DTIC ADA583916: Persistent and High-Level Expression of Human Liver Prolidase in Vivo in Mice Using Adenovirus

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Persistent and high-level expression of human liver prolidase in vivo in mice
using adenovirus 5b. GRANT NUMBER
D0003_09_RC_C
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6. AUTHOR(S) 5d. PROJECT NUMBER
Aleti, V, Reddy, GB, Parikh, K, Arun, P, Chilukuri, N
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Published in Chemico-Biological Interactions 203 (2013) 191–195. This research was supported by the Defense Threat Reduction Agency – Joint
Science and Technology Office, Medical S&T Division (Project # 1. D0003_09_RC_C).
14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Human liver prolidase, Adenovirus, Gene delivery, Chemical warfare nerve agents, Catalytic bioscavenger
16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON
OF ABSTRACT OF PAGES Nageswararao Chilukuri
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Chemico-BiologicalInteractions203(2013)191–195
ContentslistsavailableatSciVerseScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Persistent and high-level expression of human liver prolidase in vivo in mice
using adenovirus
⇑
Vineela Aleti, Gireesh B. Reddy, Kalpana Parikh, Peethambaran Arun, Nageswararao Chilukuri
DivisionofBiochemistry,WalterReedArmyInstituteofResearch,SilverSpring,MD20910,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Humanliverprolidase,ametal-dependentdipeptidase,isbeingtestedasapotentialcatalyticbioscaveng-
Availableonline12September2012 er against organophosphorus (OP) chemical warfare nerve agents. The purpose of this study was to
determine whether persistent and high-levels of biologically active and intact recombinant human
Keywords: (rHu)prolidasecouldbeintroducedinvivoinmiceusingadenovirus(Ad).Here,wereportthatasingle
Humanliverprolidase intravenousinjectionofAdcontainingtheprolidasegenewitha6(cid:2)histidine-tag(Ad-prolidase)intro-
Adenovirus ducedhigh-levelsofrHuprolidaseinthecirculationofmicewhichpeakedondays5–7at159±129U/
Genedelivery mL.Thislevelofprolidaseis(cid:3)120timesgreaterthanthatoftheenzymelevelinmiceinjectedwith
Chemicalwarfarenerveagents
Ad-null virus. To determine if all of Ad-prolidase-produced rHu prolidase was exported into the
Catalyticbioscavenger
circulation,enzymeactivitywasmeasuredinavarietyoftissues.Livercontainedthehighestlevelsof
rHuprolidaseonday7(5647±454U/g)comparedtobloodoranyothertissue.RecombinantHuprolidase
hydrolyzedDFP,asimulantofOPnerveagents,invitro.Invivo,prolidaseoverexpressionextendedthesur-
vivalof4outof6miceby4–8hagainstexposuretotwo1(cid:2)LD dosesofDFP.Incontrast,overexpression
50
ofmousebutyrylcholinesterase(BChE),aprovenstoichiometricbioscavengerofOPcompounds,protected
5outof6micefromDFPlethalityandsurvivingmiceshowednosymptomsofDFPtoxicity.Inconclusion,
theresultssuggestthatgenedeliveryusingAdiscapableofintroducingpersistentandhighlevelsof
humanliverprolidaseinvivo.Thegene-deliveredprolidasehydrolyzedDFPinvitrobutprovidedonly
modestprotectioninvivoinmice,delayingthedeathoftheanimalsbyonly4–8h.
PublishedbyElsevierIrelandLtd.
1.Introduction evaluating the efficacy of Hu prolidase to protect animals from
OPnerveagenttoxicityarelacking.Here,wereportthatanadeno-
Apromisingapproachtomitigatingthetoxicityoforganophos- virus (Ad) containing the gene for Hu prolidase with a 6(cid:2) histi-
phorus(OP)nerveagentsistheuseofenzymesthatcanbindand dine-tag at its carboxyl terminus (Ad-prolidase) is capable of
hydrolyzehundredsandthousandsofmoleculesofnerveagentper producingpersistentandhighlevelsoftheenzymeinvivoinmice.
eachmoleculeoftheenzyme[1,2].Human(Hu)liverprolidaseis Thevirus-inducedenzymewasstructurallyintactinmouseblood.
onesuchenzymethatcanpotentiallyhydrolyzeOP nerveagents Even though, Ad-prolidase-produced rHu prolidase hydrolyzed
invivo.Invitrostudies,usingtheenzymefromAlteromonasundina DFPinvitro,overexpressionoftheenzymeofferedmeager/modest
andAlteromonoshaloplanktishaveshownthattheenzymehydro- protection in vivo by delaying the time to death of animals by
lyzes soman, sarin, and diisopropyl fluorophosphate (DFP) [3–5]. about4–8h.
