ad5:ad48 hexon hypervariable region substitutions lead to toxicity and increased inflammatory...

original article

2268 www.moleculartherapy.org vol. 20 no. 12, 2268–2281 dec. 2012

© The American Society of Gene & Cell Therapy

The development of adenoviral vectors for intravascular (i.v.) delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions (HVRs) of the Ad5 hexon are a target for neutralizing antibodies, but also interact with factor X (FX), facilitating hepato-cyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene ther-apy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribu-tion of Ad5, Ad5HVR48(1-7), and Ad48 following i.v. delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells (KCs), but triggered a robust proinflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48 hours) and increased num-bers of periportal CD11b+/Gr-1+ cells in the livers of Ad5HVR48(1-7)-treated animals following i.v., but not intramuscular (i.m.), delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b+/Gr-1+ cells. Collectively, these findings sug-gest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require i.v. administration.

Received 5 April 2012; accepted 16 July 2012; advance online publication 28 August 2012. doi:10.1038/mt.2012.162

IntroductIonAdenoviruses (Ads) represent the most frequently used gene ther-apy vectors for clinical applications. Many therapeutic approaches, such as targeting of disseminated metastases or the vasculature, require systemic delivery of these agents. However, intravascular

(i.v.) delivery of Ad5-based vectors is limited by capsid interac-tions with molecules or cells (e.g., erythrocytes) encountered in the circulation, and within defined organs. The recent find-ing that Ad5 binds to human, but not murine erythrocytes, can complicate the interpretation of preclinical studies performed in mice.1,2 Furthermore, the rapid scavenging and degradation of i.v. delivered Ad vectors by Kupffer cells (KCs) in the liver triggers robust proinflammatory cytokine responses.3 It is well-established in multiple animal models4–6 that Ad5-mediated hepatocyte gene transfer following i.v. delivery is a result of a high affinity interac-tion between coagulation factor X (FX) and a series of amino acid residues within hypervariable regions (HVRs) of the hexon.7–10 This interaction allows bridging of the Ad5:FX complex to highly sulfated heparan sulfate proteoglycans expressed on the surface of hepatocytes.11 The hepatotropism of Ad5 is dose-limiting, result-ing in acute hepatocellular damage, elevation of serum transami-nases and rapid recruitment of inflammatory mediators which can exacerbate systemic toxicity.3

The clinical utility of Ad5 may also be hampered by widespread pre-existing immunity in humans.12 A large proportion of neutral-izing antibody (NAb) responses to Ad5 are reportedly directed against surface exposed epitopes within the HVRs of the hexon.12–

14 However, anti-fiber antibodies are also important, especially fol-lowing natural adenoviral infections.15–17 Many rare species Ads have low seroprevalence in humans.18,19 In addition, several species D Ads, including Ad48, do not bind FX7 and may therefore bypass the hepatotropism associated with Ad5. In recent years, these attri-butes have prompted the investigation of rare serotype vectors for gene therapy applications. An alternative approach to using novel whole serotype vectors is the generation of chimeric Ad5 vectors, which feature structural proteins substituted from diverse Ad spe-cies.14,15,20,21 It is believed that these modifications could result in vectors with preferential receptor interactions, reduced off-target side-effects and improved in vivo safety profiles.22 Although com-plete intra-species hexon swaps with human Ads have often proven incompatible with viral assembly (reviewed in ref. 22), Roberts and colleagues previously described the hexon-chimeric vector,

Correspondence: Andrew H Baker, Institute of Cardiovascular and Medical Sciences, MVLS, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK. E-mail: [email protected]

Ad5:Ad48 Hexon Hypervariable Region Substitutions Lead to Toxicity and Increased Inflammatory Responses Following Intravenous DeliveryLynda Coughlan1, Angela C Bradshaw1, Alan L Parker1, Hollie Robinson1, Katie White1, Jerome Custers2, Jaap Goudsmit2, Nico Van Roijen3, Dan H Barouch4, Stuart A Nicklin1 and Andrew H Baker1

1Institute of Cardiovascular and Medical Sciences, MVLS, University of Glasgow, Glasgow, UK; 2Crucell Holland BV, Leiden, The Netherlands; 3Department of Molecular Cell Biology, Vrije Universiteit Medical Centre (VUMC), Amsterdam, The Netherlands; 4Division of Vaccine Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

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Molecular Therapy vol. 20 no. 12 dec. 2012 2269

© The American Society of Gene & Cell TherapyAdenovirus Hexon-induced Toxicity

Ad5HVR48(1-7), in which all seven HVRs of the Ad5 hexon were substituted for the corresponding regions from Ad48 hexon.14 This vector-induced robust immune responses against defined antigens and circumvented pre-existing immunity to Ad5 in a vaccine-based study following intramuscular (i.m.) injection.14 Ad5HVR48(1-7) fails to bind FX and does not transduce hepatocytes following i.v. delivery in macrophage-depleted mice.7 Consequently, this vec-tor is appealing to gene therapists using i.v. delivery strategies and recently, Ad5HVR48(1-7) has been used to target breast cancer bone metastases.23

Despite vectors such as Ad5HVR48(1-7) and Ad48 being suggested by others as potentially improved vehicles for i.v. gene therapy approaches, to date no studies have compared their in vivo interactions following i.v. delivery, to assess their safety profile. In this study, we describe the in vivo biodistribution and pharma-cokinetics of Ad5, Ad5HVR48(1-7), and Ad48; quantifying viral genome biodistribution, colocalization with hepatic and splenic cell populations and an evaluation of systemic toxicity. We show that i.v. delivery of Ad5HVR48(1-7) induces unexpected and unde-sirable host cytokine responses, and, despite having profoundly reduced hepatocyte transduction, causes elevated transaminases and the appearance of leukocytic cells in hepatic portal tracks. Although interleukin-5 (IL-5), IL-6, monocyte chemotactic pro-tein 1 (MCP-1) and interferon γ-inducible protein (IP-10) were also elevated following i.m. injection of Ad5HVR48(1-7), levels were minimal and did not result in hepatic injury or toxicity. The route-specific toxicity observed following i.v. administration of Ad5HVR48(1-7) cannot be attributed to the biological character-istics of either parental vector. Therefore, we highlight that the use of vectors featuring substantial hexon substitutions to avoid pre-existing immunity and/or FX interactions, can have an unpredict-able outcome following i.v. delivery.

