Ad5 Fiber Research Articles

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1.

The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting sigma1 protein of reovirus type 3 Dearing (T3D). A sigma1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dsigma1 forms trimers and assembles onto Ad virions. Fibtail-T3Dsigma1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dsigma1, expresses Fibtail-T3Dsigma1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dsigma1, and their combined effect decreased transduction by 95%. Ad5-T3Dsigma1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dsigma1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.

Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis.

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells.

A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)-based vector-mediated gene transfer into malignant hematopoietic cells. Fiber-retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells. For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the PGK promoter with Ad35 fiber receptor specificity was generated (Ad5F35-GFP). The Ad5F35-GFP vector-mediated gene transfer efficiency was compared with a fiber non-modified Ad5-GFP vector, which also encodes the GFP gene under the control of the PGK promoter. We demonstrated that a variety of Ad5-refractory malignant myeloid and B lymphoid cell lines were highly permissive to the Ad5F35-GFP vector infection. Importantly, primary chronic myeloid leukemic (CML) cells and chronic lymphocytic leukemia (CLL) B cells were superiorly transduced by the Ad5F35-GFP vector at a multiplicity of infection (MOI) of 100 compared with the Ad5-GFP vector. Our study will facilitate the generation of fiber-retargeted adenoviral vectors and enable transient genetic manipulation of primary malignant hematopoietic cells.

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes

Most adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 ( Aviadenovirus), DAdV-1, and BAdV-4 ( Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy.

Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines.

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge.

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.

Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector

We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector.

Oncolytic Adenovirus: Getting There Is Half the Battle

Oncolytic Adenovirus: Getting There Is Half the Battle

Genetically targeted adenovirus vector directed to CD40-expressing cells.

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.

General strategy for broadening adenovirus tropism.

In spite of its broad host range, adenovirus type 5 (Ad5) transduces a number of clinically relevant tissues and cell types inefficiently, mostly because of low expression of the coxsackievirus-adenovirus receptor (CAR). To improve gene transfer to such cells, we modified the Ad5 fiber knob to recognize novel receptors. We expressed a functional Ad5 fiber knob domain on the capsid of phage lambda and employed this display system to construct a large collection of ligands in the HI loop of the Ad5 knob. Panning this library on the CAR-negative mouse fibroblast cell line NIH 3T3 resulted in the identification of three clones with increased binding to these cells. Adenoviruses incorporating these ligands in the fiber gene transduced NIH 3T3 cells 2 or 3 orders of magnitude better than the parent vector. The same nonnative tropism was revealed in other cell types, independently of CAR expression. These Ad5 derivatives proved capable of transducing mouse and human primary immature dendritic cells with up to 100-fold increased efficiency.

Identification of Adenovirus (Ad) Penton Base Neutralizing Epitopes by Use of Sera from Patients Who Had Received Conditionally Replicative Ad (Ad dl 1520) for Treatment of Liver Tumors

Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.

Flexibility of the adenovirus fiber is required for efficient receptor interaction.

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

An adenoviral vector containing an arg-gly-asp (RGD) motif in the fiber knob enhances protein product levels from transgenes refractory to expression.

Genetic manipulation of the adenovirus type 5 represents one strategy to modify viral transduction properties in vitro and in vivo. In the majority of studies to date, reporter gene activity has been monitored to assess transduction efficiency. BRCA1 is a gene whose protein product is clinically important, biologically toxic, difficult to overexpress, and difficult to detect as an untagged protein species. Thus, it represents an attractive candidate from which to evaluate the efficacy of a gene delivery system. In the present study, transgene expression was assessed employing otherwise isogenic viruses, which differed only in the presence or absence of an RGD integrin-binding motif in the HI loop of the Ad fiber knob. We utilized a combination of BRCA1 expression level comparisons among several human BRCA1/mutant BRCA1/murine Brca1 constructs and reporter gene activity following transduction of a panel of human breast and ovarian tumor cell lines representative of both sporadic and hereditary cases. A general overall concordance in efficiency was observed, whether the biological readout measured was reporter gene activity or steady-state level of ectopic BRCA1 protein produced. Importantly, the expression of full-length wild-type BRCA1 protein, clinically relevant mutant BRCA1 proteins or murine Brca1 was superior when the gene was delivered via the RGD-modified Ad. The ectopic BRCA1 stabilized endogenous BARD1 and this functional effect was evident at lower input viral doses when BRCA1 was delivered via the RGD-modified Ad. Quantitative, noninvasive, real-time image analysis of reporter gene function in nude mice harboring human ovarian tumor xenographs demonstrated a similar enhancement of expression in vivo by the RGD fiber modification, with low levels of transduction of normal mouse mesothelium. These results provide additional evidence supporting the concept that rational modification of viral vectors can result in the delivery of functionally active therapeutic proteins such as BRCA1 that present with technical difficulties with regard to their expression.