Hu prolidase expressed in Saccharomyces cerevisiae has also been
shown to hydrolyze soman [6,7]. More recently, recombinant (r)
HuprolidaseexpressedinEscherichiacoliwasfoundtohydrolyze 2.Materialsandmethods
allfourG-typenerveagentsinvitro[8].Incontrast,rHuskinand
kidney prolidases expressed in Tirchoplusia ni larva only hydro- 2.1.Recombinantadenoviruses
lyzedDFPandsarininvitro[9].However,detailedinvivostudies
Ads containing the genes that encode for Hu prolidase and
mousebutyrylcholinesterase(BChE)asfusionproteinswitha6(cid:2)
⇑
Correspondingauthor.Presentaddress:USArmyMedicalResearchInstituteof histidine-tagwereproducedasdescribedbefore[10,11].Ad-MoB-
Chemical Defense, 3100 Rickets Point Road, Research Division, Physiology &
ChE was used as a positive control in the challenge experiments
Immunology Branch, USAMRICD, Aberdeen Proving Ground, Edgewood, MD
21010,USA. using DFP. Ad-null (control) virus was purchased from Welgen
E-mailaddress:[email protected](N.Chilukuri). Inc.,Worcester,MA.
0009-2797/$-seefrontmatterPublishedbyElsevierIrelandLtd.
http://dx.doi.org/10.1016/j.cbi.2012.08.021
192 V.Aletietal./Chemico-BiologicalInteractions203(2013)191–195
2.2.Prolidaseactivityassay 2.7.ChallengewithDFP
Mouseplasmaortissuesamplesweretestedforprolidaseactiv- Ad-prolidase (n=6, dose of virus=2(cid:2)1011vp/animal), Ad-
ity by the method of Isaac et al. [12]. Samples were diluted to MoBChE (n=6, dose of virus=2.5(cid:2)1011vp/animal) and Ad-con-
100ll of 50mM Tris/HCl, pH 7.4, containing 1mM MnCl and trol (n=6, dose of virus=2(cid:2)1011vp/animal) were injected into
2
were preincubated for 24h at 37(cid:2)C. Samples were then mixed the tail vein of mice and plasma was collected daily for 5days.
with 100ll of 94mM glycyl-proline (Sigma–Aldrich, St. Louis, On day 5, mice were challenged intraperitoneally with 1(cid:2) LD
50
MO) in 50mM Tris/HCl, pH 7.4, containing 2mM MnCl . After of DFP (6mg/kg body weight). Mice were observed continuously
2
30min of incubation,the enzyme reactionwas stopped with the through3hafterchallengeforsignsofcholinergictoxicity.Mori-
additionof1mlof0.45Mtrichloroaceticacid,andthesupernatant bund mice were euthanized immediately. Animals that survived
was used for proline determination as described previously [12]. thefirst1(cid:2)LD DFPwerechallengedwithasecondLD ofDFP
50 50
One unit of prolidase activity represents the amount of enzyme 3hlaterandobservedfortoxicsymptoms.
that catalyzes the hydrolysis of one micromole of substrate, per
minuteat37(cid:2)C. 3.Resultsanddiscussion
2.3.DFPhydrolysisassay 3.1.Timecourseexpressionofprolidaseinvivoinmice
l l
Mouse plasma (5 l) or 10% liver homogenates (5 l) were The ability of Ad-prolidase to induce the expression of full-
addedto3mlof50mMTris–HCl(pH7.4)containing2mMMnCl2 lengthrHuprolidasewasfirstconfirmedinvitroin293Acells(data
and 5.74mM DFP, and the increase in fluoride ion release was notshown).TotestwhetherAd-prolidasealsoinducestheenzyme
monitoredfor10minusinganOrionmeterconnectedtoafluoride expressioninvivo,SwissWebsterfemalemicewereinjectedwith
ionselectiveelectrode.Fivemicrolitersofthesamebufferinstead Ad-prolidase(2(cid:2)1011viralparticlespermouse)viathetailvein.
