Comparison of Intramuscular, Intranasal and Combined Administration of Norovirus Virus-Like Particle Subunit Vaccine Candidate for Induction of Protective Immune Responses in Mice

Compa ison o In amuscula , In anasal and Combined Adminis a ion o No o i us Vi us-Like Pa icle Subuni Vaccine Candida e o Induc ion o P o ec i e Immune Responses in Mice Ma ia Malm, Ki si Tamminen, Timo Vesika i and Vesna Blaze ic* Vaccine Resea ch Cen e , Uni e si y o Tampe e Medical School, Bioka u 10, FI-33520 Tampe e, Finland *Co esponding au ho : Vesna Blaze ic, Vaccine Resea ch Cen e , Uni e si y o Tampe e Medical School, Bioka u 10 FI-33520 Tampe e, Finland; Tel- +358504211054; Fax- +358 3 364 1512; E-mail: [email p o ec ed] Recei ed da e: No embe 14, 2014, Accep ed da e: Janua y 13, 2015, Published da e: Janua y 20, 2015 Copy igh : © 2015 Malm M, e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Abs ac Backg ound and objec i es: No o i uses (NoVs) a e majo causa i e agen s o non-bac e ial acu e gas oen e i is in people o all ages wo ldwide. NoV capsid VP1 de i ed i us-like pa icles (VLPs) p oduced in a ious exp ession sys ems a e main accine candida es agains NoV. The aim o his s udy was o in es iga e and compa e sys emic and mucosal deli e y and a combina ion o bo h deli e ies o NoV VLPs o induc ion o immune esponses in BALB/c mice. Ma e ials and me hods: BALB/c mice we e immunized In amuscula ly (IM), In anasally (IN) o sequen ially (IM ollowed by IN) wi h a candida e NoV GII-4 VLP accine de eloped by ou labo a o y. NoV GII-4-speci ic se um and mucosal IgG and IgA an ibodies we e analyzed by ELISA. GII-4–speci ic T cell immune esponses we e in es iga ed using an ELISPOT assay measu ing p oduc ion o in e e on-γ (IFN-γ) a a single cell le el. Resul s: IM immunized mice de eloped a s ong sys emic and mucosal NoV-speci ic IgG an ibody esponse bu comple ely lacked IgA esponse. In con as , mice immunized IN had s ong sys emic and mucosal IgG and IgA p oduc ion bu lacked CD8+ T cell esponses. Sequen ial immuniza ion compensa ed o he de icien IgA and CD8+ T cell esponses induced by each deli e y alone. Conclusion: Ou esul s show ha sequen ial IM+IN immuniza ion should be conside ed o NoV VLP accine deli e y o ac i a e b oad immune esponses. Keywo ds: No o i uses; Vaccine; Immune Response In oduc ion No o i uses (NoV) a e he leading cause o non-bac e ial acu e gas oen e i is (AGE) in people o all ages. NoV in ec ions a e esponsible o o e a million hospi aliza ions and up o 200,000 dea hs annually in in an s and child en o de eloping coun ies [1]. A e in oduc ion o o a i us accines in he USA, Finland, and o he coun ies, NoVs ha e become he leading cause o medically a ended AGE in child en unde i e yea s o age [2,3]. The e o e, young child en a e an impo an a ge g oup o u u e NoV accina ion. NoVs a e highly con agious and cause la ge ou b eaks in communi y se ing such as nu sing homes, childca e acili ies, mili a y, and c uise ships. NoV genog oups GI and GII a e esponsible o mos in ec ions in humans wi h GII-4 geno ype being p edominan o mo e han wo decades [4,5]. A e exp ession in i o majo NoV capsid p o ein sel - assembles in o i us-like pa icles (VLPs) consis ing o 90 dime s o