resultsInvestigation of pre-existing immunity to Ad5 in a scottish cohort of patientsIn addition to numerous other factors, high level pre-exist-ing immunity to Ad5 is thought to limit its clinical utility.19 Depending on the route of adenoviral infection, NAb responses can be directed against the hexon HVRs,12–14 the fiber protein,15,17 or penton base.24,25 We investigated whether HVR substitutions within Ad5HVR48(1-7) could permit evasion from pre-existing anti-Ad5 immunity in a Scottish cohort of patients (Figure 1). As expected, there were negligible NAb responses to Ad48, consistent

with this vector being a “rare” serotype Ad.19 However, the neutral-ization profiles for Ad5 and Ad5HVR48(1-7) were broadly simi-lar, suggesting that the hexon HVRs play a minor role in eliciting antibody responses, at least in this study population. It is possible that this is due to differences between vaccine-acquired immu-nity versus natural adenoviral infection,16 in which the predomi-nant NAb responses are directed against the fiber, as a result of its excess production during infection.26 We were surprised to detect low (~33%) pre-existing immunity to Ad5 in this cohort (n = 63). However, it has previously been shown that seroprevalence can vary depending on geographical location.27

Accumulation of Ad5HVr48(1-7) and Ad48 genomes in liver are reduced compared to Ad5We compared the viral genome accumulation in the liver following i.v. injection of 3 × 1010 virus particles (vp) (low dose) or 1 × 1011 vp (high dose) Ad5, Ad5HVR48(1-7), or Ad48 (Figure 2a,b). The accumulation of Ad5HVR48(1-7) and Ad48 1 hour follow-ing low-dose injection was reduced approximately fourfold when compared with Ad5 (P < 0.05). Neither Ad5 nor Ad5HVR48(1-7) were affected by macrophage-depletion, whereas Ad48 was sensi-tive, with genome levels reduced ~112-fold (P < 0.01) when com-pared to the corresponding Ad5 group, and ~25-fold (P < 0.01) versus the control Ad48 group. Overall, we observed a similar pattern following i.v. injection of high dose vector (1 hour), with Ad5HVR48(1-7) genome levels reduced approximately fivefold (P < 0.05) and approximately fourfold (P < 0.05) relative to Ad5, in control and macrophage-depleted mice, respectively. Unlike the low dose, we did not detect differences in the accumulation of Ad48 genomes relative to Ad5 in untreated animals, but lev-els were greatly reduced compared to control when macrophages were depleted (~88-fold; P < 0.05).

At 48 hours postinjection of low dose, there were no differ-ences in the sequestration of Ad5 and Ad5HVR48(1-7) genomes (Figure 2a). In contrast, levels of Ad48 were lower, although this was not significant. In macrophage-depleted animals, the number of Ad5 and Ad48 viral genomes were increased, but Ad5HVR48(1-7) levels were unaffected. At the higher dose, there were ~25-fold (P < 0.05) and ~670-fold (P < 0.01) fewer Ad5HVR48(1-7) and Ad48 genomes in the liver at 48 hours com-pared to Ad5 (Figure 2b). Levels of Ad5HVR48(1-7) and Ad48 were also >100-fold lower than Ad5, in macrophage-depleted groups (P < 0.05 for both). Macrophage-depletion produced results which mirrored those observed with the lower dose (48

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Figure 1 Pre-existing neutralizing antibodies in scottish cohort of patients. A dilution of 2.5% patient sera (n = 63) was used to inhibit adeno-virus (Ad)-mediated luciferase transduction in HepG2 cells. Sera which inhibited >90% transduction compared to no serum control, was considered to be neutralizing, as described previously.15,50

2270 www.moleculartherapy.org vol. 20 no. 12 dec. 2012


hours); a modest increase in the level of genomes detected for Ad5 and Ad48, but no change observed for Ad5HVR48(1-7) when compared to control animals.

Ad5HVr48(1-7) and Ad48 have reduced interactions with Kcs in vivo following i.v. delivery of high doses (1 hour)In agreement with previously published studies,28,29 we detected each virus (green) in colocalization with F4/80+ KCs (red) in the liver, 1 hour following i.v. administration (Figure 2c; left panels). Viral particles were densely concentrated within F4/80+ cells, located within the sinusoids of the liver. Macrophage-depletion (+CD) completely eliminated F4/80+ cells from the liver (Figure 2c; right panel and Supplementary Figure S1). In these animals, signifi-cant amounts of Ad5-488 were scattered diffusely over the surface of liver cells (Figure 2c; right panels), whereas Ad5HVR48(1-7)-488 and Ad48-488 were undetectable. We quantified the amount of Alexa-488-labeled Ad in multiple liver sections (×40 magnifi-cation) using ImageJ analysis (Figure 2d). We detected ~2.5-fold (P < 0.01) and ~1.5-fold (P < 0.05) fewer Ad5HVR48(1-7)-488 and Ad48-488 compared to Ad5-488 (Figure 2d). Ad5HVR48(1-7)-488 and Ad48-488 were also approximately sixfold lower than Ad5-488 in the livers of macrophage-depleted animals (both P < 0.001). Using similar image analysis techniques, we quantified the amount of labeled virus associated with KCs versus whole liver sections (Figure 2e), using F4/80+ staining to delineate KCs (~14 KCs per image). Using this method of analysis, we found that the accumulation of Ad5HVR48(1-7)-488 and Ad48-488 in KCs was ~2-fold and ~1.2-fold lower than Ad5 (Figure 2e). We also tested the in vitro binding of each virus to murine macrophage cell lines, RAW264.7 and J774 (Supplementary Materials and Methods and Supplementary Figure S2). In the absence of FX, Ad5HVR48(1-7)-488 displayed reduced binding to RAW264.7 and J774 cells (>twofold; P = 0.001 for both), when compared with Ad5. Ad48 displayed reduced binding to RAW264.7 cells, but increased binding to J774 cells relative to Ad5. Interestingly, the presence of FX decreased the binding of Ad5 to RAW264.7 cells, whereas Ad5HVR48(1-7) and Ad48 were unaffected. In J774 cells, FX had no effect on the binding of Ad48 but did decrease binding of Ad5 and Ad5HVR48(1-7).

Accumulation of Ad48 in the spleen is high in comparison to Ad5HVr48(1-7) or Ad5The spleen is a major source of the cytokine response to adeno-viral vectors.30,31 Compared to Ad5 and Ad5HVR48(1-7), we detected significantly more Ad48 DNA in the spleen 1 hour postinjection, at both low (Figure 3a) and high doses of virus (Figure 3b). Macrophage-depletion did not affect the accumu-lation of Ad5 or Ad5HVR48(1-7) viral genomes in the spleen at either dose (Figure 3a,b). However, levels of Ad48 in the spleen were significantly reduced when macrophages were absent (more than fivefold reduction; P < 0.001). At 48 hours, vector genomes retained in the spleen were reduced when compared with 1 hour (Figure 3a,b). In macrophage-depleted groups, Ad5 genome levels were either modestly increased (low dose), or remained unaffected (high dose), whereas Ad5HVR48(1-7) genomes were decreased. The levels of Ad48 viral genomes at 48 hours were higher in the

absence of macrophages, possibly due to avoidance of vector deg-radation by scavenging macrophages (Figure 3a,b).

