A Universal Influenza A Vaccine Based on Adenovirus Expressing Matrix-2 Ectodomain and Nucleoprotein Protects Mice From Lethal Challenge

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1 originl rticle The Americn Society of Gene & Cell Therpy A Universl Influenz A Vccine Bsed on Adenovirus Expressing Mtrix- Ectodomin nd Nucleoprotein Protects Mice From Lethl Chllenge Dongming Zhou 1, Te-Lng Wu 1,, Mrcio O Lsro 1, Brin P Ltimer 1, Elizeth M Przych 1, Ang Bin 1, Yn Li 1, Hu Li 1, Jn Erikson 1, Zhiqun Xing 1 nd Hildegund CJ Ertl 1 1 The Wistr Institute, Phildelphi, Pennsylvni, USA; University of Pennsylvni School of Medicine, Phildelphi, Pennsylvni, USA A universl influenz vccine, designed to induce rodly cross-rective immunity ginst current nd future influenz A virus strins, is in criticl demnd to reduce the need for nnul vccintions with vccines chosen upon predicting the predominnt circulting virl strins, nd to meliorte the thret of cycliclly occurring pndemics tht hve, in the pst, killed tens of millions. Here, we descrie vccine regimen sed on sequentil immuniztion with two serologiclly distinct chimpnzee-derived repliction-defective denovirus (Ad) vectors expressing the mtrix- protein ectodomin (Me) from three divergent strins of influenz A virus fused to the influenz virus nucleoprotein (NP) for induction of ntiodies to Me nd virus-specific CD + T cells to NP. In preclinicl mouse models, the Ad vccines expressing Me nd NP elicit roust NP-specific CD + T-cell responses nd moderte ntiody responses to ll three Me sequences. Most importntly, vccinted mice re protected ginst moridity nd mortlity following chllenge with high doses of different influenz virus strins. Protection requires oth ntiodies to Me nd cellulr immune responses to NP. Received April 1; ccepted 1 August 1; pulished online Septemer 1. doi:1.13/mt.1. INTRODUCTION Ech yer sesonl influenz infections cuse severe illness in 3 5 million people worldwide nd kill 5, 5, humns. 1 The currently licensed influenz (flu) vccines re nnul vccines tht induce sutype-specific virus neutrlizing ntiodies, which do not protect ginst new sutypes or ntigenic vrints. Due to the unpredictility of evolving influenz viruses, the dvnced design of vccines ginst new strins using current strtegies is unfesile. A universl flu vccine to induce rodly cross-rective immunity ginst current nd future influenz viruses is thus in criticl demnd. Such vccine would lso meliorte the need for nnul vccintion nd would e expected to increse overll vccine coverge. Influenz A viruses re negtive-sense, single-strnded, segmented RNA viruses, which contin eight RNA segments, encoding for 11 proteins (HA, NA, NP, M1, M, NS1, NEP/NS, PA, PB1, PB1-F, PB). Mtrix protein (M) is tetrmeric trnsmemrne protein of influenz A virus. Its ectodomin (Me) shows conservtion mong humn influenz A virus strins. Me-specific ntiodies, lthough not neutrlizing, reduce in nimls the severity of infection with wide rnge of influenz A virus strins. 3, Influenz A nucleoprotein (NP), the mjor protein component of rionucleoprotein (RNP) complexes, is lso reltively conserved mking it n ttrctive cndidte for universl flu vccine. Although the NP protein induces n ntiody response, the role of such ntiodies in providing protection remins controversil. 5, The NP induces vigorous CD + T-cell response oth in mice nd men 7, tht, s epidemiologicl studies suggest, my contriute to resistnce ginst severe disese following influenz A virus infection. 