VP1 [6]. These VLPs a e mo phologically and unc ionally simila o he na i e i us bu lack gene ic ma e ial. Human NoVs a e uncul i able in i o and, he e o e, de elopmen o con en ional accines based on li e a enua ed o killed NoVs is no possible a p esen ime. Ins ead, NoV VLPs ha e been p oposed as accine candida es agains NoVs. As NoV is an en e ic pa hogen ha uses in es inal mucosa as a po o en y, mos o he immunogenici y s udies in animals [7,8] as well as ea ly phase clinical ials [9-11] ha e used mucosal immuniza ion (o al o in anasal; IN) o deli e y o NoV VLPs. Mo e ecen ly, in amuscula (IM) deli e y has been conside ed [12-14]. Co ela es o p o ec ion o NoV in ec ion a e la gely unknown. His o-blood g oup an igens (HBGAs) a e cellula a achmen ac o s o ecep o s o NoVs [15]. These complex ca bohyd a es a e p esen on he su ace o en e ic mucosal cells as well as ee an igens in body sec e ions. Se um an ibodies, which block binding o VLPs o he HBGAs, a e conside ed as a su oga e o neu alizing an ibodies and co ela es o p o ec ion o NoV in ec ion [16-19]. O he s udies ha e also sugges ed impo an ole o cellula and mucosal immuni y agains NoV in ec ion and gas oen e i is [20-22]. In his s udy we compa ed induc ion o po en ially p o ec i e immune esponses induced wi h NoV VLPs by sys emic IM o mucosal IN immuniza ion as well as sequen ial immuniza ion (IM ollowed by IN) in BALB/c mice. To da e, he e ha e been no s udies using combined sys emic and mucosal deli e y app oaches wi h NoV VLPs. Ou esul s show ha sequen ial IM+IN immuniza ion compensa ed o de icien NoV-speci ic se um IgA and T cell esponses induced by IM and IN deli e y alone. Malm e al., J Clin Cell Immunol 2015, 6:1 h p://dx.doi.o g/10.4172/2155-9899.1000284 Resea ch A icle Open Access J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access jou nal Volume 6 • Issue 1 • 1000284 Jou nal o Clinical & Cellula Immunology Ma e ials and Me hods Immuniza ion o expe imen al animals and sample collec ion To analyze NoV GII-4 VLP induced immune esponses 10 emale BALB/c OlaHsd mice (7 weeks old; Ha lan Labo a o ies, The Ne he lands) we e immunized a day 0 and day 21 wi h a NoV VLP and o a i us VP6 combina ion accine candida e de eloped by ou labo a o y [12,23] con aining 10 µg GII-4 VLPs by IM o IN ou e. The immunogen was adminis e ed in a 50 µl olume s e ile phospha e-bu e ed saline (PBS) in o quad iceps emo is o in a 25 µl olume by g adual inocula ion in each nos il. In addi ion, a g oup o mice was p imed wi h he combina ion accine con aining 10 µg NoV GII-4 VLPs by IM ou e a day 0 and boos ed by IN ou e a day 21, e med sequen ial immuniza ion. Naï e mice ecei ing ca ie only (s e ile PBS) ei he by IM o IN ou e we e used as nega i e con ols. Mice we e e mina ed wo weeks a e he inal immuniza ion (day 35) and blood, eces and spleen we e collec ed as desc ibed p e iously [24,25]. All p ocedu es we e pe o med in acco dance wi h he egula ions and guidelines o he Finnish Animal Expe imen Boa d. Se um an ibody ELISA Indi idual mouse se a we e se ially dilu ed s a ing a 1:200 and es ed o NoV GII-4 speci ic o al IgG an ibodies in an ELISA assay as p e iously desc ibed [23]. B ie ly, 96-well pla es (Co ning Inc. Co ning, NY) we