Viruses interact with marginal zone macrophages in the spleen (1 hour)To identify the virus-interacting cell types in the spleen (1 hour), we injected mice with 1 × 1011 vp fluorescently labeled Ad5-488, Ad5HVR48(1-7)-488, or Ad48-488 and performed colocaliza-tion by immunofluorescence (Figure 3c). Separate groups were pretreated with clodronate liposomes to deplete defined cell populations, as previously described.5 In agreement with the viral genomes data (Figure 3a,b), the accumulation of Ad48-488 was substantially higher than Ad5-488 or Ad5HVR48(1-7)-488 in control animals (Figure 3c). This was confirmed by quantifica-tion of Alexa-488 in images using ImageJ analysis (Figure 3e). The distribution of Ad5-488 and Ad5HVR48(1-7)-488 was dif-fuse, localizing to the marginal zone, although fluorescent virus was detectable in the red pulp region (Figure 3c). In contrast, Ad48-488 was largely localized at high levels within the marginal zone. In order to identify the cells involved in the accumulation of each virus we performed immunofluorescence to detect red pulp macrophages (F4/80+), marginal zone scavenging macrophages (MARCO+) and CD169+ marginal metallophilic macrophages. There was minimal colocalization with F4/80+ cells (Figure 3c; left panel). Ad48-488 accumulated to a high level in MARCO+ MZM (Figure 3c; centre). In agreement with the findings of DiPaolo and colleagues,31 we also detected colocalization between Ad5-488, Ad5HVR48(1-7)-488, and MARCO+ scavenging mac-rophages, albeit at a much lower level than Ad48-488. All viruses partially colocalized with CD169+ marginal metallophilic mac-rophages (Figure 3c; right panel). However, CD169+ cells were not the major cell type responsible for trapping Ad48-488, as evidenced by significant regions of non-colocalizing fluorescent virus surrounding the location where CD169+ cells reside.

In macrophage-depleted animals, F4/80+, MARCO+ and CD169+ cells were completely eliminated (Supplementary Figure S1). The overall levels of Ad5-488 and Ad5HVR48(1-7)-488 viri-ons in the spleen (1 hour) were not affected by the absence of mac-rophages (Figure 3b), whereas Ad48-488 genomes were reduced when compared to control groups. In the spleens of macrophage-depleted animals, the distribution of virus was more scattered and diffuse than those of control animals, and required higher mag-nification to identify the virus-interacting cell types (Figure 3d). We have previously shown that ER-TR7+ reticular fibroblasts and MAdCAM-1+ sinus-lining endothelial cells, both structural components of the splenic micro-architecture, are unaffected by pretreatment with clodronate liposomes.5 Interestingly, we have demonstrated that these cell are responsible for a large proportion of viral transgene expression at late time-points in macrophage-depleted animals.5,32 Therefore, we investigated whether these cells were responsible for accumulating viral particles 1 hour postin-jection (Figure 3d). All viruses were associated with ER-TR7+ reticular fibroblasts (Figure 3d; left panel). Viral particles were also detected, albeit at lower levels, within MAdCAM-1+ cells (Figure 3d; centre). Similarly, we detected isolated B220+ B-cells within the white pulp in colocalization with Ad5HVR48(1-7)-488 and Ad48-488, but not Ad5-488 (Figure 3d; right panel).



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Ad48 virions have increased blood persistence compared with Ad5 and Ad5HVr48(1-7)We quantified viral genome levels in heparinized whole blood taken 10 minutes and 1 hour postinjection of 1 × 1011 vp virus (Figure 3f). As expected, we detected low levels of Ad5 in the circulation at 10 minutes and at 1 hour.33,34 At the same time-point, levels of Ad48 in the circulation were found to be >180-fold higher than those of Ad5 (P < 0.05). Depletion of macrophages (+CD) did not increase the blood persistence of any virus relative to control at 10 minutes (Figure 3f). By 1 hour postinjection, all viruses were reduced com-pared to the levels found at 10 minutes. This was most notable for Ad5HVR48(1-7) which had ~170-fold fewer genomes in the cir-culation compared with the 10 minutes time-point. Interestingly, at the 1 hour time-point in macrophage-depleted (+CD) animals, the amount of circulating Ad5HVR48(1-7) was increased >500-fold (P = 0.03) when compared with the control group (Figure 3f). The blood persistence of Ad48, although reduced when compared to 10 minutes, remained high at 1 hour. These findings may explain why Ad5HVR48(1-7)-488 and Ad48-488 were undetectable in the livers of macrophage-depleted mice at 1 hour.

It has been shown that differential interactions of Ad vectors with human and rodent blood can affect viral transduction,2,35 potentially limiting the translational relevance of preclinical studies performed in mice. Ad5 binds human erythrocytes via the native Ad5 receptor, the Coxsackie and Adenovirus recep-tor (CAR) and complement receptor-1 (CR1), and agglutinates human but not murine erythrocytes.2 We compared the hemagglu-tination profiles of Ad5, Ad5HVR48(1-7), and Ad48 using mouse, rat and human erythrocytes (Supplementary Figure S3a–c). As expected, both Ad5 and Ad5HVR48(1-7) agglutinated human and rat erythrocytes, whereas Ad48 did not. In contrast, neither Ad5 nor Ad5HVR48(1-7) agglutinated murine erythrocytes, but Ad48 did. These data highlight the complexity of Ad interactions with blood cells derived from different species.

Ad5HVr48(1-7) induces unexpected elevations in inflammatory cytokines (6 hours)We compared the induction of cytokines following i.v. delivery of 3 × 1010 vp or 1 × 1011 vp (Figure 4a–j). At 3 × 1010 vp, cytokines induced by Ad5 remained low, although IL-1α, IL-12 and macrophage inflammatory protein-1α (MIP-1α) were increased over baseline (Figure 4b,f,i). However, multiple proinflammatory cytokines were induced by Ad5HVR48(1-7) and overall, these levels were higher than phosphate-buffered saline (PBS) or Ad5 (IL-1α, IL-12, and MIP-1α excluded). With the exception of IL-6 (~12-fold reduction;

P < 0.05), these responses were largely unaffected by the depletion of macrophages. In Ad48-treated animals, IL-2, MCP-1, IP-10, and MIP-1α were elevated in comparison to PBS- and Ad5-treated groups (Figure 4c,g,i), but did not reach the levels of Ad5HVR48(1-7) groups. Unlike Ad5HVR48(1-7), IL-2, MCP-1 and IP-10 responses induced by Ad48 were sensitive to macrophage-depletion (Figure 4c,g,h).

