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valuation of innate immunity and vector toxicity following inoculation ofovine, porcine or human adenoviral vectors in a mouse model
nurag Sharma, Dinesh S. Bangari1, Manish Tandon, Harm HogenEsch, Suresh K. Mittal ∗
epartment of Comparative Pathobiology, School of Veterinary Medicine, and Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA
r t i c l e i n f o
rticle history:eceived 19 May 2010eceived in revised form 15 July 2010ccepted 17 July 2010vailable online 24 July 2010
eywords:ovine adenovirushemokines
a b s t r a c t
Nonhuman adenovirus (Ad) vectors derived from bovine Ad serotype 3 (BAd3) or porcine Ad serotype3 (PAd3) can circumvent pre-existing immunity against human Ad (HAd). We have previously reporteddifferential transduction of human and nonhuman cells by these Ad vectors, and their distinct recep-tor usage and biodistribution. To compare the induction of innate immunity, vector toxicity and vectoruptake by Kupffer cells (KCs) following intravenous administration of PAd3, BAd3, or HAd5 vectors inmice, we determined mRNA expression levels of proinflammatory chemokines and cytokines, and Toll-like receptors (TLRs) in the liver and spleen. Tissue toxicity of these vectors was assessed by comparingserum levels of liver-specific enzymes, histopathology and Kupffer cell (KC) depletion. Compared to theHAd5 vector, PAd3 and BAd3 vectors were more potent stimulators of innate immune responses as
indicated by enhanced mRNA expression of TLRs and proinflammatory chemokines and cytokine genes.Histopathological changes in the liver were most pronounced in HAd5-inoculated mice while BAd3- orPAd3-inoculated mice revealed mild histologic changes that were confined to early time points. Inoc-ulation with HAd5 or PAd3 vectors resulted in a significant (P < 0.05) decline of the number of KCs inthe liver. Together, these results extend our previous observations regarding distinct in vivo biology ofnonhuman and human Ad vectors.
. Introduction
Vectors derived from adenoviruses (Ad) have demonstratedreat promise as gene delivery systems for both gene therapy andecombinant vaccines (Edelstein et al., 2004; Jager and Ehrhardt,007). The ongoing clinical trials of Ad-based vectors in cancerherapy and vaccination for diseases such as pandemic influenza,bola virus infection, and human immunodeficiency virus-acquiredutoimmune disease syndrome have highlighted the potential ofd vectors for clinical applications (Pandey et al., 2010; Sharma etl., 2009b). Vectors based on human adenovirus (HAd) serotype 5HAd5) and HAd serotype 2 (HAd2) are widely used for gene ther-
py applications (Edelstein et al., 2004). However, because of thendemic nature of HAd5 and HAd2, pre-existing vector immunityay inhibit the levels and duration of transgene expression (Kass-
isler et al., 1996). Additionally, the predominant hepatotropism
∗ Corresponding author at: Department of Comparative Pathobiology, School ofeterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.el.: +1 765 496 2894; fax: +1 765 494 9830.
E-mail address: [email protected] (S.K. Mittal).1 Current address: Genzyme Corporation, Department of Pathology, 5 The Moun-
of these vectors following systemic administration further limitstheir utility.
Depending on the dose and route of inoculation, Ad vectorsare known to activate innate immunity leading to vector toxic-ity and subsequent elimination of transduced cell (Hartman et al.,2008; Muruve, 2004). The innate immune response is activatedby pathogen-associated molecular patterns (PAMPs) of invadingpathogens through pattern-recognition receptors (PRRs) such asToll-like receptors (TLRs) (Hartman et al., 2008; Lee and Kim, 2007).This ensues a series of signaling events leading to the inductionof proinflammatory chemokines and cytokines, which result inthe elimination of invading pathogens and further activation ofadaptive immune responses (Lee and Kim, 2007). Furthermore, fol-lowing systemic administration, Ad vectors are rapidly removedby phagocytic cells such as Kupffer cells (KCs) in the liver, whichadditionally impairs the efficiency of gene delivery (Alemany etal., 2000; Tao et al., 2001; Wolff et al., 1997). Sequestration of Advectors by KCs results in the destruction of KCs and the limiteddistribution of Ad vectors to other tissues. Induction of proin-
flammatory molecules and the destruction of KCs following Adinoculation result in tissue toxicity, especially hepatotoxicity asoften indicated by elevated levels of liver enzymes such as aspar-tate aminotransferase (AST) and alanine aminotransferase (ALT)(Brunetti-Pierri et al., 2004).