e coa ed wi h 50 ng/well o GII-4 VLPs in PBS and bound an ibodies de ec ed wi h HRP-conjuga ed goa an i-mouse IgG (Sigma-Ald ich). G oupwise pooled se a o each expe imen al g oup was 2- old se ially dilu ed (s a ing a a 1:20 dilu ion) and es ed o GII-4 speci ic IgA an ibodies using HRP-conjuga ed goa an i-mouse IgA (Sigma-Ald ich) a a dilu ion o 1:4000. Op ical densi y (OD) a 490 nm was measu ed by Vic o 2 1420 eade (Pe kin Elme ) and a sample was conside ed posi i e i he OD was abo e he mean OD o con ol mice +3SD. End-poin i e s we e exp essed as he highes se um dilu ion gi ing a posi i e eading. Se a o each mouse a a dilu ion 1:200 we e es ed o IgG an ibody a idi y using ELISA as desc ibed abo e wi h an ex a u ea incuba ion s ep [19,26] whe e an ibodies bound o GII-4 VLP coa ed pla es we e ea ed wice wi h 8M u ea be o e addi ion o HRP-conjuga ed an i-mouse IgG. A idi y index was calcula ed as (OD wi h u ea/OD wi hou u ea) × 100%. Mucosal an ibody ELISA Faecal d ople s om each mouse we e pooled and 10% s ool suspensions we e made as ea lie desc ibed in de ails [12]. S ool suspensions we e se ially 2- old dilu ed s a ing a 1:5 and es ed o NoV GII-4-speci ic IgG and IgA wi h he ELISA as desc ibed abo e. Se um an ibody blocking assay Human ype A sali a om a sec e o posi i e indi idual and syn he ic bio inyla ed H- ype-3 ca bohyd a e we e used as a sou ce o HBGAs in blocking assays. Sali a blocking assay was pe o med essen ially as ea lie desc ibed by ou labo a o y [23]. 96-well pla es we e coa ed wi h sali a ype A a a 1:3000 dilu ion and incuba ed o e nigh a 37°C. Fo syn he ic HBGA blocking assay Supe Block p e ea ed High Binding Capaci y Neu A idin pla es (Pie ce) we e incuba ed wi h 2.5 µg/ml o he syn he ic bio inyla ed H ( ype 3)- PAA-Bio in (Glyco ech) o 1 hou a oom empe a u e [27]. GII-4 VLPs we e p e-incuba ed wi h se ially wo- old dilu ed (1:100–1:3200) expe imen al and con ol mouse se a o 1 h a 37°C and added o he sali a o H- ype-3 coa ed pla es. Sali a pla es we e u he incuba ed o 1.5 hou a 37°C and Neu A idin pla es o 2 hou a +4°C. The bound VLPs we e de ec ed wi h NoV an ibody posi i e human se um and an i-human IgG-HRP (In i ogen) ollowed by he OPD subs a e. VLPs lacking he se um we e used as he maximum binding con ol. The blocking index (%) was calcula ed as 100% – (OD wells wi h VLP se um mix/OD wells wi hou se um; maximum binding) × 100%. Cell media ed immune esponse NoV GII-4-speci ic T cell esponses we e measu ed wi h an ELISPOT assay by quan i ica ion o in e e on (IFN)-γ p oducing splenocy es [25]. Mul isc een 96-well HTS-IP il e pla es (Millipo e) we e coa ed wi h an i-mouse IFN-γ an ibody AN18 (2.5 µg/ml, Mab ech). Splenocy es (0.1×106/well) om he expe imen al o con ol mice we e s imula ed wi h a GII-4 capsid de i ed 15-me syn he ic pep ide named NP-4 a 5 µg/ml (P oImmune L d., amino acids CLLPQEWVQHFYQEA) o GII-4 VLPs a 2.5 µg/ml. Cells incuba ed in cul u e media alone and cells s imula ed wi h 10 µg/ml Conca alin A (ConA, Sigma-Ald ich) se ed as a backg ound and cell iabili y con ols. A e o e nigh incuba ion a 37°C IFN-γ sec e ion was de ec ed wi h bio inyla ed an i-mouse IFN-γ an ibody R4-6A2 (2.5 µg/ml, Mab ech) and s ep a idin-ALP (Mab ech). The spo s de eloped wi h BCIP/NBT subs a e (Mab ech) we e coun ed by ImmunoSpo ® au oma ic CTL analyze (CTL-Eu ope GmbH). The esul s a e exp essed as mean spo o ming cells (SFC)/106 cells o duplica e wells. To de e mine which cell ype is esponsible o he IFN-γ p oduc ion, splenocy es we e p eincuba ed (1 hou a 37°C) wi h he unc ional blocking an ibodies a an i-mouse CD4 o a an i-mouse CD8 (bo h om eBiosciences) a a 30 µg/ml concen a ion p io o s imula ion wi h he GII-4-speci ic pep ide o GII-4 VLPs. S a is ical analyses Fishe ’s exac es was used o compa ison o GII-4 VLP-speci ic IgG endpoin i e s. P<0.05 was conside ed s a is ically signi ican . All hypo hesis es ing was wo- ailed. Resul s Se um NoV-speci ic an ibody esponses Se um GII-4-speci ic IgG i e s o indi idual mice immunized a day 0 and day 21 wi h a candida e accine con aining 10 µg NoV GII-4 VLPs by IM o IN deli e y ou e a e shown in Figu es 1A and 1B. Rega dless o he deli e y ou e each mouse de eloped a s ong IgG an ibody esponse wi h endpoin i e s o 5log10 (p>0.05). In all o he se a he GII-4 speci ic IgG an ibodies had high a idi y wi h an a idi y index >50% (Figu e 1C). As hese esul s indica ed uni o m success o immuniza ion o each mouse in he expe imen al g oup, he se a we e pooled g oupwise and GII-4 speci ic IgA i e s we e de e mined by ELISA (Figu e 1D). IN immunized mice gene a ed a ema kable se um IgA an ibody esponse (mean OD 0.40, i e 1:20) while IM immunized mice did no (mean OD 0.07, i e 1:20). Ci a ion: Malm M, Tamminen K, Vesika i T, Blaze ic V (2015) Compa ison o In amuscula , In anasal and Combined Adminis a ion o No o i us Vi us-Like Pa icle Subuni Vaccine Candida e o Induc ion o P o ec i e Immune Responses in Mice. J Clin Cell Immunol 6: 284. doi:10.4172/2155-9899.1000284 Page 2 o 7 J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access jou nal Volume 6 • Issue 1 • 1000284 Figu e 1: NoV GII-4-speci ic se um an ibody esponses in BALB/c mice. Mice (10 mice/g oup) we e immunized wice (a day 0 and day 21) wi h a NoV accine candida e con aining 10 µg GII-4 VLPs. (A) Te mina ion se a o in amuscula ly (IM) immunized mice was assayed o NoV GII-4-speci ic IgG wi h wo- old dilu ions s a ing a 1:200. Shown a e indi idual i a ion cu es o each immune se a and i a ion cu e o pooled con ol mice se a (6 mice/g oup) immunized wi h he ca ie (PBS) only (dashed line). (B) GII-4-speci ic IgG i a ion cu es o in anasally (IN) immunized mice and pooled con ol mice se a. (C) A idi y o GII-4-speci ic IgG an ibodies o each immunized mouse was analyzed in modi ied ELISA assay a a dilu ion o 1:200 as desc ibed in he Ma e ials and Me hods. Shown is a idi y index ((OD wi h u ea/OD wi hou u ea) x 100) o each mouse se a and he mean o he g oup. Dashed line indica es he cu -o o high a idi y an ibodies (>50%). (D) GII-4-speci ic se um IgA i e s we e assayed wi h wo- old dilu ions (s a ing a a 1:20) o g oup wise pooled se um. Shown a e mean OD alues o he IM, IN and co esponding con ol mouse g oups wi h he s anda d e o s o he mean (SEM) o he eplica es. Mucosal IgG and IgA an ibodies G oupwise pooled ecal samples o IM and IN immunized mice we e es ed o GII-4 speci ic IgG and IgA an ibody con en . Bo h deli