At 1 × 1011 vp, we detected increases in the cytokine responses to all viruses. In general, Ad5 levels were lower than those induced by Ad5HVR48(1-7) and only IL-1α and MIP-1α levels were affected by macrophage-depletion (Figure 4b,i). High levels of cytokines induced by Ad5HVR48(1-7) were not further increased relative to the low dose and only serum levels of IL-6 and IP-10 were found to be reduced upon macrophage-depletion in Ad5HVR48(1-7)-treated mice (Figure 4e,h). Ad48 also triggered robust cytokine responses. These responses were similar to the levels induced by Ad5HVR48(1-7) and only IL-2 was found to be higher in Ad48-injected animals than in Ad5HVR48(1-7)-injected groups (~1.5-fold; P = 0.02). With the exception of IL-1α and MIP-1α, cytokine responses induced by Ad48, were again highly sensitive to macrophage-depletion.

With the exception of IL-5, IL-6, MCP-1, and IP-10, serum cytokines remained unaffected following i.m. delivery of 1 × 1011 vp Ad5, Ad5HVR48(1-7) or Ad48 (Supplementary Materials and Methods and Supplementary Table S1). When compared to PBS control animals, we noted minimal increases in IL-5 (~3-fold) and IL-6 (~1.5-fold) in Ad5HVR48(1-7)-treated animals. Increases in MCP-1 and IP-10 were more pronounced, with increases of ~7-fold and ~750-fold, respectively. However, induction following i.m. injection was significantly lower than the i.v. route. This indi-cates that the atypical inflammatory response to Ad5HVR48(1-7) which we have observed following i.v. delivery is largely depen-dent on the route of administration.

Ad5HVr48(1-7) elevates transaminases and triggers the periportal accumulation of granulocytic cells in the absence of significant liver gene transferWe performed immunohistochemistry for luciferase transgene expression in frozen liver sections (Figure 5a). High level expres-sion was detected in the livers of animals which received Ad5 (1 × 1011 vp) and expression was marginally increased in macrophage-depleted groups. We detected isolated luciferase positive cells in the livers of animals which received Ad5HVR48(1-7), but not in macrophage-depleted animals (+CD). We did not detect any luciferase positive cells in the livers of Ad48-treated groups.

The elevation of serum transaminases can be an indica-tor of acute toxicity. Aspartate aminotransferase (AST) was

Figure 2 detection of virus in the liver by quantitative Pcr (qPcr) and immunofluorescence. (a) MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) Ad5, Ad5HVR48(1-7), or Ad48 and livers harvested for analysis (1 hour and 48 hours). Animals were pre-treated with 200 μl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours prior. Genomes were detected by real-time qPCR. (b) Quantification of viral genomes in the liver following i.v. injection of 1 × 1011 vp. Data represent the mean ± SEM (n = 5–8 per group), ***P < 0.001, **P < 0.01, *P < 0.05. Significance indicators directly above histogram bars indicate comparison to Ad5, within same +CD treatment group. (c) Immunofluorescence detection of Alexa-488-labeled virus in the liver 1 hour postinjection of 1 × 1011 vp (+CD). Kupffer cells (KCs) were identified by F4/80+ staining (red) and Ad5-488, Ad5HVR48(1-7)-488 (abbreviated to HVR48-488 in figure) or Ad48-488 are shown in green. Nuclei were counterstained with DAPI (blue). Right hand columns of each treatment group represent magnifications of the boxed areas (outlined in white). Images are representative of multiple fields of view from different animals. White arrows indicate virus within KCs (–CD) or on the surface of hepa-tocytes (+CD). (d) Quantification of Alexa-488 in liver images. A total of 6–15 separate images from different animals (n = 3 animals/group; mean ± SD) were thresholded, analyzed and regions corresponding to Alexa-488-labeled virus quantified using ImageJ analysis software. (e) Quantification of Alexa-488 within KCs in liver images. KCs (F4/80+) were manually segmented and the amount of Alexa-488 within summed using ImageJ software (average number of KCs = 14 per image). Graphical representation of whole liver in e is the same as –CD in d, for ease of comparison (n = 3 animals/group; mean ± SD). HVR, hypervariable region.



Figure 3 detection of virus in the spleen by quantitative Pcr (qPcr) and immunofluorescence. (a) MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) Ad5, Ad5HVR48(1-7), or Ad48 and spleens harvested for analysis (1 hour and 48 hours). Animals were pretreated with 200 μl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours before virus delivery and genomes detected by qPCR. (b) Quantification of viral genomes in the spleen following i.v. injection of 1 × 1011 vp. Data represent the mean ± SEM (n = 5–8 per group), ***P < 0.001, **P < 0.01, *P < 0.05. *Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group. (c) Immunofluorescence detection of Alexa-488-labeled virus (1 × 1011 vp) in the spleen (1 hour). Note: Alexa-488 labeled Ad5HVR48(1-7) is abbreviated to HVR48-488 in figure. Spleen sections were stained for F4/80, MARCO or CD169 (red) and images captured under a ×10 objective (–CD). (d) Markers to cell types which are unaffected by macrophage-depletion (B220, ER-TR7, MAdCAM-1) were used for colocalization. Images shown were captured using a ×40 objective and are representative of multiple fields of view (n = 3 animals/group; mean ± SD). Areas of colocalization are indicated by white arrows. (e) Quantification of Alexa-488 in spleen sections. A total of 6–10 separate images from different animals were analyzed (×10 magnification) and regions corresponding to Alexa-488-labeled virus quantified using ImageJ analysis (n = 3 animals/group; mean ± SD). (f) Quantification of viral genomes from blood 10 minutes and 1 hour postinjection of 1 × 1011 vp by qPCR, using 10 ng total DNA for analysis (n = 5–8 animals/group; mean ± SEM), *P < 0.05. Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group. HVR, hypervariable region.

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Figure 4 Quantification of serum cytokines/chemokines (6 hours). (a–j) Various cytokines/chemokines analyzed in the serum. MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) or 1 × 1011 vp Ad5, Ad5HVR48(1-7), or Ad48. Separate groups were pretreated with 200 μl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours prior to virus delivery. Cytokine/chemokine levels were quantified from serum (6 hours) using a multiplex luminex kit. Data represent the mean ± SEM (n = 5–8 per group), **P < 0.01, *P < 0.05, NS, not significant. Significance indicators with a straight bar above histogram indicates comparison with corresponding Ad5-injected groups. HVR, hypervariable region.



increased approximately threefold and approximately five-fold (P < 0.02) over baseline in all Ad5 and Ad5HVR48(1-7)-injected groups following i.v. delivery (Figure 5b,c). AST was not elevated in animals treated with Ad48. Macrophage-

depletion did not alter serum AST. There were minimal changes in alanine aminotransferase (ALT) levels; though small increases in the corresponding clodronate liposome-treated groups were observed.