To circumvent some of these limitations and to expand theepertoire of Ad vectors, vectors based on less prevalent HAd,erotypes such as HAd3, HAd11, and HAd35 and nonhuman Adsuch as bovine Ad (BAd), porcine Ad (PAd), ovine Ad, canine Ad,imian Ad, and fowl Ad, are being developed as alternatives or sup-lements to HAd5 vectors (Bangari and Mittal, 2006; Stone andieber, 2006). We have earlier demonstrated that vectors based onAd3 or BAd3 can evade anti-HAd5 immunity and have distincteceptor usage and in vivo tropism compared to those of HAd5Bangari and Mittal, 2005; Bangari et al., 2005b; Li et al., 2009;
offatt et al., 2000; Sharma et al., 2009a).In the present study, we assessed the induction of innate
mmune responses and tissue toxicity in mice following intra-enous inoculation with a replication-defective PAd3 or BAd3ector. Expression levels of genes coding for proinflammatoryytokines, chemokines and TLRs in the liver and spleen, and tissueoxicity were compared among PAd3, BAd3 and HAd5 vector-noculated groups. We observed significant differences in thenduction of innate immune response and tissue toxicity betweenonhuman Ad and HAd5 vectors.
. Materials and methods
.1. Adenoviral vectors
Replication-defective HAd-GFP (Bangari and Mittal, 2004), PAd-FP (Bangari and Mittal, 2004), and BAd-GFP (Bangari et al.,005a,b) vectors with deletions in early 1 (E1) region and carry-
ng the green fluorescent protein (GFP) gene under the control ofhe human cytomegalovirus (CMV) promoter were propagated in93 (human embryonic kidney cells expressing HAd5 E1) (Grahamt al., 1977), FPRT HE1-5 (fetal porcine retina cells expressing HAd51) (Bangari and Mittal, 2004), or FBRT HE1 (fetal bovine retina cellsxpressing HAd5 E1) (van Olphen et al., 2002), respectively. Virusurification was done by cesium chloride-density gradient cen-rifugation (Bangari and Mittal, 2004). The physical particle countsf purified stocks of HAd-GFP, PAd-GFP and BAd-GFP were esti-ated by spectrophotometry and expressed as vector particles (VP)
er ml using a previously described method (Maizel et al., 1968).ince plaque assays for these vectors were carried out in differentell lines, the efficiency of plaques formation varied widely withhe virus and cell type combination. Furthermore, as the capsidroteins of Ads are mostly implicated in the induction of innate
mmunity, a dosage of equal particles of each virus was critical foromparative studies. Therefore, VP was selected instead of plaque-orming units for vector quantification to maintain consistency inector dosage.
.2. Animal inoculation
Eight-to-ten-week-old female FVB/n mice were obtained fromarlan Laboratories (Indianapolis, IN). FVB/n mice were selected
or the current study because of the availability of an immunocom-etent tumor model with this mouse strain (Noblitt et al., 2005).he use of this mouse strain would allow us to extend our studyor investigation of Ad vectors for cancer gene therapy. All ani-
al inoculations were conducted in accordance with the guidelinesnd approval from Institutional Biosafety Committee and Insti-utional Animal Care and Use Committee. Mice were inoculatedntravenously via tail vein with HAd-GFP, PAd-GFP, or BAd-GFP at
dose of 1010 VP per mouse in a volume of 100 �l PBS++ (phosphateuffered saline supplemented with 0.01% MgCl2 and 0.01% CaCl2).ice inoculated with PBS++ served as negative controls. Mice (3
nimals per group) were euthanized at various time points (0.25,.5, 1, 2, 4, 8, and 16 days) post-inoculation, and serum samples
ch 153 (2010) 134–142 135
were collected and evaluated for AST or ALT enzyme levels. Theliver, spleen, lungs, heart and kidneys were either collected in 10%neutral buffered formalin or snap frozen and stored at −80 ◦C.
