Introduction

Adenovirus (Ad) vectors have been used for vaccination in preclinical models (for a review see¹) and, recently, also in humans². Ad-based vaccines have a number of advantages over naked DNA vaccines or vaccines based on other viruses such as poxvirus or alphavirus. These advantages include the ability to prepare high-titer stocks of purified virus easily and the remarkable efficiency of each step in the Ad cell/nucleus entry process leading to high-level transgene expression. Furthermore, it is thought that Ad's can provide an adjuvant effect in stimulation of antigen-specific immune responses. Interaction of Ad capsids with cellular receptors induces expression of proinflammatory cytokines/chemokines, such as TNF-, IFN-, IL-1, IL-6, IL-12, and MCP-1 and 2, which results in recruitment of effector cells of the innate and adaptive immune system to the site of infection³. These cytokines also activate functions of antigen-presenting cells (APCs). Furthermore, presentation of Ad proteins from the incoming Ad particle and/or de novo expression of Ad proteins in APC could help in activation of T cells specific to the vaccination antigen.

A critical factor in each vaccination strategy is the efficient uptake of vaccination vectors and antigen expression in professional APCs, such as dendritic cells (DCs). DCs are located strategically at the interface of potential pathogen entry sites. They capture antigens and migrate into secondary lymphoid tissues, where they activate both helper T cells and cytotoxic T lymphocytes. They also interact with B cells and probably NK cells. Current vectors for gene transfer into human DCs, including the commonly used serotype 5 Ad vectors, are inefficient or cytotoxic. The poor transduction of human DCs with Ad5-based vectors is due to low-level expression of the coxsackie and adenovirus receptor⁴. In contrast, earlier studies have shown that Ad vectors containing B-group Ad serotype 35 fibers (Ad5/35) efficiently transduce human DCs ex vivo⁴ and appear to target APC after intravenous injection into CD46-transgenic mice and baboons⁵. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination.

Vectors containing B-group Ad fibers, including Ad5/35 vectors, use CD46 for initial cellular attachment⁶. CD46 is a membrane glycoprotein that protects cells from complement damage. There are four major isoforms of CD46 (BC1, BC2, C1, and C2), depending on the alternative splicing of a region encoding an extracellular domain and the choice between one or two cytoplasmic tails, Cyt-1 and Cyt2. CD46 is also a receptor for measles virus laboratory strains, for human herpesvirus 6, and for certain pathogenic bacteria⁷. In humans, CD46 is expressed on all nucleated cells at a low level. Although Ad5/35 vectors efficiently transduce DC in vitro and potentially in vivo, a number of findings argue against the utility of these vectors for in vivo vaccination. (i) CD46 signaling (upon binding of CD46 monoclonal antibodies, recombinant complement factor C3b, or measles virus hemagglutinin) can induce immunosuppression depending on the nature of its cytoplasmic tail, with Cyt-1 (C1 isoform) engagement suppressing inflammation and Cyt-2 (C2 isoform) increasing it⁸. (ii) Studies of an Ad35 outbreak leading to pneumonia and sepsis revealed a transient neutropenia, which could have been caused directly by Ad35 infection⁹. (iii) Measles virus and HHV6, which also use CD46 as a receptor, can cause transient immunosuppression at the level of dendritic cell precursors, bone marrow stromal cells, or CD34⁺ myeloid progenitors¹⁰.

The majority of published in vivo studies with B-group fiber-containing Ad vectors have been done in mice^{11,12,13,14,15,16}. Since mice do not express CD46 in a human-like pattern¹⁷, these studies might not be predictive of Ad5/35 behavior in humans. We therefore studied the in vivo properties of Ad5/35 in transgenic mice that express CD46 in a pattern and at a level similar to humans. This strain of CD46 transgenic mice has been extensively used in studies with measles virus (which also uses CD46 as receptor) and in studies on the role of CD46 activation on immune responses^18,19. Specifically, we evaluated the effect of Ad5/35 on the host's immune system and the ability of an Ad5/35 vector to induce immune responses against a model antigen.

