Systems biology of vaccination for seasonal influenza in humans

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1 Systems biology of vccintion for sesonl influenz in humns Helder I Nky,, Jens Wrmmert,3, Ev K Lee, Luigi Rcioppi 5,6, Stephnie Mrie-Kunze,, W Nichols Hining 7, Anthony R Mens 6, Sudhir P Ksturi,, Nooruddin Khn,, Gui-Mei Li,3, Megn McCuslnd,3, Vibhu Knchn,3, Kenneth E Kokko 8, Shuzho Li,, Rivk Elbein 9, Aneesh K Meht 9, Aln Aderem, Knt Subbro, Rfi Ahmed,3 & Bli Pulendrn,, Here we hve used systems biology pproch to study innte nd dptive responses to vccintion ginst influenz in humns during three consecutive influenz sesons. We studied helthy dults vccinted with trivlent inctivted influenz vccine (TIV) or live ttenuted influenz vccine (LAIV). TIV induced higher ntibody titers nd more plsmblsts thn LAIV did. In subjects vccinted with TIV, erly moleculr signtures correlted with nd could be used to ccurtely predict lter ntibody titers in two independent trils. Notbly, expression of the kinse CMKIV t dy 3 ws inversely correlted with lter ntibody titers. Vccintion of CMKIV-deficient mice with TIV induced enhnced ntigen-specific ntibody titers, which demonstrted n unpprecited role for CMKIV in the regultion of ntibody responses. Thus, systems pproches cn be used to predict immunogenicity nd provide new mechnistic insights bout vccines. Annul vccintion is one of the most effective methods for preventing influenz. At present, two types of vccines for sesonl influenz re licensed for use in the USA: trivlent inctivted influenz vccine (TIV), given by intrmusculr injection; nd live ttenuted influenz vccine (LAIV), dministered intrnslly. These vccines contin three strins of influenz viruses tht re usully chnged nnully on the bsis of the results of globl influenz surveillnce dt. The efficcy of vccine ginst influenz, therefore, depends on the mtch of ntigenicity between the vccine nd circulting influenz strins 3. Additionlly, other fctors such s the ge nd immunocompetence of vccinees, s well s preexisting mounts of ntibody derived from prior infection or vccintion, contribute to mechnisms tht medite the efficcy of vccines ginst influenz,,. Systems vccinology hs emerged s n interdisciplinry field tht combines systems-wide mesurements plus network nd predictive modeling pplied to vccinology 5. A systems biology pproch hs been used to identify erly gene signtures tht correlte with nd cn be used to predict lter immune responses in humns vccinted with the live ttenuted vccine YF-7D ginst yellow fever 6,7. YF-7D is one of the most successful vccines ever developed 8,9 ; it stimultes polyvlent innte immune responses nd dptive immune responses tht cn persist for decdes fter vccintion. Although systems biology pproches hve been used to predict the immunogenicity of YF-7D 6,7, which is live replicting virus, the extent to which such pproches cn be pplied to the prediction of the immunogenicity of inctivted vccines is unknown. Furthermore, it remins uncler whether systems pproches cn be used to predict the immunogenicity of recll responses. In the cse of influenz, the immune response to vccintion is gretly enhnced by the pst history of the vccine recipient, both by prior infections nd vccintions. Notbly, whether such pproches cn provide insight into the immunologicl mechnisms of ction of vccines nd help with the discovery of new correltes of protective immunity is untested. To ddress these issues, we did series of clinicl studies during the nnul influenz sesons in 7, 8 nd 9, in which we vccinted helthy young dults with TIV. Our gol ws to undertke detiled chrcteriztion of the innte nd dptive responses to vccintion with TIV to identify puttive erly signtures tht correlted with or could be used to predict lter immunogenicity nd to obtin new insight into the mechnisms tht underlie immunogenicity. The results of our studies demonstrte tht systems biology pproches cn indeed be used to predict the immunogenicity of n inctivted vccine such s TIV with up to 9% ccurcy. Notbly, the expression t dy 3 of one of the genes in the predictive Emory Vccine Center, Emory University, Atlnt, Georgi, USA. Yerkes Ntionl Primte Reserch Center, Emory University, Atlnt, Georgi, USA. 3 Deprtment of Microbiology nd Immunology, Emory University, Atlnt, Georgi, USA. Center for Opertions Reserch in Medicine & Helthcre, School of Industril & Systems Engineering, Georgi Institute of Technology, Atlnt, Georgi, USA. 5 Deprtment of Phrmcology nd Cncer Biology, Duke University, Durhm, North Crolin, USA. 6 Deprtment of Cellulr nd Moleculr Biology nd Pthology, University of Nples Federico II, Nples, Itly. 7 Dn-Frber Cncer Institute, Boston, Msschusetts, USA. 8 Deprtment of Medicine, Division of Nephrology, Emory University School of Medicine, Atlnt, Georgi, USA. 9 Division of Infectious Diseses, Deprtment of Medicine, School of Medicine, Emory University, Atlnt, Georgi, USA. Institute for Systems Biology, Settle, Wshington, USA. Lbortory of Infectious Diseses, Ntionl Institute for Allergy nd Infectious Diseses, Ntionl Institutes of Helth, Bethesd, Mrylnd, USA. Deprtment of Pthology, Emory University School of Medicine, Atlnt, Georgi, USA. Correspondence should be ddressed to B.P. (bpulend@emory.edu). Received April; ccepted 6 June; published online July ; doi:.38/ni VOLUME NUMBER 8 AUGUST nture immunology

2 signture, encoding the kinse CMKIV, ws inversely correlted with plsm hemgglutintion-inhibition (HAI) ntibody titers t dy 8. Vccintion of CMKIV-deficient (Cmk / ) mice with TIV induced enhnced ntigen-specific ntibody titers, which demonstrted n unpprecited role for CMKIV in the regultion of ntibody responses. Together our results demonstrte the utility of systems biology not only in the prediction of vccine immunogenicity but lso in offering new insight into the moleculr mechnism of influenz vccines. RESULTS Antibody responses induced by TIV nd LAIV We evluted the ntibody responses of 56 helthy young dults vccinted with either LAIV (n = 8) or TIV (n = 8) during the 8 influenz seson. We determined HAI titers for ech of the three influenz strins in LAIV nd TIV in the plsm of vccinees t bseline (dy ) nd t 8 d fter vccintion. We clculted the mgnitude of ntibody responses to the vccine (HAI response) s the mximum difference between the HAI titer t dy 8 nd the bseline titer (dy ) for ny of the three influenz strins contined in the vccine (Fig. ). The men HAI response of subjects vccinted with TIV ws sixfold higher thn tht of those vccinted with LAIV (Fig. ), consistent with mny published reports,,3. Furthermore, mong the subjects vccinted with TIV, there ws considerble vrition in the mgnitude of the HAI response (>-fold; Fig. ). According to the US Food nd Drug Administrtion Guidnce for Industry document for this