2.3. Quantification of cytokines, chemokines and TLRs specificmRNA transcripts
Frozen tissue samples were used for RNA extraction. Totalcellular RNA was isolated from 50 mg of the liver and spleen sam-ples using RNA miniprep kit (Stratagene, Cedar Creek, TX). RNAsamples were treated with DNase I to remove the residual DNA,if any. TaqMan® Gene Expression Assays (Applied Biosystems,Foster City, CA) mixture consisting of forward primer, reverseprimer, and Taqman® minor groove-binding probe (labeled with6-carboxyfluorescein dye) specific to mouse CCL2, CCL3, CCL4,CCL5, CXCL2, CXCL10, IP-10, IFN�, IL-6, TNF�, TLRs (TLR1 throughTLR9), myeloid differentiation primary response gene 88 (MyD88)or TIR-domain-containing adapter-inducing interferon-� (TRIF)were used for the quantification of chemokines, cytokines andTLRs. Two hundred ng of total cellular RNA was processed forreal-time RT-PCR using specific primers and probe and one-stepBrilliant qRT-PCR Master Mix Kit (Stratagene). For normalizationof the target gene expression, similar real-time RT-PCR reactionstargeting the endogenous 18S rRNA were simultaneously carriedout in separate tubes. Reaction mixture consisted of 2× qRT-PCRmaster mix, 250 nM each of respective forward and reverseprimers, and 100 nM of Taqman probe along with other standardkit components. Each reaction was carried out in duplicate. Thereal-time RT-PCR was performed using the Mx3000 Thermocycler(Stratagene). The reaction conditions included cDNA synthesisstep at 50 ◦C for 30 min, followed by polymerase activation (95 ◦Cfor10 min), and 45 cycles of denaturation (95 ◦C for 15 s) andannealing/extension (60 ◦C for 1 min). The Ct values for individualreactions were determined and data were analyzed with MxProsoftware (Stratagene) to obtain the relative expression levels of var-ious chemokines, cytokines, or TLRs. Quantification of expressionlevels of mRNA transcripts in the liver and spleen tissue sampleswas done by ��Ct method (Winer et al., 1999) and expressed inrelation to the mean expression levels of respective genes observedin tissues from mock-inoculated mice at respective time points(referred to as calibrators). The difference in expression levels inrelation to the time-matched calibrator was calculated as 2−��Ct
(where ��Ct = [Cttarget gene(unknown) − Ct18S rRNA(unknown)] −[Cttarget gene(calibrator) − Ct18S rRNA(calibrator)], Ct is the cyclenumber at which fluorescence signal crosses the threshold).
2.4. Histopathology and immunohistochemistry
Formalin-fixed tissues (liver, spleen, kidneys, lungs and heart)were paraffin-embedded and used for routine histopathologyand immunohistochemistry. For histopathology, formalin-fixed,paraffin-embedded tissue blocks were sectioned at 5 �m, stainedwith hematoxylin and eosin and examined microscopically by twoboard-certified veterinary pathologists (HH and DSB).
For immunohistochemistry, formalin-fixed, paraffin-embeddedtissue sections were deparaffinized and rehydrated in xylene andalcohol according to standard procedures. For antigen retrieval,sections were immersed in hot (95 ◦C) target retrieval solutionpH 6.0 (DakoCytomation, Carpinteria, CA) for 30 min. Endogenousperoxidase activity was quenched by incubating sections in 3%hydrogen peroxide solution for 10 min. Endogenous biotin and
avidin were blocked by sequential incubation in avidin and biotinsolutions (Vector Laboratories, Burlingame, CA) for 15 min each.Subsequently, sections were incubated overnight at 4 ◦C in a block-ing buffer (PerkinElmer, Waltham, MA) supplemented with 5% goatserum.
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The following day, sections were incubated with a mono-lonal rat anti-mouse F4/80 (pan-macrophage marker) primaryntibody (Abcam, Cambridge, MA) at 1:10 dilution for 2 h fol-owed by incubation with a goat anti-rat biotinylated secondaryntibody (1:2500) for 1 h at room temperature in a humidifiedhamber. This step was followed by incubation with streptavidine-orseradish peroxidase conjugate (DakoCytomation) (1:100) for0 min at room temperature. The signal was amplified by tyra-ide signal amplification kit (PerkinElmer). Color developmentas performed by aminoethyl carbazole (Red) substrate kit (Zymed
aboratories Inc., San Francisco, CA). The specimens were counter-tained with hematoxylin. Slides were mounted using Clearmountolution (Zymed Laboratories), dried and coverslipped with per-ount (Fisher Scientific, Pittsburgh, PA). For quantification of
4/80-positive cells (KCs), seven randomly selected overlappingelds were counted at 600× magnification and the averages werealculated.