ofplasmawasusedasablank.ActivityisreportedasU/ml,where The expression of rHu prolidase was determined in plasma over
l
1Urepresents1 moleoffluorideionliberated/min/ml. a 15day period using glycine-proline (Gly-Pro) as the substrate
(Fig. 1). Each time point represents mean±SD of 6–8 mice. Two
2.4.SDS–polyacrylamidegelelectrophoresis(SDS–PAGE)andWestern peaks were observed following plotting of the prolidase activity
blotting (U/ml)againstthedayaftervirusinjection:aminorpeakondays
2and3andamajorpeakondays5,6,and7.Overall,prolidaselev-
SDS–PAGEwascarriedoutwithprecast10%gels.Afterelectro- elswere100-to150-foldhigher(159±129U/ml)inmiceinjected
phoresis, the proteins were transferred to PVDF membrane. The withAd-prolidasethaninmiceinjectedwithAd-nullvirus(1–2U/
membranewasblockedin4%powderedmilkfor1h,washedonce ml)ondays5–7.Theseresultsdemonstratethatasingleinjection
withTrisbufferedsalinecontainingTritonX-100(TTBS)andincu- ofAd-prolidasewascapableofintroducingpersistent,high-levels
bated overnight in primary antibody (monoclonal anti-6(cid:2) histi- of biologically active rHu prolidase into the systemic circulation
dineantibody,1:1000dilution,Cat#15149,AbCam,Cambridge, ofmice.
MA) in 0.5% milk powder containing 0.01% sodium azide. The To determine if Ad-prolidase-produced enzyme was of full-
membrane was washed with TTBS five times with intermittent length, plasma from the mouse expressing rHu prolidase at
shaking for 8min and was incubated with a secondary antibody 256U/ml on day 6 was analyzed by Western blotting with anti-
conjugatedwithhorseradishperoxidase(Cat#Ab6721,AbCam, Hisantibody.Theantibodyrecognizedasingle54kDabandinall
Cambridge,MA;100ng/ml)in0.5%milkpowderfor1h.Themem-
branewaswashedagainasdescribedaboveandtheproteinbands
were detected using ECL-Plus reagent. Chemiluminescence was
measuredinaBio-Radimagereader.
2.5.Animalstudies
ResearchwasconductedincompliancewiththeAnimalWelfare
Actandotherfederalstatutesandregulationsrelatingtoanimals
andexperimentsinvolvinganimalsandadherestoprinciplestated
in the Guide for the Care and Use of Laboratory Animals, NRC
Publication, 1996 edition. Ad-prolidase and Ad-null (2(cid:2)1011 vp/
animal)wereinjectedinto6-to7-weekoldfemaleSwissWebster
mice through the tail vein. Blood samples were collected on the
daybeforevirusinjectionanddailyfor15daysaftervirusinjection
andwereassayedforHuprolidaselevelsbyWesternblottingand
Fig.1. Expressionlevelsofhumanliverprolidaseinmousebloodfollowingasingle
enzymaticactivity[12].Insomeexperiments,threeanimalswere
tailveininjectionoftheviruscontainingthegenefortheenzyme.NAd-prolidase
sacrificedonday4,day7,day10andday15,anddifferenttissues virus,jAd-Nullvirus.
(i.e.,brain,heart,lung,diaphragm,liver,kidney,spleen,andmuscle)
werecollectedaftersalineperfusionandassayedforHuprolidase
levels.
2.6.Humanprolidaselevelsinmousetissues
Tenpercenthomogenatesofthetissuesweremadeusingtissue
extraction reagent (T-PER, Pierce Chemical Company) containing
proteaseinhibitorcocktailreagent,andwereassayedforHuproli-
daselevelsbyenzymeactivityassay. Fig.2. Westernblotofrecombinanthumanprolidaseinmouseplasma.