field, seroconversion cn be defined by n HAI titer of : or more nd minimum fourfold increse in ntibody titer fter vccintion. Thus, we opertionlly clssified the vccinees s low HAI responders or high HAI responders bsed on whether or not fourfold increse occurred fter vccintion (Fig. ). Most of the subjects vccinted with TIV ( of 8) were clssified s high HAI responders; only six HAI response (dy 8/dy ) High responders Low responders were clssified s low HAI responders. In contrst, most subjects vccinted with LAIV ( of 8) were clssified s low HAI responders nd only four were clssified s high HAI responders (Fig. ). Antibodies re produced by ntibody-secreting B cells in the blood (plsmblsts) or bone mrrow nd secondry lymphoid orgns (fully differentited plsm cells). High frequencies of ntigen-specific plsmblsts in the blood within few dys of vccintion, reching pek t dy 7, hve been documented 5. To determine whether the erly plsmblst response to influenz vccintion correlted with the lter HAI response, we ssessed the frequency of influenz-specific plsmblsts t bseline nd 7 d fter vccintion (Fig. b,c). As reported before 5, we observed rpid clonl expnsion of influenzspecific plsmblsts 7 d fter vccintion with TIV, s mesured by enzyme-linked immunospot (ELISPOT) ssy (Fig. b) nd by flow cytometry (Fig. c). We further found tht the popultion expnsion of circulting plsmblsts secreting immunoglobulin G (IgG) ws lso greter in subjects vccinted with TIV thn in those vccinted with LAIV (Fig. b,c). We obtined similr results for IgA-secreting plsmblsts t dy 7 fter vccintion (Supplementry Fig. ), nd very good correltion ws evident between the frequency of plsmblsts s mesured by ELISPOT nd their frequency s mesured by flow cytometry (Fig. d nd Supplementry Fig. b). As we detected very low HAI response fter vccintion with LAIV, we considered only subjects vccinted with TIV in further correltion nlyses. There ws modest positive correltion between the number of IgG-secreting plsmblsts t dy 7 nd the HAI response t dy 8 fter vccintion (Fig. e). Becuse the frequency of plsmblsts returns to brely detectble mount by dy fter vccintion 5, this correltion suggested tht the lter ntibody response ws ssocited with erly circultion of plsmblsts in the blood of vccinees 3. However, given the modest correltion (r =.3), there ws clerly need for more robust correltes of immunogenicity. TIV LAIV Dy Dy 7 Dy Dy 7 c TIV LAIV d 5 e Dy Dy 7 Dy Dy CD38 Totl plsmblsts (%) CD7 b Influenz-specific plsmblsts (per 6 PBMCs) Influenz-specific plsmblsts (per 6 PBMCs) 3 TIV Influenz-specific plsmblsts (per 6 PBMCs) LAIV HAI response (fold) 6 Figure Anlysis of humorl immunity to influenz vccintion. () HAI titers in plsm on dy 8 fter vccintion with TIV or LAIV, reltive to bseline (dy ); results re the highest HAI response mong ll three influenz strins in the vccine: low responders, no increse bove twofold; high responders, fourfold or more bove bseline. P <., men HAI response, TIV versus LAIV (t-test). (b) ELISPOT ssy of influenz-specific IgG secreting plsmblsts mong PBMCs from ll vccinees t nd 7 d fter vccintion. Ech symbol represents n individul donor; smll horizontl lines indicte the medin (numbers djcent medin vlues); dotted lines re the limit of detection. (c) Flow cytometry nlysis of plsmblsts in the plsmblst gte (CD3 CD lo neg CD9 + CD7 hi CD38 hi ) in blood from subjects vccinted with TIV or LAIV. Numbers djcent to outlined res indicte percent cells in the plsmblst gte. (d) Frequency of plsmblsts, ssessed by flow cytometry, versus the number of influenz-specific IgG secreting plsmblsts, ssessed by ELISPOT, t dy 7 fter vccintion with TIV (blue) or LAIV (blck). r =.58 (Person); P <. (for Person correltion; two-tiled test). (e) Influenz-specific IgG secreting plsmblsts t dy 7 versus the ntibody response t dy 8 fter vccintion with TIV. r =.3 (Person); P =. (for Person correltion; two-tiled test). Dt re from one experiment with 56 subjects ssyed in duplicte (), 6 subjects ssyed in duplicte (b) or 59 subjects ssyed once (c) or were generted from dt in c (d,e). nture immunology VOLUME NUMBER 8 AUGUST 787

3 PSMB CD CTSB TNFSF3B MX MX CIITA OAS Down SMURF OAS PSMB8 E () TNFSF3 Up Expression (dy X/dy ) ILRB PSMB9 TNFSF OAS3 XAF IFI35 CD86 Dy 3 GATA3 CASP PLSCR PECAM TNFAIP3 TICAM IFITM APOBEC3G Moleculr signtures of influenz vccines We first determined whether TIV nd LAIV induced moleculr signtures tht were detectble in the blood. To identify such signtures of immunogenicity, we first mesured by multiplex ssy the concentrtions of key cytokines in the plsm of vccinees on dys, 3 nd 7 fter vccintion (Supplementry Fig. ). We selected ten cytokines or chemokines on the bsis of their importnce s key meditors of host immune responses (CCL5 (RANTES), interleukin α, interferon-α (IFN-α), CCL3 (MIP-α), CCL (eotxin), interleukin subunit p7, IFN-γ, interleukin β, CXCL (IP-) nd CCL (MCP-)). Among those, only the chemokine CXCL (IP-) ws significntly induced by TIV on dy 3 reltive to its expression on dy (P =.89 (t-test); Supplementry Fig. b). None of those cytokines were significntly induced or repressed by vccintion with LAIV. The concentrtion of CXCL (IP-) t dy 3 reltive to its bseline concentrtion ws negtively correlted to the HAI response t dy 8 fter vccintion (Supplementry Fig. c), which suggested possible involvement of CXCL (IP-) in the ntibody response. However, the correltion coefficient ws modest (r =.8), which gin emphsized the need for more robust correltes of immunogenicity. To determine in n unbised wy the expression chnges induced by vccintion ginst influenz on genome-wide scle, we did microrry nlysis using peripherl blood mononucler cells (PBMCs) collected from ll 56 vccinees on dys, 3 nd 7 fter vccintion. We clculted the chnge in expression by subtrcting the log expression vlue t dy from its corresponding vlue dy 3 or 7, nd we filtered out genes if we observed no increse or decrese greter thn 5% (.5-fold) in t lest % of the vccinees. After tht step, IRF7 STAT TLR8 STAT TRIM TC-PTP IRF5 MYD88 TLR7 JAK CYLD IFNB IFNA IFNAR UNC93B KATB STAT6 IKBKB IL7RA CXCL DNAJA3 UBA7 IL8 CSNKA CDKNB JUN SOCS3 MAP3K7 OSM AZI IRF3 EIFS PMAIP MAVS TRADD IRAK3 ICAM ICAM BCL ITGA IRF8 HDAC CREBBP Dy 7 PTPN6 CD TP53 SMARCA CSFR TRAF6 IL6R ITGB ERAP HDAC IFI6 C3AR ITGAL we pplied three independent sttisticl tests to the remining genes nd considered only genes identified by ll three nlyses s being differently expressed. Trnscriptome nlysis of vccinees showed tht LAIV nd TIV induced very different gene signtures (Supplementry Fig. 3). However, the expression of,5 probe sets ws ltered similrly by both vccines (Supplementry Fig. 3). Among these common differentilly expressed genes (DEGs), ingenuity pthwy nlysis identified network composed of severl genes relted