we aimed
o es he sa e y and immunogenici y o he H4:IC31 accine in
a p ime-boos accina ion s a egy in heal hy, p e iously BCG-
accina ed indi iduals who ecei ed di e en an igen-adju an
dose combina ions o he s udy accine, in wo phase I clinical i-
als pe o med in Sweden (Ae as p o ocol C-005-404) and Finland
(Ae as p o ocol C-006-404), espec i ely. We designed he i s -
in-human C-005-404 s udy o op imize he dose o he IC31 adju-
an wi h a ixed dose o H4 an igen, and he subsequen C-006-
404 s udy o op imize he dose o H4 an igen wi h a ixed dose
o IC31 adju an .
2. Ma e ials and me hods
2.1. Subjec s and s udy design
We conduc ed wo phase I andomized, double-blind, placebo-
con olled s udies a he Depa men o In ec ious Diseases,
Ka olinska Uni e si y Hospi al Huddinge in S ockholm, Sweden
(Ae as p o ocol C-005-404; ClinicalT ials.go ID NCT02066428)
and a he Vaccine Resea ch Cen e , Uni e si y o Tampe e in Fin-
land (Ae as p o ocol C-006-404; ClinicalT ials.go ID
NCT02074956). Inclusion c i e ia we e: p e iously BCG-
accina ed (P5 yea s), males o emales ( emales equi ed o be
s e ile o C-005-404), age 18-50 yea s, HIV-unin ec ed, good
heal h based on medical his o y, no mal BMI (19–33), no e idence
o an ongoing TB in ec ion and w i en in o med consen com-
ple ed. Bo h s udies employed dose-escala ion, wi h inc easing
amoun s o he H4 an igen (5, 15, 50 o 150
l
g) adminis e ed in
he p esence o inc easing amoun s o he IC31 adju an (0, 100
o 500 nmol) (Table 1). S a ens Se um Ins i u (SSI) in Copenhagen,
Denma k manu ac u ed he H4 usion p o ein and he IC31 adju-
an . Fo mula ion bu e was used as placebo con ol. Subjec s
ecei ed accina ions ia in amuscula injec ion wi h H4:IC31 o
placebo on s udy days 0 and 56. T ea men assignmen s we e
based on a andomly-gene a ed sequence o subjec iden i ica ion
numbe s on a andomiza ion schedule, p o ided by an unblinded
s a is ician o he s udy pha macis in a sealed ampe -e iden
en elope.
P o ocol C-005-404 was app o ed by he Regional E hical
Re iew Boa d (S ockholm) and he Medicinal P oduc Agency in
Sweden, while p o ocol C-006-404 was app o ed by he Hospi al
Dis ic o Pi kanmaa E hics Commi ee (Tampe e) and he
Na ional Agency o Medicines in Finland (Finnish Medicines
Agency). The s udies we e conduc ed in acco dance wi h he Dec-
la a ion o Helsinki and applicable local egula ions o conduc ing
clinical ials on medicinal p oduc s in humans.
2.2. Ad e se e en s
Sa e y o he H4:IC31 accine ea men egimens we e based
on he induc ion o ad e se e en s (AEs) ha ep esen ed bo h
clinical and labo a o y e alua ions (see Supplemen a y ma e ials).
AE se e i y was g aded acco ding o he US Food and D ug Admin-
is a ion (FDA) oxici y ables o Heal hy Adul and Adolescen
Volun ee s En olled in P e en i e Vaccine Clinical T ials [17] using
c i e ia ha we e p e-speci ied in he s udy p o ocols p o ided by
he sponso i.e. A eas. We eco ded solici ed and unsolici ed AEs
du ing he i s 28 days a e each accina ion i.e. s udy days
0–28 and 56–84, and se ious AEs (SAEs) du ing he 6 mon h s udy
pe iod.
2.3. Immunogenici y es ing
Pe iphe al blood mononuclea cells (PBMC) we e isola ed om
blood collec ed on s udy days 0, 7, 14, 28, 56, 63, 70, 84, and 182
o in acellula cy okine s aining (ICS) pe o med a Ae as [18]
and on s udy days 0, 56, 84, and 182 o assessmen o M b-
speci ic IFN-
c
p oduc ion using ELISpo pe o med a Leiden
Uni e si y Medical Cen e acco ding o P o ocol S2 p e iously
desc ibed by D . S e en Smi h e al. [19] (see Supplemen a y
Table 1
A dose ma ix o he H4 and IC31 dose combina ions adminis e ed o he s udy subjec s in he C-005-404 and C-006-404 ials.
a
One accina ion Two accina ions
Day 0 Day 0 and 56
H4 dose 50
l
g 150
l
g5
l
g15
l
g50
l
g 150
l
g To al (n = 124)
No adju an 8 8 – – 8 – 24
100 nmol IC31 8 – 9 9 8 – 34
500 nmol IC31 8 – 8 8 16
b
848
Placebo – 18
c
18
a
A o al o 124 pa icipan s we e included in he wo ials.
b
Two doses o 50
l
g H4 in 500 nmol IC31 we e adminis e ed o 8 pa icipan s in each o he C-005-404 and C-006-404 ials.
c
A o al o 18 pa icipan s ecei ed wo placebo accina ions in he wo ials.
M. No by e al. / Vaccine 35 (2017) 1652–1661 1653
ma e ials). In s udy C-005-404, blood was also collec ed on s udy
days 0, 7, 28, 63, 84, and 182 o Flow-cy ome ic Assay o Speci ic
Cell-media ed Immune- esponse in Ac i a ed whole blood assay
(FASCIA pe o med a he Public Heal h Agency o Sweden)
[20,21] (see Supplemen a y ma e ials). Subjec s we e sc eened
o ongoing TB in ec ion using Quan iFERON and he ube culin
skin es (TST) (see Supplemen a y ma e ials).