e y ou es induced simila le els o IgG an ibodies in he in es ines (mean OD 0.57 o IM and mean OD 0.48 o IN, i e 1:50) (Figu e 2A). Simila o he se um samples (Figu e 1D, espec i ely), mice immunized wi h NoV GII-4 VLP by IM deli e y did no de elop in es inal IgA an ibodies (mean OD 0.04, i e 1:5) (Figu e 2B) while Ci a ion: Malm M, Tamminen K, Vesika i T, Blaze ic V (2015) Compa ison o In amuscula , In anasal and Combined Adminis a ion o No o i us Vi us-Like Pa icle Subuni Vaccine Candida e o Induc ion o P o ec i e Immune Responses in Mice. J Clin Cell Immunol 6: 284. doi:10.4172/2155-9899.1000284 Page 3 o 7 J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access jou nal Volume 6 • Issue 1 • 1000284 mucosal IN deli e y induced conside able le el o in es inal IgA an ibodies (mean OD 0.49, i e 1:5) (Figu e 2B). Figu e 2: NoV GII-4-speci ic mucosal an ibody esponses in BALB/c mice. Mice we e immunized wice wi h a NoV accine candida e con aining 10 µg GII-4 VLPs. In es inal IgG (A) and IgA (B) an ibodies we e analyzed om 10% ecal suspensions o IM and IN immunized mice (solid lines) o he con ol mice (dashed lines). Fecal samples o i e mice/expe imen al g oup we e pooled o analysis. Shown a e mean ODs wi h he s anda d e o s o he mean (SEM) o wo independen expe imen s. Figu e 3: Blocking o NoV GII-4 VLP binding o he HBGA ecep o s by immune mouse se a. G oup wise pooled ( i e mice/ g oup), wo- old dilu ed se a o mice immunized by IM o IN ou e wi h a NoV accine candida e con aining 10 µg GII-4 VLPs we e assayed o blocking ac i i y. Co esponding con ol g oup se um was used as a non-speci ic blocking con ol. (A) Blocking o GII-4 VLP binding o human sec e o posi i e sali a ype A. (B) Blocking o GII-4 VLP binding o he H- -3 syn he ic HBGA. The blocking index (%) was calcula ed as 100% - (OD wells wi h se um/OD wells wi hou se um, a maximum binding) × 100%. Resul s a e shown as he mean blocking index o duplica e wells wi h simila esul s om a minimum o wo independen expe imen s. Blocking abili y o NoV-speci ic an ibodies Se a o GII-4 VLPs immunized and con ol mice we e pooled g oupwise and es ed o blocking o GII-4 VLP binding o human ype A sali a (Figu e 3A) and syn he ic H- ype-3 ca bohyd a e (Figu e 3B). Bo h IM and IN immuniza ion induced s ong blocking an ibodies in he se a wi h 100% blocking up o a dilu ion 1:800 in he sali a blocking assay (Figu e 3A). Blocking o he GII-4 VLPs’ binding o he syn he ic H- ype-3 was used o con i m he esul s o he sali a blocking assay (Figu e 3B). Bo h immuniza ions induced compa able blocking esponses in he se a, which co ela ed o he blocking esponses seen in he sali a assay. Nei he o he con ol g oups’ (c l IM o c l IN, espec i ely) se a con ained an ibodies able o block GII-4 VLP binding o he HBGAs (Figu e 3A and 3B). Figu e 4: NoV GII-4-speci ic in e e on-γ (IFN-γ esponses. (A) Splenocy es o mice immunized by IM o IN ou e wi h a NoV accine candida e con aining 10 µg GII-4 VLPs o con ol mice ecei ing PBS we e s imula ed in i o wi h GII-4 capsid de i ed 15-me pep ide (NP-4) o wi h he homologous GII-4 VLPs. IFN-γ p oduc ion a a single cell le el was de ec ed by an ELISPOT assay. (B) T cell es ic ion o he NoV GII-4-speci ic IFN-γ p oduc