to inflmmtory nd ntimicrobil responses (Supplementry Fig. 3b; complete list of DEGs fter vccintion with TIV or LAIV, Supplementry Tble ). This indicted tht processes relted to innte immunity my hve influenced the immunogenicity of ech vccine. The expression of severl interferon-relted genes ws ltered fter vccintion with LAIV but not fter vccintion with TIV (Fig. ). Type interferons re centrl components of the innte immune response to virus 6. Therefore, the higher expression of type I interferon relted genes my be ttributed to the repliction competence of LAIV. Our nlysis identified genes encoding molecules closely ssocited with the interferon signling pthwys, such s STAT, STAT, TLR7, IRF3 nd IRF7 (Fig. ). Notbly, the difference in expression for mny interferon-relted genes ws gretest t dy 3 fter immuniztion with LAIV (Fig. ). We lso compred the gene signtures of the two influenz vccines with tht of nother live ttenuted vccine, the YF-7D vccine ginst yellow fever 6. For consistency with tht publiction 6, we pplied the sme stringency nd criteri to identify genes with differences in b OAS RNA (reltive) IRF7 RNA (reltive) MX RNA (reltive) STAT RNA (reltive) Medium LAIV TIV YF-7D Figure Moleculr signture induced by vccintion with LAIV. () Interferon-relted genes upregulted (Up) or downregulted (Down) on dy 3 or 7 ( X in key) fter vccintion with LAIV reltive to their expression t dy (colors in key): solid lines indicte direct interctions; dshed lines indicte indirect interctions. (b) Quntittive RT-PCR confirmtion of the induction of key interferon-relted genes (OAS, IRF7, MX nd STAT) in PBMCs obtined from helthy subjects nd left unstimulted (Medium) or stimulted for h in vitro with LAIV, TIV or YF-7D; results re normlized to the expression of GAPDH (glycerldehyde phosphte dehydrogense) nd re presented reltive to those of unstimulted PBMCs. Dt re representtive of one experiment () or three independent experiments with one subject ech (b; error brs, s.d.). 788 VOLUME NUMBER 8 AUGUST nture immunology

4 Figure 3 Moleculr signtures induced by vccintion with TIV. () Het mp of gene signtures of cells of the immune response, identified by met-nlysis. Expression of ech gene (rows) is presented s s.d. bove (red) or below (blue) the verge vlue for tht gene for ll smples (columns). mdc, myeloid DC; pdc, plsmcytoid DC; NK, nturl killer. (b) Enrichment for genes upregulted by TIV mong genes with high expression in ny PBMC subset (numbers in plot indicte enrichment (fold)). (c) Enrichment for genes upregulted by TIV mong genes with high expression in B cells nd lso in specific B cell subset. (d) Het mp of genes upregulted fter vccintion with TIV nd lso with high expression in B cells (PBMCs) nd ASCs (B cell subsets); indictes probe sets mpping to ntibody vrible regions, nd Affymetrix probe identifiers re provided for probe sets not nnotted. (e) Enrichment for genes upregulted by LAIV mong genes with high expression in ny PBMC subset. *P < (two-tiled Fisher s exct test). Dt re representtive of 8 experiments with 8 smples. expression in subjects vccinted with YF-7D, s follows: we filtered out genes if we found no increse or decrese in expression (on dy 3 or 7 reltive to bseline) greter thn.-fold in t lest 6% of the vccinees; we used one-wy nlysis of vrince with the Benjmini nd Hochberg flse-discovery-rte method with cutoff of.5; nd genes hd to hve difference in expression in both YF-7D trils 6. However, this time we did the nlysis t the level of the probe set insted of defining genes bsed on the UniGene dtbse (Ntionl Center for Biotechnology Informtion). Although subjects vccinted with YF-7D hd gene-expression profile distinct from tht of those vccinted ginst influenz, mny interferon-relted genes were commonly induced by YF-7D nd LAIV (dt not shown). RT-PCR nlysis of RNA from PBMCs stimulted in vitro with LAIV, TIV or YF-7D confirmed tht interferon-relted genes were upregulted h fter tretment with LAIV or YF-7D but not fter stimultion with TIV (Fig. b). Together these dt demonstrted tht vccintion with TIV or LAIV induced distinct moleculr signtures in the blood. Moleculr signtures of sorted cell subsets We did microrry nlyses of the gene-expression profiles of PBMCs isolted from the blood of vccinees t bseline nd t dys 3 nd 7 fter vccintion. One confounding vrible here ws tht the observed trnscriptionl chnges my hve resulted from new induction of gene expression or my hve simply reflected the chnging cellulr composition of the PBMC comprtment. To overcome this issue, we used the pproch of isolting nd identifying the genomic signtures of ech subset in the PBMC pool. We did microrry experiments with the following four different cell types, obtined from subjects vccinted with LAIV (n = 6) or TIV (n = 6) nd sorted by flow cytometry: CD9 + B cells, CD + monocytes, CDc hi CD3 lo myeloid dendritic cells (DCs) nd CD3 hi CDc lo plsmcytoid DCs. We extrcted, mplified nd lbeled totl RNA from 96 sorted cell smples t bseline nd dy 7 nd hybridized the RNA on microrry chips (Supplementry Fig. ). We did significnce nlysis of microrrys 7 for ech subset, seprtely compring the vlues t dy 7 with the corresponding bseline vlues. This pproch identified hundreds to thousnds of probe sets with differences in expression fter vccintion with TIV or LAIV (Supplementry Fig. b nd Supplementry Tble ), which demonstrted tht vccines ginst influenz produced globl expression chnges for ech cell type. In subjects vccinted with TIV, myeloid DCs nd B cells hd the most DEGs (Supplementry Fig. b). Notbly, there ws n enrichment for DEGs ssocited with plsmblsts (Supplementry Fig. c). However, becuse substntil proportion of plsmblsts die fter being frozen nd thwed (dt not shown), the DEGs observed in the s.d. Memory GC TIVupregulted genes B cell * Monocyte T cell Nive mdc NK cell pdc PBMCs Monocyte mdc pdc B cell T cell NK cell ASC Nive d b c TIV- e upregulted genes (high expression in B cells) ASC 5 5 * * Memory 3 7 B cell T cell NK cell pdc mdc Monocyte ASC GC Memory Nive LAIVupregulted genes B cell Monocyte T cell * * GC mdc NK cell B cell comprtment were probbly n underestimtion of the DEGs ssocited with plsmblsts (Fig. 3). Nevertheless, we were still ble to identify DEGs relted to ntibody-secreting cells (ASCs) nd the unfolded protein response in sorted B cells fter immuniztion with TIV (Supplementry Fig. c,d). To cope with the lrge mount of immunoglobulin proteins tht re produced, ASCs must gretly increse the function of their secretion mchinery, which my led to the ccumultion of misfolded proteins in the endoplsmic reticulum 8,9. In response to such stress, the cells ctivte intrcellulr signl-trnsduction pthwys nd the unfolded protein response, which protects the cells by enhncing the cpcity of the secretory pprtus nd by diminishing