9 Influenz vccines sed on Me, NP, or oth hve een tested extensively in niml models, where they hve shown sufficient promise tht some of them dvnced to clinicl trils ( 3,,1 13 However, results from efficcy trils, which hve commonly only shown limited efficcy even for licensed vccines,,15 re not yet ville. Here, we introduce different vccine pltform tht is uniquely suited to induce potent nd sustined immune responses. Specificlly, we generted E1-deleted denovirus (Ad) vectors from chimpnzee serotypes C (AdC) or C (AdC, ref. 1) expressing, in tndem, three Me sequences from diverse strins of influenz A virus (H1N1, H5N1, nd H7N) fused to H1N1 NP. Ad vccines expressing Me nd NP elicit roust NP-specific CD + T-cell responses nd moderte ntiody responses to the three Me sequences in mice. Most importntly, vccinted young mice re protected ginst mortlity following chllenge with high doses of different influenz viruses. This protection is dependent upon oth ntiodies to Me nd cellulr immune responses to NP. Old mice develop roust immune response upon vccintion ut fil to e protected. RESULTS Trnsgene product expression The Me(3)-NP chimeric gene encodes the Me of A/PR/, n H1N1 virus, pthogenic H5N1 virus tht evolved in 1997, nd Correspondence: Hildegund CJ Ertl, The Wistr Institute, Phildelphi, Pennsylvni 19, USA. E-mil: ertl@wistr.upenn.edu 1 vol. 1 no. 1, 1 19 dec. 1

2 The Americn Society of Gene & Cell Therpy A Novel Influenz A Vccine Bsed on Me nd NP SLLTEVETPIRNEWGCRCNGSSD SLLTEVETLTRNGWGCRCSDSSD SLLTEVETPTRNGWECKCSDSSD H1N1-Me (A/Puerto Rico//193) C57BL/ H5N1-Me (A/Hong Kong/3/1997) H7N-Me (A/Duck/Tsmni/77/7) 1 c SLLTEVETPIKNEWECRCNDSSD P CMV AdCr.gp H1N1 H5N1 H7N SS Me Me Me AdCMe(3)-NP H1N1-Me (A/Fort Monmouth/1/197) H1N1 NP AdCMe(3)-NP 1 9 vp 1 9 vp 1 vp 1 7 vp 1 9 vp 1 vp 1 7 vp Poly(A) Antiody titers ( g/ml) 1 1 H1N1-M H5N1-M H7N-M Anti-Me ntiody -Actin Prime Prime-oost Figure 1 Me(3)-NP fusion protein. () Amino cid sequences of three Me in the construct nd of A/Fort Monmouth/1/7 virus. () Schemtic representtion of the chimeric Me(3)-NP gene. (c) Expression of Me(3)-NP protein y different vectors in infected cells in comprison to -ctin. Sme sustrin. CMV, cytomeglovirus; Me, mtrix- protein ectodomin; NP, nucleoprotein. n vin H7N strin isolted in 7 (Figure 1). The three Me sequences were comined with the full-length NP sequence. Linker sequences, encoding three lnine residues, were inserted etween ech gene nd signl sequence from HSV-1 glycoprotein D ws plced upstrem of the chimeric gene (Figure 1). Western lotting ws conducted with monoclonl ntiody to Me termed C-S Results show tht AdCMe(3)-NP nd AdCMe(3)-NP express comprle levels of the chimeric protein in vitro (Figure 1c). Similr results were otined upon lotting with n ntiody to NP (dt not shown). Antiody responses to Me Groups of young C57Bl/ mice were vccinted with virus prticles (vp) of AdCMe(3)-NP; some of them were oosted months lter with vp of AdCMe(3)-NP. Ser were hrvested from individul mice 5 weeks fter the oost, nd together with nive control ser or, in seprte experiments, ser from mice vccinted with vectors expressing the ries virus glycoprotein (r.gp), tested for ntiodies to Me on the different M-trnsfected or shm-trnsfected HeL cell lines (Figure ). Antiody titers were comprle upon testing on the three cell lines nd incresed fter the oost. Ser from mice vccinted with the control vectors showed ckground rectivity similr to tht of ser from nive mice (e.g., verge ntiody titers to Me in nive ICR mice, 1. μg/ml; verge ntiody titers in ICR mice fter n AdCr.gp prime oost regimen:.99 μg/ml). To ensure tht the vccine induced response in geneticlly distinct strins of mice, outred ICR mice were tested using the sme vccine regimens. Antiody titers chieved fter priming were lower thn those in C57Bl/ mice, ut comprle fter the oost (Figure ). NP-specific CD + T-cell responses Vccine-induced CD + T-cell responses to NP were tested t different time points fter vccintion y intrcellulr cytokine stining for interferon-γ (Figure 3). After priming with AdCMe(3)-NP, ll of the mice developed detectle frequencies of NP-specific CD + T cells in lood, which grdully declined. A ooster immuniztion with AdCMe(3)-NP Antiody titers ( g/ml) given months fter priming ffected n increse in circulting NP-specific CD + T cells. Mice were killed months fter priming nd frequencies of NP-specific CD + T cells were determined from lymphocytes isolted from lood, spleens, nd lungs of individul mice (Figure 3). Frequencies were higher in mice tht hd received the prime-oost regimens; frequencies were highest in lungs nd lowest in spleens. Higher frequencies in peripherl tissues tht primry ttrct effector/effector memory cells thn lymphtic tissues such s spleen is typicl for Ad vector induced T-cell responses s hs een descried previously. 