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Figure 5 Quantification of liver gene transfer and hepatotoxicity (48 hours). (a) Luciferase expression in frozen liver sections (4 μm) was detected by immunofluorescence. (b) Serum transaminases, aspartate aminotransferase (AST) and (c) alanine aminotransferase (ALT) were quantified 48 hours postinjection of 1 × 1011 virus particles (vp). (d) Paraffin-embedded liver sections (5 μm) were stained with hematoxylin and eosin (H&E) and assessed by a pathologist. Images were captured using a ×10 and a ×40 objective. Data represent the mean ± SEM (n = 5–8 per group), **P < 0.01, *P < 0.05. Significance indicators directly above histogram bars indicate comparison to phosphate-buffered saline (PBS) control.



We assessed the histopathology of hematoxylin and eosin-stained liver sections 48 hours following i.v. delivery of 1 × 1011 vp (Figure 5d). Acute hepatic injury has previously been reported in animals receiving high doses of Ad5.3,30 We did not observe obvi-ous pathological changes in the livers of the Ad5 group (Figure 5c; left panel), although there were sporadic areas with cellular swell-ing and mild hepatic lipidosis (marked by black arrows). In con-trast, hepatic injury was more severe in macrophage-depleted Ad5 groups, where we noted hepatocyte degeneration and necrosis, identified by cells with shrunken, fragmented nuclei (Figure 5d; right panel). Furthermore, numerous mitotic figures (some with an abnormal tripolar or quadripolar appearance) were identified in the livers of macrophage-depleted (+CD) Ad5-treated animals (marked by black arrows), indicative of hepatocyte regenerative responses following damage (Figure 5d; right panel). In the livers of both Ad5HVR48(1-7)-injected groups, there was evidence of widespread pleiocellular granulocytic hematopoiesis, with hetero-geneous cells of mixed maturity identified in the portal tracks and also forming isolated small clusters within the sinusoids. This fea-ture was not detected in the livers of control animals, or animals treated with Ad5 or Ad48. We also detected mild hepatic injury in the livers of macrophage-depleted, Ad5HVR48(1-7)-injected ani-mals, with isolated regions of ballooning hepatocyte degeneration with mild lipidosis, but abnormal mitotic activity was not detected in the livers of these animals.

The hepatic toxicity induced by Ad5HVR48(1-7) was associ-ated with the i.v. route of administration and these effects were not observed following i.m. delivery of the same dose of virus (Supplementary Materials and Methods and Supplementary Figure S4a,b). Although serum AST levels in Ad5HVR48(1-7) animals were elevated over baseline and when compared to Ad5 or Ad48 following i.m. injection, these levels remained within the normal range for mice (Supplementary Figure S3a). Furthermore, we did not observe any pathological changes in the livers of animals receiving i.m. delivery of virus, again indicating that these effects are specific to i.v. administered Ad5HVR48(1-7) (Supplementary Figure S4b).

In order to phenotype the periportal cells observed in the hematoxylin and eosin-stained livers of AdHVR48(1-7)-injected animals (Figure 5d), we performed immunofluores-cence for myeloid cell markers, CD11b and Gr-1 (Figure 6a,b). We detected increased accumulation of CD11b+ and Gr-1+ cells within the livers of animals which received Ad5HVR48(1-7). We quantified the amount of CD11b+ and Gr-1+ cells in multiple liver sections using ImageJ analysis (Figure 6c,d). Ad5HVR48(1-7)- and Ad48-injected animals had more than threefold and approximately twofold higher CD11b+ cells than PBS- and Ad5-injected groups (both P < 0.01), although their localization differed. In the livers of Ad48-injected groups, detection of CD11b+ cells was limited to the hepatic sinusoids, whereas in animals which received Ad5HVR48(1-7), CD11b+ cells also accumulated to high levels within, and surrounding portal vessels. Interestingly, macrophage-depletion did not affect the presence of CD11b+ cells in Ad5HVR48(1-7)-treated animals (P = 0.17). However, macrophage-depletion reduced the number of CD11b+ in Ad48-injected groups to basal levels (~1.5-fold reduction; P = 0.0001). We detected >5-fold greater

numbers of Gr-1+ cells in the livers of Ad5HVR48(1-7) injected animals compared with other groups (Figure 6b,d). The levels of Gr-1+ cells were also increased in clodronate-treated mice (+CD); although these levels were lower than control, indicat-ing a partial sensitivity to macrophage-depletion. Gr-1+ cells were not increased in the livers of PBS-, Ad5- or Ad48-injected groups.

dIscussIonThe profound hepatocyte transduction associated with Ad5-based vectors once represented a major limitation to successful extra-hepatic delivery. Since the identification of the FX:hexon interac-tion and its important role in bridging hepatocyte transduction in vivo,7–9 there have been significant advances in the generation of genetically modified Ad5 vectors which do not bind FX.5,10 It is well established that dose-limiting acute liver toxicity is commonly observed in small animal models following i.v. delivery of high doses of Ad5. We previously have demonstrated the importance of the FX interaction in mediating hepatocyte transduction in mice, rats, and nonhuman primates.4,5,10 However, the importance of the Ad5:FX interaction in determining liver transduction in humans has not been conclusively determined. Although numerous clini-cal studies with therapeutic oncolytic vectors have shown that Ad5-based vectors are well tolerated, low-grade transient liver tox-icities and transaminitis are commonly reported side-effects.36,37 Furthermore, hepatocyte transduction, portal or lobular lympho-cytic inflammation in liver biopsies as well as dose-dependent elevations in cytokines, namely IL-6, have been reported following intra-arterial delivery of Ad5 in the clinic.36,37 Therefore, efforts to limit any potential toxicity or off-target effects are critical when designing and assessing optimal therapeutic Ad vectors.

The chimeric Ad5HVR48(1-7) vector was originally designed as a candidate vector for vaccination approaches, exploiting the finding that anti-Ad5 NAb responses are often directed against epitopes within the hexon HVRs.14 Subsequent studies dem-onstrated that the FX:hexon interaction also involved defined residues located within HVRs.7,10 Others have now capitalized on this, proposing Ad5HVR48(1-7) as an attractive vector for i.v. applications.23 In contrast, we report herein that i.v. delivery of Ad5HVR48(1-7) triggers unpredictable systemic effects, with elevated cytokines, transaminases and the appearance of granulo-cytic hematopoiesis in the liver, despite the absence of significant hepatocyte transduction.

