659 TLR4 and TRIF play positive and negative roles in ad induced innate immunity in vivo

659 TLR4 and TRIF Play Positive and Negative Roles in Ad Induced Innate Immunity In Vivo 657 Construction of a High Performance Lentiviral cDNA Library Derived from Human Fetal Liver Ryo Kurita,'''' Tats[.]

657 Construction of a High Performance

Lentiviral cDNA Library Derived from Human Fetal

Liver

Ryo Kurita,' Tatsuo Oikawa,' Michiyo Okada,ITomoko Yokoo,'

NorioKomatsu,'YoshikuniTanioka,'Erika Sasaki,'Ken zaburo

Tani.'

I Department ofMolecular Genetics, Inst ofBioregulation,

K Ylls11/I Univ , Fukuoka, Japan; 2 Department ofHematology,

Yamanashi University School ofMedicine, Kohfu, Japan ;J

Divi-sion ofAnimal Experimentation, Central Institute ofExperimental

Animals, Kawasaki , Japan

(Background) Recent developmentof the gene

therapeuticstrate-gies is remarkable and will certainly open new era to cure various

intractable diseases Based on current gene therapeutic strategies,

regenerative medicine has also been developed Namely, some

specific gene transfer has been reported to facilitate the

differentia-tion of stem cells including embryonic stem (ES)cells to various

functional cells We have recently reportedlentiviralgene transfer

oftaillscl gene differentiate nonhuman primate commonmarmoset

ES cells to hematopoietic cells efficiently without the support of

any stromal cells (Kurita et ai,Stem Cells 2006) Translation of the

result to human ES cells,however, requires further in vitro and in

vivo experiments Also,we will possibly need to add the second

or third gene to more efficiently differentiate primate ES cells to

various hematopoieticcells and functionalgene screening targeting

all-inclusive candidate genes strongly associated with early

hema-topoiesis of primates For this purpose,in this study we constructed

high performance human fetal liver eDNAexpression library using

Icntiviralvector.(Materialsand Methods) Human fetal liver mRNA

was purchased from BDclonetechand lentiviral human fetal liver

eDNAlibrarywas constructedutilizinggateway stystem(Invitrigen)

following the manufacturer's protocol 293'1' cells, adenovirus E1A

transformedhuman renal epithelial cell line, were tranduced by the

lentiviral human fetal liver eDNA library Transduced 293'I'cells

were plated by limiting-dilution method and each clone was picked

up and examined for the integrated eDNA using standard

genomic-PCR method (Results) The library consisted of more than IOA8

individual clones and the average insert size was more than 2 kb

DNAsequence analysis for each insertedcDNA revealed that more

than 60% of them contained full-length coding region of proteins

includingcytokinereceptors,cytoplasmicproteins,proteininhibitors

and nuclear factors Transduction efficiency targeting 293'I'cells

was 100% and average size ofthc integrated cDNAs was about 1.1

kb (for the present).(0iseussion)Our previousstudiesdemonstrated

thelentivirusvector can transducer human hematopoietic stem

cells as well as nonhuman primate ES cells Also, lentivirus vector

can transduce proliferating cells as well as donnant cells, and our

Ientivirus human fetal liver eDNA expression library is considered

to be very helpful tool to accelerate the discovery of novel genes

strongly related to early hematopoiesis and possibly hepatopoiesis

Our nexttarget cells includehuman ES cells and cytokinc-depcndent

cell lines to identify novel hematopoiesis-related genes

658 The Erythrocyte Immunity Research on

Chronic Venous Insufficiency of Lower Limb

Zhang Lan,Zhang Baigen

I Vascular Department, Jiaotong University School ofMedicine,

Shanghai, China

Chronic venous insufficiency(CVI) of lower limb is among

the most prevalent of vascular discases.A large body of

sub-sequent work indicates that CVI is the result of the systemic

inflammatory processes.Otherwise,there have been rheological

abnormalities reported in CVI,mainlyan increase of erythrocyte

aggregability,which probably take part in the pathophysiology of

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the disease.It is clear that erythrocytes.as the largestquanitity blood cells in ciucluation,expressingvarious innate immunereceptor,play

an important role in innate immune reaction and specific immune reaction.additionally participate in controlling immunity Blood cell innate immune reaction and mechanism of CVI To determine the CD35,Fy6,CD44 expression on erythrocytes,CDI Ib expres-sion on neutrophils,CD14 expression on rnonocytes in patients with CVI by FCM,then compare them with the normal control IL-8 and IL-IO levels were measured by ELISA analysis from blood plasma.These changes will have great clinical value tojudge the genesis,development and turnover of CVI.The interest in the inflammatory reaction participation in the mechanism of CVI is helpful to offer theoretic basic for the research of venous active drug on therapeutic effect Our result shows that the fluorescence intensity expression of CD35 and Fy6 onerythrocytesare lower associated with the development of CVI Meanwhile,CDII b ex-pression on neutrophils and CDI4 exex-pression onmonocytesare higher at the later stage of CVI,accompaniedby an inbalance of proinflammatory mediator IL-8 andanti-inflammatory mediator IL-IO.These excessiveinflammatoryresponse may lead to the lo-cal tissue and microvascular damage of lower limb;Thesymptom oflipodennatosclerosis belong to C4can play an important role to devide the severity degree of CVI.On the other hand,the capability

of erythrocytes innate immunity in CVI is lower than innormal control,it become more obvious by added with inactive S.gn tumor cell which acted as antigen.Less expression of CD35 and Fy6 on erythrocytes weakened the modulation function leading to impro-priety inflammatory intensity of neutrophils andcytokine.Vcnous endothelium and venous valve were damaged progressively.Once deep venoussystem occurred reflux,theimmuneadherence function and inflammatoryregulating capacity of erythrocyte were reduced