the endoplsmic reticulum lod. After vccintion with TIV, upregultion of genes encoding two trnscription fctors, XBP- nd ATF6B, which re centrl orchestrtors of the unfolded protein response, ws detectble in sorted B cells but not in PBMCs (Supplementry Fig. d). In subjects vccinted with LAIV, in contrst to results obtined with those vccinted with TIV, the plsmcytoid DC subset generted the most DEGs (Supplementry Fig. b). Of the mny interferon-relted genes induced by LAIV (Fig. ), we found tht 37 were induced in t lest one subset of the sorted cells. Of those, 7 nd were upregulted in monocytes nd plsmcytoid DCs, respectively (Supplementry Fig. e). In ddition, there were interferon-relted genes tht were induced or repressed in t lest one subset of the sorted cells but not in the PBMCs (Supplementry Fig. e). Most were upregulted in myeloid nd plsmcytoid DCs (Supplementry Fig. e). These dt suggest tht ntigen-presenting cells my be importnt in the innte response to vccintion with LAIV. The lrge number of interferon-relted genes missing from the PBMC nlysis my hve been due to the fct tht myeloid DCs nd plsmcytoid DCs together represent <% of totl PBMCs. The observtions reported here indicted the type of informtion tht cn be obtined by exmintion of the gene-expression profiles TIV Time (d).66.5 Expression reltive to dy (fold) PBMCs s.d. B cell subsets pdc SLC7A7 CAV CLPTM ECE IGHD APOBEC3B SELL3 IGJ IGHE 836_x_t MGC956 TPD5 TPD5 TPD5 IGHG3 TNFRSF7 DMAP DTNB IGHE IG LOC3888 IGHG LOC33956 IGHD IGKsimilr 6576_x_t nture immunology VOLUME NUMBER 8 AUGUST 789

5 of sorted cell types. However, evluting the gene-expression signtures of individul subsets of cells isolted by flow cytometry presents considerble chllenge. The prcticl use of such n pproch is very limited, both logisticlly (tht is, the need to use freshly isolted smples to prevent the preferentil loss of certin cell types, such s plsmblsts nd effector T cells) nd finncilly (tht is, the need for lrge numbers of gene chips). Therefore, s described below, we devised n lterntive strtegy. Met-nlysis of cell type specific signtures Humn PBMCs consist of mny different cell types, ech with distinct trnscriptome. A published study hs demonstrted the use of deconvolution method to nlyze cell type specific gene expression differences in complex tissues. We devised n independent strtegy to discern cell type specific trnscriptionl signtures with the results of the PBMC microrry nlyses. We did met-nlysis of publicly vilble microrry studies in which the gene-expression profiles of isolted individul cell types of PBMCs (such s T cells, B cells, monocytes, nturl killer cells nd so on) or B cell subsets (such s nive, memory nd germinl center B cells nd ASCs from blood or tonsils) hd been nlyzed (Supplementry Fig. 5,b). To void issues of cross-pltform normliztion nd probe selection, we used only smples hybridized to Affymetrix Humn Genome U33 Plus. Arrys or Affymetrix Humn Genome U33A Arrys in our met-nlysis. Additionlly, for ech study, we mnully removed smples bsed on the severity of the disese or tretment nd/or the method of cell purifiction (smples nd studies, Supplementry Tble 3). We included in our met-nlysis microrry dt of flow cytometry sorted plsmcytoid nd myeloid DCs obtined from PBMCs of subjects before nd fter vccintion with TIV or LAIV (Supplementry Fig. ). We compred the expression profile of given cell subset with the expression profile of ll other subsets by t-test (P <.5; men chnge, over twofold). We designted gene s hving high expression in prticulr cell type by determining the number of times the gene ws upregulted in the cell type by ll possible pirwise comprisons with its expression in other cell types (Supplementry Fig. 5b nd Supplementry Methods). We then compred the genomic signtures of cells of the immune response obtined by this pproch (Fig. 3 nd Supplementry Tble ) with the genomic signtures of subjects vccinted ginst influenz. Our met-nlysis confirmed tht the group of genes upregulted by TIV ws enriched for genes with high expression in B cells (Fig. 3b) nd, mong those, genes with high expression in ASCs (Fig. 3c). We prepred het mp of the genes upregulted in ASCs fter vccintion with TIV (Fig. 3d). Among the genes upregulted were those encoding ntibody prts (rerrnged vrible-diversity-joining immunoglobulin gene segments) nd severl other genes encoding prts of immunoglobulins (, IGHE, IGHG3, IGHG nd IGHD), s well s TNFRSF7 (which encodes BCMA, member of the BAFF-BLyS fmily of receptors 3, nd whose expression hs been shown before to be key feture of the best predictive signtures of neutrlizing ntibody responses to YF-7D 6 ). These results confirmed the results obtined by flow cytometry nd ELISPOT, with which we observed greter frequency of IgG + nd IgA + ASCs in the blood of vccinees t dy 7 fter vccintion with TIV (Fig. nd Supplementry Fig. ). In ddition to the ASC signture, we observed signture composed of severl genes encoding molecules tht orchestrte the unfolded protein response 9, (dt not shown). The lrge number of XBP- trget genes with differences in expression fter vccintion ws consistent with role for XBP- in orchestrting the differentition of plsm cells 9. Among those, genes such s ATF6, MANF, CREB3, PDIA, DNAJB, HSP9B, HERPUD nd DNAJB9 encode molecules re lredy known to be involved in the unfolded protein response 6. In contrst to results obtined for TIV, nlysis of the trnscriptionl signture induced by LAIV by met-nlysis showed considerble enrichment for genes with high expression in T cells nd monocytes (Fig. 3e). We lso found mny genes with high expression in nturl killer cells, lthough these results did not rech sttisticl significnce (dt not shown). Among the interferon-relted genes upregulted fter vccintion with LAIV (Fig. ), most hd high expression in monocytes nd nturl killer cells (dt not shown). Tht result ws similr to our microrry nlysis of flow cytometry sorted cells obtined from subjects vccinted with LAIV, in which most interferon-relted genes with differences in expression in PBMCs nd t lest one cell subset hd high expression in monocytes (Supplementry Fig. e). These results indicte tht the innte immune responses cn hve n importnt role in the mechnism of ction of this live ttenuted virus vccine. Signtures tht correlte with the ntibody response Vccintion with TIV induced considerble vrition in the mgnitude of the HAI response (Fig. ). To gin insight into the potentil mechnisms underlying tht vrition nd to identify gene signtures with which we could predict the mgnitude of the HAI response, we serched for erly gene signtures tht correlted with the B cell responses t dys 7 nd 8 fter vccintion with TIV (complete list, Supplementry Tble 5). Person correltion nlysis identified 6, probe sets tht