Live baculovirus acts as a strong B and T cell adjuvant for monomeric and oligomeric protein antigens

Con en s lis s a ailable a ScienceDi ec Vi ology jou nal homepage: www.else ie .com/loca e/y i o Li e baculo i us ac s as a s ong B and T cell adju an o monome ic and oligome ic p o ein an igens Su i Heinimäki, Ki si Tamminen, Ma ia Malm, Timo Vesika i, Vesna Blaze ic ⁎ Vaccine Resea ch Cen e , Uni e si y o Tampe e, Finland ARTICLE INFO Keywo ds: Adju an Baculo i us Immunogenici y Vi us-like pa icle ABSTRACT Recombinan p o eins p oduced by baculo i us (BV) exp ession sys ems con ain esidual BV a e c ude pu ifica ion. We s udied adju an effec o BV on an ibody and T cell esponses agains wo model an igens, monome ic o albumin (OVA) p o ein and oligome ic no o i us (NoV) i us-like pa icles (VLPs). BALB/c mice we e immunized in ade mally wi h OVA alone o OVA o mula ed wi h li e o inac i a ed BV, and VLP o mula ions comp ised o ch oma og aphically pu ified NoV GII.4 VLPs alone o mixed wi h BV, o o c ude pu ified VLPs con aining BV impu i ies om exp ession sys em. Li e BV imp o ed immunogenici y o NoV VLPs, spa ing VLP dose up o 10- old. Mo eo e , soluble OVA p o ein induced IgG2a an ibodies and T cell esponse only when co-adminis e ed wi h li e BV. BV adju an effec was comple ely ab oga ed by emo al o inac i a ion o BV. These findings suppo he usage o c ude pu ified p o eins con aining esidual BV as accine an igens. 1. In oduc ion Nume ous cu en ly licensed accines a e based on li e a enua ed o inac i a ed pa hogens, bu he e is a endency owa ds de elopmen o sa e non- eplica ing subuni p o ein accines. The e o e, no el accines unde de elopmen a e o en based on ecombinan p o eins. P oduc ion o ecombinan p o eins esul s in subs an ial amoun s o impu i ies ela ed o he exp ession echnology u ilized in he p oduc- ion p ocess. Impu i ies de i ed om baculo i us (BV) exp ession sys em include li e BV, baculo i al dsDNA genome, and baculo i al p o eins, pa icula ly en elope glycop o ein gp64 (He as-S ubbs e al., 2007; Huh i e al., 2013; Lappalainen e al., 2016). A p esen , se e al ecombinan p o ein accines based on BV exp ession echnology a e comme cially a ailable, including human accines agains human papilloma i us (Ce a ix®, GlaxoSmi hKline) and influenza i us (Flublok®, P o ein Sciences), as well as e e ina y accines agains classical swine e e i us (Po cilis Pes i®, MSD Animal Heal h) and po cine ci co i us ype 2 (Po cilis®PCV, MSD Animal Heal h; Ci coFLEX®, B. Ingelheim). Mo eo e , o he po en ial accine candida es a e in ad anced s ages o clinical ials, such as hose di ec ed agains no o i us (NoV) (Vesika i and Blaze ic, 2015; A ma e al., 2016). Because o a s ic egula o y con ol, subs an ial effo s a e unde aken o pu i y p o eins o use as human accine an igens. On he o he hand, impu i ies ela ed o BV exp ession sys em ha e commonly been associa ed wi h s ong immunos imula- o y effec s. BV has been epo ed o possess adju an p ope ies, p omo ing humo al and cellula immune esponses agains o eign an igens as well as ac i a ion o inna e immune esponses by inducing ype I and II IFNs (G onowski e al., 1999; Abe e al., 2005; He as- S ubbs e al., 2007; Suzuki e al., 2010). Mo eo e , wild- ype BV has con ibu ed o p o ec ion in mice om a le hal challenge o encepha- lomyoca di is i us (EMCV) (G onowski e al., 1999), influenza i us H1N1 (Abe e al., 2003) and oo -and-mou h disease i us (FMDV) (Molina i e al., 2011; Qua occhi e al., 2013), whe e p o ec ion was di ec ly associa ed wi h immune esponses elici ed by BV. In he his o y o human accines, he mos success ul in disease con ol and e adica ion ha e been hose employing li e a enua ed i uses (e.g. polio, measles, yellow e e , influenza i us, o a i us) (Mino , 2015). As hese i uses can s ill in ec cells and eplica e o a ce ain limi , he li e a enua ed accines a e able o induce as and du able p o ec i e immuni y. Ins ead, ecombinan p o eins a e gen- e ally weakly immunogenic unless adminis e ed wi h an ex e nal adju an (Bachmann and Jennings, 2010; Jose sbe g and Buckland, 2012). Due o he adju an and an i i al p ope ies o BV desc ibed abo e, esidual BV o igina ing om he exp ession sys em could be conside ed as an adju an o imp o e immunogenici y o hese p o eins. In he p esen s udy, we in es iga ed adju an effec o BV on h p://dx.doi.o g/10.1016/j. i ol.2017.08.023 Recei ed 16 May 2017; Recei ed in e ised o m 15 Augus 2017; Accep ed 16 Augus 2017 ⁎ Co espondence o: Vaccine Resea ch Cen e , Uni e si y o Tampe e, Bioka u 10, 33520 Tampe e, Finland. E-mail add esses: su i.heinimaki@u a.fi(S. Heinimäki), ki si. amminen@u a.fi(K. Tamminen), ma ia.malm@u a.fi(M. Malm), imo. esika i@u a.fi(T. Vesika i), esna.blaze ic@u a.fi(V. Blaze ic). Vi ology 511 (2017) 114–122 A ailable online 24 Augus 2017 0042-6822/ © 2017 The Au ho s. Published by Else ie Inc. 