It is well established that i.v. delivery of Ad5 results in rapid scavenging by resident hepatic macrophages.3 This interaction trig-gers the release of cytokines/chemokines from activated or dying KCs,3 which in turn affect the antiviral status of local parenchymal and endothelial cells.38 These early responses not only contribute to the elimination of virally infected cells and the development of adaptive immune responses, but they also play a causative role in the development of acute hepatic injury following i.v. delivery of high doses of Ad vectors.28,39 Immune effector cells, including neutrophils, natural killer cells and monocyte-macrophage pop-ulations, which are rapidly recruited to the Ad-infected liver in response to chemotactic signals, participate in these processes.39 Therefore, differential interactions between vector and KCs at early time-points could alter the transcription of defined cytokines



or chemokines, potentially modulating the outcome of the inflam-matory process and ensuing toxicity.

We observed reduced colocalization between Ad5HVR48(1-7) and KCs 1 hour postinjection and reduced binding to cultured murine macrophage cells, RAW264.7 and J774. Similar results have been reported by Khare and colleagues who reported that an Ad5:Ad6 hexon-chimeric vector, Ad5/6GL, can evade KCs in vivo.21 In another recent publication, the authors have

subsequently proposed that the charge of certain HVRs can affect recognition by scavenging receptors,40 which preferentially inter-act with negatively charged particles.41,42 KC-mediated recognition of Ad5 via scavenging receptors has also been reported to involve CAR-independent interactions with the fiber knob.28,43,44 Despite a reduced interaction with KCs compared to Ad5, Ad5HVR48(1-7) triggered robust cytokine responses which culminated in increased toxicity (AST) and the periportal accumulation of CD11b+/

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Figure 6 Phenotypic identification of accumulating cells in periportal areas. (a) Frozen liver sections were stained by immunofluorescence using antibodies to broad myeloid marker CD11b or (b) granulocyte marker Gr-1. Images were captured using a ×10 objective. (c) The amounts of CD11b+ or (d) Gr-1+ cells were quantified in multiple images from several different animals using ImageJ software. All thresholding and adjustments were applied equally across compared images. Data represent the mean ± SEM (n = 3–5 animals/group), ***P < 0.001, **P < 0.01. Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group.



Gr-1+ cells, 48 hours postinjection. Although Ad48 also displayed reduced KC uptake relative to Ad5 and induced elevated cytokine levels, there was no hepatoxicity or infiltration of CD11b+/Gr-1+ cells. The precise reasons for these differences remain unclear and the determinants are likely multifactorial.

The repertoire of proinflammatory cytokines triggered in response to the Ad capsid cannot be attributed solely to interac-tions with KCs and likely involve multiple cell types. The mecha-nisms which underlie the accumulation of Ad vectors in the liver are thought to be redundant, sequential and synergistic, involv-ing interactions with KCs, endothelial cells, and hepatocytes.29 It is known that Ad vectors can induce cytokine transcription in epithelial cells following the activation of NF-κB, ERK1/2, or p38MAPK signaling pathways.45,46 Additionally, hemodynamic changes induced by KC-endothelial cell interactions,38 contribute to the retention of viral particles in hepatic sinusoids or the space of Disse, placing viral particles in close proximity with endothelial cells which line the sinusoids.28 Recently, liver sinusoidal endothe-lial cells have been proposed to play a major role in the rapid scav-enging and elimination of i.v. delivered Ad vectors.34 The authors showed that 1 minute postinjection, ~90% of Ad5 colocalized with liver sinusoidal endothelial cells as compared with ~10% of KCs.34 In support of this, it was previously reported that KCs are not the major reservoir for the accumulation of Ads in the liver within the first 30 minutes.28 Therefore, it may be possible that the reduced accumulation of Ad5HVR48(1-7) in KCs, coupled with its lack of FX-binding and minimal hepatocyte transduction, serves to increase its interaction with liver sinusoidal endothelial cells at very early timepoints postinjection. Interactions of Ads with surface expressed integrins on endothelial cells contributes to the activation and secretion of inflammatory molecules follow-ing i.v. delivery.47 Liver sinusoidal endothelial cells are also known to express SR, Fc-γ and mannose receptors.34 However, the roles these receptors play in the uptake and potentially rapid elimina-tion of Ad in vivo are currently unclear.

The acute infiltration of neutrophils has been reported 1–6 hours postinjection of Ad5.39 Immune effector cells are recruited to sites of injury in response to chemokines such as IP-10, MCP-1, MIP-1α, MIP-1β, and RANTES.39 These signals correlate directly with the infiltration of inflammatory cells and the degree of hepatic injury.39 Interestingly, depletion of neutrophils can attenuate Ad-induced liver injury and the elevation of transaminases.39 In this study, we detected increases in IP-10, MCP-1, and MIP-1α following high doses of vector, with levels highest in animals receiving Ad48, fol-lowed by Ad5HVR48(1-7)-treated animals. However, despite the high levels of cytokines induced, we did not detect any pathologi-cal changes in the livers of Ad48-injected animals. In contrast, we observed a pronounced increase in the accumulation of peripor-tal CD11b+ and Gr-1+ cells in Ad5HVR48(1-7)-injected groups, as well as some mild hepatic injury (swollen cells and sporadic lipidosis) and elevations in serum AST. The characteristic pheno-type observed following i.v. injection of Ad5HVR48(1-7), was not observed following i.m. delivery. Although selected cytokines (IL-5, IL-6, MCP-1, and IP-10) were elevated following i.m. injection, these were minimal in comparison to those induced following i.v. injection. Furthermore, we did not detect evidence of hepatic injury induced following i.m. administration of Ad5HVR48(1-7).

The in vivo biodistribution of i.v.-administered Ad vectors can directly affect the degree of interaction with defined cell types. In addition to cells of the reticuloendothelial system, Ads have been shown to interact with macrophage populations and den-dritic cells in the spleen, which also contribute to innate immune responses to Ads.31 High-level splenic accumulation has been associated with vector-induced toxicity in vivo, as a result of trig-gering more robust inflammatory profiles.30 The high levels of Ad48 detected in the spleen provided a rational explanation for the increased inflammatory response to this vector. However, we did not detect increased levels of Ad5HVR48(1-7) in the spleen. The use of clodronate liposomes to deplete defined hepatic/splenic macrophages can help dissect the complex interactions between Ads and antigen presenting cells in vivo.5 Overall, we found that cytokine responses to Ad48 were highly sensitive to macrophage depletion whereas, with the exception of IL-1α, IL-6 and IP-10, responses to Ad5HVR48(1-7) were largely unaffected. This sug-gests that distinct cell types contribute to the antiviral response to Ad48 and Ad5HVR48(1-7).