correlted, either directly or inversely, with the mgnitude of the HAI response (Fig. ). Among those were severl genes known to be regulted by XBP- nd to be involved in the differentition of plsm cells nd the unfolded protein response (Fig. b). Ingenuity pthwy nlysis of the genes tht were either positively or negtively correlted with HAI titers showed enrichment for genes relted to the cell-medited immune response nd to the infection mechnism nd inflmmtory response, respectively (Supplementry Fig. 6,b). The identifiction of genes such s TLR5, CASP, PYCARD, NOD nd NAIP suggested previously unknown mechnistic links between host innte immunity nd humorl responses to influenz vccintion. In fct, reserch hs shown tht cndidte vccine ginst influenz composed of recombinnt fusion protein linking influenz ntigens to the Toll-like receptor 5 lignd flgellin my induce potent immunogenicity in mice 7 nd humns 8,9. In ddition, cnonicl pthwys, such s T cell receptor ntigen receptor signling nd CTLA- signling in cytotoxic T lymphocytes, included mny of the genes present in the cell-medited immune response network nd were mong those with the highest enrichment score by ingenuity pthwy nlysis (Supplementry Fig. 7,b). Although further experimenttion is needed, these dt indicted possible ssocition between cellulr responses nd humorl responses to vccintion with TIV 3. Among the top cnonicl pthwys enriched for genes positively correlted to HAI response (Supplementry Fig. 7c), we found networks ssocited with innte immunity, such s the nturl killer cell signling network, nd network for the production of nitric oxide nd rective oxygen species in mcrophges (Supplementry Fig. 7d). Our nlysis lso showed tht the expression of interferonrelted genes (including those encoding the receptors for interferon-α nd interferon-γ) on dy 3 fter vccintion ws correlted to the HAI response (Supplementry Fig. 8), which suggested link between the interferon response nd the ntibody response VOLUME NUMBER 8 AUGUST nture immunology

6 Figure Moleculr signtures tht correlte with titers of ntibody to TIV. () Het mp of probe sets (rows) nd subjects (columns) whose bseline-normlized expression t dy 3 (top) or dy 7 (bottom) correlted with bseline-normlized ntibody response t dy 8 fter vccintion with TIV (colors in mp indicte gene expression t dy 3 or 7 reltive to expression t dy ). Right mrgin, number of probe sets with negtive correltion (blue) or positive correltion (red). Probe sets tht correlted with the HAI response on both dy 3 nd dy 7 were considered dy 7. P <.5 (Person). (b) HAI response correlted genes ssocited with the unfolded protein response (purple shding) or ASC differentition (tn shding) nd/or regulted by XBP- (solid nd dshed lines s in Fig. ). P <.5 (Person). (c) Enrichment for genes (mong those with high expression in ny PBMC subset) whose expression on dy 3 or 7 fter vccintion with TIV ws positively or negtively correlted with HAI titers (cutoff, P <.5 (Person)). *P < (two-tiled Fisher s exct test). (d) Het mp of probe sets with high expression in B cells nd ASCs whose bseline-normlized expression correlted with the bselinenormlized HAI response. P <.5 (Person). Dt re representtive of one experiment with 8 subjects. Next we compred the genes whose expression correlted with the HAI response t dy 8 fter vccintion of subjects with TIV with the genomic signtures of the cells of the immune response defined by our metnlysis. This pproch showed tht the set of genes positively correlted to HAI response ws enriched for genes with high expression in B cells (Fig. c) nd, more specificlly, in the ASC subset (Fig. d). The genes with negtive correltion to the HAI response were substntilly enriched mong the genes with high expression in T cells (Fig. c), which supported the identifiction of the T cell pthwys by ingenuity pthwy nlysis (Supplementry Fig. 7,b). Together these dt demonstrted the identifiction of erly signtures tht correlted with lter HAI titers induced by TIV. Moleculr signtures to predict ntibody responses Once we hd delineted signtures tht correlted with the mgnitude of HAI response, our next step ws to identify the minimum sets of genes we could use to predict such response. Idelly, such sets of genes must be ble to be used to ccurtely clssify high responders versus low responders in dditionl nd independent TIV trils. For this, we used DAMIP (discriminnt nlysis vi mixed integer progrmming 3,33 ), which is very powerful supervised-lerning clssifiction method for predicting vrious biomedicl nd biobehviorl phenomen 3,33. In initil nlyses, we clssified the subjects vccinted with TIV into two extreme groups: very low HAI responders, nd very high HAI responders. The former group consisted of subjects with n increse of twofold or less in HAI titers ginst ny of the three influenz strins of the vccine (Fig. ). The ltter group consisted of subjects with n increse of eightfold or more in the HAI response for HAI response (dy 8/dy ) Dy 3 Dy 7.5 Expression (fold) b c MANF CALR Monocyte mdc Unfolded protein response PDIA IGHM ATF6B CD38 Correltion HSP9B ATF6 POUAF FCGR3A PECAM B cell * POUF TNFRSF3B pdc HAI response t dy 8 Positive correltion (dy 7) Negtive correltion (dy 7) * SMC3 IGKC IGHA t lest one of the three influenz strins of the vccine. We did not nlyze subjects with intermedite HAI response (between twofold nd eightfold) nd subjects for whom microrry dt were not vilble t either dy 3 or dy 7 fter vccintion (n = 7). We used tht tril (the 8 9 tril) to trin the DAMIP model to estblish n unbised estimte of correct clssifiction. We used second, independent tril to evlute the predictive ccurcy of the clssifiction rules identified in the first tril (Fig. 5). The second tril (the 7 8 tril) consisted of the microrry gene-expression profiles of subjects (n = 9) vccinted with TIV in the previous yer. With this pproch, DAMIP model identified sets of genes contining two to four genes ech (ech set ssocites with one predictive rule) from 8 9 tril with tenfold cross-vlidtion ccurcy over 9%. The resulting blind prediction ccurcy of the 7 8 tril (predicting low or high responders) ws over 9%. Furthermore, some of the 7 sets of discrimintory genes offered n ccurcy of over 9% in both tenfold cross-vlidtion in the trining tril nd blind prediction ccurcy (Fig. 5 nd Supplementry Tble 6). We then used rel-time RT-PCR to confirm tht genes from the DAMIP gene signtures encoded molecules with potentil biologicl relevnce nd/or utility s predictor of influenz d T cell NK cell GOPC XBP HAI response (dy 8/dy ) TNFSF3 BAX TNFRSF PIGA IGL@ HYOU IL6ST KDELR RPN CAV SECC SSR SRPR SEC3B ASCs RPN SECD MOGS SPCS PDIA6 SLC7A9 DTNB CAV 736_x_t DMAP APOBEC3B MGC956 78_x_t 7378_x_t 6576_x_t TNFRSF7.5 Expression (fold) nture immunology VOLUME NUMBER 8 AUGUST 79