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/). MARK an ibodies and T cell media ed immune esponses agains wo diffe en model an igens, a soluble monome ic o albumin (OVA) p o ein and NoV i us-like pa icles (VLPs). We demons a e he e ha li e BV p omo ed immunogenici y o bo h OVA and NoV VLPs, and he effec was abolished by i us inac i a ion. 2. Ma e ials and me hods 2.1. An igenic o mula ions 2.1.1. O albumin Lyophilized OVA (Albumin om chicken egg whi e; Sigma) was dissol ed a 2 mg/mL in phospha e-buffe ed saline (PBS; Lonza, Ve ie s, Belgium). The s ock was s e ilized wi h a 0.2 µm fil e p io o use. 2.1.2. P oduc ion o mock BV Li e BVs we e gene a ed in Bac- o-Bac BV exp ession sys em (In i ogen, Ca lsbad, CA) in S 9 insec cells and pu ified on suc ose g adien s and ul acen i uga ion as p e iously desc ibed (Huh i e al., 2013). The in ec ious BV i e o he mock BV, exp essed as plaque- o ming uni pe mL (p u/mL), was de e mined using a BacPAK™ Rapid Ti e Ki (Clon ech Labo a o ies, Moun ain View, CA) acco ding o he manu ac u e 's ins uc ions. The inac i a ed BV (IBV) was p epa ed om li e BV by hea inac i a ion a 60 °C o 1 h. The inac i a ion o li e BVs was confi med by BacPAK™Rapid Ti e Ki . 2.1.3. P oduc ion o ecombinan NoV VLPs NoV GII.4 (Re e ence s ain accession numbe AF080551) VLPs we e p oduced in BV–insec cell exp ession sys em, as desc ibed in de ails elsewhe e (Huh i e al., 2010). GII.4 VLPs we e c udely pu ified on wo discon inuous suc ose g adien s and ul acen i uga ion as desc ibed ea lie (Huh i e al., 2010). In o de o ob ain highly pu ified p o eins, he c ude GII.4 VLPs we e u he pu ified using a combina- ion o wo-s ep anion exchange (HiT ap Q, GE Heal hca e, Uppsala, Sweden) ch oma og aphy p ocedu es (Huh i e al., 2013). The pu i y, iden i y and mo phology o he c ude and ch oma o- g aphically pu ified NoV GII.4 VLPs we e de e mined using p e iously published p ocedu es (Huh i e al., 2013; Blaze ic e al., 2016), such as SDS-PAGE ollowed by PageBlue™s aining (The mo Fishe Scien ific Inc., Rock o d, IL) o immunoblo ing wi h an i-BV gp64 (San a C uz Bio echnology Inc, San a C uz, CA), Quan -i dsDNA B oad-Range Assay Ki (In i ogen), Limulus Amebocy e Lysa e Assay (Lonza, Walke s ille, MD), BacPAK™Rapid Ti e Ki , and ansmission elec on mic oscopy (FEI Tecnai F12, Philips Elec on Op ics, Holland) a e nega i e s aining. 2.1.4. Syn he ic pep ides A mu ine CD4 + T cell epi ope ( 323 ISQAVHAAHAEINEAGR 339 , In i oGen, San Diego, CA) de i ed om chicken OVA (McFa land e al., 1999) was used o es OVA-specific T cell esponses by enzyme- linked immunospo (ELISPOT) in e e on gamma (IFN-γ) assay. Ro a i us-specific 14-me R6-1 pep ide ( 289 RLSFQLMRPPNMTP 302 ) syn he ized by P oimmune LTd. (Ox o d, UK) was used as an i ele an nega i e con ol pep ide in he assays. Se en y-six 18-me o e lapping pep ides ep esen ing he en i e 539 amino acid (aa) sequence o GII.4–1999 NoV VP1 we e cus om syn he ized (Synpep ide Co. L d, Shanghai, China) and pooled (Malm e al., 2016b, 2016c). A comple e pep ide pool (named 99 pool) as well as an indi idual pep ide 99-50 ( 344 TRAHKATVSTGSVHFTPK 361 ) co esponding o he p e iously iden ified mu ine 18-me NoV-specific CD4 + T cell epi ope (Malm e al., 2016c) we e used o es NoV-specific T cell esponses by he ELISPOT. 2.2. Animal immuniza ion and sample p epa a ion Female BALB/c OlaHsd mice, aged 6 weeks, we e pu chased om En igo (Ho s , he Ne he lands). The mice we e andomly di ided in o eigh g oups (G I–VIII), acclima ized unde con olled specific pa ho- gen- ee condi ions o one week p io o he s a o he expe imen , and main ained h oughou he s udy pe iod wi h ood and wa e p o ided ad libi um. Animals (3–4 mice/expe imen al g oup) we e immunized wice (a s udy weeks 0 and 3) in ade mally (i.d.) a he base o he ail wi h 50 µl o diffe en an igenic o mula ions, each o mula ion dilu ed in s e ile PBS o con ain he indica ed dose o immunogen (Table 2). Con ol g oup ecei ed no an igen (PBS only). Immuniza ions we e pe o med unde gene al anes hesia induced wi h a mix u e o Hypno m®(Ve aPha ma Limi ed, Leeds, UK) and Do micum®(Roche Pha ma AG, G enzach-Wyhlen, Ge many). To es he kine ics o he an ibody esponses in se a, ail blood samples (dilu ed 1:200 in PBS a he ime o collec ion) we e collec ed a s udy weeks 0 (p e-bleed, non-immune se a) and 2 o 3. Mice we e sac ificed 35 days a e he fi s immuniza ion (a s udy week 5) by decapi a ion, when whole blood and lymphoid issues we e collec ed. P epa a ion o blood samples and a single-cell suspension om he spleen o each mouse was conduc ed acco ding o he published p ocedu es o ou labo a o y (Tamminen e al., 2012). All o he expe imen al p ocedu es ca ied ou we e in acco dance wi h he egula ions and guidelines o he Finnish Na ional Expe imen Boa d (pe mission numbe s ESLH-2009–06698/Ym-23 and ESAVI/4106/ 04.10.03/2012) and all effo s we e made o minimize animal suffe - ing. Animals we e moni o ed o physical condi ions h oughou he expe imen . 