1 Protective immunity C57Bl/ mice were vccinted nd then infected with 1 men lethl dose (LD 5 ) of A/PR/ virus. Lung virus titers were mesured from the right inferior loe of the lungs of individul mice 5 dys fter chllenge (Figure ). This time point ws chosen for the following resons. Influenz virus repliction cn e detected rpidly within hours in lungs of mice. Depending on the dose of chllenge virus, mice tht re le to fend off the infection then show decline in titers round dy Vccines tht induce neutrlizing ntiodies would e expected to decrese virus titers from the onset while vccine such s ours tht induces immune mechnisms directed ginst infected cells would e expected to ct with dely. Mice tht hd een primed with AdCMe(3)-NP ICR H1N1-M H5N1-M H7N-M Prime Prime-oost Figure Humorl responses in C57Bl/ mice or ICR mice. () Three cell lines expressing M of H1N1, H5N1, or H7N were used in cellulr enzyme-linked immunosorent ssys to mesure Me-specific ntiody titers in ser of C57Bl/ mice (n = 1) hrvested 3 months fter priming or 5 weeks fter oosting. () Me-specific ntiody titers in ser of ICR mice (n = 1) hrvested 3 months fter priming or 5 weeks fter oosting. P <.5; P <.1. Moleculr Therpy vol. 1 no. 1 dec. 1 13

3 A Novel Influenz A Vccine Bsed on Me nd NP The Americn Society of Gene & Cell Therpy % IFN CD /CD cells % tet CD /CD cells Boosting Time (weeks) Prime showed reduction in men titers this did not rech significnce compred to control mice (P =.). A significnt reduction in lung virl titers ws chieved upon prime-oosting (P =.3). Lungs of individul mice from given group showed rther surprisingly lrge rnge of virus titers; PCR rections rther neutrliztion ssys were used to mesure virl titers. Although PCR-sed ssys hve the dvntge of higher sensitivity, they not only mesure infectious virus ut lso defective genome- contining prticles. Another fctor tht my hve contriuted to the high vriility in virus titers ws the use of single time point tht my hve missed pek virus titers in some of the mice. To ensure tht the reduction in virl titers resulted in clinicl enefit, the experiment ws repeted with the prime-oost regimen nd mice were chllenged with 1LD 5 of A/PR/ or A/ Fort Monmouth virus. Vccinted, nïve, or shm-vccinted mice lost weight fter chllenge. Weight loss of vccinted mice peked y dys fter chllenge nd then mice egn to gin weight nd y 1 dys fter chllenge most mice hd returned to their prechllenge weight. or shm-vccinted control mice continued to lose weight fter chllenge till they died or required euthnsi (Figure,c). Upon chllenge with either virus strin, 9% of the vccinted mice survived while ll of the control mice died (Figure d,e). Lung loes hrvested 5 dys fter chllenge were stined with hemtoxylin nd eosin nd nlyzed for signs of inflmmtion using the scoring system descried in the Mterils nd Methods section. Most of the unvccinted Prime-oost Lung Blood Spleen Figure 3 NP-specific CD + T-cell responses in C57Bl/ mice () Intrcellulr IFN- stining of NP-specific CD + T cells ws crried out on peripherl lood mononucler cells from mice (n = 1) t weeks, 5,, 1, nd 1, fter vccintion. Prime-oosted mice (filled circles), primed mice (empty squres). () Frequencies of NP-specific CD + T cells in lung, lood, nd spleen tested months fter priming or months fter oosting y tetrmer stining. P <.5; P <.1. IFN, interferon; NP, nucleoprotein. mice hd perivsculr infiltrtes in their lungs nd hlf of them hd interstitil infiltrtes with n verge pthology score of.35 (Figure f). Pthology ws less pronounced in mice tht hd received the prime-oost vccintion, nd the verge pthology score of their lungs ws 1.5 (P =.). Mice were in infected t months fter priming. Those tht received second dose of vccine were oosted t months fter priming nd then chllenged months lter. This