HigerCD14expressionof'rnonocytesand theinbalanceoflL-8 and IL-IOaggravatelyinjured vein valve function

IMMUNE RESPONSES TO AoENOVIRAL VECTORS

659 TLR4 and TRIF Play Positive and Negative Roles in Ad Induced Innate Immunity In Vivo

Daniel M Appledorn,' Zachary Hartman,' Jeannine M.Scott,' AndreaAmalfitano.'

'Microbiology and Molecular Genetics, Michigan State Univer -sity, East Lansing; 2Division ofMedical Genetics, Department of Pediatrics, Duke University Medical Center; Durham

The use of Adenovirus (Ad) based vectors for both gene transfer applications and vaccine development continues to be promising Our recent studies have shown that aggressive innate immune responses following intravenous administration of adenovirus are dependent on both alternative and classical complement pathways

as well as the TLR adaptor protein MyD88.This response is in part characterized by thrombocytopenia, and increased plasma levels of numerouscytokinesand chemokines Recent studies in TLR9 and MyD88 deficient mice have revealed their partial role in regulating many of these Ad induced innate immune responses In this study,

we show that TRIF,a TLR adaptor protein mediating both TLR3 and TLR4 signaling responses to other pathogens,also plays a significant role in Ad induced thrombocytopenia,induction of cy-tokine andchemokines, aswell as profound changes in the mouse transcriptome response to Ad mediated gene transfer.Specifically,

in response to high titer adenovirus administration, we show that TRIF deficient mice have reduced serum levels of IL-Ia, IL-12, G-CSF,MCP-I and RANTES at both high (1.5 x10"vp/mouse)

and low (7.5 x 1010vp/mousc) doses of adcnoviral administration, relativeto identicalAd treatmentsofTRIF+ mice Wealso observed that TRIF is required for high serum IL-6 levels induced after high dose Ad treatment, but is not required for this response at low