2.3. De ec ion o se um IgG and IgG sub ypes by ELISA 2.3.1. OVA- and NoV GII.4-specific IgG, IgG1 and IgG2a esponses Se a o expe imen al mice we e es ed in ELISA o he p esence o OVA- o NoV GII.4-specific IgG, IgG1 and IgG2a an ibodies as desc ibed in de ail elsewhe e (Blaze ic e al., 2011; Tamminen e al., 2012). B iefly, 96-well hal -a ea polys y ene pla es (Co ning Inc, Co ning, NY) we e coa ed wi h OVA (200 ng/well o IgG, 100 ng/well o IgG sub ypes) o GII.4 VLPs (20 ng/well). Se um samples a 1:200 dilu ion o se ially dilu ed wo- old we e added on he pla es, and he bound an ibodies we e de ec ed wi h ho se adish pe oxidase (HRP)- conjuga ed an i-mouse IgG (Sigma-Ald ich, S . Louis, MO), IgG1 (In i ogen) o IgG2a (In i ogen) and SIGMA FAST OPD subs a e (Sigma-Ald ich). Op ical densi y (OD) alues a 490 nm (OD 490 ) we e measu ed by a mic opla e eade (Vic o 2 1420; Pe kinElme , Wal ham, MA). A sample was conside ed posi i e i he OD 490 was abo e he cu -off alue (mean OD 490 o he con ol mice + 3 × SD) and OD 490 > 0.1. The end-poin i e s we e defined as he ecip ocal o he highes dilu ion wi h an OD 490 abo e he cu -off alue. A i e o 100 was assigned o nega i e samples, being a hal o he s a ing se um dilu ion. 2.3.2. BV-specific IgG esponse Induc ion o BV-specific IgG esponse was e alua ed by es ing 1:200 dilu ed se um samples o indi idual expe imen al mice by ELISA as desc ibed abo e o OVA- and NoV-specific esponses, bu he mic o i e pla es we e coa ed wi h mock BV (100 ng/well). 2.4. An ibody a idi y assay The a idi y o OVA- and NoV-specific IgG an ibodies was de e - mined in 1:200 dilu ed se um samples acco ding o he p e iously published a idi y assay (Tamminen e al., 2012) using an ex a u ea incuba ion s ep o emo e he low-a idi y an ibodies. Resul s we e exp essed as a idi y index: (OD 490 wi h u ea/OD 490 wi hou u ea) × 100%. S. Heinimäki e al. Vi ology 511 (2017) 114–122 115 2.5. NoV blocking assay To examine he abili y o se um an ibodies o block he binding o NoV VLPs o a cellula his o-blood g oup an igen (HBGA) ecep o , pig gas ic mucin (PGM) ype III (Sigma Chemicals) was used as a sou ce o HBGAs acco ding o he published p ocedu e (Lindesmi h e al., 2012) wi h sligh modifica ions (Malm e al., 2017). G oup-wise pooled wo- old se um dilu ions (s a ing a 1:50) we e p e-incuba ed o 1 h a 37 °C wi h 0.1 µg/mL o NoV GII.4 VLPs be o e pla ing on 96- mic owell pla es coa ed wi h 2.5 µg/mL o PGM. Maximum binding was de e mined wi h NoV VLPs wi hou se um. Bound VLPs we e de ec ed wi h human NoV an ise um, an an i-human IgG-HRP (No ex) and Sigma FAST OPD subs a e, ollowed by de e mina ion o OD 490 eadings wi h a mic opla e eade (Vic o 2 1420). Blocking index (%) was calcula ed as ollows: 100% −[OD 490 (wells wi h se um)/ OD 490 (wells wi hou se um, maximum binding) × 100%]. Resul s we e exp essed as he blocking i e 90 (BT90), he ecip ocal o he highes se um dilu ion blocking ≥90% o he VLPs binding o he HBGAs. 2.6. Cell-media ed immune esponses 2.6.1. NoV and OVA-specific ELISPOT IFN-γ An igen-specific T cell esponses we e analyzed using a sligh ly modified ELISPOT IFN-γassay (Tamminen e al., 2013). G oup-wise pooled splenocy es (0.2×10 6 cells/well) we e pla ed on Mul isc een HTS-IP fil e pla es (Millipo e, Bille ica. MA) coa ed wi h a monoclonal an i-mouse IFN-γan ibody (Mab ech AB, Nacka S and, Sweden) a 5 µg/mL. Fo de ec ion o OVA-specific IFN-γp oducing cells, cell we e s imula ed in duplica es wi h OVA pep ide (0.2, 1, 2, and 4 µg/mL) o R6-1 pep ide (nega i e con ol, 4 µg/mL). To de ec NoV-specific IFN- γp oducing cells, cells we e s imula ed in duplica es wi h GII.4–99 pep ide pool (2 µg/mL), GII.4 99-50 pep ide (4 µg/mL) o OVA pep ide (nega i e con ol, 4 µg/mL). Backg ound con ol (cul u e medium; RPMI 1640 supplemen ed wi h 10% FBS, 100 U/mL penicillin, 100 µg/mL s ep omycin, 50 µM 2-me cap oe hanol, and 2 mM L- glu amine; all om Sigma-Ald ich) and cell iabili y con ol (10 μg/ mL T cell mi ogen Concana alin A; Sigma-Ald ich) we e es ed in each assay. A e o e nigh incuba ion a 37 °C, IFN-γsec e ion was de ec ed wi h bio inyla ed an i-mouse IFN-γmonoclonal an ibody and alkaline phospha ase -conjuga ed s ep a idin (bo h om Mab ech AB). The spo s de eloped wi h BCIP/NBT (5-b omo-4- chlo o-3-indolyl-phospha e) subs a e (Mab ech AB) we e coun ed by ImmunoSpo ®au oma ic CTL analyze (CTL-Eu ope GmbH, Bonn, Ge many). The esul s we e exp essed as mean spo - o ming cells (SFC)/10 6 splenocy es o duplica e wells. An inc ease o a leas wice abo e he nega i e con ol pep ide SFC coun s was conside ed as a posi i e esul . 