2.4. Da a analysis
The sample size o each ial was selec ed as adequa e o an
ini ial e iew o he sa e y p o ile o H4:IC31. Basic desc ip i e
analysis was pe o med o each ea men egimen o examine
AEs and immune esponses measu ed by ICS, IFN-
c
ELISpo and
FASCIA. Compa isons be ween ea men egimens o ICS and
IFN-
c
ELISpo immune esponses we e conduc ed by a ea unde
he cu e (AUC) analyses. The apezoidal ule was used o calcu-
la e he AUC o each subjec , wi h nega i e and posi i e peaks el-
a i e o each subjec ’s baseline da a included o calcula e ne peak
a ea. O e all p- alues o median AUC among ea men egimens
we e ob ained using he K uskal-Wallis es . Pai wise compa isons
o median AUC be ween ea men egimens we e conduc ed using
a Mann-Whi ney exac es . The Holm me hod was used o co ec
o mul iple compa isons.
3. Resul s
3.1. En ollmen and demog aphy
We sc eened a o al o 206 heal hy BCG- accina ed indi iduals
o eligibili y, en olled 125 indi iduals and andomized hem in o
he di e en in e en ion g oups as desc ibed in Fig. 1. Demo-
g aphic cha ac e is ics we e gene ally simila ac oss ea men
egimens wi hin each ial (Supplemen a y Tables 1A and 1B). In
he C-005-404 s udy, all 64 andomized subjec s ecei ed he
s udy day 0 accina ion and all subjec s comple ed he s udy. In
he C-006-404 s udy, 60 o 61 andomized subjec s ecei ed he
s udy day 0 accina ion and all subjec s excep wo comple ed
he s udy.
3.2. Ad e se e en s (AEs)
The majo i y (83%) o subjec s ac oss bo h s udies had AEs
g aded as mild o mode a e (Tables 2a and 2b), and h ee (2%) sub-
jec s had no AEs. n = 18 (15%) subjec s had a leas one se e e AE
(Tables 2a and 2b) o which ou we e conside ed ela ed o he
s udy accine: e e (150/0 one dose egimen), inc eased p o ein
in u ine (15/100 wo dose egimen), TST si e eac ion (50/100
wo dose egimen, discussed below), and inc eased in e na ional
no malized a io (INR) (150/500 wo dose egimen). The o e all
incidence and se e i y o he AEs we e no di e en compa ing
H4:IC31 accina ions wi h placebo (Tables 2a and 2b). Fou SAEs
we e epo ed, none o which was conside ed ela ed o s udy ac-
cina ion: men al s a us changes (50/0 one dose egimen); mesen-
e ic lymphadeni is (50/500 wo dose egimen); ileus (50/500
wo dose egimen); and subdu al hemo hage (placebo).
Mos subjec s had a leas one solici ed o unsolici ed AE
eco ded (Tables 3a and 3b). Among solici ed AEs, myalgia, a h al-
gia, and e e (py exia) occu ed a a highe equency in subjec s
who ecei ed he H4:IC31 accine compa ed o placebo in he C-
005-404 s udy (Table 3a). All cases o e e occu ed wi hin 1–
2 days o he i s accina ion, esol ed wi hin 2–3 days and did
no ecu a e he second s udy accina ion. The e was a end
owa ds an inc eased equency o some sys emic solici ed AEs a
he 150
l
g H4 dose le el, pa icula ly when combined wi h
500 nmol IC31 (Tables 3a and 3b). Pain a he injec ion si e was
inc eased in subjec s who ecei ed H4 oge he wi h he IC31 adju-
an , as compa ed o placebo (Tables 3a and 3b) o H4 alone
(Table 3a). Fo o he solici ed AEs and all unsolici ed AEs (excep
TST si e eac ion, discussed below), he AE p o iles we e simila
ac oss he H4:IC31 and placebo ea men egimens (Tables 3a
and 3b). Fo each egimen, he AE p o iles iden i ied a e he sec-
ond accina ion we e simila o hose de ec ed a e he i s ac-
cina ion (da a no shown). See Supplemen a y Tables 2A and 2B,
o a comple e lis o AEs in he ials.
In he C-005-404 s udy, some deg ee o pos -s udy accina ion
in lamma ion occu ed a he sc eening TST injec ion si e in 14 o
he i s 21 (66.7%) TST-nega i e subjec s who ecei ed he H4:
IC31 accine. Among hese 14 subjec s, TST si e eac ions we e eli-
ci ed by he H4 an igen alone (11/14, 78.6%) o H4 combined wi h
a low dose (100 nm) o IC31 adju an (3/14, 21.4%) (Table 3a). One
subjec (50/100 wo dose egimen) expe ienced a se e e eac ion
a he TST si e wi h onse he day o he i s s udy accine admin-
is a ion. The subjec had no sc eening TST eac i i y o a eac ion
a he accine si e (Supplemen a y ma e ials).