ion. Cells we e s imula ed wi h he NP-4 pep ide o GII-4 VLPs in he p esence o absence o CD4 and CD8 speci ic an ibodies o block he T cell ac i a ion. Resul s a e exp essed as he mean Spo Fo ming Cells (SFC)/106 cells o a leas wo independen expe imen s wi h s anda d e o s. NoV GII-4-speci ic CD8+ T cell esponses NoV GII-4-speci ic T cell esponses measu ed by IFN-γ p oduc ion a a single cell le el in ELISPOT assay we e di e en in mice immunized by IM o IN ou e. IM immunized mice had high equency o IFN-γ p oducing cells in spleen in esponse o he GII-4 capsid de i ed 15-me pep ide NP-4 (438 ± 119 SFC/106 cells) while IN immunized mice comple ely lacked hese esponses, as did he he con ol mice (Figu e 4A). The T cell esponses we e also es ed using NoV GII-4 VLPs as an in i o s imuli and compa able esponses we e de ec ed in mice immunized by IM (140 ± 40 SFC/106 cells) and IN deli e y ou e (156 ± 42 SFC/106 cells) (Figu e 4A). In o de o de e mine which T cells esponded o he pep ide and GII-4 VLPs, blocking an ibodies o CD4 and CD8 cell su ace an igens we e used o show he es ic ion o he NoV GII-4-speci ic IFN-γ esponses (Figu e 4B). The T cell esponses o he NP-4 pep ide we e only blocked by an i-CD8 an ibody (mean 68 % inhibi ion), while GII-4 VLP esponses we e blocked by an i-CD4 an ibody only (mean 77% inhibi ion). These esul s indica e impai ed unc ionali y o CD8+ T cells in mice immunized by IN deli e y in con as o IM immunized mice. Fu he mo e, he esul s also show ha bo h deli e y ou es induced unc ional CD4+ T cells by esponding o GII-4 VLPs. Immune esponses by sequen ial immuniza ion As IM and IN immuniza ions alone induced de icien immune esponses we nex combined he wo o de e mine combined e ec o he deli e y ou es. Immuniza ion o mice wi h NoV GII-4 VLPs i s IM (a day 0) and sequen ially IN (a day 21) induced se um and mucosal GII-4 speci ic IgA an ibodies (Figu e 5A) and NP-4 pep ide Ci a ion: Malm M, Tamminen K, Vesika i T, Blaze ic V (2015) Compa ison o In amuscula , In anasal and Combined Adminis a ion o No o i us Vi us-Like Pa icle Subuni Vaccine Candida e o Induc ion o P o ec i e Immune Responses in Mice. J Clin Cell Immunol 6: 284. doi:10.4172/2155-9899.1000284 Page 4 o 7 J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access jou nal Volume 6 • Issue 1 • 1000284 speci ic CD8+ T cell IFN-γ esponses (Figu e 5B) compa able o he op imal esponses induced by each ou e sepa a ely (Figu e 1D, 2B and 4A espec i ely). Se um and ecal GII-4 speci ic IgG an ibodies as well as blocking an ibody ac i i y we e compa able o each ou e sepa a ely as well. Se um GII-4 IgG endpoin i e was 102400 and he mean a idi y index was 94.5 ± 0.2 %. Se um dilu ion o 1:800 blocked 92% o GII-4 VLP binding in he sali a blocking assay. Figu e 5: NoV GII-4-speci ic humo al and cell-media ed immune esponses induced by sequen ial immuniza ion. G oup o mice we e immunized wi h a NoV accine candida e con aining 10 µg GII-4 VLPs a day 0 by IM deli e y and day 21 by IN deli e y. (A) NoV GII-4-speci ic IgA an ibodies we e analyzed om se um and ecal suspensions. Se um samples we e wo- old dilu ed s a ing a a 1:20 dilu ion and 10% ecal suspension we e analyzed s a ing a a 1:2 dilu ion. Shown a e