Results and discussion

Ad5/35 transduction of dendritic cells in vitro

To corroborate the use of Ad5/35 for immunotherapy, we performed transduction studies with human DC that were generated by in vitro differentiation of CD34⁺ cells or CD14⁺ monocytes. We showed that immature human DC can be efficiently transduced at low m.o.i. with Ad5/35 vectors (Fig. 1A). Ad5/35 transduction did not affect the expression of maturation markers (data not shown). Ad5/35 infection of CD34⁺-derived DCs could be efficiently blocked by soluble CD46, indicating that CD46 can mediate DC transduction (Fig. 1B). We also performed transduction studies with bone marrow-derived DC from C57Bl/6 (C57) and C57Bl/6-CD46 transgenic (C57-CD46) mice (Fig. 1C). While transduction of DC with the Ad5 vector was inefficient in both wild-type and transgenic strains, Ad5/35 efficiently transduced DC from C57-CD46 but not from C57 mice.

Figure 1.

Assessment of Ad5/35 vectors for transduction of dendritic cells. (A) Transduction of human dendritic cells (DCs) with first-generation Ad5 and Ad5/35 vectors containing a CMV-GFP expression cassette. Immature human DCs (>95% positive for CD11c) derived from either CD34⁺ cells (left) or peripheral CD14⁺ monocytes (right) were infected at different m.o.i. for 3 h. GFP expression was analyzed by flow cytometry 24 h postinfection. (B) Transduction of DCs in the presence of soluble CD46. CD34⁺-derived DCs were transduced with Ad5-GFP or Ad5/35-GFP at an m.o.i. of 10 pfu/cell in the presence of an excess of soluble CD46 (as a competitor) and GFP expression was analyzed 24 h later by flow cytometry. (C) Transduction of DCs from CD46 transgenic mice with Ad vectors. Myeloid DCs from C57 and C57-CD46 mice were infected with first-generation Ad5 and Ad5/35 vectors at an m.o.i. of 5 and 50 pfu/cell. The percentage of GFP-expressing cells was analyzed 24 h after infection by flow cytometry.

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Ad5/35 vector biodistribution after intramuscular (im) injection

To study the impact of CD46 signaling on the host's immune response to Ad5/35 or to an antigen expressed from Ad5/35 vectors, we performed the following studies in both C57 and C57-CD46 mice and also included Ad5 vectors (that do not interact with CD46) as controls. As a standard route for vaccination, we applied Ad5/35 vectors im. We assessed biodistribution of transgene expression on organ sections 3 days after im vector injection (Fig. 2A). While Ad5.GFP efficiently transduced myocytes around the injection site in C57 and C57-CD46 mice, Ad5/35 transduced muscle cells in C57-CD46 mice but not in C57 mice. As expected we found Ad5 transduction in livers and spleens of both C57 and C57-CD46 mice at higher levels than Ad5/35 transduction. Interestingly, transduction of splenic and hepatic cells of C57-CD46 mice with Ad5/35 was slightly less efficient than in C57 mice. Furthermore, Ad5/35 appeared to transduce cells in the inguinal lymph nodes (adjacent to the injection site) in C57-CD46 mice. Ad5/35 transduction of mesenteric and lumbar lymph node cells was not detectable (data not shown). Overall, the amounts of viral genomes present in analyzed organs correlated with the observed transduction efficiency based on -galactosidase expression (Fig. 2B). Although we saw no significant Ad5/35-mediated -Gal expression in muscles of C57 mice, Ad5/35 genomes were detected. The observation that Ad5/35 transduction of liver, lung, and spleen was less efficient in C57-CD46 mice than in C57 mice was corroborated by the genome distribution data. We speculate that a large portion of im-injected Ad5/35 particles is taken up or sequestered by CD46 transgenic muscle cells, while in C57 mice (without apparent Ad5/35 muscle transduction), more Ad5/35 particles leak into the circulation. This speculation is supported by the finding that more Ad5/35 viral particles can be found in CD46 transgenic muscles around the injection site than in muscles from nontransgenic mice (Fig. 2C). Clearly, questions such as whether the interaction between Ad5/35 and CD46 on tissues changes the kinetics of viral clearance from the blood circulation or influences viral degradation have to be further studied. Importantly, upon im injection into C57-CD46 mice, Ad5/35 transduced myocytes and lymph nodes, which potentially allows for efficient antigen presentation.

Figure 2.