It is clear that differences also exist between Ad5HVR48(1-7) and Ad48, in terms of their biodistribution and inflammatory outcome. Ad5HVR48(1-7)-based vectors have previously been shown to be more immunogenic than Ad48 in naive mice following i.m. injec-tion.14 In order to achieve long-term protective immunity, vaccina-tion strategies favor the use of vectors capable of stimulating robust immune responses, but the precise mechanisms which contribute to the natural immunogenicity of certain Ad serotypes remain unclear. Early immune responses to i.v. delivered Ads are capsid-dependent, so it seems logical that rare serotype, or capsid-modi-fied vectors could alter the coordinated and synergistic contribution of diverse cell types to the inflammatory process. Additionally, Ads are known to stimulate multiple innate signaling pathways, includ-ing TLR-dependent and TLR-independent pathways.48 Therefore, the underlying mechanisms are likely to be complex and currently are poorly understood. Importantly, the criteria which determine the suitability and potency of vectors for vaccination-based studies and i.v.-based therapeutic strategies differ greatly, both in terms of their mechanism of action and their downstream, off-target effects. Therefore, novel vectors undergoing development for each applica-tion will need to be assessed independently and systematically, to ensure their safety and efficacy in vivo.

In addition to these factors, further investigations into the impact of differential interactions between Ad vectors and blood cells are warranted. The binding of Ad5 to human erythrocytes, but not murine erythrocytes, is thought to limit the translation of Ad5-based treatments to the clinical setting. However, tran-sient thrombocytopenia and lymphopenia are frequently reported side effects in clinical studies,36 which are also observed in ani-mal models.43 Reassuringly, in recent years, experimental studies with Ad vectors have successfully identified numerous virus-host interactions, and have succeeded in developing improved capsid-modified vectors with the ability to evade these undesirable associations. Indeed, point mutations to ablate interactions with CAR and prevent the hemagglutination of CAR-expressing eryth-rocytes can easily be incorporated into the Ad5 capsid,6 thereby improving the translational relevance and potentially, the safety profile of Ad vectors.



The unpredictable nature of Ad5HVR48(1-7) and increased inflammation relative to Ad5 outlined in this study, suggest that Ad5HVR48(1-7) may be undesirable for i.v. delivery strategies, despite its demonstrated safety and success in vaccination strate-gies. Many vectors now exist which feature refined point mutations in HVR5/7 which completely ablate hepatocyte tropism,5,10 with-out triggering unwarranted inflammatory sequelae. These may represent more attractive platform vectors for future investigation, although it is clear that additional modifications will be required to further optimize and evade immune recognition and/or off-target interactions. Our findings have shown that the immune response to Ad48 following i.v. delivery was largely predictable, a reflec-tion of its high level uptake in the spleen. Reassuringly, previous studies have shown that it is possible to redirect the tropism of modified Ad vectors from the spleen to defined target tissues, thus circumventing the inflammatory response induced in this organ.5 Furthermore, we have recently show that mutation of the penton base RGD motif can reverse the increased inflammation induced by FX-ablated vectors which exhibit increased splenic uptake.32 Ad48 does not mediate hepatocyte transduction, induce idiopathic systemic hematopoiesis or cause hepatic injury despite the induc-tion of high levels of cytokines/chemokines. Collectively, these characteristics suggest that it may be worth developing Ad48 for future retargeting purposes using high affinity targeting entities.

MAterIAls And MetHodsVirus production and quality control. The viruses used in this study were E1/E3 deleted replication-incompetent Ad5Luc, Ad5HVR48(1-7)Luc, and Ad48Luc with the luciferase transgene under the control of the pClip cyto-megalovirus promoter.14 Viruses were amplified to high titers on HEK293 cells and purified by CsCl ultracentrifugation. Viral particle titers were deter-mined using the micro-bicinchoninic acid Protein Assay (Thermo Scientific, Loughborough, UK), calculated using the formula 1 μg protein = 4 × 109 vp, as described previously.11 Viral structural integrity (Supplementary Figure S5) was assessed following SDS-PAGE analysis and PageSilver Silver Staining (Fermentas, York, UK). Infectious titers (plaque-forming unit /ml) were determined by TCID50 end-point dilution.49 Viral genome copies (GC/ml) were quantified using the Adeno-X quantitative PCR (qPCR) Titration Kit (Clontech, Saint-Germain-en-Laye, France) or by modifying the assay to include primers directed against conserved regions of the hexon; forward TGGCGCATCCCATTCTCC and reverse CGCGGTGCGGCTGGTG. qPCR was carried out on ABI 7900HT Fast Real-Time PCR machine using the following cycling conditions; 50 °C for 2 minutes, 95 °C for 10 minutes followed ×40 cycles of 95 °C for 15 minutes and 60 °C for 1 minute. In the custom method, total adenoviral genomes were calculated using a stan-dard curve of 101 to 109 Ad5 genomes. GC titers calculated using the same method recommended by the manufacturer (Clontech). Viruses were tested for endotoxin using the Kinetic-QCL assay (Lonza, Slough, UK), according to the manufacturer’s instructions. Detailed information about the vectors used in this study and their quality control is presented in Table 1.

Neutralization inhibition assay. Serum samples (n = 63) were obtained from a Scottish cohort of patients, with ethical approval. We used an estab-lished luciferase transduction inhibition assay to evaluate the levels of pre-existing immunity against (A) Ad5, (B) Ad5HVR48(1-7), and (C) Ad48.50 HepG2 cells were seeded at 2 × 104 cells per well and were transduced in triplicate with 5,000 vp/cell for 3 hours in the presence or absence (con-trol) of 2.5% serum, using a previously described and validated method.15,50 Luciferase expression was quantified 48 hours later and normalised to pro-tein concentration. Sera which exhibited >90% inhibition of transduction was considered to be neutralizing, as described previously.15

Generation of Alexa-488 labeled viruses. Adenoviral capsids were fluores-cently labeled using an AlexaFluor-488 (green) protein labeling kit accord-ing to the manufacturer’s instructions (Invitrogen, Paisley, UK) and quality control performed as described previously.11 Alexa-488 labeled viruses are defined as Ad5-488, Ad5HVR48(1-7)-488, and Ad48-488.