protocol llowed us to prime nd chllenge mice together thus reducing experimentl vriility. One could mke the rgument tht differences in the time intervl etween vccintion nd chllenge my hve ised the results. We, therefore, in dditionl experiments, tested mice tht only received one dose of the AdCM(e)3-NP vccine t months fter vccintion. Protection ws comprle to tht oserved in mice chllenged t months fter immuniztion. The experiment ws repeted with ICR nd BALB/c mice. Mice were chllenged months fter priming or months fter the oost with 1LD 5 of A/PR/ virus. By dy 5 fter chllenge, lung virus titers were significntly reduced in ICR mice tht were primed (P =.) or primed nd oosted (P =.3; Figure 5). In 3% of primed mice nd 5% of mice tht received the prime-oost regimen, virus hd een clered completely from their lungs while ll of the mice of the two control groups hd titers in excess of 1 5 genome copies. Similr results were otined with BALB/c mice (Figure 5). Next, vccinted ICR mice were chllenged with n incresed dose of 15LD 5 of A/PR/ virus (Figure 5c,d). In spite of this very severe chllenge, which killed 9% of the control mice, 7% of the vccinted mice survived. Histologicl nlyses (Figure 5e) of lung section of ICR mice conducted 5 dys fter chllenge reveled significnt reduction in inflmmtion in vccinted mice with n verge pthology score of.7 in nive mice nd 1.9 in vccinted mice (P =.9). Influenz viruses cuse deth minly in the ged nd commercilly ville vccines re commonly poorly immunogenic in this popultion. To test whether the AdM(e)3-NP vectors induce protection in ged mice, we immunized group of -month-old C57Bl/ mice with AdCM(e)3-NP nd oosted them months lter with AdCM(e)3-NP. Mice were chllenged 3 months fter the oost with 3LD 5 of A/Fort Monmouth virus nd virl titers were determined from lungs 5 dys lter. Groups of young mice were tested in prllel. Antiody responses to Me were comprle in old nd young vccinted mice lthough old nive mice hd slightly higher ckground titers (verge ntiody titers to Me in old mice: 17 μg/ml; verge ckground titers in old mice: μg/ml). Frequencies of NP-specific CD + T cells were higher in ged mice t the time of chllenge (% NP-specific CD + T cells/ll CD + T cells: old mice: 1.%; young mice: 5.9%, P =.3). By 5 dys fter chllenge, lung virus titers in young vccinted mice were significntly elow those of young nive mice, while such difference ws not seen in ged mice, lthough they hd on verge lower titers compred to the young (Figure ). In ddition, young vccinted mice showed reduced weight loss following chllenge compred to young nive mice, which gin ws not seen for the ged mice indicting tht the vccines, lthough they induced immune responses in ged mice, nevertheless, lcked efficcy in this popultion. 1 vol. 1 no. 1 dec. 1

4 The Americn Society of Gene & Cell Therpy A Novel Influenz A Vccine Bsed on Me nd NP 1.E 9 1.E f Prime-oost 1.E 7 1.E 1.E 5 1.E 1.E 3 Prime Prime-oost 11 c 11 % of prechllenge weight % of prechllenge weight d % Survivl 1 1 Vccinted e % Survivl 1 1 Vccinted Figure Protection ginst chllenge. () C57Bl/ mice were chllenged with 1LD 5 of A/PR/ virus months fter priming or months fter oosting. Five dys lter lung virus titers were determined. Grph shows titers of virl genomes (vgs) per grm of tissue of individul mice (open circles) or s geometric men titers ( ). Prime-oost mice (n = 15) versus nive mice (n = 1), P =.3, primed mice (n = 1) versus nive mice (n = 1), P =.. (,c) Weight loss fter chllenge with () 1LD 5 A/PR/ or (c) A/Fort Monmouth virus. Vccinted mice (filled circles), nive mice (open circles). (d,e) Survivl curve of the mice fter chllenge with (d) A/PR/ or (e) A/Fort Monmouth virus. In oth experiments, vccinted (n = 9) versus nive mice (n = 9), P =.. (f) Stined lung sections from mouse tht received the prime-oost nd from nive mouse oth hrvested 5 dys fter chllenge with 1LD 5 A/PR/ virus. Hemtoxylin nd eosin stined sections re shown t 1 mgnifiction. Immune correltes of protection To elucidte the immune mechnisms tht