2.6.2. BV-specific ELISPOT IFN-γ BV-specific T cell esponses we e assessed by ELISPOT IFN-γassay as desc ibed abo e, excep g oup-wise pooled splenocy es (1×10 5 cells/ well) o immunized and con ol mice we e s imula ed wi h mock BV (1×10 4 –10 6 p u/well). 2.7. S a is ical analyses The Mann-Whi ney U- es and K uskal-Wallis es we e employed o de e mine he s a is ical diffe ences be ween he non-pa ame ic obse a ions o wo o mo e independen g oups. All analyses we e conduc ed by IBM SPSS S a is ics o Windows (IBM Co p., A monk, NY), Ve sion 23.0. The s a is ically significan diffe ence was defined as p≤0.05. 3. Resul s 3.1. Cha ac e iza ion o NoV VLPs used o immuniza ion C ude and ch oma og aphically pu ified NoV GII.4 VLPs we e cha ac e ized o pu i y, iden i y and mo phology p io o use o immunizing animals. Fig. 1A shows SDS-PAGE, whe e NoV capsid p o ein appea ed as a double band, ypical o he capsid GII.4 p o ein (Huh i e al., 2010), in bo h VLP p epa a ions. Ins ead, a ain band co esponding o he size o he BV en elope gp64 p o ein, was obse ed only in he c ude p epa a ion (Fig. 1A). The p esence o gp64 in he c ude pu ified VLPs was confi med by immunoblo ing (Table 1) wi h an i-BV gp64. Fu he mo e, he p esence o BV was u he es ed by de e mining he in ec ious BV i e s (Table 1). Al hough c ude VLPs a e p oduced by he s anda d me hods as desc ibed abo e, diffe en p oduc ion lo s ha e inconsis en quan i y o BV (da a no show). Mo eo e , c ude and ch oma og aphically Fig. 1. Cha ac e iza ion o NoV GII.4 VLPs used o immuniza ions. (A) Pu i y and in eg i y analysis o c ude (lane 1) and ch oma og aphically pu ified (lane 2) NoV GII.4 VLPs wi h SDS-PAGE ollowed by PageBlue s aining. Lane M, molecula weigh ma ke . (B) Elec on mic oscopy images o c ude (panel 1) and ch oma og aphically pu ified (panel 2) NoV GII.4 VLPs co esponding o he espec i e SDS-PAGE lanes 1 and 2 (A). P o ein s uc u es we e examined a e nega i e s aining wi h 3% u anyl ace a e. Images we e obse ed a 18500 × magnifica ion. Table 1 Speci ica ions and analysis o impu i ies ela ed o he exp ession sys em in he c ude pu i ied and ch oma og aphically pu i ied NoV GII.4 VLPs. Specifica ion C ude GII.4 VLPs Pu e GII.4 VLPs Mo phology a VLPs (~38 nm) VLPs (~38 nm) In ec ious BV (p u/mL) b 10 7 0 BV gp64 c +– To al DNA (ng dsDNA/10 µg p o ein) d <30 <10 Endo oxin (EU/10 µg p o ein) e < 0.1 < 0.1 a Elec on mic oscopy a e nega i e s aining. b BacPAK™Rapid Ti e Ki . c SDS-PAGE ollowed by immmunoblo ing wi h an i-BV gp64. d Quan -i dsDNA B oad-Range Assay Ki . e Limulus Amebocy e Lysa e Assay. S. Heinimäki e al. Vi ology 511 (2017) 114–122 116 pu ified NoV GII.4 VLPs we e mo phologically simila as indica ed by EM analysis (Fig. 1B). Hence, he ch oma og aphic pu ifica ion did no affec mo phology o in eg i y o VLPs, bu emo ed impu i ies ela ed o he BV exp ession sys em, as i ually no aces o dsDNA genome, li e BV o baculo i al p o eins we e de ec ed in he pu e VLP p epa a ions (Table 1). 3.2. Assessmen o physical condi ion Rega dless o he an igenic o mula ion used o immuniza ion, all animals emained heal hy and gained weigh du ing he s udy pe iod (Table 2). No diffe ence in body weigh be ween he expe imen al g oups was obse ed (p=0.98). 3.3. Enhancemen o OVA-specific se um an ibody esponses by BV Possible influence o BV on OVA-specific immune esponses was in es iga ed by immunizing he expe imen al mice on a wo-dose schedule wi h 45 μg o OVA alone o oge he wi h li e BV o IBV a an in e al o h ee weeks (Table 2). Two immuniza ions o each o mula ion elici ed conside able le el o o al an i-OVA IgG an ibodies (Fig. 2A). Simila IgG esponses wi h end-poin i e s o 12800 we e induced, when OVA was adminis e ed alone o oge he wi h IBV. Ins ead, co-adminis a ion o OVA wi h li e BV esul ed in 16- old inc ease in he IgG i e (end-poin i e 204800). No OVA-specific an ibodies we e de ec ed in se a o con ol mice. De e mina ion o OVA-specific IgG sub ype IgG1 and IgG2a i e s, ep esen ing Th2- and Th1- ype esponses, e ealed induc ion o IgG1 an ibodies by each OVA o mula ion (Fig. 2B) bu induc ion o significan IgG2a an ibodies only by he OVA o mula ions con aining li e BV (Fig. 2C). Co-adminis a ion o OVA wi h BV gene a ed ≥16- old highe le els o IgG1 (end-poin i e 409600) compa ed o adminis a ion o OVA alone o oge he wi h IBV. No IgG2a was de ec ed a e immuniza ion o mice wi h OVA alone while o mula ion wi h IBV induced e y low le el o IgG2a, p obably due o incomple e inac i a ion o BV (Table 2) con aining 10 3 p u. In con as , combina- ion o OVA and li e BV induced e y high an i-OVA IgG2a an ibodies (end-poin i e 102400). Se um samples om indi idual mice we e u he assayed o he a idi y o OVA-specific IgG an ibodies. Immuniza ion wi h OVA o combina ion o OVA and IBV induced IgG an ibodies wi h low a idi y ( espec i e a idi y indices 21.0 ± 5.8% and 10.4 ± 1.3%), bu inclusion o BV in