mean ODs wi h s anda d e o s o he eplica es. (B) NoV GII-4-speci ic IFN-γ esponses a e s imula ing he splenocy es wi h GII-4 capsid de i ed 15-me pep ide (NP-4) o cul u e media (CM) only. Shown a e he mean spo o ming cells (SFC)/106 cells o he indi idually es ed mice wi h he s anda d e o s. Discussion NoV VLPs a e excellen accine candida es as hey esemble na i e i ions mo phologically and an igenically and a e highly immunogenic [6], in addi ion o being sa e. NoV VLPs can be gi en by mucosal deli e y o ally o IN, o pa en e ally (IM o in ade mally, ID). As NoV is an o ally ansmi ed en e ic pa hogen and na u al immuni y o NoV is o a sho du a ion [28-30] i emains o be de e mined i sys emic immuniza ion wi h NoV VLP accine migh be a be e choice han mucosal one o induce long las ing p o ec i e immuni y. In he e we s udied di e en deli e y o NoV VLP accine by compa ing immune esponses induced by IN and IM immuniza ion. Ou esul s indica e ha he e a e inhe en di e ences in immune sys em ac i a ion by NoV VLPs adminis e ed sys emically o mucosally. We ha e p e iously shown ha sys emic deli e y (IM and ID) o NoV VLPs ei he alone o in a combina ion wi h o a i us VP6 p o ein induces obus humo al and cell media ed immune esponses speci ic o NoV [12,23,25]. The esul s in his s udy con i m he ea lie indings ha IM immuniza ion wi h GII-4 VLPs induces bo h IgG an ibody and T cell esponses (CD4+ and CD8+, espec i ely) bu in addi ion show he absence o se um and mucosal IgA al hough signi ican le els o IgG we e de ec ed in he gu mucosa. T ans e o se um IgG in o he gu lumen is likely an impo an p o ec i e mechanism agains gu in ec ion [31]. On he con a y, IN deli e y induced simila IgG an ibody esponses o NoV in he se um and he gu as obse ed wi h IM deli e y, bu he T cell esponses, speci ically CD8+ T cells, we e de icien . Velasquez e al. [8] ha e shown low le els o IgG2a an ibodies (a ma ke o a Th1 ype immune esponse) by IN deli e y o NoV VLPs which is in a suppo o ou indings. The impo ance o T cells in NoV in ec ion is no well known and he e is a limi ed numbe o s udies add essing cellula immuni y [21,22,32]. I is belie ed ha in con as o li e i al accines subuni p o ein accines equi e T cells o induce p o ec ion [33]. In gene al, CD8+ T cells, namely cy o oxic T lymphocy es (CTL), a e c i ical o clea ance o i ally in ec ed cells [34,35]. A di ec e idence o he ole o T cells in NoV in ec ion comes om a s udy o mu ine no o i us (MNV) showing ha CD8+ and CD4+ T cells clea ed in ec ion in mice [36]. IN deli e y o NoV GII-4 VLPs in his s udy induced high sys emic and mucosal GII-4 speci ic IgA an ibodies. Con adic o y indings a e ela ed o he ole o NoV-speci ic mucosal immuni y. Lindesmi h e al. [20] epo ed a signi ican ole o mucosal immuni y, especially sali a y IgA in a p o ec ion om in ec ion. On he con a y, o he esea che s ha e epo ed insigni ican ole o se um IgA and in es inal IgA in a chimpanzee model o a NoV in ec ion [37] and na u al in ec ion in young child en [30]. Compa able le el o blocking/neu alizing ac i i y o se um de i ed om IM and IN immunized mice ha we e de ec ed in his s udy (Figu e 3, espec i ely) would also sugges a negligible ole o se um IgA an ibodies, as IgA was