In vivo studies. All animal experimentation was approved by University of Glasgow Animal Procedures and ethics committee and was performed under UK Home Office license in strict accordance with UK Home Office guidelines. Male outbred MF1 immunocompetent mice aged between 7-9 weeks (Harlan, Blackthorn, UK) were used for all in vivo studies. To deplete macrophages, 200 μl of PBS or clodronate encapsulated liposomes (a gift from Roche Diagnostics, Mannheim, Germany) were administered 48 hours before virus administration. Matched control groups were injected with PBS at the same time-point. All treatment groups were injected with low dose (3 × 1010 vp) or high dose (1 × 1011 vp) Ad5, Ad5HVR48(1-7) and Ad48 on day 0 and tissues harvested for qPCR and immunohistochemistry at 1 hour or 48 hours postinjection (N = 5–8 mice). A 10 minutes and 1 hour heparinized blood sample (20 μl) was obtained from the 1-hour treatment groups for analysis of viral blood kinetics by qPCR. Blood was taken from 48 hours groups at 6 and 48 hours and serum separated from whole blood using BD SST Microcontainer with clot activator (BD Diagnostics, Oxford, UK). Cytokine/chemokine profiles were determined from serum (>60 μl) using a cytokine mouse multiplex assay (Invitrogen). Serum transami-nases, ALT, and AST were quantified from 80 μl serum by the Veterinary Diagnostic Service at University of Glasgow. Separate groups (n = 3) were treated similarly but were injected with low doses and high doses of Alexa-488 fluorescently labeled Ads. Animals were perfused to exsanguination with PBS and their livers and spleens used for immunohistochemistry on frozen tissue (IHC-Fr) and viral biodistribution validated by qPCR.

Quantification of viral genomes by real-time qPCR. Viral DNA was extracted from murine tissue using the QIAamp DNA mini kit (Qiagen, Crawley, UK) and DNA concentrations determined using a nanodrop spectrophotometer (Thermo Scientific). SyBR green qPCR was used to cal-culate viral biodistribution using 20 ng total DNA per reaction and primers designed to detect the hexon region of the viral genome as described previ-ously.10 Samples were analyzed in triplicate on two separate occasions on a 7900HT Sequence Detection System (Applied Biosystems, Warrington, UK) and were compared to a standard curve of 101–108 viral particles. For quantification of viral genomes in heparinized blood, viral DNA was extracted using the Nucleospin Blood kit (Machery-Nagel) as directed by the manufacturer and 10 ng total DNA used for SyBR qPCR.

table 1 Virus quality control

Virus VP/ml PFu/ml VP/PFu ratioGc/ml

(Adeno-X)VP/Gc ratio (Adeno-X)

Gc/ml (custom)

VP/Gc ratio custom

endotoxin (eu/ml)

Ad5 3.90 × 1010 4.77 × 1010 81.76 2.46 × 1010 158.50 5.53 × 1010 70.52 0.05Ad5HVR48(1-7) 1.27 × 1012 3.60 × 1012 35.28 8.90 × 1009 142.70 2.21 × 1010 57.46 0.08Ad48 2.40 × 1012 1.88 × 1008 12,766 2.56 × 1007 93,750 4.34 × 1010 55.29 0.09Abbreviations: Ad, adenovirus; GC, genome copy; HVR, hypervariable region; PFU, plaque-forming unit; qPCR, quantitative PCR; VP, virus particle.VP titers were determined by micro BCA assay, PFU titers by TCID50 by end-point dilution and GC titers determined using the Adeno-X titration kit, or a customized qPCR protocol using primers to detect conserved regions within the adenoviral genome corresponding to the hexon.



Histology and immunofluorescence. For histological analysis of liver, 5-μm paraffin sections were stained with hematoxylin and eosin and indepen-dently assessed by a pathologist. Staining was visualized using an Olympus BX40 microscope coupled to a Qimaging MicroPublisher 3.3 RTV camera and images captured using QCapture Pro 6.0 software. Tissue samples were also taken from liver and spleen 1 hour and 48 hours postinjection of virus and frozen immediately in OCT compound. Sections were cut, fixed and stained using antibodies to F4/80, MARCO, CD169, ER-TR7, B220, and CD11b exactly as described previously.5 Gr-1+ cells were detected using rat anti-mouse IgG2b antibody (Ly-6G; eBiosciences, Hatfield, UK) at a final concentration of 0.00125 μg/ml followed by goat anti-rat Alexa-488 (Invitrogen). Sections were fixed with 4% paraformaldehyde for 10 minutes at room temperature. Immunohistochemistry for luciferase expression was achieved by staining 4μm frozen liver sections with a mouse anti-luciferase (Luci17; Santa Cruz Biotechnology, Heidelberg, Germany) IgG1, used at a final concentration of 1.33 μg/ml. Sections were fixed with ice-cold acetone at –20 °C for 10 minutes and allowed to air dry for 1 hour. Endogenous avi-din/biotin activity was blocked using a commercial kit (Vector Laboratories, Peterborough, UK). Staining was performed using the M.O.M Fluorescein Immunodetection kit (Vector Laboratories), as recommended by the manu-facturer. Fluorescence was visualized using an Olympus BX60 fluorescence microscope (×10 or ×40 objectives) and images acquired using Cell’M software (Olympus, London, UK). Isotype control antibodies were used at identical final concentrations for all staining protocols.

Image adjustments and analysis. Fluorescent images were processed to reduce background using ImageJ (National Institutes of Health) and adjust-ments were applied equally to all compared images. Quantification of Alexa-488 positive regions was calculated as follows: 10–30 separate images (n = 3 animals/group) were processed under binary, selecting find maxima. Noise tolerance was set uniformly at 10 and output set to identify single pixels while excluding edge maxima. We quantified the proportion of Alexa-488 associ-ated with KCs relative to the entire image exactly as described by Ganesan and colleagues, by delineating the borders of KCs using F4/80+ staining.34 An equivalent number of KCs (~14) were identified in each ×40 image.

Statistical analysis. Statistical analysis was performed using GraphPad Prism version 4.0 (GraphPad Software). Unless otherwise stated, data show the mean ± SEM of n = 3–8 per group (specific n numbers are indicated in each figure legend). Statistical significance was determined using one-way way ANOVA with a post-hoc Tukey’s test. *P value of <0.05, **P < 0/01, ***P < 0.001, **** P < 0.0001, NS = not statistically significant, P > 0.05.

suPPleMentArY MAterIAlFigure S1. Depletion of defined cell types in liver and spleen using clodronate liposomes.Figure S2. Alexa-488 virus binding to murine macrophage cells.Figure S3. Hemagglutination assay.Figure S4. Hepatic toxicity following im delivery of virus.Figure S5. Virus silver stain.Table S1.Cytokine responses following intramuscular injection (6 hours).Materials and Methods.

AcKnoWledGMentsThis work was funded by the BBSRC (BB/G016844/1, awarded to A.H.B). We thank Nicola Britton and Gregor Aitchison for their in-valuable technical assistance, Dr Richard Harbottle for his protocol for luciferase immunohistochemistry and Janet C. Patterson-Kane for pathological assessment of tissue.

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4. Alba, R, Bradshaw, AC, Mestre-Francés, N, Verdier, JM, Henaff, D and Baker, AH (2012). Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus). Gene Ther 19: 109–113.

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