he o mula ion ele a ed he a idi y o an ibodies (a idi y index 47.5 ± 8.8%) (Fig. 2D). No s a is ical diffe ence (p=0.083) was de ec ed in he a idi y indices be ween he g oups immunized wi h OVA alone and a combina ion o OVA and BV, bu he a idi y was significan ly g ea e (p=0.021) in mice co-adminis e ed wi h OVA and BV compa ed o he mice co-adminis e ed wi h OVA and IBV. 3.4. Induc ion o OVA-specific T cell esponses by li e BV The diffe ence in induc ion o Th1 esponses by OVA o mula ions wi h o wi hou BV was u he s udied measu ing Th1- ype cy okine IFN-γp oduc ion om he splenocy es o immunized mice (Fig. 3). Cells om he mice ecei ing OVA and BV esponded wi h conside able IFN-γ elease o ex i o s imula ion wi h he 17-me OVA-specific pep ide, ep esen ing a CD4 + T cell epi ope (Fig. 3). On he con a y, immuniza ion wi h OVA alone o oge he wi h IBV did no induce OVA-specific IFN-γp oduc ion by T cells (p=0.020 o bo h) (Fig. 3). No esponse o he nega i e con ol pep ide R6-1 was de ec ed in any o he s udy g oups (da a no shown). 3.5. Imp o ed immunogenici y o NoV VLPs by BV To examine effec o BV on NoV GII.4-specific humo al immune esponses, mice we e immunized wice wi h 1 o 10 µg doses o pu e NoV GII.4 VLPs, 1 µg dose o pu e VLPs combined wi h BV o 1 µg dose o c ude-pu ified VLPs con aining BV (Table 2). A e he fi s immuniza ion, 1 µg o pu e VLPs induced significan ly lowe IgG esponse in compa ison wi h o he VLP o mula ions (p=0.003), whe eas 10 µg dose o pu e VLPs as well as bo h VLP o mula ions con aining BV esul ed in simila esponses (p=0.221) a s udy week 2 o 3 (Fig. 4A). Two immuniza ions o each an igenic o mula ion con aining NoV GII.4 VLPs elici ed high le el o o al an i-GII.4 IgG an ibodies a week 5 (Fig. 4A). Con ol mice emained nega i e o GII.4-specific o al IgG du ing he s udy pe iod (Fig. 4A). De e mina ion o IgG sub ype i e s showed induc ion o bo h Th2- and Th1- ype esponse by each GII.4 VLP o mula ion. Simila IgG1 esponses we e elici ed, i espec i e o he p esence o absence o BV (Fig. 4B). Ins ead, adminis a ion o he GII.4 VLPs wi h BV esul ed in 8- old highe IgG2a i e s (end-poin i e s o 204800) compa ed wi h adminis a ion o 1 o 10 µg doses o pu e VLPs (end-poin i e s o 25600) (Fig. 4C). No GII.4-specific IgG sub ype an ibodies we e de ec ed in se a o con ol mice (Fig. 4B and C). 3.6. Effec o BV on unc ionali y o GII.4-specific an ibodies Tes ing o indi idual immune se a o he a idi y o GII.4-specific IgG an ibodies indica ed, ha immuniza ion wi h 1 µg o pu e VLPs induced an ibodies wi h a conside ably lowe (p=0.029) a idi y (a idi y index 18.4 ± 7.9%) as compa ed wi h an ibodies induced wi h a combina ion o VLPs and BV (67.2 ± 10.3%) o c ude-pu ified VLPs wi h esidual BV (60.9 ± 3.9%) (Fig. 5A). No s a is ical diffe ence (p=0.624) was obse ed be ween he g oups o mice immunized wi h 10 µg o pu e VLPs (a idi y index 48.7 ± 8.9%) and he wo VLP o mula ions con aining BV (Fig. 5A). The unc ionali y o he GII-4-specific an ibodies was u he examined by measu ing he blocking po en ial o he pooled immune se a, whe e PGM was employed as he HBGA sou ce o GII.4 VLP binding. Each VLP o mula ion induced an ibodies able o block ≥90% o he VLP binding (Fig. 5B). Howe e , 4- old highe blocking i e s BT90 we e obse ed in mice immunized wi h VLPs combined wi h BV o c ude-pu ified VLPs con aining BV, compa ed wi h mice immunized wi h 1 µg dose o pu e VLPs (Fig. 5B). Mo eo e , adminis a ion wi h 10 µg dose o pu e VLPs esul ed in an ibodies wi h 2- old g ea e BT90 as compa ed wi h an ibodies induced wi h lowe dose o pu e VLPs (Fig. 5B). 3.7. Enhancemen o GII.4-specific T cell esponses by BV Induc ion o T cell esponses by NoV GII.4 VLP o mula ions was Table 2 An igenic o mula ions used o immuniza ion o expe imen al mice g oups and body weigh s. Expe imen al g oup Immunogen Injec ion dose S a weigh (g) a Te mina ion weigh (g) b I OVA 45 µg 17.9 ± 0.3 21.0 ± 0.8 II OVA + BV 45 µg + 10 7 p u 17.4 ± 1.1 20.6 ± 1.2 III OVA + IBV 45 µg + 10 3 p u 17.9 ± 1.4 21.4 ± 1.8 IV GII.4 VLPs 1 µg 17.9 ± 0.9 20.5 ± 1.0 V GII.4 VLPs 10 µg 17.5 ± 0.4 20.5 ± 0.5 VI GII.4 VLPs + BV 1 µg + 10 7 p u 17.3 ± 4.3 21.1 ± 3.9 VII C ude GII.4 VLPs 1 µg (10 5 p u) 17.2 ± 1.0 20.8 ± 2.1 VIII PBS –17.8 ± 0.6 20.7 ± 0.5 a Mean weigh +/−SD pe expe imen al g oup o mice a s udy week 0 (p io o immuniza ion). b Mean weigh +/−SD pe expe imen al g oup o mice a s udy week 5. S. Heinimäki e al. Vi ology 511 (2017) 114–122 117 cha ac e ized by analyzing Th1- ype cy okine IFN-γp oduc ion om he splenocy es o expe imen al mice. Splenocy es om he mice ecei ing 1 µg o pu e GII.4 VLPs did no p oduce IFN-γin esponse o any s imula ion (Fig. 6). Ins ead, immuniza ion o mice wi h all o he VLP o mula ions elici ed a obus IFN-γ esponse, when s imula ed wi h he 18-me 99-50 pep ide ep esen ing a NoV-specific CD4 + T cell epi ope, o wi h 99 pep ide pool ep esen ing he en i e GII.4–1999 NoV VP1 (Fig. 6 and da a no shown). Ten µg dose o pu e VLPs induced simila quan i ies o IFN-γsec e ing cells o 1 µg o VLPs combined wi h BV (p=0.08) as well as 1 µg o c ude-pu ified VLPs con aining BV (p=0.885). Nega i e con ol OVA pep ide s imula ed no IFN-γp oduc ion by he cells om any o he expe imen al g oups (Fig. 6). 