.5. Evaluation of serum liver enzymes
Serum levels of liver-specific enzymes (AST and ALT) wereetermined using the VITROS 5.1 FS Chemistry System (Johnson &
ohnson Gateway) at the Clinical Pathology Laboratory, Veterinaryeaching Hospital, School of Veterinary Medicine, Purdue Univer-ity.
.6. Statistical analyses
A two-way ANOVA model was used to test the statistical signif-cance between or within the groups. All statistical analyses werepplied in PROC GLM with CONTRAST option in SAS 9.1. For all tests,< 0.05 was considered significant.
. Results
.1. Induction of proinflammatory chemokines and cytokinesxpression in mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP
Intravenous inoculation of a host with Ad vectors results inctivation of innate immune response which is often associatedith toxicity and rapid vector clearance. Previous studies have
eported the induction of a variety of proinflammatory cytokinesnd chemokines following the systemic administration of Adectors (Muruve et al., 1999; Muruve, 2004; Zaiss et al., 2002). Var-ous serotypes of human Ad vectors differentially activate innatemmune responses (Appledorn et al., 2008a; Iacobelli-Martinez andemerow, 2007). We have developed nonhuman Ad vectors basedn PAd3 and BAd3 to circumvent some of the limitations associatedith HAd vectors (Bangari and Mittal, 2004; Mittal et al., 1995).
To investigate the differences in the induction of innate immuneesponses following systemic administration of these vectors,VB/n mice were inoculated intravenously with 1010 vector par-icles (VP) of replication-deficient HAd-GFP, PAd-GFP or BAd-GFP,ach of which carries the GFP transgene as a reporter. At 6, 12,4 and 48 h post-inoculation, animals were euthanized and multi-le tissues including the liver and spleen were collected. We havereviously reported the in vivo biodistribution of HAd5-, BAd3-nd PAd3-based vectors and their efficiency to express transgeneSharma et al., 2009a). Since the majority of intravenously inoc-lated human or nonhuman Ads localize to the liver and spleenSharma et al., 2009a), these tissues were selected for assessment of
ytokine and chemokine genes implicated in the induction of innatemmune responses. Total cellular RNA extracted from the liver andpleen samples was analyzed for expression of various cytokinesnd chemokines mRNA by real-time reverse transcriptase poly-erase chain reaction (RT-PCR). Significant (P < 0.05) increases
ch 153 (2010) 134–142
in mRNA expression levels of various chemokines (CCL2, CCL3,CCL4, CCL5, CXCL2, CXCL10, and IP-10) and cytokines, (TNF�, IFN�and IL-6) were observed in both the liver (Fig. 1) and spleen(data not shown) of vector-inoculated groups compared to thePBS-inoculated control group. In Ad-vector-inoculated animals, rel-ative mRNA expression levels of proinflammatory cytokines andchemokines peaked at 6 or 12 h post-inoculation and declinedgradually thereafter. Relative mRNA expression levels of almost allthe investigated cytokines and chemokines at 6 h post-inoculationwere significantly (P < 0.05) higher in the nonhuman Ad-inoculatedmice than in those HAd-GFP-inoculated mice. Between the twononhuman Ad vectors, relative mRNA expression levels of thesecytokines and chemokines were either comparable or marginallyhigher in mice inoculated with PAd-GFP.
3.2. Induction of TLR expression in mice inoculated withHAd-GFP, PAd-GFP, or BAd-GFP
TLRs interact with various viral components to stimulate thehost innate immune responses (Janssens and Beyaert, 2003). Fol-lowing activation, TLRs initiate a signaling cascade through adaptormolecules such as MyD88 and/or TRIF that subsequently resultsin activation of innate as well as adaptive immune responses. Inthe present study, we examined relative mRNA expression levelsof TLRs (TLR 1 through TLR 9), MyD88 and TRIF in the liver andspleen of mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP.A significant (P < 0.05) increase in relative mRNA expression lev-els of TLRs 2, 3, 4, 7 and 9, together with MyD88 and TRIF wasobserved in both the liver (Fig. 2) and spleen (data not shown) ofAd vector-inoculated mice compared to mock-inoculated controls.No significant changes were observed in mRNA expression levelsof TLRs 1, 5, 6 and 8 compared to mock-inoculated mice. RelativemRNA expression of several of the TLRs peaked at 6 or 12 h post-inoculation and gradually declined thereafter. Significantly highermRNA expression levels of TLR 4 and 7 were observed in mice inoc-ulated with nonhuman Ad vectors compared to those inoculatedwith HAd-GFP. Similar to mRNA expression levels of proinflamma-tory chemokines and cytokines, relative mRNA expression levels ofTLRs in PAd-GFP-inoculated mice were either comparable or higherthan those in BAd-GFP-inoculated mice.