Biodistribution of Ad-mediated transgene expression and vector genomes in C57 and C57-CD46 mice after intramuscular injection of Ad5.GFP and Ad5/35.GFP. (A) GFP expression in organ sections 72 h after intramuscular injection of 5 10⁹ pfu of Ad5.GFP and Ad5/35.GFP. Tissue sections were analyzed for GFP expression by immunohistochemistry with anti-GFP antibodies. (B) Quantitative comparison of viral genomes present in major organs at 72 h after intramuscular Ad injection. The genome concentration was expressed as the number of viral genomes per 10⁷ cells (assuming that the mass of a diploid human genome is 6 pg). qPCR results for vector genomes were equalized based on qPCR data for an endogenous (two copies/genome) mouse GAPDH gene. The averages of three independent tissue samples are shown. Standard deviation was less than 10%. (C) Detection of Ad5/35 particles around the injection site. Mice were injected im with 5 10⁹pfu Ad5/35.GFP in 40 l of PBS. One hour after injection, mice were sacrificed. Sections (8 m) of muscle tissue around the injection site were analyzed by immunofluorescence. Viral particles were detected using anti-Ad5 hexon–FITC antibodies (green). Muscle cells were stained with anti-laminin antibodies (red). Cell nuclei are blue.

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Innate immune responses after im Ad injection

In vivo application of Ad5 vectors results in the release and expression of proinflammatory cytokines, which in turn can cause hepatotoxicity, increased vascular permeability, leukocyte infiltration into sites of infection, and other symptoms of acute inflammation. On the other hand, increased levels of proinflammatory cytokines and chemokines are thought to contribute to the so-called "adjuvant" effect of Ad vectors in vaccination studies²⁰. To assess the properties of Ad5/35 as a vaccination vector, we measured serum proinflammatory cytokine and chemokine levels upon im injection of Ad vectors (Fig. 3). In preliminary studies, we found that peak levels occurred at 6 h postinfusion (data not shown), in accordance with levels seen after iv injection into mice or baboons^5,21. Compared to animals injected with an Ad5 vector, im injection of Ad5/35 vectors into C57 and C57-CD46 mice caused less elevation of proinflammatory cytokines (IL-6, TNF-, IFN-) and chemokines (MCP-1). For Ad5/35-injected animals, there was no significant difference between the levels in C57 and C57-CD46 mice, indicating that CD46 is not involved in Ad uptake into cytokine-producing cells. This is in agreement with earlier studies in which we found that Ad uptake into Kupffer cells is mediated by blood factors and that the latter process is inefficient for Ad vectors containing short-fiber shafts, such as Ad5/35^21,22.

Figure 3.

Innate immune responses upon im injection of Ad5/35 into mice. Plasma levels of MCP-1, TNF-, IFN-, and IL-6 at 6 h after im injection of Ad vectors are shown. Mice were injected with PBS (Mock) or with 5 10⁹ pfu of Ad5.GFP or Ad5/35.GFP. Plasma samples from three individual mice per virus were collected and analyzed in duplicate by cytometric bead array for cytokine and chemokine levels.

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Assessment of the immune status after Ad5/35 injection

In an attempt to assess the host's immune response to Ad5/35 vectors, we studied two types of T-cell-dependent inflammatory reaction: delayed type hypersensitivity (DTH) to keyhole limpet hemocyanin (KLH) mediated by CD4⁺ T cells and contact hypersensitivity (CHS) induced by epicutaneous exposure to the hapten dinitrofluorobenzene (DNFB) and mediated by CD8⁺ T cells. Six hours after Ad5 and Ad5/35 injection into C57 and C57-CD46 mice, we injected the animals with DNFB or KLH. We measured ear and foot pad thickness upon rechallenge with the hapten (Fig. 4A). After rechallenge, we saw no Ad5/35-mediated inhibition of CHS and DTH responses.

Figure 4.