3.3. Immunogenici y
We conside ed de ec ion o an igen-speci ic T cell p oli e a ion
and cy okine p oduc ion (single- o co-exp ession o IFN-
c
, TNF-
a
and/o IL-2) in PBMC samples in esponse o accine-an igens as
po en ially impo an co ela es o immune p o ec ion. T cell
expansion in each o he H4:IC31 one dose egimens was limi ed
o he CD4
+
T cell subse ha esponded o he accine an igen
Ag85B in some subjec s, al hough hese esponses we e no sus-
ained (Fig. 2A, Supplemen a y Table 3A). In each o he H4:IC31
wo dose egimens, he s udy accine induced Ag85B-speci ic bu
also TB10.4-speci ic CD4
+
T cell esponses, wi h a boos ing e ec
seen a e adminis a ion o he second dose (Fig. 2B and Fig. 2C,
Supplemen a y Tables 3A and 3B). These esponses peaked a 2
and 4 weeks a e he second accina ion and we e sus ained up
o 18 weeks a e he las accina ion (Fig. 2B and Fig. 2C). The
mos s ong and long-li ed median CD4
+
T cell esponses com-
pa ed o placebo we e seen a e s imula ion wi h Ag85B in he
5/500 (p < 0.01), 15/500, and 50/500 (p < 0.01) wo dose egimens,
while he p opo ion o an igen-speci ic CD4
+
T cells was lowe
using he highe H4 dose 150/500 (Fig. 2B and Fig. 2C, Supplemen-
a y Tables 3A, 3B and 4B). These esul s we e suppo ed by he
whole blood FASCIA assay, whe e he wo dose 50/500 ea men
was shown o be he supe io egimen ha induced signi ican
expansion o Ag85B-speci ic CD4
+
T cells (p = 0.02) (Supplemen a y
Fig. 1A) and CD8
aa
+
T cells (p < 0.001) (Supplemen a y Fig. 1B)
compa ed o he wo dose placebo egimen.
IFN-
c
ELISpo esponses we e ele a ed in esponse o bo h
Ag85B and TB10.4, bu only o he wo dose egimens oge he
wi h he IC31 adju an (Fig. 3A and Fig. 3B). A boos ing e ec
was seen a e he second H4:IC31 dose, pa icula ly in he p es-
ence o he highe IC31 adju an dose, and hese esponses we e
sus ained up o 18 weeks a e he las accina ion
(Fig. 3B and Fig. 3C). Simila o an igen-speci ic CD4
+
T cell expan-
sion (Fig. 2B and Fig. 2C), he mos po en IFN-
c
ELISpo esponses
we e seen in he 5/500 (p < 0.01), 15/500 (p = 0.02) and he 50/500
(p = 0.02) wo dose egimens compa ed o placebo
(Fig. 3B and Fig. 3C, Supplemen a y Tables 4C and 4D). ICS analysis
o Ag85B-speci ic CD4
+
T cells a 4 weeks a e he second accina-
ion (s udy day 84) con i med ha only he wo dose ea men
egimens e icien ly induced cy okine p oducing cells (Fig. 4A–C)
These p ima ily included bi- unc ional IL-2/TNF-
a
p oducing and
mul i unc ional IFN-
c
/IL-2/TNF-
a
p oducing T cells and, o a lesse
ex en , mono- unc ional T cells (Fig. 4B and Fig. 4C). See Supple-
men a y Tables 4A–4D, o comple e analyses on he s a is ical di -
e ences be ween he accine g oups.
1654 M. No by e al. / Vaccine 35 (2017) 1652–1661
4. Discussion
These phase I accine ials we e he i s o explo e a sa e and
immunogenic dose and dosage ange and o iden i y po en ial side
e ec s o he H4:IC31 accine candida e in humans. Ou p incipal
indings demons a ed ha all accine doses and dosage combina-
ions es ed we e well ole a ed in p e iously BCG- accina ed
s udy subjec s and mos o he epo ed local and sys emic AEs
Assessed o eligibili y
(n=200) Excluded (n=75)
No mee ing inclusion c i e ia (n=66)
Abno mal labo a o y alue (n=22)
Posi e/inde e mina e QFT/PPD (n=20)
O he (n=24)
Declined o pa icipa e (n=9)
Analysed (n=18)
Excluded om analysis (n=0)
Los o ollow-up (n=0)
Discon inued in e en ion
(
n=0
)
Alloca ed o placebo (n=18)
Recei ed alloca ed in e en ion (n=18)
C-005-404 C-006-404
G oup 1
Placebo: 2 doses (n=3)
G oup 2
Placebo: 2 doses (n=3)
G oup 3
Placebo: 2 doses (n=2)
To al Placebo (n=8)
G oup 1
Placebo: 2 doses (n=2)
G oup 2
Placebo: 2 doses (n=2)
G oup 3
Placebo: 2 doses (n=2)
G oup 4
Placebo: 2 doses (n=2)
G oup 5
Placebo: 2 doses (n=2)
To al Placebo (n=10)
Did no ecei e alloca ed in e en ion (n=0)
Los o ollow-up (n=0)
Discon inued in e en ion (n=6)
C-005-404 C-006-404
eac aon o G a e’s
disease, eco ded as
hype hy oidism (n=1)
wi hd ew consen (n=1)
labo a o y alues ou side
e e ence ange (n=4)
Discon inued om s udy (n=2)
C-005-404 C-006-404
n=0 wi hd ew consen (n=1)
did no ecei e alloca ed
in e enon(n=1)
Alloca ed o H4:IC31 ( g H4/nmol IC31; n=107)
Recei ed alloca ed in e en ion (n=106)
C-005-404 C-006-404
G oup 1
50/0: 1 dose (n=8)*
50/0: 2 doses (n=8)
G oup 2
50/100: 1 dose (n=8)*
50/100: 2 doses (n=8)
G oup 3
50/500: 1 dose (n=8)*
50/500: 2 doses (n=8)
150/0: 1 dose (n=8)*
* Recei ed placebo on
s udy day 56
G oup 1
5/500: 2 doses (n=8)
G oup 2
15/500: 2 doses (n=8)
G oup 3
50/500: 2 doses (n=8)
G oup 4
150/500: 2 doses (n=8)
G oup 5
5/100: 2 doses (n=9)
15/100: 2 doses (n=9)
Did no ecei e alloca ed in e en ion (due o abno mal
labo a o y alue [C-006-404]; n=1)
Analysed (n=106)
Excluded om analysis (n=1; subjec ha did no
ecei e alloca ed in e en ion)
Alloca ion
Anal
y
sis
Follow U
p
Randomized (n=125)
C-005-404 C-006-404
26/No /2007
11/No /2008
13/May/2008
15/Ap /2009
En ollmen
Fig. 1. Conso diag am o heal hy p e iously BCG- accina ed indi iduals, om sc eening o analysis. All subjec s in C-005-404 comple ed he s udy. One C-005-404 subjec
(150/0 one dose ea men egimen) wi h a his o y o G a e’s disease did no ecei e he s udy accine on s udy day 56 due o onse o hype hy oidism. In he C-006-404
s udy, all subjec s excep wo comple ed he s udy, one who e ac ed consen , and one who was no accina ed and who was excluded om all analyses. Fi e C-006-404
subjec s did no ecei e he s udy day 56 accina ion; one subjec (150/500 wo dose ea men egimen) wi hd ew consen and 4 subjec s (one in each o he 5/100, 15/500,
50/500, and 150/500 wo dose ea men egimens) had labo a o y alues ou side he e e ence anges o he local labo a o y.