de ec ed only in se a o IN immunized animals. Two human challenge s udies ha e been published ecen ly in subjec s immunized wi h candida e NoV VLP accines adminis e ed IN [16] and IM [14]. In e es ingly, IN deli e y o GI.1 VLP accine (wi h an adju an ) esul ed in signi ican p o ec ion agains homologous NoV in ec ion [16], whe eas IM deli e y o GII.4 accine did no [14]. Howe e , IM immunized subjec s had signi ican p o ec ion agains se e e NoV gas oen e i is [14]. These esul s sugges di e en mechanism o p o ec ion agains NoV in ec ion, induced by IN and IM immuniza ion, espec i ely. P o ec ion agains NoV in ec ion equi es immuni y a he mucosal su aces whe eas p o ec ion agains he se e e disease may equi e di e en media o s o p o ec ion. Ou indings in mice may shed ligh on he in e p e a ion o he esul s o he human challenge s udies. Ou esul s show ha sequen ial IM+IN immuniza ion o mice wi h NoV GII-4 VLPs compensa e o de iciencies esul ing om IM and IN deli e ies alone. This schedule was chosen as immunogenici y s udies o hepa i is B su ace an igen in mice [38] showed ha he s onges sys emic immune esponse associa ed wi h a s ong mucosal esponse was induced by IM p ime ollowed by IN boos and no ice e sa. Sequen ial immuniza ion induced GII-4 speci ic T cells o bo h pheno ypes (CD4+ and CD8+) and IgA an ibodies in se um and mucosa. I is possible ha ex e nal adju an in combina ion wi h NoV VLP accine migh di ec immune esponse in a desi able pa hway. Howe e , in accine de elopmen in gene al, NoV VLP accine wi hou an adju an would be p e e ed pa icula ly o use in child en. I emains o be de e mined why he e is no induc ion o e ec o CD8+ T cells p oducing IFN-γ a e IN deli e y o NoV VLPs. I may be ha hese cells a e ac i a ed a he mucosal deli e y si e bu do no dissemina e o seconda y lymphoid issue, as has ecen ly been sugges ed [39,40]. Ou labo a o y has wo ked on he de elopmen o NoV and o a i us combina ion accine agains he wo mos de as a ing Ci a ion: Malm M, Tamminen K, Vesika i T, Blaze ic V (2015) Compa ison o In amuscula , In anasal and Combined Adminis a ion o No o i us Vi us-Like Pa icle Subuni Vaccine Candida e o Induc ion o P o ec i e Immune Responses in Mice. J Clin Cell Immunol 6: 284. doi:10.4172/2155-9899.1000284 Page 5 o 7 J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access jou nal Volume 6 • Issue 1 • 1000284 en e ic pa hogens in child en [12,23]. As na u al immuni y o NoV is o a sho du a ion, we pos ula ed ha IM immuniza ion migh induce s onge and mo e du able immune esponses han mucosal deli e y [13]. The esul s in his s udy show ema kable di e ences in he immune esponses induced by IM and IN deli e y o NoV VLPs in mice; absence o mucosal immuni y, speci ically in es inal IgA in IM immunized mice and lack o NoV-speci ic CD8+ T cell immune esponses by IN deli e y. I emains o be de e mined which esponses a e ele an o p o ec ion agains NoV in ec ion and/o disease and he e o e o choosing o he ou e o NoV VLP accine deli e y. Be o e he exac co ela es o p o ec ion a e iden i ied, sequen ial immuniza ion migh be a good choice o ensu e ha di e en e ec o s/media o s o he immune esponse a e ac i a ed. 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