contriute to protection in vccinted mice, the vccines were tested in β-microgloin knockout mice, which lck CD + T cells. Mice received primeoost regimen or were left nive. Antiody titers mesured t 5 weeks fter the oost were comprle to those chieved in wildtype C57Bl/ mice (men titer of 1.7 μg of ntiodies to Me/ml). Upon chllenge of mice with 1LD 5 A/PR/, only 33.3% (/) of vccinted mice survived, while ll of the nive mice succumed the infection (Figure 7). This difference ws not significnt suggesting tht ntiodies lone filed to provide protection ginst severe chllenge. In second experiment, the role of ntiodies ws ssessed y doptive trnsfer studies. Donor C57Bl/ mice received the primeoost regimen with the AdMe(3)-NP vectors nd 1 ml of their pooled ser, which t the time of hrvest contined.3 μg of Mespecific ntiodies/ml, ws trnsferred to nive C57Bl/ recipients, which were chllenged hours lter with 1LD 5 of A/PR/. Control mice received ser from nive donors. Trnsfer of Me immune ser protected 5% of the recipients, while ll of the mice injected with the control ser died following chllenge (Figure 7). To further ssess correltes of protection, mice were vccinted with n AdC vector expressing NP only (AdCNP) or they were primed with AdCNP nd then oosted months lter with AdCNP. Frequencies of NP-specific CD + T cells were mesured from lood months lter (Figure 7c) nd were found to e comprle to those chieved with the AdCMe(3)-NP vccine or prime-oost regimen with the two heterologous Ad vectors. It should lso e noted tht AdNP-vccinted mice developed ntiodies to NP. Two months fter vccintion, one group of AdCNP-vccinted mice received 1. ml of ser otined from C57Bl/ mice tht hd received the prime-oost regimen with the AdMe(3)-NP vectors, the other group received 1. ml of ser from nive mice. Mice were chllenged hours lter with 1LD 5 of A/PR/ virus. All of the control nimls died, while 33.3% of AdCNP-vccinted mice trnsferred with nive serum survived (Figure 7d). This degree of survivl did not rech sttisticl significnce. Survivl ws significnt t 55.% in AdCNP-vccinted mice tht received the immune serum gin suggesting tht vccine-induced protection ginst high-dose chllenge requires oth ntiodies to Me nd CD + T cells to NP. Enhncing NP-specific CD + T cells y ooster immuniztion did not result in incresed Moleculr Therpy vol. 1 no. 1 dec. 1 15

5 A Novel Influenz A Vccine Bsed on Me nd NP The Americn Society of Gene & Cell Therpy 1.E 1.E 7 IOR 1.E 7 1.E BALB/c 1.E 1.E 5 1.E 1.E 3 1.E 1.E 1 1.E c % of prechllenge weight Prime Prime-oost AdC-r.gp d % Survivl 1.E 5 1.E 1.E 3 1.E 1.E 1 1.E 1 1 Prime-oost Vccinted AdCr.gp control e Prime-oost Figure 5 Protection of ICR nd BALB/c mice. () ICR mice were chllenged with 1LD 5 A/PR/ months fter priming or months fter the oost. Five dys fter chllenge, lung virus titers were determined. Grph shows titers of vgs per grm of tissue of individul mice (open circles) or s geometric men titers ( ). () Virus titers in BALB/c mice. (c) Weight loss nd (d) the survivl curves of ICR mice fter chllenge with 15LD 5 of A/PR/ virus. Vccinted mice (filled circles); control mice (open circles). Survivl of vccinted (n = 1) versus AdCr.gp immune mice (n = 1), P =.1. (e) Hemtoxylin nd eosin stined sections of lungs hrvested 5 dys fter chllenge with 1LD 5 of A/PR/ virus ( 1 mgnifiction). protection, ut mice rther showed trend towrd ccelerted deth potentilly suggesting tht potent NP-specific CD + T-cell response my excerte disese. In ddition, these results show tht comintion of T cells nd ntiodies to NP does not suffice for protection ut tht ntiodies to Me re essentil. DISCUSSION Novel nd more efficcious flu vccines tht induce rod nd sustined protection ginst wide rnge of influenz A viruses re necessry to reduce the need for nnul vccintion cmpigns with vccines sed upon virl strins predicted to e the predominnt circulting strins nd to meliorte the thret of future pndemics tht cn potentilly kill millions. Vrious strtegies hve een tested to develop universl flu vccine. Most of these