Absence of immunosuppression upon Ad5/35 infection. (A) CD4- and CD8-mediated hypersensitivity reactions. C57 or CD57-CD46 mice were injected intramuscularly with saline or 5 10⁹ pfu of Ad5.GFP or Ad5/35.GFP. Top: Contact hypersensitivity. Six hours after virus injection mice were sensitized with dinitrofluorobenzene. Six days later, mice were challenged and the ear thickness was measured before challenge and 24 and 48 h after challenge. Bottom: Delayed-type hypersensitivity. Six hours after virus injection, mice were sensitized with KLH. Six days later, mice were challenged and the foot pad thickness was measured before challenge and 48 h after challenge. To assess potential long-term effects of Ad injection, mice were rechallenged with KLH and foot pad thickness was measured before and after rechallenge (N = 3). (B) Analysis of regulatory T cells. C57 or CD57-CD46 mice were injected with 5 10⁹ pfu of Ad5.GFP or Ad5/35.GFP. Seven days later splenocytes were harvested and analyzed by flow cytometry for the percentage of CD4⁺CD25⁺ and FoxP3⁺ cells (N = 3).

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Kemper et al. reported that coengagement of CD3 and CD46 in the presence of IL-2 can induce regulatory T cells (Treg's), which potentially can suppress adaptive immune responses or induce tolerance²³. Human and murine Treg's are CD4⁺CD25⁺ and express a number of other markers including Forkhead P3 (FoxP3). We therefore analyzed whether Ad5/35 interaction with CD46 can induce Treg's in mice. We injected C57 and C57-CD46 mice with 5 10⁹ pfu of Ad5.GFP or Ad5/35.GFP and 7 days later analyzed cell suspensions from spleen and draining lymph nodes by flow cytometry for the presence of CD4⁺CD25⁺ and FoxP3⁺ cells (Fig. 4B). There were no significant differences between the experimental groups, indicating that Ad5/35 does not increase the frequency of Treg's in CD46 transgenic mice.

T cell response against a model antigen and Ad5/35 vector particles

To study the ability of Ad5/35 vectors to induce an immune response against a test antigen, we constructed Ad5 and Ad5/35 vectors that expressed HBeAg under the control of the CMV promoter (which is known to be active in DCs). HBeAg is the secreted form of the hepatitis B core antigen and is considered to be a target for immunotherapy of HBV-associated hepatocarcinoma²⁴.

We confirmed HBeAg expression upon in vitro infection of DCs from C57-CD46 mice by immunoprecipitation/Western blot analysis (data not shown). We mock-injected C57 and C57-CD46 mice or im injected them with 5 10⁹ pfu of Ad5.HBeAg or Ad5/35.HBeAg. Three weeks later, we isolated splenocytes and analyzed the frequency of IFN--producing T cells by ELISpot assay upon in vitro sensitization with HBeAg and, as a negative control, HBsAg (Fig. 5A). Injection of Ad5/35.HBeAg into C57-CD46 mice resulted in a significantly higher frequency of HBeAg-specific T cells, compared to all other groups (60 vs <15 spots per 10⁶ peripheral blood mononuclear cells (PBMCs), P < 0.05). We also determined the frequency of anti-Ad5-specific T cells in splenocytes of test mice (Fig. 5A, right). For in vitro sensitization we infected splenocytes with Ad5.GFP at an m.o.i. of 500 vp/cell. All Ad-injected animals, regardless of the Ad vector or genetic background, demonstrated a high frequency of anti-Ad T cells, which was 5- to 10-fold higher than the frequency of HBeAg-specific T cells. Furthermore, we used intracellular cytokine (IFN-) staining to assess whether anti-HBeAg T cell responses observed in ELIspot assays were CD4⁺ or CD8⁺ mediated. Fig. 5B shows that Ad5/35.HBeAg injection into C57-CD46 mice induces both antigen-specific CD4 and CD8 responses.

Figure 5.

Analysis of frequencies of IFN--producing T cells specific to a model antigen or adenovirus vector. (A) ELIspot analyses of IFN--producing T cell frequencies. C57 or C57-CD46 mice were injected intramuscularly with 5 10⁹ pfu of Ad5.HBeAg, Ad5/35.HBeAg, or PBS. Twelve days later, spleen cells of naïve syngeneic animals were obtained and pulsed with 5 g/ml recombinant HBeAg and HBsAg (as a negative control) (left) or transduced with 25 pfu (=500 viral particles) of Ad.5GFP (right). On day 14, vaccinated animals were sacrificed, splenocytes were collected, and 1 10⁶ cells were mixed with 1 10⁶ ex vivo-pulsed splenocytes for in vitro sensitization. After 24 h of incubation in 96-well plates, cells were plated in anti-IFN--coated wells of an ELIspot plate. Twenty-four hours later, plates were washed and the spots of IFN--producing T cells were counted. The number of spots was expressed as the average the standard deviation. N = 3 animals per group. (B) Frequency of IFN-⁺CD4⁺ cells (left) and IFN-⁺CD8⁺ cells (right) analyzed by intracellular cytokine staining. C57-CD46 mice were injected intramuscularly with 5 10⁹ pfu of Ad5/35.HBeAg or PBS. Twelve days after immunization, splenocytes were harvested and cultured with recombinant HBeAg and (control) HBsAg for 5 h in the presence of brefeldin. Cells were then stained for CD4 and CD8 and intracellular IFN-. Shown is the percentage of IFN-⁺CD4⁺ cells in all CD4⁺ cells and IFN-⁺CD8⁺ cells in all CD8⁺ cells (N = 3).