M. No by e al. / Vaccine 35 (2017) 1652–1661 1655
we e mild o mode a e. The H4:IC31 accine was able o elici pe -
sis en an igen-speci ic CD4
+
T cell esponses in he pe iphe al ci -
cula ion including enhanced T cell p oli e a ion and IFN-
c
p oduc ion, and also induc ion o mul i unc ional Th1 cells. Two
accina ions wi h he H4 an igen using he lowe doses anging
om 5 o 50
l
g (i.e. 5, 15, o 50
l
g) in combina ion wi h he highe
dose (500 nmol) o he IC31 adju an induced he s onges T cell
esponses o Ag85B, while he H4 an igen alone esul ed in e y
low T cell esponses. This suppo s he ac ha adju an p ope -
ies a e equi ed o he induc ion o a s ong and sus ained
immune esponse [22]. Impo an ly, a second accina ion wi h
H4:IC31 did no inc ease he equency o se e i y o any epo ed
AEs as compa ed o a single accina ion.
The equency and se e i y o AEs appea ed o be independen
o he numbe (one o wo) o dose (5, 15 o 50
l
g) o H4 an igen in
he lowe dose ange, while an inc eased equency o some sys-
emic solici ed AEs was seen a he 150
l
g H4 dose le el. The AE
p o iles among subjec s who ecei ed he H4 an igen in he p es-
ence o absence o he IC31 adju an we e simila , excep o injec-
ion si e pain, which sugges ed ha he adju an did no con ibu e
Table 2a
Ad e se e en s by highes se e i y o each subjec : C-005-404.
H4:IC31 (
l
g H4/nmol IC31)
50/0 50/100 50/500 150/0
Placebo 1 Dose 2 Doses 1 Dose 2 Doses 1 Dose 2 Doses 1 Dose
(n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8)
Se e i y n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Mild 3 (37.5) 4 (50.0) 2 (25.0) 4 (50.0) 2 (25.0) 1 (12.5) 2 (25.0) 1 (12.5)
Mode a e 4 (50.0) 2 (25.0) 5 (62.5) 2 (25.0) 4 (50.0) 6 (75.0) 4 (50.0) 4 (50.0)
Se e e 1 (12.5) 1 (12.5) 1 (12.5) 2 (25.0) 2 (25.0) 1 (12.5) 2 (25.0) 3 (37.5)
Table 2b
Ad e se e en s by highes se e i y o each subjec : C-006-404.
H4:IC31 (
l
g H4/nmol IC31)
Placebo 5/100 5/500 15/100 15/500 50/500 150/500
2 Doses 2 Doses 2 Doses 2 Doses 2 Doses 2 Doses 2 Doses
(n = 10) (n = 9) (n = 8) (n = 9) (n = 8) (n = 8) (n = 8)
Se e i y n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Mild 4 (40.0) 3 (33.3) 4 (50.0) 3 (33.3) 2 (25.5) 1 (12.5) –
Mode a e 5 (50.0) 6 (66.7) 3 (37.5) 5 (55.6) 5 (62.5) 6 (75.0) 6 (75.0)
Se e e – – 1 (12.5) 1 (11.1) 1 (12.5) – 2 (25.0)
Table 3a
Ad e se e en s: C-005-404.