3.8. Induc ion o BV-specific an ibodies and T cell esponses Induc ion o BV-specific IgG esponse was e alua ed in se um samples o mice immunized wi h OVA o mula ions (g oups I-III, Table 2). As expec ed, addi ion o li e BV o IBV o he OVA o mula ion induced BV-specific esponses (p=0.015). No BV esponse was elici ed immunizing wi h OVA alone, bu conside able le els o BV- specific an ibodies we e induced, when OVA was co-adminis e ed wi h BV o IBV (Fig. 7A). No diffe ence in an ibody le els was no ed whe he OVA was adminis e ed wi h BV o IBV (p=0.149). Induc ion o BV-specific T cell esponses was s udied measu ing IFN-γp oduc ion om he splenocy es o he expe imen al g oups. Cells om he mice ecei ing OVA alone did no p oduce IFN-γin esponse o s imula ion wi h BV (Fig. 7B). Ins ead, immuniza ions o mice wi h o mula ions con aining li e BV elici ed high IFN-γ esponse (Fig. 7B). Mo eo e , low le els o IFN-γwe e induced by co-adminis- a ion o OVA wi h IBV, which we e subs an ially lowe compa ed wi h he esponses induced by li e BV o mula ion (p=0.019). No BV- specific IFN-γ esponse was de ec ed by he cells o nega i e con ol mice (Fig. 7B). 4. Discussion Se e al epo s ha e shown li e BV o ha e s ong adju an p ope ies, p omo ing adap i e immune esponses agains co-adminis- e ed an igen and ac i a ion o inna e immuni y (Abe e al., 2003; Fig. 2. OVA-specific se um IgG esponses. End-poin i a ions o an i-OVA IgG (A), IgG1 (B) and IgG2a (C) an ibodies o g oup-wise pooled e mina ion se a o mice immunized wi h OVA alone o o mula ed wi h BV. Con ol mice ecei ed PBS only. Mean i a ion cu es wi h s anda d e o s o duplica e wells a e shown. (D) Mean a idi y indices (%) wi h s anda d e o o he means o se um OVA-specific IgG an ibodies. Fig. 3. OVA-specific T cell esponses. Diffe en concen a ions o an OVA-specific pep ide we e employed o s imula e IFN-γp oduc ion om he g oup-wise pooled splenocy es o mice immunized wi h OVA alone o oge he wi h BV o IBV. Mean IFN-γ spo - o ming cells (SFC)/10 6 splenocy es o duplica e wells wi h s anda d e o s o he means a e shown. S. Heinimäki e al. Vi ology 511 (2017) 114–122 118 He as-S ubbs e al., 2007; Molina i e al., 2011; Ma gine e al., 2012; Qua occhi e al., 2013). The adju an and an i i al p ope ies o BV make i an in e es ing ool o be conside ed in imp o emen o immunological esponses o accine an igens. This s udy was designed o in es iga e adju an effec o BV on adap i e immune esponses agains wo diffe en model an igens, monome ic OVA p o ein and oligome ic NoV VLPs. We ound significan diffe ences be ween he immune esponses igge ed by hese p o eins alone o o mula ed wi h BV in e ms o an igen-specific an ibody kine ics and i e s, IgG an ibody sub ypes, induc ion o T cell esponses, and impo an ly, unc ionali y o he induced an ibody esponses. The diffe ences we e clea ly associa ed wi h he p esence o li e BV in he p epa a ions and no he s uc u al o mo phological diffe ences (Fig. 1), as emo al and inac i a ion o BV ab oga ed he esponses. The exac mechanism h ough which BV exe s i s adju an beha io is no ully elucida ed. Acco dingly, mannose-binding esi- dues in gp64 ha e been p oposed o in e ac wi h he mannose ecep o exp essed on mac ophages and dend i ic cells (DCs) (Abe e al., 2003). Al hough mannose ecep o plays an impo an ole in hos de ense and induc ion o inna e immuni y, se e al e idence con adic s gp64 as he main ac o in he s imula o y ac i i ies o BV (Abe e al., 2005; He as-S ubbs e al., 2007). Ins ead, gp64-media ed up ake o BVs and ecogni ion o unme hyla ed CpG sequences wi hin baculo i al DNA by Toll-like ecep o 9 (TLR9)/MyD88 dependen and independen pa h- ways ha e been sugges ed o be necessa y o he ac i a ion o inna e immune esponses (Abe e al., 2005, 2009). BV is a po en induce o IFN-αand IFN-β(G onowski e al., 1999; Abe e al., 2005; He as- S ubbs e al., 2007) bu also o he cy okines o ele ance, mos no ably IFN-γ(Suzuki e al., 2010). Adju an ac i i y is p ima ily media ed by he p oduc ion o IFN-αand IFN-βby immune cells (Abe e al., 2005; Fig. 4. NoV-specific se um IgG esponses. (A) Kine ics o NoV GII.4-specific o al IgG an ibodies in se um samples o mice immunized wi h 1 o 10 µg o pu e NoV GII.4 VLPs, 1 µg o pu e VLPs combined wi h BV o 1 µg o c ude-pu ified VLPs con aining BV a weeks 0 and 3. Con ol mice ecei ed PBS only. G oup mean OD 490 alues wi h s anda d e o o he means o ail blood samples collec ed a s udy weeks 0 (p e-immune