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Memory anti-Ad T cell responses in humans

A potential problem for the application of Ad vectors (including Ad5/35) for induction of T cell responses, particularly against relatively weak antigens such as tumor-associated antigens, is the high immunogenicity of Ad vectors, resulting in induction of anti-Ad T cells at a frequency that potentially might affect the T cell response to the vaccination antigen. The latter may be even more problematic in humans as a result of preexisting anti-Ad T memory responses²⁵. To illustrate this, we analyzed the frequencies of IFN--producing Ad-specific T cells in human PBMCs from eight donors (Fig. 6). In this study, we compared E1/E3-deleted Ad5/35 vectors (which are known to express viral proteins in transduced cells) with helper-dependent Ad5/35 vectors (which are devoid of all viral genes). Since most T cells are thought to be directed against immunodominant Ad5 hexon epitopes²⁵, Ad5/35 vectors (containing Ad5 hexons) will probably induce the same T cell responses. In our study, we found strong T cell responses in all samples upon infection of human PBMCs with Ad5 and Ad5/35 vectors. The responses to the first-generation Ad5/35 vectors were comparable to those induced by the HD-Ad5/35 vector. The latter indicates a response to Ad capsid epitopes from the incoming particles. Frequencies of anti-Ad11 IFN--producing T cells were comparable to those seen for the other vectors. Previous studies reported memory T cell responses against Ad hexon protein^25,26,27,28. Using an experimental approach similar to that described for Fig. 6 and recombinant Ad5 and Ad35 fiber knob domains for pulsing of PBMCs, we found memory T cell also against the fiber knob domains, whereby the frequency of anti-Ad35 knob T cells was significantly less than that of Ad5 knob-specific T cells (Fig. 7A). Another experiment with recombinant fiber knobs suggested that Ad5 and Ad35 knob-specific T cells did not cross-react (Fig. 7B). This was not surprising considering that the homology between Ad5 and Ad 35 fiber knobs is less than 30% (on the amino acid level).

Figure 6.

Analysis of memory T cells against Ad vectors in human PBMCs by ELIspot for IFN--expressing T cells. PBMCs were obtained from normal donors that have never done research with Ad vectors, from normal donors that have worked with Ad vectors, and from Senegalese Stage III breast cancer patients (before chemotherapy). Cells were transduced with the indicated Ad vectors at an m.o.i. of 200 viral particles/cell and the number of IFN--expressing T cells was analyzed by ELIspot 24 h later (N = 3).

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Figure 7.

Cross-reactivity of anti-fiber knob T cells. (A) Memory T cells against fiber knob domains. PBMCs from two normal donors that are involved in adenovirus research were incubated with 0.2 g/ml recombinant Ad5 or Ad35 fiber knob domains (produced in Escherichia coli) or control peptide (HBeAg) and the frequency of IFN--expressing T cells was analyzed by ELIspot 24 h later (N = 3). (B) Cross-reactivity of T cells against fiber knobs. T cells were primed by incubating PBMCs from a healthy donor with recombinant Ad5 or Ad35 fiber knob. Subsequently T cells were incubated for 7 days in the presence of IL-2 and then boosted with dendritic cells pulsed with control peptide (Co), Ad5, or Ad35 fiber knob. The frequency of IFN--expressing T cells was analyzed by ELIspot 24 h later (N = 3).

