Fiber Shaft Research Articles

Development of an AdEasy‐based system to produce first‐ and second‐generation adenoviral vectors with tropism for CAR‐ or CD46‐positive cells

The AdEasy system has acquired preeminence amongst the various methods for producing first-generation, early region 1 (E1)-deleted human adenovirus (HAdV) vectors (AdVs) as a result of the fast and reproducible recovery of full-length AdV genomes via homologous recombination in Escherichia coli. From the classical AdEasy system, a new production platform was derived to assemble first- and second-generation [i.e. E1- plus early region 2A (E2A)-deleted] AdVs displaying on their surface HAdV serotype 5 (HAdV5) fibers (F5) or chimeric fibers (F5/50) comprising the tail of F5 and the fiber shaft and knob of HAdV serotype 50 (HAdV50). The CD46-interacting chimeric fibers allow for the high-level transduction of various human primary cell types of clinical interest with low or no surface expression of the Coxsackievirus and adenovirus receptor. A new set of pAdEasy plasmid 'backbones' with or without E2A and encoding F5 or F5/50 was constructed and recombined in E. coli strain BJ5183 with a 'shuttle' plasmid coding for β-galactosidase. The resulting clones yielded AdV preparations with similar high titers following their rescue and propagation in producer cells. The AdVs with F5/50 were superior to those carrying F5 with respect to transducing human skeletal myocytes and mesenchymal stem cells. In the present study, an AdEasy system tailored for the production of not only first-, but also second-generation AdVs equipped with the receptor-interacting fiber domains of the prototypic species C HAdV5 or of the species B member HAdV50 is presented. This system expands the range of applications for this robust and versatile AdV production platform.

Conserved Fiber-Penton Base Interaction Revealed by Nearly Atomic Resolution Cryo-Electron Microscopy of the Structure of Adenovirus Provides Insight into Receptor Interaction

Adenovirus (Ad) cell attachment is initiated by the attachment of the fiber protein to a primary receptor (usually CAR or CD46). This event is followed by the engagement of the penton base protein with a secondary receptor (integrin) via its loop region, which contains an Arg-Gly-Asp (RGD) motif, to trigger virus internalization. To understand the well-orchestrated adenovirus cell attachment process that involves the fiber and the penton base, we reconstructed the structure of an Ad5F35 capsid, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber, at a resolution of approximately 4.2 Å. The fiber-penton base interaction in the cryo-electron microscopic (cryo-EM) structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5, indicating that the fiber-penton base interaction of adenovirus is conserved. Our structure also confirms that the C-terminal segment of the fiber tail domain constitutes the bottom trunk of the fiber shaft. Based on the conserved fiber-penton base interaction, we have proposed a model for the interaction of Ad5F35 with its primary and secondary receptors. This model could provide insight for designing adenovirus gene delivery vectors.

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated from AIDS patients

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated between 1986 and 1997 from AIDS patients has been performed. Sequencing the L2 part of the hexon gene of 51 adenoviruses isolated between 1986 and 1997 from AIDS patients revealed only one type each from species A and C and two types from species B with all the remaining isolates from species D. Further sequencing and phylogenetic analysis of the fiber knob region of these species D adenoviruses revealed that 28/46 were intermediate strains with conflicting hexon and fiber sequences. When the penton regions of these intermediate strains were sequenced, it became clear that some had originated from a third adenovirus type presumably by intergene recombination events. Evidence from sequencing the L1 hexon and fiber shaft regions showed no evidence of intragene recombination but penton sequences showed that recombination between the hypervariable region (HVR) and RGD regions was common. Six isolates appear to be from three new adenovirus types. Five AIDS patients showed sequential infection with different adenovirus variants and six such variants were isolated from a single patient in 2 years.

Association of RrgA and RrgC into the Streptococcus pneumoniae Pilus by Sortases C-2 and C-3

Pili are surface-exposed virulence factors involved in the adhesion of bacteria to host cells. The human pathogen Streptococcus pneumoniae expresses a pilus composed of three structural proteins, RrgA, RrgB, and RrgC, and requires the action of three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3, to covalently associate the Rrg pilins. Using a recombinant protein expression platform, we have previously shown the requirement of SrtC-1 in RrgB fiber formation and the association of RrgB with RrgC. To gain insights into the substrate specificities of the two other sortases, which remain controversial, we have exploited the same robust strategy by testing various combinations of pilins and sortases coexpressed in Escherichia coli. We demonstrate that SrtC-2 catalyzes the formation of both RrgA-RrgB and RrgB-RrgC complexes. The deletion and swapping of the RrgA-YPRTG and RrgB-IPQTG sorting motifs indicate that SrtC-2 preferentially recognizes RrgA and attaches it to the pilin motif lysine 183 of RrgB. Finally, SrtC-2 is also able to catalyze the multimerization of RrgA through the C-terminal D4 domains. Similar experiments have been performed with SrtC-3, which catalyzes the formation of RrgB-RrgC and RrgB-RrgA complexes. Altogether, these results provide evidence of the molecular mechanisms of association of RrgA and RrgC with the RrgB fiber shaft by SrtC-2 and SrtC-3 and lead to a revised model of the pneumococcal pilus architecture accounting for the respective contribution of each sortase.