H4:IC31 (
l
g H4/nmol IC31)
50/0 50/100 50/500 150/0
Placebo 1 Dose 2 Doses 1 Dose 2 Doses 1 Dose 2 Doses 1 Dose
(n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8)
MedDRA P e e ed e m n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Subjec s wi h a leas one solici ed AE 6 (75.5) 6 (75.5) 5 (62.5) 7 (87.5) 6 (75.0) 7 (87.5) 7 (87.5) 8 (100.0)
A h algia – 1 (12.5) 2 (25.0) 3 (37.5) 2 (25.0) 4 (50.0) 1 (12.5) 4 (50.0)
Dia hoea 1 (12.5) – – 1 (12.5) 1 (12.5) – – –
Fa igue 5 (62.5) 4 (50.0) 3 (37.5) 5 (62.5) 3 (37.5) 6 (75.0) 4 (50.0) 6 (75.0)
Headache 5 (62.5) 3 (37.5) 4 (50.0) 4 (50.0) 5 (62.5) 4 (50.0) 6 (75.0) 7 (87.5)
Injec ion si e e y hema 1 (12.5) 1 (12.5) – – – – 1 (12.5) –
Injec ion si e pain 1 (12.5) – – 2 (25.0) 3 (37.5) 1 (12.5) 5 (62.5) 2 (25.0)
Injec ion si e swelling 1 (12.5) – – – 1 (12.5) 1 (12.5) 3 (37.5) –
Myalgia 1 (12.5) 4 (50.0) 4 (50.0) 6 (75.0) 3 (37.5) 3 (37.5) 2 (25.0) 5 (62.5)
Py exia – – 2 (25.0) 2 (25.0) 1 (12.5) 4 (50.0) – 4 (50.0)
Subjec s wi h a leas one unsolici ed AE 7 (87.5) 7 (87.5) 8 (100.0) 8 (100.0) 7 (87.5) 7 (87.5) 8 (100.0) 8 (100.0)
Aspa a e amino ans e ase inc eased – – 1 (12.5) – 1 (12.5) 3 (37.5) – 3 (37.5)
Blood c ea ine phosphokinase inc eased – – 2 (25.0) 1 (12.5) 1 (12.5) 5 (62.5) – 1 (12.5)
Blood p essu e sys olic inc eased 1 (12.5) – – 2 (25.0) 2 (25.0) – 2 (25.0) –
Haemoglobin dec eased – 1 (12.5) – 1 (12.5) 1 (12.5) 1 (12.5) 3 (37.5) 1 (12.5)
Hea a e dec eased 3 (37.5) – – – 6 (75.0) 3 (37.5) 5 (62.5) 2 (25.0)
Nasopha yngi is 2 (25.0) 1 (12.5) 2 (25.0) 1 (12.5) 1 (12.5) – 2 (25.0) 2 (25.0)
Nausea – 1 (12.5) 1 (12.5) 2 (25.0) 1 (12.5) 1 (12.5) – 1 (12.5)
Neu ophil coun dec eased 2 (25.0) – 2 (25.0) 3 (37.5) 2 (25.0) 1 (12.5) 2 (25.0) 3 (37.5)
Red blood cells u ine – 2 (25.0) 1 (12.5) 2 (25.0) 1 (12.5) 3 (37.5) 1 (12.5) 1 (12.5)
TST si e eac ion
a
– 5 (62.5) 5 (62.5) 1 (12.5) 2 (25.0) – – 1 (12.5)
No e: Indi idual unsolici ed AEs a e shown o AEs epo ed in P10% o subjec s ac oss he combined H4:IC31 egimens. A ull lis o AEs is p esen ed in Supplemen a y
Table 2A.
a
Applica ion si e hype sensi i i y o in lamma o y eac ions a he TST applica ion si e. No e ha only he i s 26 subjec s andomized ecei ed a TST a sc eening.
1656 M. No by e al. / Vaccine 35 (2017) 1652–1661

signi ican ly o he obse ed AEs. Among he unsolici ed AEs, a
pos - accina ion in lamma o y eac ion was obse ed a he TST
injec ion si e in some o he subjec s who we e sc eened wi h
he TSTs a he ime o inclusion. Mos o hese TST eac ions we e
mild o mode a e, al hough one subjec de eloped a mo e se e e
TST si e eac ion. Thus, TST sc eening was emo ed om he s udy
p o ocol and should be omi ed om subsequen s udies o H4:
IC31. Acco dingly, TST was no pe o med du ing sc eening in
ano he ial es ing sa e y and immunogenici y o H4:IC31 in
BCG- accina ed adul s en olled in a high-endemic se ing in Sou h
A ica (Ae as p o ocol C-011-404) [18]. The TST eac ion mos ly
occu ed upon accina ion wi h he H4 an igen alone, which sug-
ges ed ha his local in lamma ion was caused by he M b-
speci ic an igens and no by he adju an . A simila pos -
accina ion eac ion a he si e o skin es ing was epo ed in a
clinical ial wi h ano he ecombinan p o ein adju an TB accine
candida e, M72F/AS02 [23]. This delayed- ype hype sensi i i y is
likely caused by PPD-speci ic T cells ha will apidly mig a e o
he TST injec ion si e in esponse o accine-an igens ha a e also
p esen in PPD. Thus, he isk o pos - accina ion TST si e eac-
ions needs o be conside ed upon adminis a ion o simila TB
accine cons uc s.
The e a e no well-de ined co ela es o bioma ke s o p o ec-
i e immuni y in TB, al hough CD4
+
Th1 cells ha exp ess IFN-
c
, IL-2 and TNF-
a
a e conside ed necessa y [24,25]. Ag85B-
TB10.4 esponses may p o ide he mos consis en p o ec ion in
popula ions wi h a high TB bu den [26]. The TB10.4 an igen is
ecognized by T cells om bo h BCG- accina ed and M b-
in ec ed indi iduals [7], and TB10.4-speci ic CD4
+
T cells co e-
la ed wi h p o ec ion agains TB in mice [27]. Adop i e ans e
o Ag85B/TB10.4-speci ic memo y CD4
+
T cells om immunized
mice also con e ed p o ec ion agains M. bo is BCG challenge in
ecipien mice [28]. Likewise, he H4:IC31 accine was able o eli-
ci expansion o and cy okine-p oduc ion in M b-speci ic CD4
+
T
cells in BCG- accina ed adul s, simila o he low accine-
speci ic CD4
+
T cell esponses demons a ed in a ela ed andom-
ized ial conduc ed in Sou h A ica (C-011-404) [18]. O e all, he
magni ude and quali y o he CD4
+
T cell esponses induced by
H4:IC31 seemed e y simila be ween he Sou h A ica ial and
he dose-escala ion ials conduc ed in a low-TB-endemic se ing
in Sweden and Finland. Bo h IFN-
c
ELISpo and ICS esul s con-
i med ha a e wo doses o H4:IC31, cy okine p oducing T cells
we e de ec ed p ima ily in esponse o he Ag85B componen o
H4 [18]. In mice, compa able T cell esponses ha e been de ec ed
agains bo h Ag85B and TB10.4 a e accina ion wi h an Ag85B/
TB10.4 subuni accine [8]. Despi e he dominance o Ag85B-
speci ic T cell esponses, i is possible ha e en e y low
esponses o TB10.4 in H4:IC31 accina ed indi iduals could be
expanded upon exposu e o M b, especially since TB10.4 epi opes
a e p esen ed du ing ac i e TB in ec ion [7].