7 Figure 5 Signtures tht cn be used to predict the ntibody response induced by TIV. () Experimentl design used to identify the erly gene signtures tht cn be used to predict ntibody responses to vccintion with TIV: the 8 9 tril ws used s trining set to identify predictive signtures with the DAMIP model; those signtures were then tested on the dt from the 7 8 tril (the testing set). The expression of subset of genes in the DAMIP predictive signtures of the 7 8 nd 8 9 trils ws then quntified by RT-PCR in third independent tril (9 tril); the DAMIP model ws gin used to confirm the predictive signtures. (b) RT-PCR confirmtion of the expression of subset of genes in the predictive signtures generted by the DAMIP model. Ech symbol represents single gene t given time point. P <, microrry versus RT-PCR (Person); r =.68; n =,897 XY pirs. Dt re representtive of one experiment with genes from 8 subjects t two time points. (c) DAMIP gene signtures identified with the 8 9 tril s the trining set nd the 7 8 nd 9 trils s the vlidtion sets (DAMIP model 3); the ccurcy represents the number of subjects correctly clssified s low responders or high responders (Fig. ). Dt re representtive of three independent experiments. vccine immunogenicity. We found significnt positive correltion (r =.679; P = 3.5 ) for chnges in expression on dy 3 or 7 reltive to bseline expression s detected by microrry nd RT-PCR (Fig. 5b nd Supplementry Tble 7), which confirmed the correctness of the microrry dt. More notbly, tht result gve us confidence to test some of the cndidte predictors of immunogenicity in third nd independent influenz vccine tril (Fig. 5). We collected RNA from PBMCs of subjects (n = 3) vccinted with TIV during the 9 influenz seson nd nlyzed this RNA by rel-time RT-PCR. We then used the expression of the genes selected from the initil DAMIP gene signtures to confirm their utility in predicting the mgnitude of ntibody response in this third TIV tril (Fig. 5). To void the identifiction of over-trined rules, we re-rn the DAMIP nlysis using the 8 9 tril s the trining set nd the 7 8 nd 9 trils s the blind predictive sets. This pproch identified 7 sets of genes; some of these we used to correctly clssify >85% of the vccines s being very low HAI responders or very high HAI responders in ny of the three trils (Supplementry Tble 8). Becuse seroconversion fter vccintion is widely defined s fourfold increse in HAI titers 3, we rn n dditionl DAMIP nlysis using cutoff of fourfold to clssify the vccinees (Fig. 5). Thus, we clssified subjects with n increse of fourfold or greter in the HAI titers fter vccintion s high responders nd those with n increse of twofold or less s low responders. With the 8 9 tril s trining set nd 7 8 nd 9- trils s blind predictive sets, the DAMIP model generted sets of gene signtures (Fig. 5c), ech composed of three to four discrimintory genes, some of which hd n unbised estimte of correct clssifiction bove 85%, s determined by tenfold cross-vlidtion nd blind prediction (Supplementry Tble 9). One of the genes present in the DAMIP gene signtures, TNFRSF7, ws lso identified in DAMIP models used to predict ntibody responses to vccintion with YF-7D 6. Among the genes in the TIV DAMIP models, we found five members of the leukocyte immunoglobulin-like receptor fmily (Supplementry Tble 9). These genes re expressed by immuneresponse cells of both myeloid nd lymphoid lineges nd the molecules they encode re thought to hve n immunomodultory role in the innte nd dptive immune systems by regulting T cells b RT-PCR (dy X/dy ) DAMIP nlysis (eightfold cutoff) Trining set 8 9 tril >9% Testing set DAMIP gene 7 8 signtures tril DAMIP nlysis (eightfold cutoff) Trining set 8 9 tril c 87% Testing set DAMIP gene 9 signtures tril DAMIP nlysis 3 (fourfold cutoff) Trining set 8 9 tril >9% DAMIP gene signtures >9% 9% >9% 85% Microrry (dy X/dy ) Testing set 7 8 tril Testing set 9 tril GGH LILRB LILRA3 TNFRSF7 TNFRSF7 GGH LST LILRA3 CFD ERLEC NLRP NLRP SLAMF7 ERLEC MANF SIGLEC7 LILRA LILRB CLPTML ERLEC 99 8 TNFRSF7 APOBEC3B GGH PYCARD LST LST SLAMF7 HSP9B LILRA Dy 3 versus dy Dy 7 versus dy Accurcy (%) nd utoimmunity Our met-nlysis showed tht these genes hd high expression in monocytes nd myeloid DCs t dy 3 fter vccintion (dt not shown). These results nd the presence of five members of this fmily mong mrkers of ntibody responses to influenz vccintion rised the possibility of previously unknown roles for these innte immune receptors in regulting ntibody responses. CMKIV regultes the ntibody response To demonstrte tht the gene signtures identified in our study could be used to generte new hypotheses, we selected one gene in the predictive signture, CAMK, for functionl confirmtion experiments. CMKIV is involved in severl processes of the immune system, such s T cell development 38, inflmmtory responses, nd the mintennce of hemtopoietic stem cells 3. However, nothing is known bout the possible role of CMKIV in B cell responses. The chnge in CAMK expression on dy 3 fter vccintion with TIV ws negtively correlted with the ntibody response on dy 8 fter vccintion in two independent trils (Fig. 6). Additionlly, the chnge in CAMK expression ws negtively correlted with the popultion expnsion of IgG-secreting plsmblsts t dy 7 (Fig. 6b), which suggested possible role for CMKIV in the regultion of ntibody responses to vccintion ginst influenz. In vitro stimultion of mouse splenocytes with TIV resulted in phosphoryltion of CMKIV (Fig. 6c), which suggested tht this vccine my trigger ctivtion of CMKIV. Tht finding ws further demonstrted in humn PBMCs, in which in vitro stimultion with influenz vccine resulted in phosphoryltion of CMKIV s erly s h fter stimultion (Fig. 6d). The mechnism by which this occurs remins to be identified. To check if CMKIV regultes the ntibody response to influenz vccine, we immunized wild-type nd Cmk / mice with TIV nd mesured serum concentrtions of IgG nd IgGc on dys 7, nd 8 fter vccintion (Fig. 6e). After immuniztion, Cmk / mice hd significntly greter ntibody response thn tht of wild-type mice (Fig. 6e). The biggest difference ws on dy 7, with 3- to 6.5-fold higher ntibody titers in Cmk / mice thn in wild-type mice (Fig. 6e). These results supported our prediction bsed on the microrry results nd suggested tht CMKIV is importnt in the regultion of B cell response VOLUME NUMBER 8 AUGUST nture immunology