strtegies focused on vccines expressing the M1, M, or NP of influenz virus ( gov/ct/show/nct9933), 3,,1 13,1 which re reltively conserved etween different virl strins. Both the M1 nd the NP re dominnt trgets of CD + T cells in humns, while the Me inds non-neutrlizing ntiodies tht provide some, leit limited, protection. Although epidemiologicl evidence supports the 1.E 7 1.E 1.E 5 1.E 1.E 3 1.E 1.E 1 Old vccinted Young vccinted Old control Young control Figure Protection of ged mice. Old nd young C7BL/ mice were primed with AdCMe(3)-NP nd months lter oosted with AdCMe(3)-NP. Three months lter vccinted nd ge-mtched nive control mice were chllenged with 3LD 5 A/Fort Monmouth virus. Five dys fter chllenge, virus titers in the lungs were determined. Old vccinted mice (n = ) versus old control mice (n = 1), P =.3. Young vccinted mice (n = 15) versus young control mice (n = 1), P = vol. 1 no. 1 dec. 1

6 The Americn Society of Gene & Cell Therpy A Novel Influenz A Vccine Bsed on Me nd NP 1 1 Vccinted 1 1 Immune ser ser % Survivl % Survivl c% tet CD /CD cells 1 1 d % Survivl 1 1 AdCNP Immune ser AdCNP AdCNP/AdCNP AdCNP AdCNP/AdCNP Figure 7 Correltes of protection. () Protection of -microgloin knockout mice. The grph shows the survivl curves of nive nd vccinted (prime/oost regimen) mice upon chllenge with 1LD 5 of A/PR/ virus. Vccinted mice (n = ) versus nive mice (n = ), P =.. () Protection y doptive trnsfer of immune ser. C57Bl/ mice were given 1. ml of ser hrvested from C57Bl/ mice vccinted with AdCMe(3)-NP/ AdCMe(3)-NP vectors. mice received 1. ml of nive ser. hours lter, mice were chllenged with 1LD 5 of A/PR/ virus. The grph shows the survivl curves. Recipients of immune ser (n = 1) versus controls (n = 1), P =.. (c) Frequencies of NP-specific CD + T cells mesured 1 dy efore chllenge from the lood of the sme mice shown in d determined y tetrmer stining. (d) Protection of AdCNP-vccinted mice with or without immune ser trnsfer. The grph shows the survivl curves. Groups of AdCNP-immune C57Bl/ mice (n = 1 in ech group) were injected with 1. ml of ser from C57Bl/ mice prime-oosted with AdCMe(3)-NP/AdCMe(3)-NP. Another AdCNP-vccinted group ws given 1. ml of nive ser/mouse. The control group received 1. ml of nive serum/mouse. AdCNP+immune ser mice versus nive mice, P =.1. AdCNP-vccinted mice versus nive mice, P =.9. AdCNP/AdCNP-vccinted mice versus nive mice, P =.. development of CD + T-cell vccines to influenz A viruses, 9 in niml models, the effectiveness of CD + T cells in protecting ginst influenz A viruses remins controversil; while some investigtors reported induction of protection, 1 others reported lck of efficcy or even excertion of disese following virl chllenge. 3 In our hnds, vccines tht induce very high frequencies of NP-specific CD + T cells such s Ad vectors expressing the NP provided only mrginl protection. Me is poorly immunogenic during nturl influenz A virus infection due to the pucity of its expression on virions. Humns develop Me-specific ntiodies following infection ut titers re low nd not sustined. 5 Severl studies conducted in mice nd ferrets hve shown tht Me-specific ntiodies cn restrict susequent virus repliction nd reduce moridity nd mortlity to rod rnge of influenz A virus strins. 3, Although Me is reltively conserved, vriility etween different strins hs een descried nd vccines tht express only one version of Me my thus lck efficcy ginst strins with Me muttions. By the sme token it ws shown tht ntiodies to Me select for escpe mutnts. 7 A numer of vccine pltforms hve een tested ( 3,,1 13 nd severl of those such s the Me-heptitis B core protein vccine susequently underwent erly clinicl testing, which confirmed their sfety nd immunogenicity. 3 We decided to explore universl flu virus vccine sed on Ad vectors for the following resons. Production, purifiction, nd qulity control procedures for Ad vectors re well estlished. 