Safety and immunogenicity of the novel H4:IC31 tuberculosis vaccine candidate in BCG-vaccinated adults: Two phase I dose escalation trials

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