3.3. Evaluation of vector toxicity in mice inoculated withHAd-GFP, PAd-GFP, or BAd-GFP
To examine vector toxicity in mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP, the liver, spleen, lungs, kidneys andheart were collected at 0.25, 0.5, 1, 2, 4, 8, and 16 days post-inoculation. The tissue samples were analyzed for histologicalchanges by histopathology and for the number of KCs in theliver sections by immunohistochemistry. At earlier time points(6–24 h post-inoculation), histologic changes in the liver wereminimal to mild in all Ad vector-inoculated mice (Fig. 3). Thesechanges included scattered apoptotic hepatocytes with occasionalinfiltration by macrophages and fewer granulocytes. Apoptotichepatocytes were observed at increased frequency in BAd-GFP-inoculated mice followed by PAd-GFP or HAd-GFP-inoculated mice(Fig. 3B–D). The most striking liver histopathologic changes wereobserved in HAd-GFP-inoculated mice at later time points (4 or 8days post-inoculation). At these time points, the livers of HAd-GFP-inoculated mice revealed multifocal hepatocellular degeneration,scattered apoptotic hepatocytes, portal lymphohistiocytic inflam-
mation as well as scattered foci of inflammation (Fig. 3F andJ). In mice inoculated with PAd-GFP, BAd-GFP or PBS, the liverhistology was unremarkable at these time points (Fig. 3). Rare clus-ters of macrophages (Fig. 3G) were observed within the hepaticparenchyma of some mice from all groups. Histologic changes in
A. Sharma et al. / Virus Research 153 (2010) 134–142 137
Fig. 1. Expression levels of proinflammatory cytokines and chemokines mRNA at different time points post-inoculation in the liver of mice inoculated with HAd-GFP, PAd-G ether2 expreo are shv rsus H
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FP, or BAd-GFP. Real-time RT-PCR assays for various cytokines and chemokines tog00 ng of total RNA and comparative quantification of cytokines and chemokinesbserved with mock-inoculated mice at each time point post-inoculation. Valuesersus expression level in PBS-inoculated mice. †P < 0.05 for PAd-GFP or BAd-GFP ve
he spleen were comparable among all Ad vector-inoculated mice.rominent germinal centers were present in the splenic white pulpf all Ad-inoculated mice at 8 and 16 days post-inoculation (dataot shown). As expected, no significant histologic changes werebserved in the control group at any time point.
Immunohistochemical staining for KCs in the liver sectionsndicated significant decreases in the number of F4/80 immuno-ositive KCs in HAd-GFP and PAd-GFP inoculated mice at 12nd 24 h post-inoculation compared to mock-inoculated controlsFig. 4a and b). In HAd-GFP-inoculated mice, KCs declined bypproximately 50, 90 and 80% at 6, 12 and 24 h post-inoculation,espectively. At the same time points, the declines in KCs inAd-GFP-inoculated mice were approximately 5, 60 and 70%,
espectively. The decline in KCs in HAd-GFP-inoculated mice sam-les was significantly higher (P < 0.05) compared to that of mice
noculated with PAd-GFP at 12 h post-inoculation. No significanteduction (P > 0.05) in KCs was observed in BAd-GFP-inoculatedice at any time point (Fig. 4b).
with 18S rRNA (as an endogenous control for normalization) were performed usingssion levels was achieved. Values are reported relative to mean expression levelsown as the mean ± standard deviation for three mice at each time point. *P < 0.05
Ad-GFP at each time point.