V.Aletietal./Chemico-BiologicalInteractions203(2013)191–195 193
thesamples(Fig.2,days1through15).Theprebleedsample(day
0) did not contain any antibody reactive protein. These results
demonstratethatAd-prolidaseexpressedenzymeisoffull-length
andintactinthecirculationofmiceforaslongas15days.
3.2.Recombinanthumanliverprolidaseexpressioninmousetissues
WedeterminedwhetherAd-prolidase-producedenzymeisre-
tainedinanyofthemousetissuesorfullyexportedintocirculation.
On day 4after virusinjection,threeAd-prolidase miceand three
Ad-control mice were euthanized for determination of prolidase
activitylevelsintissuesincludingliver,diaphragm,kidney,brain,
lung,heart,spleenandmuscle.Livercontainedthehighestamount
ofrHuprolidasecomparedtoanyothertissue.Prolidaseactivityof Fig. 4. Time course of recombinant human liver prolidase expression in mouse
2487±137U/gintheliversofmiceinjectedwithAd-prolidasewas liver.
200-to300-foldgreaterthantheactivityoftheenzymeinthelivers
ofanimalsinjectedwiththecontrolvirus(Fig.3).
Sinceliveristheprimarytissueofvirusactivityandcontained and pesticidecompounds, is mostlyexportedintothe circulation
most of the virus-produced enzyme, prolidase levels were deter- [11]. Similarly, human paraoxonase1, a promising enzyme in the
mined in the livers of mice on days 4, 7, 10, and 15 following categoryofcatalyticbioscavengers,isfullyfoundinthecirculation
Ad-prolidaseinjection(Fig.4).Recombinantprolidaselevelswere [13].Incontrast,gene-deliveredrHuprolidaselargelyremainedin
highest on day 7 (5647±454U/g tissue) compared to day 4 and theliver(Figs.3–5),andonlyasmallamount(3–5%)crossedover
day 10 and returned closer to baseline levels on day 15. On day intoplasma.Nevertheless,thisamountofrHuprolidaseis 50-to
7, the plasma rHu prolidase levels were 140±56U/ml, which is 150-fold higher than the prolidase baseline activity in mouse
40-foldlowerthanthoseintheliver.Theseresultssuggestthata blood. We considered that such high levels of rHu prolidase in
large amount of Ad-prolidase-produced enzyme was retained in mouseplasmawouldbesufficienttoprovideprotectiontotheani-
theliverandwasnotexportedintothecirculation. mal, provided that the catalytic activity/efficiency of the enzyme
was high enough for the enzyme to hydrolyze the DFP prior to
3.3.HydrolysisofDFPbyrecombinanthumanliverprolidase its escape from circulation which leads to cholinergic crisis and
RecombinantHuprolidasewastestedforitsabilitytohydrolyze eventualdeath.
DFP,asimulantcompoundofOPnerveagents,byaninvitroDFP First,wedeterminedtheLD50doseofDFPinSwiss-Websterfe-
hydrolysis assay. DFPase activity was undetected in the plasma malemice.Sixmicewerechallengedintraperitoneallywithoneof
of mice injected with Ad-control compared to a value of thethreefollowingDFPdoses:6, 9,and 12mg/kg.Threemicein
5.67±2.58U/mlintheplasmaofmiceinjectedwithAD-prolidase the 6/mg/kg group, 1 mouse in the 9mg/kg group, and 0 in the
on day 4 (Fig. 5, inset). Similarly, DFPase activity in the livers of 12mg/kg group survived the DFP challenge, suggesting that the
mice injected with Ad-prolidase was higher by 50-fold on day 4, LD50doseofDFPforthisstrainofmiceis(cid:3)6mg/kg.Thisvalueis
90-fold on day 7, 12-fold on day 10 and 10-fold on day 15 than consistentwiththeLD50valuesfoundinotherstrainsofmice[14].