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In summary, our data indicate that, compared to Ad5 vectors, Ad5/35 has a better safety profile (less induction of proinflammatory cytokines) and induces stronger CD4 and CD8 T cell responses to a model antigen (which is most likely due to a more efficient transduction of APCs). Hyposensitivity assays as well as analysis of Treg and T cell responses did not indicate that Ad5/35 injection induces immunosuppression in CD46 transgenic mice. Several explanations might account for the discrepancies between the reports listed above and our data. (i) The binding domains of CD46 ligands within CD46 used in the study by Marie et al. are different (C3b, anti-CD46 antibodies) or only partially overlap (measles H) with the Ad35 binding domain²⁹, which might have different downstream effects in terms of signaling. (ii) Ad5/35 uses CD46 for internalization, while the other ligands do not, which again might result in different intracellular signaling. In agreement with our results in C57-CD46 mice, a recent study in baboons demonstrated no suppression of host immune responses upon Ad5/35 vector injection^12,16. (iii) Immunosuppression by measles virus and wt Ad35 might be the result of viral replication in and cytolysis of infected cells, which does not occur with the first-generation vectors used in our model.

In this study we also showed that Ad5, as well as Ad5/35, vectors induce a strong anti-Ad T cell response that is at least an order of magnitude higher than the anti-HBeAg response. Considering the concept of competition between T cell clones and space in the thymus³⁰, this might be problematic if T cell responses against weak antigens such as tumor-associated antigens have to be induced. In an attempt to address this problem we tested a vector derived from another Ad serotype (Ad11) with less seroprevalence of neutralizing antibodies in the human population; however, we found the same T cell responses in humans. This is probably due to cross-reactivity of T cells with Ad serotypes, specifically to immunodominant hexon epitopes, which are conserved among Ad serotypes³¹. To address this problem, we are currently attempting to modify immunodominant epitopes within the Ad capsid. Notably, the immunodominance of Ad antigens might not be as problematic if T cell responses against strong antigens such as HIV antigens to be raised.

Materials and methods

Cells

PBMCs from normal donors were obtained after leukapheresis and provided by Dr. Nora Disis (Tumor Vaccine Group, Department of Oncology, University of Washington, Seattle, WA, USA). PBMCs from Senegalese cancer patients were purified from heparinized blood by density gradient centrifugation using Ficoll–Hypaque gradients. To obtain monocyte-derived DCs, PBMCs were resuspended in Iscove modified Dulbecco medium (Invitrogen, Carlsbad, CA, USA) and plated in 6-well plates at 1 10⁷ cells/well for 1 h. After 1 h, nonadherent cells were washed off and AIMV medium containing 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 800 U/ml GM-CSF (Avigen, Seattle, WA, USA), and 500 IU/ml interleukin-4 (R&D Systems, Minneapolis, MN, USA) was added. Cells were left for 5–7 days and medium was replaced on day 3. Human CD34⁺ peripheral blood-derived cells were provided by Dr. Shelly Heimfeld (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). Standard protocols for ex vivo differentiation of CD34⁺ progenitors into DCs with GM-CSF, IL-4, flt3-ligand, and TNF- were used. To obtain mouse DCs from bone marrow, mouse bone marrow cells were cultured in RPMI 1640, 50 M -mercaptoethanol, 10% inactivated FCS, and 200 U/ml rmGM-CSF for 8 days. On the day of the experiment, 1 10⁵ DCs were plated in 24-well plates and incubated with the indicated viruses overnight. At 24 h postinfection, GFP expression was assessed by flow cytometry.

Viruses

Ad5.GFP and Ad5/35.GFP are first-generation E1/E3-deleted adenovirus vectors expressing GFP³². Ad5.HBeAg and Ad5/35.HBeAg express the HBV core antigen under the control of the CMV promoter³³. Ad11.GFP is an E1-deleted Ad serotype 11 vector, expressing GFP under the control of the CMV promoter³⁴. HD-Ad5/35.GFP is a helper-dependent (HD) Ad5/35 vector expressing GFP under the control of the CMV promoter³⁵. The contamination with helper virus genomes in the HD-Ad5/35.GFP preparation used in this study was less than 0.1%. Viral vectors were produced and propagated following standard procedures³⁶. Viruses were banded in CsCl gradients, dialyzed, and stored in aliquots as described previously. Titers were determined by plaque titration in 293 cells as described³⁶ and the contamination level of wild-type (wt) virus was examined by real-time PCR as described³². For all first-generation Ad5 and Ad5/35 vectors used in this study, the ratio of viral particles to pfu was 20:1. Only vector preparations that contained less than 1 wt viral genome in 10⁶ genomes were used in these studies. All vectors were free of endotoxin.