1 Ad vectors induce innte immune responses meliorting the need for ddition of djuvnts. They lso induce very potent B nd CD + T-cell responses, which, due to low-level persistence of the vectors, re remrkly sustined. Pre-existing neutrlizing ntiodies to common humn serotypes of Ad viruses such s serotype 5, which impct vccine efficcy, 1 cn redily e voided y the use of serotypes from other species such s chimpnzees, which typiclly neither circulte in humns nor cross-rect with humn serotypes. 9 In cses where prime-oost regimens re needed to chieve immune responses of sufficient potency, vectors sed on distinct Ad serotypes re ville. 1 Ad viruses nd Ad vectors hve een used extensively in the clinic where they were well tolerted. They cn e pplied through vriety of routes including mucosl routes such s the irwys 3 or even orlly upon encpsidtion s ws shown with vccine to Ad viruses nd 7 used y the US militry. 31 In order to develop vccines tht induce rodly crossrective immunity, we expressed fusion protein composed of Moleculr Therpy vol. 1 no. 1 dec. 1 17

7 A Novel Influenz A Vccine Bsed on Me nd NP The Americn Society of Gene & Cell Therpy three divergent versions of Me for induction of ntiodies nd the NP for stimultion of CD + T cells in two serologiclly distinct Ad vectors derived from chimpnzee isoltes. The vccines induce modest titers of ntiodies to Me in ll mouse strins tested. Antiody titers were very similr to ll three versions of Me including tht derived from n H7N virus, which, ut for one mino cid (serine), is identicl to tht of the currently pndemic H1N1 virus. Whether this reflects tht ll three sequences re eqully immunogenic, or whether most ntiodies cross-rect, remins to e tested. The vccines lso induced potent CD + T-cell response tht could e detected in lood s well s in lungs. Although the vccines pprently filed to induce sterilizing immunity ginst high-dose virl chllenge, they resulted in lowering of virl lods y dy 5 following infection, reduction of infectionssocited lung pthology, nd they prevented influenz A virus ssocited mortlity. Protection ws chieved ginst A/PR//3 virus from which the NP s well s one of the Me sequences originted s well s ginst A/Fort Monmouth/1/7, which crries n Me sequence tht is divergent from those expressed y the vccines. Solid vccine-induced protection ginst chllenge required oth ntiodies nd CD + T cells. Either of these two immune mechnisms could provide some protection s ws shows y pssive trnsfer of vccine-induced immune ser, y the use of mice lcking CD + T cells, or y the AdNP vccines, ut the level of protection y only one of the immune mechnisms ws well elow tht chieved with oth. Not surprisingly our studies show tht preventtive vccine tht ims to induce protective immunity to rpidly mutting pthogen such s influenz virus my require comprehensive pproch in which multiple ntigens re provided for stimultion of different rms of the immune system rther thn the reductionist pproch of single epitope vccines. MATERIALS AND METHODS Ad vectors. AdC nd AdC vectors expressing the Me(3)-NP chimeric protein were generted s follows: the 3 Me-encoding sequences with signl peptide ws synthesized y Integrted DNA Technologies (Corville, IA) nd cloned into pshuttle (Clonetech, Mountin View, CA). The NP gene, upon deletion of the strt codon, ws cloned in frme downstrem of the Me sequences. Upon digestion with I-CeuI nd PI-SceI, the fusion gene ws cloned from pshuttle into the E1 domin of the moleculr clones of AdC nd AdC, respectively. Recominnt Ad vectors (AdCMe(3)-NP nd AdCMe(3)-NP) were rescued y trnsfection of plsmid DNA into HEK 93 cells. The Ad vectors were purified y cesium chloride density-grdient centrifugtion nd virus prticle content ws determined y spectrophotometry t nm. Vectors were titrted to determine infectious units nd vector tches hd vp to infectious unit rtios elow nd were clered for endotoxin contmintion. Other vectors encoding NP only or the glycoprotein of ries virus (r.gp) were generted nd qulity controlled using the sme methods. Expression of the vccine ntigen. HEK 93 cells were infected with 1 1, vp/cell. At hours fter infection, western lots were