Improved systemic antitumor therapy with oncolytic adenoviruses by replacing the fiber shaft HSG-binding domain with RGD

Retargeting oncolytic adenoviruses from their systemic preeminent liver tropism to disseminated tumor foci would highly improve the efficacy of these agents at eradicating tumors. We have replaced the KKTK fiber shaft heparan sulfate glycosaminoglycan-binding domain with an RGDK motif in order to achieve simultaneously liver detargeting and tumor targeting. When inserted into a wild-type backbone, this mutation palliated liver transaminase elevation and hematological alterations in mice. Importantly, when tested in a backbone that redirects E1A transcription towards pRB pathway deregulation, RGD at this novel shaft location also improved significantly systemic antitumor therapy compared with the broadly used RGD location at the HI-loop of the fiber knob domain.

Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon∶FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.

Amyloid-like self-assembly of a dodecapeptide sequence from the adenovirus fiber shaft: Perspectives from molecular dynamics simulations

Peptide and protein self-assembly is related to the fundamental problems of protein folding and misfolding and has potential applications in medicine, materials science and nanotechnology. Sequence repeats from self-assembling proteins may provide useful elementary building blocks of peptide-based nanostructures. Sequences from the adenovirus fiber shaft self-assemble into amyloid-like fibrils outside their native context. In earlier simulations we studied the self-assembly of two shaft sequences, the octapeptide NSGAITIG and the hexapeptide GAITIG. Based on these simulations, cysteine residues were substituted at the first two positions of the octapeptide, yielding amyloid fibrils capable of binding to silver, gold and platinum nanoparticles. Here, we study by implicit-solvent replica-exchange simulations the self-assembly of a longer shaft sequence, the dodecapeptide LSFDNSGAITIG. The simulations provide insights on the molecular organization of the corresponding fibers. Individual molecules tend to adopt hairpin-like conformations in the observed intermolecular β-sheets, in line with the experimentally determined amyloid fiber diameters and the conformation of the peptide in the adenovirus fiber shaft. By analyzing the arrangement of individual peptides in the intermolecular sheets, we suggest possible structural models of the corresponding fibers and interpret their stability by energetic calculations.

Adenoviruses with an αvβ integrin targeting moiety in the fiber shaft or the HI-loop increase tumor specificity without compromising antitumor efficacy in magnetic resonance imaging of colorectal cancer metastases

BackgroundColorectal cancer is often a deadly disease and cannot be cured at metastatic stage. Oncolytic adenoviruses have been considered as a new therapeutic option for treatment of refractory disseminated cancers, including colorectal cancer. The safety data has been excellent but tumor transduction and antitumor efficacy especially in systemic administration needs to be improved.MethodsHere, the utility of αvβ integrin targeting moiety Arg-Gly-Asp (RGD) in the Lys-Lys-Thr-Lys (KKTK) domain of the fiber shaft or in the HI-loop of adenovirus serotype 5 for increased tumor targeting and antitumor efficacy was evaluated. To this end, novel spleen-to-liver metastatic colorectal cancer mouse model was used and the antitumor efficacy was evaluated with magnetic resonance imaging (MRI).ResultsBoth modifications (RGD in the HI-loop or in the fiber shaft) increased gene transfer efficacy in colorectal cancer cell lines and improved tumor-to-normal ratio in systemic administration of the vector.ConclusionsAntitumor potency was not compromised with RGD modified viruses suggesting increased safety profile and tumor specificity.

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

Phylogeny and primary structure analysis of fiber shafts of all human adenovirus types for rational design of adenoviral gene-therapy vectors

The fiber shaft of human adenoviruses (HAdVs) is essential for bringing the penton base into proximity to the secondary cellular receptor. Fiber shaft sequences of all 53 HAdV types were studied. Phylogeny of the fiber shaft revealed clustering corresponding to the HAdV species concept. An intraspecies recombination hot spot was found at the shaft/knob boundary, a highly conserved sequence stretch. For example, HAdV-D20 clustered with HAdV-D23 in the fiber shaft, but with HAdV-D47 in the fiber knob. Although all shafts exhibited the typical pseudorepeats, amino acid sequence identity was found to be as high as 92 % (interspecies) and 54 % (intraspecies). In contrast to a previous study, a flexibility motif (KXGGLXFD/N) was found in eight HAdV-D types, whereas the putative heparan sulfate-binding site (KKTK) was only found in species HAdV-C. Our results suggest that pseudotyping of gene-therapy vectors at the shaft/knob boundary is feasible, but that flexibility data of shafts should be considered.