Hepatic toxicity due to vector administration was assessed byevaluating serum AST and ALT levels. At 0.5 and 1 day post-inoculation, serum AST (Fig. 5) and ALT (data not shown) levelswere mildly to moderately elevated in PAd-GFP and BAd-GFP inoc-ulated mice. Although significantly higher (P < 0.05) than those inmock-inoculated mice, these values were increased by less thantwo-fold in PAd-GFP inoculated mice or less than four-fold in BAd-GFP inoculated mice compared to the reported reference range (AST95.6 ± 63.6 IU/L, ALT 59.5 ± 12 IU/L) for these enzymes in the FVB/nmouse strain (Mouse Phenome Database, 2010).
4. Discussion
In the current study, we examined the induction of innateimmune response and vector toxicity in mice inoculated intra-venously with human or nonhuman Ad vectors. Induction of avariety of proinflammatory chemokines and cytokines includingCCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10, IP-10, IFN�, IL-6, and TNF�
138 A. Sharma et al. / Virus Research 153 (2010) 134–142
Fig. 2. mRNA expression levels of various TLRs and adaptor molecules (Myd88 and TRIF) at different time points post-inoculation in the liver of mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP. Real-time RT-PCR assays for various TLRs, Myd88, or TRIF together with 18S rRNA (as an endogenous control for normalization) were performedusing 200 ng of total RNA and comparative quantification of TLRs, Myd88 and TRIF expression levels was achieved. Values are reported relative to mean expression levelsobserved with mock-inoculated mice at each time point post-inoculation. Values are shown as the mean ± standard deviation for three mice at each time point. *P < 0.05versus expression level in PBS-inoculated mice. †P < 0.05 for PAd-GFP or BAd-GFP versus HAd-GFP at each time point.
Fig. 3. Representative photomicrographs of the liver sections of mice inoculated with PBS, HAd-GFP, PAd-GFP, or BAd-GFP. Blue asterisks indicate lumen of portal veinsand insets represent a high magnification of area indicated by blue arrows. Note scattered apoptotic hepatocytes (black arrows) in Ad-vector-inoculated mice liver at 0.5day post-inoculation. Multifocal, random lymphocytic infiltration and hepatocellular degeneration/apoptosis as well as portal inflammation were observed only in HAd-GFP-inoculated mice at 4 and 8 days post-inoculation (panels F and J). Rare clusters of macrophages (inset, panel G) were occasionally found in mice from all groups. Stain:hematoxylin and eosin. Original magnification: 200× (insets 600×).
A. Sharma et al. / Virus Research 153 (2010) 134–142 139
Fig. 4. (a) Immunohistochemistry for Kupffer cells (KCs) in the liver sections of mice inoculated with PBS, HAd-GFP, PAd-GFP, or BAd-GFP. Formalin-fixed, paraffin-embeddedl in mat( riousf on forP
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iver tissue sections were processed for immunohistochemical analysis as described600× magnification). (b) Quantitative estimation of KCs in the liver sections at varom each mouse liver section. Values are reported as the mean ± standard deviatiBS-inoculated mice at each time point.
ollowing systemic administration of Ad vectors has previouslyeen reported (Muruve, 2004; Muruve et al., 1999; Zaiss et al.,002). Several studies have demonstrated the role of TLRs in innate
mmune response to Ad vectors (Appledorn et al., 2008b; Hartmant al., 2007a). In the present study, PAd-GFP was the most potentnducer of various cytokines and chemokines followed by BAd-GFP,
hile the HAd-GFP-induced mRNA expression levels of cytokinesnd chemokines were the lowest of the three vectors.
The observed differences in innate immune responses follow-ng systemic administration of PAd3, BAd3, or HAd5 vectors further
ighlight their distinct biological properties such as receptor usage,ector genome sequence, vector structural (capsid) proteins, inivo tropism, etc. It is believed that the molecular events leadingo the induction of immunoregulatory genes are triggered due toirus–cell interaction (Tibbles et al., 2002). Ad serotypes that uti-
erial and methods. Anti-F4/80 labeled KCs stained in brown color could be observedtime points. KCs in seven randomly selected non-overlapping fields were countedthree mice at each time point. †P < 0.05 for PAd-GFP, BAd-GFP or HAd-GFP versus
lize distinct cellular receptors differ in their intracellular traffickingand localization (Leopold and Crystal, 2007; Rogee et al., 2007).For instance, CD46-utilizing Ads tend to accumulate in lysosomesunlike subgroup C Ads that traffic rapidly to the nuclear envelope(Iacobelli-Martinez and Nemerow, 2007). Consequently, CD46-utilizing Ads preferentially activate TLR9 that is also located inlysosomes. Moreover, the CD46-utilizing Ads show altered expres-sion and/or activity of transcription factors responsible for theactivation of proinflammatory cytokine genes (Iacobelli-Martinezand Nemerow, 2007).