performed nd memrnes were lotted with monoclonl ntiody to Me (C- S1-.). Influenz virus. Influenz viruses A/PR//3 nd A/Fort Monmouth/1/7 were grown in the choriollntoic fluid of emryonted chicken eggs nd titrted in dult mice upon their intrnsl infection to determine the LD 5. Mice. Femle C57Bl/, BALB/c, nd ICR mice were purchsed t weeks of ge from ACE Animls (Boyertown, PA). Femle C57Bl/J mice (βm /, strin B.19P-Bm tm1 Unc ) were purchsed t weeks of ge from Jckson Lortory (Br Hror, ME). All mice were housed in the Wistr Institute Animl Fcility. C57Bl/ mice were ged t the Animl Fcility of the Wistr Institute nd used once they were > months old. Animl procedures in this study were conducted in ccordnce with Institutionl Animl Cre nd Use Committee guidelines. Immuniztion of mice. Groups of 15 mice were vccinted with vp of the AdCMe(3)-NP vector given intrmusculrly. Two months lter, some groups of mice were oosted with vp of the AdCMe(3)-NP vector given intrmusculrly. Chllenge of mice. Two months fter vccintion, mice were nesthetized nd then chllenged intrnslly with either 1 or 15 LD 5 of influenz A/ PR//3 virus or 3 or 1 LD 5 of influenz A/Fort Monmouth/1/7 virus, diluted in 3 μl phosphte-uffered sline. Mice were weighed dily. They were killed if they lost in excess of 3% of their prechllenge weight. In some experiments, mice were euthnized 5 dys fter chllenge. Virus titrtion. The ssy ws dopted from previously pulished method 3 tht ws vlidted ginst the stndrd plque ssy. Lung tissue smples were excised from experimentl mice, nd their weight ws recorded. After tissue smples were mechniclly homogenized, RNAs were isolted y using TRIzol regent (Invitrogen, Crlsd, CA) nd were resuspended in 5 μl of diethylpyrocronte-treted wter (Amion, Austin, TX). The RNA concentrtion of ech smple ws determined spectrophotometriclly t n sornce of nm. From the entire 5 μl RNA solutions, complementry DNA (cdna)ws otined using 1 μl rection volumes with the mnufcturer-specified component proportions of High-Cpcity cdna Archive Kit (Applied Biosystems, Foster City, CA). Rections were run on therml cycler (Eppendorf Mstercycler; Hmurg, Germny) in one cycle t 5 C for 1 minutes, 37 C for 1 minutes, nd 5 C for 5 minutes. Concentrtions of cdna were stndrdized to 5 ng/5 ml, nd influenz A/PR cdna ws used to crete stndrd curve y seril dilution, rnging from ng/5 ml to. ng/5 ml. Virl cdna ws quntified using TqMn rel-time PCR ssy on n ABI Prism 7 Sequence Detector (Applied Biosystems). The primers for virl cdna quntifiction were specific to the influenz A mtrix protein gene (MP), which were MP sense (5 -AAGACC AATCCTGTCACCTCTGA-3 ) nd MP ntisense (5 -CAAAGCGTCTACGCTGCAGTCC-3 ). The reporter proe ws TqMn TAMARA (Applied Biosystems) of sequence [-FAM]-5 - TTTGTGTTCACGCTCACCGTT-3 -[TAMARA]. The cdna smples were quntified in triplicte. Ech rection totled 5 μl, nd included 1.5 μl TqMn Universl PCR Mster Mix, 5 pmol/l reporter proe, pmol/l MP sense primer, pmol/l MP ntisense primer, nd 5μl (5 ng) cdna smple templte. Rections were run t 5 C for minutes, 95 C for 1 minutes, nd then cycled times etween 95 C for 15 seconds nd C for 1 minute. In nlyzing the spectrl curves, the cycle threshold ws defined just ove the emission seline to sty within the exponentil mplifiction phse of the PCR. Virl copy numers were normlized with the originl tissue smple msses, nd clculted sed on the molr mss of influenz A/PR genome. Antiody titers to Me. Antiody responses specific to Me were mesured from ser of individul mice y cellulr enzyme-linked immunosorent ssy tht ws modified from previously pulished procedure. 17 We cloned the three full-length M sequences from which the Me sequences of the vccines hd originted, into lentivirus vectors. Lentivirus ws rescued in 93T cells nd used to infect HeL cells to generte stle cell lines tht express full-length M. A control cell line ws generted y infection of 93T cells with empty lentivirus. These cell lines were used s immunosorents in n enzyme-linked immunosorent ssy s descried vol. 1 no. 1 dec. 1