8 HAI response (fold) tril 7 8 tril 5 5 Time (h) 56 8 TIV (µg/ml) 6 p-cmkiv CMKIV Microrry (log dy 3/dy ) Microrry (log dy 3/dy ) Microrry (log dy 3/dy ) HAI response (fold) b Influenz-specific plsmblsts (per 6 PBMC) c Mouse e..8 d Time (min) p-cmkiv CMKIV Cmk / WT LPS Humn TIV 36 7 ** * ** Figure 6 CMKIV regultes the ntibody response to vccines ginst influenz. () HAI response t dy 8 versus microrry nlysis of CAMK mrna in PBMCs t dy 3 fter vccintion with TIV in the 8 9 tril (left; r =.7 (Person); P =.6 (for Person correltion; two-tiled test) or the 7 8 tril (right; r =.73 (Person); P =. (for Person correltion; two-tiled test). (b) ELISPOT nlysis of influenz-specific IgG secreting plsmblsts t dy 7 versus microrry nlysis of CAMK mrna on PBMCs t dy 3 fter vccintion with TIV. (c) Immunoblot nlysis of the phosphoryltion (p-) of mouse CMKIV fter in vitro stimultion of splenocytes for or h with vrious doses of TIV (bove lnes). (d) Immunoblot nlysis of the phosphoryltion of CMKIV fter in vitro stimultion of humn PBMCs for 7 min with lipopolyscchride (LPS) or TIV. (e) Serum ntigen-specific IgG (top) nd IgGc (bottom) responses of wild-type nd Cmk / mice t dys 7, nd 8 fter immuniztion with TIV (symbols represent individul mice), presented s bsorption t 5 nm (A 5 ). *P <.5 nd **P <. (Student s t-test). Dt re representtive of one tril ech with 6 subjects (8 9) or 9 subjects (7 8; ), one experiment with 6 subjects (b), three experiments (c,d) or t lest four independent experiments (e). DISCUSSION Despite the gret success of vccines, little is understood bout the mechnisms by which effective vccines stimulte protective immune responses. Two developments re beginning to offer such understnding: incresing pprecition of the key roles of the innte immune system in sensing vccines nd tuning immune responses, nd emerging dvnces in systems biology. A systems biology pproch hs been used to obtin globl picture of the immune responses in humns to vccine YF-7D ginst yellow fever, one of the most successful vccines ever developed. This pproch hs identified unique biomrkers (moleculr signtures) used to predict the mgnitude of the ntigen-specific CD8 + T cell nd ntibody responses induced by YF-7D 6,7 nd hs resulted in the formultion of new hypotheses bout the mechnism of ction of this vccine. However, whether such n pproch could hve brod utility in the identifiction of signtures of immunogenicity of other kinds of vccines, prticulrly inctivted vccines, nd whether such signtures would be informtive bout the underlying mechnisms of immunity remin unknown. To ddress these issues, we did series of studies over three consecutive influenz sesons. The gol of these studies ws to nlyze in detil the innte nd dptive immune responses to vccintion with two vccines ginst influenz, TIV nd LAIV, to identify erly moleculr signtures tht cn be used to predict lter immune responses nd to obtin insight into the mechnisms tht underlie immunogenicity. According to guidelines estblished by the US Food nd Drug Administrtion, seroconversion cn be defined s n HAI titer of : or more nd minimum increse of fourfold in ntibody titer fter vccintion. However, it often tkes severl weeks fter vccintion to chieve this titer; therefore, the bility to predict seroconversion just few dys fter vccintion nd identify nonresponders would be of gret vlue from public helth perspective. We thus used systems biology pproches to identify erly signtures tht we used to predict HAI titers weeks fter vccintion. To ccomplish this gol, we used n interdisciplinry pproch, including gene-expression profiling by microrry, RT-PCR nd computtionl methods, combined with cellulr nd moleculr biologicl pproches, s well s experiments involving geneticlly deficient mice. Our dt hve demonstrted tht such systems biology pproch cn indeed lgg (A 5 ) lggc (A 5 ) be used not only to identify predictive signtures but lso to obtin new insights bout the immunologicl mechnisms involved. Although the clinicl effectiveness of both vccines is similr, LAIV induces lower serum ntibody response in dults thn does TIV,3,5. This probbly reflects the lower tke of LAIV becuse of preexisting mucosl IgA tht cn neutrlize the virus 3. Nevertheless, our microrry nlysis identified lrge number of genes with differences in expression, most relted to the type I interferon response, in the PBMCs of subjects vccinted with LAIV. Future studies should focus on nlyzing chnges in the trnscriptome of the nsl mucos fter vccintion with LAIV nd how tht correltes with or cn be used to predict locl ntibody responses. Among the genes induced by vccintion with TIV, we found enrichment for genes with high expression in ASCs. This result my hve reflected the rpid prolifertion of plsmblsts t dy 7 fter vccintion 5 ; however, our microrry nlysis of B cells sorted from subjects vccinted ginst influenz indicted tht the chnges in expression observed in PBMCs could lso hve been derived from rel trnscriptionl chnges in B cells. The trnscription fctor XBP-, which is essentil for the differentition of ASCs nd the unfolded protein response 8, nd its trget genes were upregulted fter vccintion with TIV nd correlted with IgG nd HAI responses. The genes identified by our study my offer new opportunities for studying the complex mechnisms involved in the unfolded protein response nd its link to ASC differentition 8. A key question ws whether the signtures tht cn be used to predict the T cell nd B cell response to one vccine cn lso be used to predict such responses to nother vccine. Notbly, of the 33 genes present in the 7 DAMIP gene signtures tht we used to predict the ntibody response to vccintion with TIV, 7 were lso predictors of the ntibody response to vccintion with the YF-7D vccine ginst yellow fever 6. Key genes in the predictive signtures were TNFRSF7, which encodes BCMA, receptor for the B cell growth fctor BLyS (known to hve key role in B cell differentition 3 ), nd CD38, which encodes surfce protein importnt in lymphocyte development 6,7. BCMA belongs to fmily of molecules (BAFF, APRIL, BAFF-R nd TACI) tht regulte the differentition of plsm cells nd ntibody Unvccinted 7 8 Time fter immuniztion (d) ** * ** Unvccinted 7 8 Time fter immuniztion (d) nture immunology VOLUME NUMBER 8 AUGUST 793