intheliversofmiceinjectedwithAd-control(Fig.5).Theseresults To determine the protection offered by rHu prolidase against
suggest that Ad-prolidase-produced rHu prolidase is capable of DFP toxicity, three groups of mice (Control, Prolidase, and BChE)
hydrolyzingDFPinvitro. with6animalsineachgroupwereinjectedwithAd-null,Ad-pro-
lidase and Ad-MoBChE, respectively and 5days later challenged
3.4.RecombinantHuprolidasefailedtooffersubstantialprotection intraperitoneally with 1(cid:2) LD50 dose of DFP. Mice injected with
againstDFPinvivo Ad-BChE were included as a positive control group since BChE is
The most notable nerve agent stoichiometric bioscavenger, expectedtofullyprotectagainstallOPcompounds[1,2].Surviving
BChE, which offers protection against all types of nerve agents miceineachgroupwerechallengedwithasecond1(cid:2)LD50doseof
DFPthreehoursafterthefirstchallenge(Table1).Fourof6animals
inthecontrolgroup,and6of6animalsintheprolidaseandBChE
groupssurvivedthefirstLD doseofthetoxicant.ThesecondLD
50 50
injectionofDFP3hlaterresultedinthedeathofalltheanimalsin
thecontrolgroupwithin15minofthetoxicantinjection.Bycon-
trast,only2ofthe6animalsintheprolidasegroupand1ofthe
6animalsintheBChEgroupdied.Intheprolidasegroup,the4sur-
vivingmicedemonstratedsignsofnerveagentpoisoning,includ-
ing piloerection, salivation, and splayed hind limbs, etc. These
fourmicewerefounddeadthefollowingmorning,suggestingthat
prolidase overexpression failed to offer protection against DFP
invivo.Incontrast,the5miceintheBChEgroupshowednoDFP
toxicitysymptomsat24h.TheoneanimaldeathintheBChEgroup
mayhavebeentheresultofaninadequateamountofBChEexpres-
sioninitsbloodbecauseitdidnotreceiveasufficientamountof
Ad-BChE. Taken together, these data suggest that rHu prolidase,
at best,offered meager protection against DFP toxicityin vivo by
extendingthesurvivalof4outof6micebyabout4–8h.Thelack
ofhumanliverprolidasetoofferprotectionagainstDFPinvivoin
micemaynotbeduetoinadequateenzymeactivity.Theenzyme
Fig.3. Expressionlevelsofrecombinanthumanliverprolidaseinmousetissues. totoxicantratioonthedayoftoxicantchallengewas 1:100–200
194 V.Aletietal./Chemico-BiologicalInteractions203(2013)191–195
Fig.5. HydrolysisofDFPbyrecombinantHuliverprolidaseexpressedinmouseliver(Bargraph)andplasma(inset).
theGuidefortheCareandUseofLaboratoryAnimalsandtheAni-
Table1
malWelfareActof1966(P.L.89-544),asamended.
Group DFPchallenge Survival DFP Overnight
1st(aanimals) challenge survivalafter Conflictofintereststatement
2nd thechallenge
Ad-Control 6 4/6 4 0/4 Nonedeclared.
Ad-Prolidase 6 6/6 6 0a/6
Ad-MoBChE 6 6/6 6 5b/6
Acknowledgments
a UnlikeAd-controlanimalswhichdied15minafterthe2ndDFPchallenge,Ad-
prolidaseinjectedanimalstook4–8htooktodie.
b MiceinjectedwithMoBChEexpressingviruswerealive24hafterthe2ndDFP This research was supported by the Defense ThreatReduction
challengewithoutanysignsofDFPpoisoning. Agency–JointScienceandTechnologyOffice,MedicalS&TDivision
(Project#1.D0003_09_RC_C).WethankDrs.DouglasM.Cerasoli,
CarolynP.Chambers,andBrianM.KeyserandCPTCarlSmithfor
(4–8nmolesoftheenzymevs800nmolesofDFP).Mostlikely,the
critical evaluation of the manuscript. We also thank the staff at
catalytic efficiency of recombinant human liver prolidase is not
Washington Biotechnology Inc., Columbia, MD for their technical
highenoughtorapidlyhydrolyzeDFPandloweritsbloodconcen-
helpwiththeanimalexperiments.
tration to non-lethallevels. A similar conclusionhas been drawn
basedonitsinvitrocatalyticefficiencyfortheenzymeexpressed
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Adenovirus mediated human paraoxonase1 gene transfer to provide