Recombinant fiber knob domains

Ad5 and Ad35 knobs were produced in E. coli and purified as described elsewhere³⁷. The knob domains were dialyzed against 5 mM KCl, 10% glycerol, 10 mM MgCl₂ and stored at -20°C at a stock concentration of 5 mg/ml.

sCD46 competition assays

sCD46 expression plasmid (10 g; courtesy of J. P. Atkinson) was transfected onto 293-T cells. The supernatant was collected 72 h posttransfection. This supernatant was diluted with fresh growth medium 1:1 and incubated with virus in a volume of 200 l at room temperature for 1 h before being added to cells for 30 min at 4°C. Cells were washed once, plated, and examined 24 h later for GFP expression by flow cytometry.

Mice

All experiments involving animals were conducted in accordance with the institutional guidelines set forth by the University of Washington. All mice were housed in specific-pathogen-free facilities. C57Bl/6 mice ("C57") were obtained from Charles River (Wilmington, MA, USA). The CD46 transgenic C57Bl/6 mouse line MCP8B (C57-CD46) has been described elsewhere¹⁸. (This line has been crossed into the C57Bl/6 background for at least nine generations.) These mice express CD46 at levels similar to those in human cells. The transgene is a CD46 C1 isoform under the control of the ubiquitously active hydroxymethylglutaryl coenzyme A reductase promoter. CD46 DNA-positive mice were identified by PCR on tail DNA using the following primers: F-CD46, 5'-GGTCAAATGTCGATTTCCAGT-3'; R-CD46, 5'-AATCACAGCAATGACCCAAA-3'. For intramuscular injection, Ad vectors in 50 l of PBS were injected into the hamstring muscle.

Quantitative PCR (qPCR) for vector genomes

Three days after virus injection, mice were anesthetized, the heart was cannulated, and mice were exsanguinated and flushed with 20 ml PBS. Tissue samples were harvested under conditions that minimize cross-contamination. Organ samples were processed for histological analyses and snap frozen in liquid nitrogen. DNA was subsequently extracted from snap-frozen tissues and analyzed for viral DNA using primers specific for viral DNA. Samples were analyzed using a Lightcycler and the SYBR green kit, and the following primers were used for viral DNA: F-L4, 5'-TGCAAGATACCCCTATCCTG-3'; R-L4, 5'CCTGTTGCAGAGCGTTTGC-3'. Purified Ad DNA was used as external standards. Samples were equalized for DNA input using control primers against mouse GAPDH: F-mGAPDH, 5'-ATCACTGCCACCCAGAAGAC-3'; R-mGAPDH, 5'-CACATTGGGGTAGGAACAC-3'.

Histological analyses

GFP expression was visualized on paraffin sections by immunohistochemistry using a mouse anti-GFP antibody (1:200; Clontech, BD Biosciences, San Diego, CA, USA). Binding was visualized using the ABC method (Vector Laboratories, Burlingame, CA, USA). Ad particles were detected on muscle sections using goat anti-Ad5 hexon–FITC antibodies (Chemicon, Temecula, CA, USA). Muscle cells were stained with rabbit anti-laminin (DAKO, Denmark) and goat anti-rabbit–AlexaFluor568 (Molecular Probes, Eugene, OR, USA). Cell nuclei were stained with DAPI (Sigma, St. Louis, MO, USA).

Cytokines

Serum samples were analyzed by Cytometric Bead Array, using a Mouse Inflammatory Cytometric Bead Array (BD Biosciences, Palo Alto, CA, USA) as described elsewhere²¹.

Hypersensitivity assays

CHS and DTH were analyzed 6 h after virus injections. For CHS, DNFB was diluted in acetone:olive oil (4:1) before use, and 25 l of 0.5% DNFB solution was applied to a shaved ventral skin area (sensitization phase). After 6 days, mice received 10 l of a nonirritant concentration of DNFB applied on both sides of the left ear and the solvent alone on the right ear (effector phase). Ear thickness was monitored before challenge and 24 and 48 h after challenge, in a blinded study. DTH response to KLH was measured by a conventional foot-pad swelling assay. Mice were sensitized by subcutaneous injection with 200 l of 300 g KLH emulsified with complete Freund's adjuvant (1:1, v/v). After 6 days, mice were challenged by subcutaneous injection in the left hind foot pad with 150 g of KLH diluted in PBS. The right foot pad was injected with PBS alone. Foot-pad thickness was measured before challenge and after challenge.