Mul i unc ional T cells, co-exp essing se e al cy okines includ-
ing IFN-
c
, IL-2, and TNF-
a
, ha e been associa ed wi h immune con-
ol o pa asi es [28], and i al [29] and bac e ial in ec ions [30].I
has been sugges ed ha he IFN-
c
/IL-2/TNF-
a
iple posi i e cells
ep esen e ec o memo y T cells, he IL-2/TNF-
a
double posi i e
cells cen al memo y cells, and he IFN-
c
single posi i e cells e -
minally di e en ia ed e ec o T cells [24]. In he expe imen al
mouse model, he TB subuni accine Ag85B-ESAT-6/CAF01 was
shown o induce high le els o mul i unc ional memo y CD4
+
T
cells wi h p o ec i e e icacy ha accumula ed a he si e o in ec-
ion a e M b challenge, while ewe mul i unc ional T cells we e
e iden upon BCG accina ion [31]. Impo an ly, wo doses o he
Ag85B-ESAT-6/CAF01 subuni accine induced long- e m memo y
esponses [22]. The H4:IC31 accine induced p ima ily IFN-
c
/IL-2/
TNF-
a
iple posi i e and IL-2/TNF-
a
double posi i e CD4
+
Th1
subse s ha co ela ed wi h p o ec ion agains subsequen chal-
lenge wi h M b in mice [14,16]. An inc eased p opo ion o
TB10.4-speci ic mul i unc ional CD4
+
T cells we e also ound a
he si e o in ec ion when mice ecei ed H4:IC31 as a boos o
he BCG accine compa ed o accina ion wi h BCG alone [14].
Ou obse a ions sugges ha he H4:IC31 accine induced simila
mul i unc ional CD4
+
T cell p o iles in BCG- accina ed human sub-
jec s, which is also consis en wi h he H4:IC31 ial in Sou h A ica
[18]. Such mul i unc ional CD4
+
T cells we e also obse ed in
Table 3b
Ad e se e en s: C-006-404.
H4:IC31 (
l
g H4/nmol IC31)
Placebo 5/100 5/500 15/100 15/500 50/500 150/500
2 Doses 2 Doses 2 Doses 2 Doses 2 Doses 2 Doses 2 Doses
(n = 10) (n = 9) (n = 8) (n = 9) (n = 8) (n = 8) (n = 8)
MedDRA P e e ed e m n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Subjec s wi h a leas one solici ed AE 8 (80.0) 9 (100.0) 6 (75.0) 8 (88.9) 7 (87.5) 6 (75.0) 8 (100.0)
A h algia – 1 (11.1) 1 (12.5) 1 (11.1) – – 2 (25.0)
Dia hoea 1 (10.0) 3 (33.3) 1 (12.5) 1 (11.1) 2 (25.0) – –
Fa igue 6 (60.0) 6 (66.7) 2 (25.0) 6 (66.7) 5 (62.5) 4 (50.0) 7 (87.5)
Headache 6 (60.0) 5 (55.6) 2 (25.0) 4 (44.4) 5 (62.5) 5 (62.5) 6 (75.0)
Injec ion si e e y hema – 2 (22.2) – – – – 1 (12.5)
Injec ion si e swelling – 1 (11.1) – – 1 (12.5) – 2 (25.0)
Injec ion si e pain 2 (20.0) 8 (88.9) 3 (37.5) 5 (55.6) 6 (75.0) 4 (50.0) 5 (62.5)
Myalgia 4 (40.0) 6 (66.7) 1 (12.5) 5 (55.6) 3 (37.5) 4 (50.0) 5 (62.5)
Py exia 2 (20.0) – 2 (25.0) 1 (11.1) 3 (37.5) 1 (12.5) 2 (25.0)
Subjec s wi h a leas one unsolici ed AE 8 (80.0) 9 (100.0) 7 (87.5) 8 (88.9) 8 (100.0) 6 (75.0) 8 (100.0)
Aspa a e amino ans e ase inc eased – 1 (11.1) 1 (12.5) 1 (11.1) 1 (12.5) 2 (25.0) –
B adyca dia – – 2 (25.0) – 3 (37.5) – –
Haemoglobin dec eased 2 (20.0) 1 (11.1) – 1 (11.1) 3 (37.5) 1 (12.5) 2 (25.0)
Hea a e dec eased 1 (10.0) 1 (11.1) – 3 (33.3) – 2 (25.0) 2 (25.0)
Nausea – 1 (11.1) – – – 1 (12.5) 3 (37.5)
Neu ophil coun dec eased 2 (20.0) 3 (33.3) 1 (12.5) 3 (33.3) 4 (50.0) 1 (12.5) 3 (37.5)
Pha yngola yngeal pain – 1 (11.1) – – – – 4 (50.0)
P o ein in u ine 1 (10.0) 1 (11.1) 1 (12.5) 2 (22.2) 1 (12.5) – 2 (25.0)
Respi a o y ac in ec ion 4 (40.0) 2 (22.2) 4 (50.0) 4 (44.4) 4 (50.0) 2 (25.0) 1 (12.5)
No e: Indi idual unsolici ed AEs a e shown o AEs epo ed in P10% o subjec s ac oss he combined H4:IC31 egimens. A ull lis o AEs is p esen ed in Supplemen a y
Table 2B.