se a) and 2 o 3 as well as e mina ion se a a week 5 a e shown. End-poin i a ions o an i- GII.4 IgG1 (B) and IgG2a (C) sub ype an ibodies o g oup-wise (4 mice/g oup) pooled e mina ion se a. Mean i a ion cu es wi h s anda d e o s o duplica e wells o up o 2 independen expe imen s a e shown. Fig. 5. A idi y and blocking an ibody i e s o mice immunized wi h NoV GII.4 VLP o mula ions. (A) Mean a idi y indices (%) wi h s anda d e o o he means o GII.4-specific IgG an ibodies in e mina ion se a o immunized g oups. (B) An ibody i e s o g oup-wise pooled se a blocking ≥90% (BT90) o he VLPs binding o he HBGAs. Fig. 6. NoV GII.4-specific T cell esponses.NoV GII.4–1999 –specific 99 pep ide pool and a single 99-50 pep ide we e used o s imula e IFN-γp oduc ion om he g oup-wise pooled splenocy es o mice immunized wi h 1 µg o pu e NoV GII.4 VLPs o 1 µg o pu e VLPs combined wi h BV. Cul u e medium (CM) as well as an OVA pep ide we e used as nega i e con ols. Mean IFN-γspo - o ming cells (SFC)/10 6 splenocy es o duplica e wells wi h s anda d e o s o he means a e shown. S. Heinimäki e al. Vi ology 511 (2017) 114–122 119 He as-S ubbs e al., 2007), p obably due o he abili y o ype I IFNs o imp o e B cell as well as T cell esponses (Le Bon e al., 2001, 2003; Deal e al., 2013). The cu en s udy demons a es ha li e BV has desi able ea u es a ibu ed o classical adju an s, which wo k o spa e he dose o he an igen and imp o e he immune esponses. We de ec ed a conside - ably highe immunogenici y o bo h an igens, OVA and NoV VLPs, when o mula ed wi h BV. Impo an ly, BV was able o spa e he dose o he NoV VLPs used o immuniza ion. In addi ion, BV ac ed o p omo e bo h Th1- and Th2- ype esponses wi hou skewing he o e all immune esponse in any pa icula di ec ion. Howe e , BV could be conside ed a po en Th1- ype adju an , as OVA p o ein was capable o inducing an igen-specific IgG2a immunoglobulin sub ype only when co-adminis e ed wi h BV. Consis en wi h his obse a ion, Ma gine and co-wo ke s (Ma gine e al., 2012) epo ed, ha esidual BV in an igenic o mula ions biases he iso ype dis ibu ion o he an ibody esponse owa ds he IgG2a immunoglobulin sub ype, sug- ges ing s imula ion o cell-media ed esponses. In he e, he BV adju an effec was s ongly affec ed by he emo al o inac i a ion o BVs, indica ing ha he adju an p ope ies we e due o he p esence o li e BV. This is no su p ising, as BV inac i a ion has been shown o comple ely ab oga e immunopo en ia ion effec , pa ly due o abol- ished p oduc ion o ype I IFNs (He as-S ubbs e al., 2007), which a e gene a ed in esponse o li e i uses (Goodbou n e al., 2000). An ibodies wi h high a idi y ha e been shown o p omo e efficien i us neu aliza ion (Rockx e al., 2005; Puschnik e al., 2013). These an ibodies ha e also been associa ed wi h p o ec ion om some i al in ec ions, including esicula s oma i is i us (VSV) (Salmi, 1991; Bachmann e al., 1997). Mo eo e , p e ious da a by ou g oup demons a ed ha child en wi h high-a idi y an i-NoV IgG an ibodies had ewe NoV in ec ions han child en wi h low-a idi y an ibodies (Nu minen e al., 2011). The p esen s udy shows ha BV was able o induce unc ionally efficien humo al esponse, as li e BV inc eased he a idi y o se um IgG an ibodies agains co-adminis e ed OVA p o ein and NoV VLPs. Fu he mo e, NoV has been shown o use HBGAs, complex ca bohyd a es ound on ed blood cells, mucosal epi helial cells and as sec e ed ee an igens in body fluids, as cellula a achmen ac o s o ecep o s (Ha ing on e al., 2002; Ma ionneau e al., 2002; Huang e al., 2003). An i-NoV an ibodies which block binding o NoV VLPs o he HBGAs a e conside ed co ela e o p o ec ion agains NoV in ec ion (Ha ing on e al., 2002; Reeck e al., 2010; Nu minen e al., 2011; Malm e al., 2014). Ou esul s demons a e ha adminis a ion o GII.4 VLPs in a combina ion wi h BV conside ably inc eased he blocking po en ial o hese an ibodies. Al hough he p esen s udy did no explo e he induc ion o inna e immuni y, we hypo hesize ha ollowing i.d. immuniza ion BV is aken up by imma u e DC in he skin, and induces ma u a ion and ac i a ion o hese an igen p esen ing cells (APC) as p e iously demons a ed (Molina i e al., 2011), up egula ing co-s imula o y molecules as well as p oduc ion o p o-inflamma o y cy okines (He as-S ubbs e al., 2007; Suzuki e al., 2010; Molina i e al., 2011; Qua occhi e al., 2013). In conco dance wi h hese findings, we ha e ecen ly shown ha esidual BV in he c ude pu ified p o ein p epa a ions induces TNF-α p oduc ion by monocy es/mac ophages in i o (Malm e al., 2016a). TNF-α igge s he ec ui men and ac i a ion o APCs a he injec ion si e and acili a es mig a ion o APCs o lymph nodes (S oi zne e al., 1999), hus imp o ing he an igen up ake and p esen a ion o T cells. P e ious epo s ha e demons a ed ha BV enhances T cell esponses agains co-adminis e ed an igens (He