Molecular Therapy Yo hunc 1 5 S upplcrncnr t, ~b )' 2007

C opyright © O ll i e 'u uericm Soci ety o f Gen e Therap y

doses Furthermore, we found that TRIF plays a significant role in

attenuating secretion of KC shortly after higher dose Ad injection

Ad induced thrombocytopenia was also avoided in TRIF deficient

mice compared to TRIF+ miceinjected at low doses Array based

analysis ofthe mouse liver transcriptome after Ad injection revealed

over 1900 gene expression differences in Ad treated TRIF deficient

mice,relative to Ad treated normal mice These genes include many

expected changes i.e TRAF and TAK-binding proteins as well as

numerous transcripts not otherwise implicated in immune response

pathways Since our previous publication confirmed MyD88 as

an-other TLR adaptor protein important in several Ad induced immune

responses', and the TLR4 receptor utilizesboth TRIF and MyD88

as adaptor proteins for TLR4 mediated signaling after LPS

expo-sure, we next asked whether TLR4 mediates the TRIF and MyD88

dependent Ad induced immune responses we were studying While

thrombocytopenia and most cytokinc and chernokinc responses were

not significantly altered in Ad treated mice deficient in TLR4

signal-ing,we actually observed an inhibitory role forTLR4 in secretion of

both IL-6 and IL-12(p70) This study clearly reveals the complexity

of innate immune responses to high titer Ad injection,and suggests

negative roles of TLR4,and both positive and negative roles for

TRIF in these responses These roles must be taken into account

when considering usc of inhibitors or activators ofTLR signaling

in gene transfer applications or vaccine development generally, and

specifically in the context of Ad based gene transfer approaches

'Hartman et.al: J Virology(81)pp:1796-1812:2007

Responses Are Mediated in Part by the Presence

of Natural Antibodies in Ad NaIve Mice

Jeannine M Scott,ITyler T Voss,IDaniel M Appledom,IAndrea

Amalfitano.1 2

I Department ofMicrobiology and Molecular Genetics, Michigan

State University, East Lansing, M/; 2Department ofPediatrics,

Michigan State University, East Lansing, Ml

Intravenous,high-dose Adenovirus (Ad) injection leads to a rapid

and robust innate immune response, resulting in plasma elevations of

several proinflammatory chemokines and cytokines, and

thrombo-cytopenia Our previous studies have confirmed that the molecular

basis of these responses is dependent upon Ad interactions with the

complement system The complement dependent anti-Ad immune

response is complex and dependent upon interactions with both

the classical and alternative pathways of complement activation

(see Scott et.al.this meeting) It is known that antibodies (specific

and natural) are involved in the activation of complement systems,

eg antigen-antibody complexes interact with Clq and activate

the elassical pathway of complement, while natural antibodies are

involved in stabilizingan alternative pathway C3 convertase We

wishedto determine if natural antibodies present in Ad narve mice

may be directly involved in the innate immune response to Ad

vectors We therefore injected an Ad vector expressing LacZ into

immunoglobulin deficient (SCID) mice, and assessed several known

Ad induced innate immune responses shortly after infection, relative

to identical injections into wild-type (WT) mice Interestingly, Ad

injections intoscmmice did not result in thrombocytopenia This

correlated with significantly higher levels ofLacZ expression in the

livers ofscmmice,relative to Ad-injected WT mice

Investiga-tion of this latter phenomenon,found no significant difference in

the Ad genome copy number per hepatocyte, (nor hepatotoxicity)

suggesting the CMV promoter utilized to drive LacZ expression in

this construct had enhanced activity in the livers ofscmmice We

also noted higher plasma levels of IL-6 at 1 hpi in the Ad injected

scmmice, but no increase in IL-12 or G-CSF at 1,6or 24 hpi,

again relative to Ad injected WT mice In an attempt to directly

implicate immunoglobulins as causative in some ofthese responses,

Molecular Therapy Volume15 SupplementI ~ br 2007

Copyright © The American So ci etyo r GeneTherapy

we pretreatedscmmice with immunoglobulins derived from Ad nalve mice Immunoglobulin reconstitutedscmmice now devel-oped thrombocytopenia after Ad injection,similar to Ad-injected

WT mice, demonstrating a requirement for circulating antibody in this important Ad induced response Our previous studies have im-plicated the C3 and Factor B proteins ofthe alternative complement pathway as also necessary fortheAd induction ofthrombocytopenia These new results indirectly suggest that Ads are interacting with C3,Factor B, and natural antibodies to cause thrombocytopenia in

Ad naive mice These studies suggest that natural antibodies present

in Ad narve subjects may both positively and negatively regulate several pro-inflammatory innate immune responses rapidly elicited

by Ad vectors

Systemic Administration of Non-Human Primate Derived Adenoviral Vectors to Cynomolgus Macaques

Suryanarayan Sornanathan,' Guang-Ping Gao,'Martin Lock; Peter Bell,IRebecca Grant,ISoumitra Roy,' Arbansjust Sandhu,' James M Wilson:

'Gene Therapy Program Department ofPathology and Labora-tory Medicine, University ofPennsylvania, Philadelphia PA.

Non-human primate derived adenoviral vectors are currently being pursued in pre-clinical trials as immunizing agents against diseases such as HIV,Ebola, and SARS However, adenoviral vec-tors based on human adenovirus type 5, a sub-group B virus,induce

a dose-dependent systemic inflammatory response when adminis-tered intravenously in species ranging from mice to humans In the present study we compared the inflammatory response following intravenous administration of ElIE3-deleted non-human primate derived adenoviruses, AdC6 and AdC7, expressing b-galactosidase

in cynomolgus macaques (Macaca fascicularis) The hematological, biochemical, histochemical and pathological findings were com-pared to those obtained from animals administered HuAd5 vectors Animals were dosed with increasing amounts ofAdC6 vectors and with higher doses of(2:5* 1012p/kg) ofAdC7 and HuAd5 vectors The actual administration of vee tor was uneventful and the only symptoms observed upon recovery from anesthesia were nausea, hypothermia or an elevated heart rate A striking difference between non-human primate and HuAd5 vector administered animals was the development ofpetechiae by 24 hr in the latter that resolved by day

3 All vectors induced a robust inflammatory response characterized

by transient leukopenia, thrombocytopenia,and elevations in serum levels of' pro-inflammatory cytokines, When compared to HuAd5, the non-human primate derived adenoviruses induced much higher levels of pro-inflammatory cytokines IL-6 and lFN-a Two animals that received high dose AdC6 vectors died or had to be euthanized within 6 hrs of vector for reasons that were not clear;all other animals survived to the scheduled necropsy time Bio-distribution

of vectors indicated that infectious AdC6 and AdC7 viral partieles were rapidly cleared from peripheral blood (24 hr) when compared

to HuAd5 (>day 3) Furthermore, liver transaminases (AST, ALT) were elevated in all animals In addition,vector genomes could

be detected in several tissues including bone marrow, spleen, skeletal muscle, heart and brain Histochemical analysis indicated that only AdC7 and HuAd5 vectors transduced cells in the liver and spleen,although,all three vectors induced infiltrates in liver (HuAd5>AdC7o;AdC6) Markers for disseminated intravascular coagulopathy (DIC) were increased following all three vector ad-ministrations These data indicate a disconnect between the extent of transduction and the onset ofa systemic inflammatory response and strongly suggest that the initial inflammatory response is primarily vector induced These studies represent an important step towards understanding the safety or non-human primate derived adenoviral vector administrations

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