9 production 3. Notbly, there were strong correltions between the expression of genes encoding APRIL, BAFF-R nd TACI nd the mgnitude of the HAI titers in response to vccines ginst influenz nd the mgnitude of neutrlizing ntibody response to YF-7D (dt not shown), which suggested tht this network my be criticlly involved in regulting ntibody responses to different vccines. The functionl relevnce of this network in mouse models remins to be determined. It lso remins to be seen whether this network represents common predictor of ntibody responses induced by mny vccines. A second issue ws whether the dt generted from such studies would be useful in providing new biologicl insights into the regultory mechnisms tht underlie vccine immunogenicity. Our experiments with Cmk / mice demonstrted tht such dt cn indeed identify unexpected biologicl trgets, which cn be mechnisticlly confirmed by mouse models. Although the dt demonstrted potent role for CMK in regulting ntibody responses to vccines ginst influenz, further work is needed to delinete the cellulr mechnisms involved. Third, whether signtures tht cn be used to predict immunogenicity cn lso be used to predict efficcy must be considered. Severl studies hve shown tht serum HAI ntibody concentrtions correlte with protection ginst influenz 8 5. Seroconversion fter vccintion, commonly defined s n increse of fourfold in HAI titers 3, represents useful surrogte for vccine efficcy when pplied to popultion. However, this prmeter my not provide the optiml prediction of protection in n individul vccinee or group of vccinees. In ddition, protective concentrtions of ntibody my vry ccording to the prevlent virus subtype nd lbortory doing the ssy 5. Therefore, we used more stringent prmeter (n increse of eightfold or more in HAI response) to clssify subjects with very high ntibody responses. Using this cutoff in our nlyses, the DAMIP method ws ble to identify gene signtures tht we could use to predict the ntibody response induced by vccintion with TIV. We confirmed the vlidity of these gene signtures in three independent trils, which demonstrted the robustness of our pproch. To meet the definition of seroconversion in the US Food nd Drug Administrtion Guidnce for Industry document for this field (n HAI titer of : or more nd minimum increse of fourfold in ntibody titer fter vccintion), we re-rn the DAMIP nlysis using n increse of fourfold s cutoff for defining high HAI responders. Agin, the DAMIP method ws ble to identify sets of three to four discrimintory genes with n unbised estimte of correct clssifiction up to 9% for the three influenz trils. However, the generlity of our findings in terms of using gene signtures in PBMCs to predict the immunogenicity nd/or efficcy of other vccines such s mucosl vccines must be tested. It is likely tht different signtures could be generted by nlysis of mucosl tissues. Finlly, lthough the min gol of our study ws proof-of-concept demonstrtion of the fesibility of this pproch in predicting vccine immunogenicity, (rther thn demonstrtion of cost effectiveness), in scertining the predictive vlue of our signture in the 9 tril, we used PCR-bsed ssy (insted of n ssy with gene-expression chips) of only hndful of genes. This demonstrted the fesibility of designing cost-effective, PCR-bsed vccine chip tht cn be used to predict the immunogenicity of vccines. Thus, we hve shown how systems biology pproches cn be pplied to elucidte the moleculr mechnisms of influenz vccines. We envision tht the predictive signtures of influenz vccine induced ntibody responses my hve implictions in vccine development, in the monitoring of suboptiml immune responses (in the elderly, infnts or immunocompromised popultions) or perhps in identifying new correltes of protection. Methods Methods nd ny ssocited references re vilble in the online version of the pper t Accession codes. GEO: microrry dt, GSE969. Note: Supplementry informtion is vilble on the Nture Immunology website. Acknowledgments We thnk B.T. Rouse nd R. Compns for discussion nd comments on the mnuscript, nd H. Oluoch for technicl ssistnce. Supported the US Ntionl Institutes of Helth (U9AI93, HHSN6676C, U5AI5757, R37AI8638, RDK57665, U9AI5766 nd N AI55 for the B.P. lbortory; AI38 nd AI5766 for the R.A. lbortory; DK77 for the A.R.M. lbortory; Intrmurl Reserch Progrm of the Ntionl Institute of Allergy nd Infectious Diseses for the K.S. lbortory; nd UL RR58 from the Clinicl nd Trnsltionl Science Awrd progrm, Ntionl Center for Reserch Resources for clinicl work), the Bill & Melind Gtes Foundtion (Collbortion for AIDS Vccine Discovery 3865 to the R.A. nd B.P. lbortories), the Ntionl Science Foundtion (E.K.L. lbortory) nd the Centers for Disese Control (E.K.L. lbortory). AUTHOR CONTRIBUTIONS H.I.N. did ll the experiments nd nlyses in Figures 6 nd Supplementry Figures 8; J.W., G.-M.L., M.M. nd V.K. did the nlyses in Figure nd Supplementry Figure ; E.K.L. did the DAMIP model nlyses in Figure 5; L.R., A.R.M., S.P.K. nd N.K. did the mouse experiments in Figure 6; W.N.H. helped with the microrry nlyses in Supplementry Figure ; S.L. ssisted with the bioinformtics nlyses of the dt in Figure 3; A.A. did the microrry nlysis of smples from the 7 influenz nnul seson; S.M.-K., K.E.K., R.E. nd A.K.M. ssisted with the collection nd processing of smples; K.S. mesured HAI titers; R.A. helped conceive of nd design the study nd supervised the studies in Figure nd Supplementry Figure ; B.P. conceived of the study nd designed nd supervised the experiments nd nlyses in Figures 6 nd Supplementry Figures 8; nd B.P. nd H.I.N. wrote the pper. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Published online t Reprints nd permissions informtion is vilble online t reprints/index.html.. Sski, S. et l. Comprison of the influenz virus-specific effector nd memory B-cell responses to immuniztion of children nd dults with live ttenuted or inctivted influenz virus vccines. J. Virol. 8, 5 8 (7).. Fiore, A.E. et l. Prevention nd control of influenz with vccines: recommendtions of the Advisory Committee on Immuniztion Prctices (ACIP),. MMWR Recomm. Rep. 59, 6 (). 3. Sski, S. et l. Influence of prior influenz vccintion on ntibody nd B-cell responses. PLoS ONE 3, e975 (8).. Zemn, A.M. et l. Humorl nd cellulr immune responses in children given nnul immuniztion with trivlent inctivted influenz vccine. Peditr. Infect. Dis. J. 6, 7 5 (7). 5. Pulendrn, B., Li, S. & Nky, H.I. Systems vccinology. Immunity 33, (). 6. Querec, T.D. et l. Systems biology pproch predicts immunogenicity of the yellow fever vccine in humns. Nt. Immunol., 6 5 (9). 7. Gucher, D. et l. Yellow fever vccine induces integrted multilinege nd polyfunctionl immune responses. J. Exp. Med. 5, (8). 8. Pulendrn, B. Lerning immunology from the yellow fever vccine: innte immunity to systems vccinology. Nt. Rev. Immunol. 9, 7 77 (9). 9. Month, T.P. Yellow fever vccine. Expert Rev. Vccines, (5).. Querec, T. et l. Yellow fever vccine YF-7D ctivtes multiple dendritic cell subsets vi TLR, 7, 8, nd 9 to stimulte polyvlent immunity. J. Exp. Med. 3, 3 (6).. Brrett, A.D. & Teuwen, D.E. Yellow fever vccine how does it work nd why do rre cses of serious dverse events tke plce? Curr. Opin. Immunol., (9).. Johnson, P.R. Jr. Feldmn, S., Thompson, J.M., Mhoney, J.D. & Wright, P.F. Comprison of long-term systemic nd secretory ntibody responses in children given live, ttenuted, or inctivted influenz A vccine. J. Med. Virol. 7, (985). 3. Beyer, W.E., Plche, A.M., de Jong, J.C. & Osterhus, A.D. Cold-dpted live influenz vccine versus inctivted vccine: systemic vccine rections, locl nd systemic ntibody response, nd vccine efficcy. A met-nlysis. Vccine, (). 79 VOLUME NUMBER 8 AUGUST nture immunology