M. No by e al. / Vaccine 35 (2017) 1652–1661 1657
PPD-nega i e human subjec s a e accina ion wi h he M72/AS01
accine [32]. The induc ion o memo y cells by he H4:IC31 accine
can be a sou ce o new e ec o cells ha eme ge a e immune
con ac ion, which could be pa icula ly impo an o con ol o
ch onic in ec ions.
These ials clea ly show s onge and mo e pe sis en an igen-
speci ic CD4
+
T cell esponses upon accina ion wi h he H4 an i-
gen oge he wi h he IC31 adju an compa ed o H4 alone. The
IC31 adju an has p e iously been demons a ed o augmen sus-
ained Ag85B-ESAT-6 speci ic IFN-
c
esponses in heal hy human
subjec s [9]. We also ound a clea ad an age o wo accina ions
compa ed o only one dose. In he Sou h A ica ial, he H4:IC31
accine con aining he 15
l
g dose showed a s onge boos ing
e ec a e he second accina ion compa ed o he 5 and 50
l
g
dose le els [18], while a simila disc epancy be ween he 5, 15
and 50
l
g H4 doses was no appa en in ou ials. In addi ion,
one dose was shown o induce pos - accina ion IFN-
c
esponses
abo e p e- accina ion esponses in mos o 8 pa icipan s in he
Sou h A ican ial [18], whe eas pa icipan s ecei ing only one
dose in ou No dic ials main ained low T cell esponses. The
explana ion o hese di e ences is unclea , bu i is di icul o
make conclusions om hese smalle s udies (a ound 8 subjec s/
pe accine egimen), al hough he esul s may e lec on he di -
e en s udy popula ions i.e. A icans om a high-TB-endemic
coun y who may also become exposed o non- ube culous
mycobac e ia e sus no he n Eu opeans om low-TB-endemic
coun ies who may be mo e naï e o M b as well as en i onmen al
mycobac e ia.
In e es ingly, an inc eased H4 an igen dose om 50
l
g o
150
l
g induced weake CD4
+
T cell esponses, which is consis en
wi h o he s udies showing ha induc ion o M b-speci ic mul i-
unc ional T cells by Ag85B-TB10.4/IC31 was highly depended on
he H4 an igen dose [16,18]. In con as o highe an igen doses,
a lowe an igen dose selec i ely inc eased he numbe o
0.00
0.05
0.10
0.15
0.20
Placebo
50/0 1 dose
50/100 1 dose
50/500 1 dose
150/0 1 dose
%CD4 Response
%CD4 Response %CD4 Response
4.01BTB58gA
0.00
0.05
0.10
0.15
0.20
Placebo
50/0 2 Doses
50/100 2 Doses
50/500 2 Doses
050 100 150 200
0.00
0.05
0.10
0.15
0.20
S udy Day
050 100 150 200
Placebo
5/100 2 doses
5/500 2 doses
15/100 2 doses
15/500 2 doses
50/500 2 doses
150/500 2 doses
S udy Day
Vaccina ion(s) Vaccina ion(s)
A
B
C
Fig. 2. H4:IC31 accina ion induces ele a ed le els o an igen-speci ic CD4
+
T cells in he pe iphe al ci cula ion o BCG- accina ed s udy subjec s. F equencies o Ag85B- and
TB10.4-speci ic CD4
+
T cells induced by he H4:IC31 accine we e measu ed by 7-colo ICS assay and low cy ome y a e s imula ion o PBMCs wi h M b an igen-pep ide
pools. DMSO-sub ac ed an igen-speci ic expansion o he CD4
+
T cell subse in esponse o Ag85B o TB10.4 is p esen ed o he di e en ea men egimens in s udy C-005-
404 one dose (A) and wo dose (B) egimens and s udy C-006-404 (C). Samples o he ICS assay we e collec ed a s udy days 0, 7, 14, 28, 56, 63, 70, 84, and 182. Da a a e
p esen ed in linea g aph plo s showing he median and in e qua ile ange o each g oup.
1658 M. No by e al. / Vaccine 35 (2017) 1652–1661
mul i unc ional T cells, and was associa ed wi h a conside ably
s onge p o ec ion agains subsequen M b challenge in mice
[16]. The e o e, inding he app op ia e an igen and adju an doses
is a c ucial ac o when es ing new accines. Ou da a sugges ha
low ange H4 an igen doses in combina ion wi h he highe dose o
he IC31 adju an would be he op imal accine egimen o use in a
la ge scale.
P o ec i e immuni y in TB is also dependen on he induc ion o
CD8
+
cy oly ic T cell (CTL) esponses [33]. Using a whole blood
assay (FASCIA), we demons a ed ha simila o CD4
+
T cell
esponses, signi ican p oli e a ion o Ag85B-s imula ed CD8
aa
+
T cells we e seen wi h he wo dose 50/500 H4:IC31 accine com-
pa ed o he wo dose placebo egimen. These esul s we e consis-
en wi h p e ious da a demons a ing an inc eased in i o
p oli e a ion o CD4
+
T cells and CD8
aa
+
T cells in esponse o
Ag85B, a e immuniza ion wi h he TB accine candida e BCG
AFRO-1 in non-human p ima es [21]. I is belie ed ha CD8
aa
+
T
cells comp ise e ec o memo y cells and e minally di e en ia ed
memo y cells, and pe sis ence o hese cells may e lec he p es-
ence o con inuous an igen s imula ion [34]. IC31 is mos ly consid-
e ed a CD4
+
T cell p iming adju an ha induces ac i a ion o
majo his ocompa ibili y complex (MHC) class II
+
dend i ic cells
(DCs) including an up- egula ed exp ession o co-s imula o y
molecules [35]. I has been sugges ed ha adju an -ac i a ed
DCs may also igge CD8
+
T cells ia TLR9-dependen p oduc ion
o IFNband enhanced MHC class I an igen p esen a ion [36]. How-
e e , ew s udies ha e in es iga ed he e ec s o he IC31 adju an
on he induc ion o CD8
+
T cell esponses in i o and/o in humans
[37], which may be ela i ely lowe compa ed o CD4
+
T cells [14].