ELIspot assay

C57 or C57-CD46 mice were intramuscularly injected with 5 10⁹ pfu of Ad5.HBeAg, Ad5/35.HBeAg, or saline. Twelve days later, spleen cells of naïve syngeneic animals were obtained and pulsed with 5 g/ml recombinant HBeAg or HBsAg or transduced with 500 viral particles/cell (25 pfu/cell) of Ad5.GFP. On day 14, vaccinated animals were sacrificed, their spleen cells were collected, and 1 10⁶ cells were mixed with 1 10⁶ ex vivo-pulsed splenocytes for in vitro sensitization. After 24 h of incubation in 96-well plates, cells were plated at a concentration of 10⁵ cells/100 l into wells of 96-well hydrophobic polyvinylidene difluoride-backed plates (Millipore, Bedford, MA, USA), previously coated with 50 l of 10 g/ml anti-IFN- monoclonal antibody (1-D1K, mouse immunoglobulin G1; Mabtech, Nacka, Sweden) overnight at 4°C. As a positive control, phytohemagglutinin (Sigma) was added to 2 wells at a final concentration of 1 g/ml. All responses were tested in triplicate. Plates were incubated overnight, washed with phosphate-buffered saline containing 0.05% Tween 20, and incubated at room temperature for 2 h with biotinylated anti-IFN- monoclonal antibody at 1 g/ml (7-B6-1, mouse immunoglobulin G1; Mabtech). Binding was developed using the Vectastain ABC Elite kit (PK-6100; Vector Laboratories).

T cell cross-reactivity to fiber knobs

Human PBMCs were incubated with Ad5 or Ad35 fiber knob (1 g/ml) or PBS. Upon pulsing T cells were separated and expanded for 7 days in the presence of 5 ng/ml IL-2. Simultaneously, DC were derived from peripheral monocytes as described elsewhere³⁴. DC were incubated with 0.2 g/ml Ad5 or Ad35 fiber knobs or control peptide. A total of 5 10³ DCs were mixed with 5 10⁴ T cells and incubated for 24 h for IFN- ELIspot assays.

Intracellular cytokine staining

Splenocytes from immunized animals were incubated with 5 g/ml recombinant HBeAg or HBsAg protein for 5 h in 96-well U-bottom plates with 10⁶ cells/well in the presence of 50 U/ml human recombinant IL-2 and 1 l/ml brefeldin A (Golgistop; Pharmingen, La Jolla, CA, USA) (to block the intracellular transport processes of cytokine proteins in the endoplasmic reticulum or Golgi complex). After incubation, the cells were washed and surface-stained with an FITC-conjugated rat anti-mouse CD8 or CD4 antibody (Pharmingen) followed by fixation/permeabilization (Cytofix/Cytoperm, Pharmingen) and intracellular staining for IFN- (PE-conjugated rat anti-mouse IFN-; Pharmingen) according to the manufacturer's instructions. After being washed, cells were examined by two-color flow cytometry and CellQuestPro software. Percentage of responding cells was calculated counting a minimum of 50,000 events (CD8⁺ or CD4⁺ T cells).

Analysis of Treg's

Treg analysis was performed as described elsewhere³⁸. Briefly, splenocytes were analyzed by flow cytometry using the following antibodies: rat Mab anti-FoxP3-PE (clone FHK16s; eBioscience), rat Mab anti-CD4-PE (Pharmingen), and rat Mab anti-CD25-FITC (eBioscience). All samples were treated with Fc-block (CD16/CD32). Corresponding isotope controls yielded no significant staining.

References

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Acknowledgements

We thank Steve Roffler for critical discussions. We are grateful to Si-Yi Chen, Nora Disis, and Shelly Heimfeld for providing valuable material. This study was supported by NIH Grants CA080192, HLA078836, and HL-00-008 and grants from the Doris Duke and Avon Charitable Foundations.