as-S ubbs e al., 2007; Ma gine e al., 2012). Thus, a e obse ing he adju an effec o BV on IgG2a an ibody p oduc ion agains bo h an igens, we in es iga ed an igen-specific IFN-γcy okine p oduc ion, a hallma k o Th1 cell immuni y. BV p omo ed gene a ion o T cell esponses agains co-deli e ed an igens, as add essed by conside able sec e ion o IFN-γby splenocy es upon s imula ion wi h OVA and NoV GII.4 de i ed pep ides co esponding o he immunodominan CD4 + T cell epi opes, as well as a pep ide pool co e ing he en i e sequence o NoV VP1. Simila ly o he an ibody esponse, BV inac i a ion abolished he capaci y o BVs o induce OVA-specific T cell esponse, sugges ing ha induc ion o T cell esponse is also dependen on he p esence o li e BV pa icles. Cells o mice immunized wi h o mula ions con aining li e BV, bu no IBV, p oduced high amoun o IFN-γupon ex i o s imula ion wi h BV. This indica es ac i a ion o T cells in lymphoid issues by BV, which is in ag eemen wi h p e ious obse a ions by o he s (S auss e al., 2007; Molina i e al., 2011). Pa ac ine sec e ion o cy okines by BV-specific T cells is o a significance as hese cy okines d i e p oli e a ion and diffe en ia ion o co-deli e ed an igen p imed lymphocy es. BV has a p onounced adju an effec on T cell esponses o NoV, al hough i also imp o es unc ionali y o he an ibodies in e ms o a idi y and blocking/neu aliza ion. Howe e , mo e p onounced B and T cell adju an effec was obse ed o co-deli e ed OVA an igen, which is no su p ising due o he less immunogenic na u e o soluble monome ic p o eins in compa ison o he mo e immunogenic VLPs (Bachmann and Jennings, 2010). Supe io immunogenici y o pa icles o p o ein agg ega es o e soluble p o eins is s ongly linked o he mul i alen o ganiza ion o an igen p esen ed on pa icles (Bachmann e al., 1993; Ghosh e al., 2002). Unlike soluble p o eins, he la ge s uc u es wi hin he size ange o ~40 nm a e efficien ly in e nalized by APCs (Fifis e al., 2004), and hus ca ied o he lymphoid o gans and p esen ed o T cells. In conclusion, he p esence o li e BV in an igenic o mula ions s eng hened bo h an ibody and T cell immune esponses o hese p o eins, u he spa ing he dose o he an igen. The e o e, i is emp ing o specula e, ha c ude pu ified p o eins could be excellen candida es o e e ina y accines. Fo ins ance, a accine con aining c ude pu ified a ian influenza an igens could be used o accina e wild bi ds and domes ic poul y o limi he po en ial sp ead o pandemic influenza i uses, ex emely ha m ul o human popula ion (Noh e al., Fig. 7. BV-specific an ibodies and T cell esponses.(A) BV-specific se um IgG esponses in mice immunized wi h OVA alone o oge he wi h BV o IBV. Con ol mice ecei ed PBS only. Shown a e mean OD 490 alues wi h s anda d e o s o he means o e mina ion se a. (B) BV-specific T cell esponses induced in mice immunized wi h diffe en OVA o mula ions. BV was used o s imula e IFN-γp oduc ion om he g oup- wise pooled splenocy es o immunized and con ol mice. Mean IFN-γspo - o ming cells (SFC)/10 6 splenocy es o duplica e wells o 1–3 independen expe imen s wi h s anda d e o s o he means a e shown. S. Heinimäki e al. Vi ology 511 (2017) 114–122 120 2016; Pushko e al., 2017). High quan i ies o he accine s ocks could be p oduced ela i ely easy and a a low cos , wi hou he need o ime consuming and cos ly ch oma og aphic pu ifica ion s eps. The esidual BV in c ude pu ified p o ein p epa a ions would alle ia e he need o addi ion o ex e nal adju an s o he accine o mula ion because o he in insic immunos imula o y effec o li e BV. Significan ad an ages o BV include i s inhe en inabili y o eplica e in e eb a e cells (Tjia e al., 1983; B usca e al., 1986), hus elimina ing he need o inac i a ion, as well as low cy o oxici y and absence o p e-exis ing immuni y (Shoji e al., 1997; S auss e al., 2007). Al hough he e is a high conce n by egula o y agencies abou con amina ing i uses in human accines, BV could be conside ed as an adju an in e e ina y accines due o hei less s ingen egula o y equi emen s (Adams, 2015). Because i is difficul o ensu e he cons an le el o BV in c ude p o ein p epa a ions, he ac i i y o BV in a accine o mula ion should be e alua ed by de e mining he ange o BV concen a ions (e.g. adding exac amoun s o BV in o he pu e VLPs) exe ing adju an effec in ca e ully designed p eclinical animal expe imen s, and hus, each c ude accine ba ch needs o be es ed o mee he concen a ion ange. Since li e BVs in accine o mula ions may p o oke eac ogeni- ci y and aise sa e y conce ns, u he s udies a e needed o asce ain he eac ogenici y, oxici y and sa e y o BV con aining accine p epa a ions in ele an animal models. Acknowledgemen s We g a e ully acknowledge he echnical assis ance gi en by he labo a o y pe sonnel o he Vaccine Resea ch Cen e . 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