To enhance CD8
+
T cell ac i a ion mo e e icien ly, a po e- o ming
p o ein such as a bac e ial cy olysin, e.g. pe ingolysin [21,38] o
lis e iolysin [39], can be in oduced in he accine cons uc o pe -
mi leakage o an igens om he phagosome o he cy osolic MHC
class I pa hway.
0
100
200
300
400
500
500
1000
SFU/10
6
PBMC
Ag85B
50/100 1 dose
50/500 1 dose
Placebo
150/0 1 dose
50/0 1 dose
TB10.4
0
100
200
300
400
500
500
1000
SFU/10
6
PBMC
Placebo
50/0 2 doses
50/100 2 doses
50/500 2 doses
050 100 150 200
0
100
200
300
400
500
500
1000
S udy Day
SFU/10
6
PBMC
050 100 150 200
Placebo
5/100 2 doses
5/500 2 doses
15/100 2 doses
15/500 2 doses
50/500 2 doses
150/500 2 doses
S udy Day
Vaccina ion(s) Vaccina ion(s)
A
B
C
Fig. 3. H4:IC31 accina ion induces an igen-speci ic IFN-
c
p oduc ion in pe iphe al T cells om BCG- accina ed s udy subjec s. F equencies o Ag85B-speci ic IFN-
c
p oducing T cells induced by he H4:IC31 accine we e measu ed using he IFN-
c
ELISpo assay a e s imula ion o PBMCs wi h M b an igen-pep ide pools. Exp ession o IFN-
c
in Ag85B-speci ic T cells is shown o he di e en ea men egimens a s udy days 0, 56, 84 and 182 in s udy C-005-404 one dose (A) and wo dose (B) egimens and C-
006-404 (C). The da a illus a e medium-sub ac ed spo - o ming cells pe 10
6
PBMCs o all accina ed subjec s. Da a a e p esen ed in linea g aph plo s showing he median
and in e qua ile ange o each g oup.
M. No by e al. / Vaccine 35 (2017) 1652–1661 1659
Encou aging esul s om hese ials suppo u he clinical
e alua ion and de elopmen o he H4:IC31 accine candida e o
boos p o ec i e immuni y agains TB. In iew o ha , H4:IC31 is
cu en ly being e alua ed o he abili y o p e en M b in ec ion
in Quan iFERON-nega i e heal hy adul s compa ed o placebo
and BCG e accina ion in a andomized con olled ial conduc ed
in Sou h A ica (Ae as p o ocol C-040-404 and Clinical ial.go ID:
NCT02075203).
Funding
The ials we e unded by Ae as in he Uni ed S a es, who as he
sponso , de eloped he ial p o ocols and o e saw ial conduc .
Addi ional suppo o comple ion o his s udy was p o ided by
he Swedish Hea and Lung ounda ion, he Swedish Resea ch
Council and he Ka olinska Ins i u e in S ockholm, Sweden.
Con lic o in e es s a emen
The au ho s decla e no con lic o in e es .
Acknowledgemen s
We hank he olun ee s who pa icipa ed in hese ials. We
also hank Zhongkai Shi, Ka h yn Ru kowski and Ann Ginsbe g a
Ae as o c i ical e iew o he manusc ip and edi o ial sugges-
ions; and B idge Col in o assis ance wi h p epa a ion o he
manusc ip .
0.00
0.02
0.04
0.06
0.08
0.10
0.2
0.4
0.00
0.02
0.04
0.06
0.08
0.10
0.2
0.4
Placebo
50/0 2 Doses
50/100 2 Doses
50/500 2 Doses
Placebo
50/0 1 Dose
50/100 1 Dose
50/500 1 Dose
% CD4 Response % CD4 Response
0.00
0.02
0.04
0.06
0.08
0.10
0.2
0.4
Placebo
5/100 2 doses
5/500 2 doses
15/100 2 doses
15/500 2 doses
50/500 2 doses
150/500 2 doses
150/0 1 Dose
% CD4 Response
A
B
C
Fig. 4. H4:IC31 accina ion induces an igen-speci ic mul i unc ional CD4
+
Th1 esponses in he pe iphe al ci cula ion o BCG- accina ed s udy subjec s. F equencies o
cy okine-exp essing Ag85B-speci ic CD4
+
T cells exp essing IFN-
c
, IL-2 and/o TNF-
a
induced by he H4:IC31 accine, we e measu ed by 7-colo ICS assay and low cy ome y
a e s imula ion o PBMCs wi h M b an igen-pep ide pools. DMSO-sub ac ed cy okine esponses in Ag85B-speci ic CD4
+
T cells a e shown o he di e en ea men
egimens a s udy day 84 in s udy C-005-404 one dose (A) and wo dose (B) egimens and C-006-404 (C). Da a a e p esen ed in box and whiske s plo s showing he median,
in e qua ile ange, minimum and maximum o each g oup.
1660 M. No by e al. / Vaccine 35 (2017) 1652–1661