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector.

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34(+) cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.

Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice.

Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.

Binding of adenoviral fiber knob to the coxsackievirus-adenovirus receptor is crucial for transduction of fetal muscle.

Adenoviral (Ad) infection involves attachment mediated by the Ad fiber protein binding to the coxsackievirus-adenovirus receptor (CAR) of a target cell and internalization facilitated by the interaction of the Ad penton base protein with alpha(v) integrins. To understand the relative importance of the Ad binding and internalization steps for the transduction of fetal skeletal muscle, we used a panel of genetically modified vectors that specifically ablate the fiber-CAR interaction (AdL.F*), the penton base-alpha(v) integrin interaction (AdL.PB*), or both (AdL.PB*F*) to transduce embryonic day 16 (E-16) mouse muscle in vivo and primary E-16 muscle cells in vitro. Quantification of transgene expression and vector genome copies revealed a striking absence of E-16 muscle transduction by AdL.F* and AdL.PB*F*. In contrast, fetal muscle transduction with AdL.PB* was not significantly different than with the unmodified vector. Similar results were observed with in vitro Ad infection studies in primary E-16 muscle cells. From these data we conclude that the fiber-CAR interaction is important for the transduction of fetal muscle by Ad vectors. The high dependence on fiber-CAR binding will impact the development of strategies for Ad vector retargeting to achieve muscle-specific transduction in utero.

The interaction between the fiber knob domain and the cellular attachment receptor determines the intracellular trafficking route of adenoviruses.

Most of the presently used adenovirus (Ad) vectors are based on serotype 5. However, the application of these vectors is limited by the native tropism of Ad5. To address this problem, a series of fiber chimeric vectors were produced to take advantage of the different cellular receptors used by Ad of different subgroups. In this study we utilize an Ad5-based chimeric vector containing sequences encoding the Ad35 fiber knob domain instead of the Ad5 knob (Ad5/35L) to analyze factors responsible for selection of intracellular trafficking routes by Ads. By competition analysis with recombinant Ad5 and Ad35 knobs we showed that the Ad5/35L vector infected cells through a receptor different from the Ad5 receptor. Intracellular trafficking of Ad5 and Ad5/35L viruses was analyzed in HeLa cells by tracking fluorophore-conjugated Ad particles, by immunostaining for capsid hexon protein, by electron microscopy, and by Southern blotting for viral DNA. These studies showed that the interaction with the Ad35 receptor(s) predestines Ad5/35L vector to intracellular trafficking pathways different from those of Ad5. Ad5 efficiently escaped from the endosomes early after infection. In contrast, Ad5/35L remained longer in late endosomal/lysosomal compartments and used them to achieve localization to the nucleus. However, a significant portion of Ad5/35L particles appeared to be recycled back to the cell surface. This phenomenon resulted in significantly less efficient Ad5/35L-mediated gene transfer compared to that of Ad5. We also demonstrated that the selection of intracellular trafficking routes was determined by the fiber knob domain and did not depend on the length of the fiber shaft. This study contributes to a better understanding of the mechanisms that govern the infection of retargeted, capsid-modified vectors which have potential application for hematopoietic stem cell and tumor gene therapy.

CD40-targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain Fv antibody enhances cytotoxic T cell activation

Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially expressed bispecific conjugate, consisting of a fusion of recombinant single-chain (sc) mAb Fv fragments, which bind and neutralize the Ad fiber knob (through the S11 mAb scFv) and retarget Ad to CD40 on the DC surface (through the G28-5 mAb scFv). We show that this bispecific scFv fusion protein significantly enhances transduction efficiency of monocyte-derived DC (MoDC), reduces the amount of virus needed for a given level of transduction, and increases the ability of MoDC to activate CTL in an antigen specific manner. This single-component conjugate may prove to be a valuable immunotherapeutic tool for the targeting of Ad to DC in vivo.

Generation of an adenoviral vector containing an addition of a heterologous ligand to the serotype 3 fiber knob.

As an initial assessment of the feasibility of employing the adenovirus serotype 3 (Ad3) fiber knob as a locale for introducing a tropism-modifying motif, we generated an adenoviral vector containing a six-histidine tag genetically fused to the carboxy-terminus of the Ad3 fiber knob. The heterologous tag proved to be accessible for binding in the context of the virion and, moreover, had rendered the modified vector capable of mediating gene transfer through an artificial, non-Ad3 receptor.