Similarly, CAR-fiber (Tamanini et al., 2006) or RGD-integrin (Liuet al., 2003) interactions have been shown to result in the activationof inflammatory responses. Tropism-modified Ads with altered CARinteraction or having additional RGD-binding domains resulted indifferential gene expression following infection (Volk et al., 2005),
140 A. Sharma et al. / Virus Resear
Fig. 5. Serum levels of AST enzyme in mice inoculated with PBS, HAd-GFP, PAd-GFP,oem
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stronger inducers of innate immune response than HAd5-based
r BAd-GFP. Values are reported as the mean ± standard deviation for three mice atach time point. †P < 0.05 for PAd-GFP, BAd-GFP or HAd-GFP versus PBS-inoculatedice at each time point.
urther highlighting the influence of Ad entry pathway and the dif-erences of viral capsids on activation of immune responses. BothAd3 and PAd3 utilize cellular receptors distinct from HAd5 recep-ors (i.e., CAR and integrins) (Bangari and Mittal, 2005; Bangarit al., 2005a; Li et al., 2009). Moreover, vectors derived fromhese nonhuman Ads demonstrated distinct biodistribution follow-ng intravenous delivery, indicating their distinct receptor usageSharma et al., 2009a). Details of intracellular trafficking and local-zation of BAd3 and PAd3 following receptor binding are noturrently known.
Ad capsid proteins (e.g., hexon or fiber) have been implicatedn expression of proinflammatory mediators (Molinier-Frenkel etl., 2002; Tamanini et al., 2006). The variations in capsid compo-ents of HAd5, PAd3 and BAd3 may also be responsible for theifferential activation of proinflammatory gene expression. Ad DNAlso appears to be involved in the activation of innate immunityMuruve et al., 2008; Zhu et al., 2007). Cells infected with wildype HAd5, HAd5 with E1 and E3 deletions or helper-dependentAd5 had a differential transcriptome dysregulation compared
o uninfected cells (Martina et al., 2007). Furthermore, specificNA sequences contained within the Ad vector may influence the
nduction of adaptive immune responses (Martina et al., 2007).lthough the transgene expression cassette in all three vectors used
n our study was identical, differences in the genomic nucleotideequences of HAd5, PAd3 and BAd3 may have some contribution toheir abilities to induce chemokine and cytokine mRNA expressionollowing systemic delivery. Differential chemokine and cytokineesponse following the systemic delivery of Ad vectors may alsoesult from different concentrations of the vector preparation. Toliminate such a possibility, we used equal number of viral particlesor each of the three vectors.
As a part of the innate immune response following Ad infec-ion, a number of TLRs and associated proteins such as MyD88 andRIF are activated/upregulated (Appledorn et al., 2008a; Appledornt al., 2008b; Hartman et al., 2007a; Hartman et al., 2007b;acobelli-Martinez and Nemerow, 2007; Zhu et al., 2007). VariousLRs are activated by unique PAMPs of viral components such asingle-stranded or double-stranded RNA and CpG motifs in viralNA. Activation of TLRs initiates a signaling cascade that results
n activation of transcription factors, including NF�B, AP-1, andTAT1 (Hartman et al., 2007b). These transcription factors in turn
pregulate expression of several cytokines and chemokine genesnd stimulate innate as well as adaptive immune responses. Asxpected, upregulation of mRNA expression levels of several TLRsTLR 2, 3, 4, 7 and 9) was observed that mirrored the induction of
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cytokines and chemokines genes, indicating that Ad vector inocu-lation activated the host TLR machinery. However, relative mRNAexpression levels of TLRs 1, 5, 6 and 8 were minimally elevated inAd vector-inoculated mice, suggesting the selective involvement ofTLRs for immune activation following Ad vector inoculation.
Recent studies have also implicated the role of TLR-independentmechanisms such as RLR (RIG-I-like receptor), NALP3 (NATCHT-leucine-rich repeat- and pyrin-domain-containing protein 3 alsoknown as NLRP3 or cryopyrin) and ASC (apoptosis-associatedspeck-like protein containing a CARD) components of inflamma-somes in Ad-mediated induction of innate or adaptive immuneresponses (Cheng et al., 2007; Hartman et al., 2008; Muruve etal., 2008; Nociari et al., 2007; Zhu et al., 2007). The role of suchTLR-independent mechanisms in Ad-mediated activation of innateimmune response was not investigated in the current study.
Hepatotoxicity following systemic delivery of Ad vectors reflectsa preferential vector biodistribution to the liver. In the presentstudy, hepatotoxic changes were minimal to mild at earlier timepoints. Scattered apoptotic hepatocytes were observed in miceinoculated with Ad vectors but were slightly more frequent in BAd-GFP-inoculated mice. Serum levels of liver-specific enzymes wereonly assessed at 0.25, 0.5 and 1 days post-inoculation and wereslightly elevated in PAd-GFP- and BAd-GFP-inoculated mice. His-tologic changes indicative of hepatotoxicity were most prominentin HAd5-inoculated mice at 4 to16 days post-inoculation. At thesetime points, liver histology in mice inoculated with PAd-GFP orBAd-GFP was unremarkable. Whether these observations reflectlow hepatotoxicity of these nonhuman Ad vectors as compared tothe HAd5 vector will require further investigations at higher vectordoses.
We observed a significant reduction in the number of KCs inHAd-GFP- or PAd-GFP-inoculated mice, but there was no signifi-cant change in the KC number in BAd-GFP-inoculated mice. Thedecline in KCs in HAd-GFP-inoculated mice is in agreement withprevious reports (Manickan et al., 2006; Schiedner et al., 2003;Xu et al., 2008). Interestingly, relatively prolonged persistence aswell as efficient transgene expression by BAd-GFP was observedin various tissues in our previous study (Sharma et al., 2009a).This could be due to the reduced vector uptake and clearanceby the KCs. The clearance of HAd5-based vectors by KCs doesnot involve their interaction with HAd5 cell receptors (CAR orintegrins), virus-binding to platelets, or vitamin K-dependent coag-ulation factors (Smith et al., 2008; Xu et al., 2008). However, thescavenger receptors, known for their ability to recognize negativelycharged materials, have been implicated in Ad uptake by KCs (Xuet al., 2008). In general, Ad capsids bear an overall negative chargethat varies with amino acid composition among various serotypes.Interestingly, hexon proteins of HAd5 and PAd3 are highly neg-atively charged (−21.3 and −21.4, respectively), while the BAd3hexon carries a considerably lower negative charge (−5.6) at pH 7.The low negative charge on the BAd3 capsid might allow evasionof KCs, whereas, HAd5 and PAd3 are efficiently taken up by KCs viascavenger receptors. As we have previously reported, minimal or nosequestration of a BAd3 vector by KCs in the liver probably allows itto reach other tissues such as the lungs, kidneys and heart (Sharmaet al., 2009a). Opsonization of Ad particles by natural IgM antibodiesand complement has also been implicated in their clearance by KCs(Xu et al., 2008). However, no detectable antibodies against HAd5,BAd3 or PAd3 were present in naïve mice in the present study (datanot shown).
Our results suggest that PAd3 and BAd3-based vectors are
vectors. The activation of innate immunity and the release ofcytokines and chemokines following Ad administration create aninflammatory milieu and provide a potent adjuvant effect forimmune activation against Ad-delivered antigens. Potent induc-
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ion of chemokine and cytokine genes by BAd3 and PAd3 vectorsill be advantageous in their application as vaccine vectors. Therolonged persistence of BAd3 vectors together with their natu-al adjuvant-like properties and ability to evade sequestration byCs will make them attractive candidates for gene delivery. Consis-
ent with this hypothesis, our BAd3-vector based avian influenzaaccine study in BALB/c mice resulted in significantly higher lev-ls of humoral and cell-mediated immune responses compared toHAd5-based vaccine (Singh et al., 2008). Clearly, further investi-ation on the mechanisms underlying the observed differences innnate immunity and vector toxicity among these nonhuman anduman Ad vectors will be critical for their development for clinicalpplications.
cknowledgements
This work was supported by Public Health Service grantA110176 from the National Cancer Institute. We are thankful to
ane Kovach for her excellent secretarial assistance and Ching-Yunhang for help with statistical analyses.
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