Chimeric adenoviral vectors incorporating a fiber of human adenovirus 3 efficiently mediate gene transfer into prostate cancer cells

We have developed a range of adenoviral (Ad) vectors based on human adenovirus serotype 5 (HAdV-5) displaying the fiber shaft and knob domains of species B viruses (HAdV-3, -11, or -35). These species B Ads utilize different cellular receptors than HAdV-5 for infection. We evaluated whether Ad vectors displaying species B fiber shaft and knob domains (Ad5F3Luc1, Ad5F11Luc1, and Ad5F35Luc1) would efficiently infect cancer cells of distinct origins, including prostate cancer. The fiber chimeric Ad vectors were genetically generated and compared with the original Ad vector (Ad5Luc1) for transductional efficiency in a variety of cancer cell lines, including prostate cancer cells and primary prostate epithelial cells (PrEC), using luciferase as a reporter gene. Prostate cancer cell lines infected with Ad5F3Luc1 expressed higher levels of luciferase than Ad5Luc1, as well as the other chimeric Ad vectors. We also analyzed the transductional efficiency via monitoring of luciferase activity in prostate cancer cells when expressed as a fraction of the gene transfer in PrEC cells. In the PC-3 and DU145 cell lines, the gene transfer ratio of cancer cells versus PrEC was once again highest for Ad5F3Luc1. Of the investigated chimeric HAdV-5/species B vectors, Ad5F3Luc1 was judged to be the most suitable for targeting prostate cancer cells as it showed the highest transductional efficiency in these cells. It is foreseeable that an Ad vector incorporating the HAdV-3 fiber could potentially be used for prostate cancer gene therapy.

Amyloid-Like Self-Assembly of Peptide Sequences from the Adenovirus Fiber Shaft: Insights from Molecular Dynamics Simulations

The self-assembly of peptides and proteins into nanostructures is related to the fundamental problems of protein folding and misfolding and has potential applications in medicine, materials science and nanotechnology. Natural peptides, corresponding to sequence repeats from self-assembling proteins, may constitute elementary building blocks of such nanostructures. In this work, we study by implicit-solvent replica-exchange simulations the self-assembly of two amyloidogenic sequences derived from the naturally occurring fiber shaft of the adenovirus, the octapeptide NSGAITIG (asparagine-serine-glycine-alanine-isoleucine-threonine-isoleucine-glycine) and its hexapeptide counterpart, GAITIG. In accordance with their amyloidogenic capacity, both peptides form readily intermolecular beta-sheets, stabilized by extensive main- and side-chain contacts involving the C-terminal moieties (segments 3-8 and 2-6, respectively). The structural and energetic properties of these sheets are analyzed extensively. The N-terminal residues Asn1 and Ser2 of the octapeptide remain disordered in the sheets, suggesting that these residues are exposed at the exterior of the fibrils and accessible. On the basis of insight provided by the simulations, cysteine residues were recently substituted at positions 1 and 2 of NSGAITIG; the newly designed peptides maintain their amyloidogenic properties and can bind to silver, gold and platinum nanoparticles [Kasotakis et al. Biopolymers 2009, 92, 164-172]. Computational investigation can identify suitable positions for rational modification of peptide building blocks, aiming at the fabrication of novel biomaterials.

Replacement of Adenovirus Type 5 Fiber Shaft Heparan Sulfate Proteoglycan-Binding Domain with RGD for Improved Tumor Infectivity and Targeting

Tumor targeting on systemic adenovirus administration is key to improve the prospects of adenovirus-mediated gene therapy and virus therapy of cancer. Despite many genetic and ligand conjugation approaches this objective remains elusive. Ablation of human adenovirus type 5 (Ad5) binding to its natural receptors in airway epithelial cells, that is, the coxsackievirus and adenovirus receptor (CAR) and integrins, does not impact on the preeminent liver tropism of adenovirus in the bloodstream. This is explained by a distinct entry pathway mediated by blood factors and heparan sulfates. Mutation of the KKTK heparin sulfate-binding domain of the fiber shaft to GATK results in liver transduction detargeting, but it is not compatible with otherwise useful HI-loop tumor-targeting ligand insertions such as the insertion of Arg-Gly-Asp (RGD). To circumvent this problem we have mutated the KKTK domain to RGDK, and analyzed the liver-detargeting and tumor-targeting transduction properties of this replacement mutant. Similar to RGD at the HI-loop, RGD at this new shaft location efficiently enhances the infectivity of adenovirus and improves the tumor-to-liver transduction ratio in vivo.

Concomitant use of Ad5/35 chimeric oncolytic adenovirus with TRAIL gene and taxol produces synergistic cytotoxicity in gastric cancer cells

Chimeric adenoviral vectors possessing fiber derived from human adenovirus subgroup B (Ad35) have been developed for their high infection efficiency in cell types which are refractory to adenovirus serotype 5 (Subgroup C). The present study constructed an E1B-deleted chimeric oncolytic adenovirus, SG235-TRAIL, which carries a human TRAIL gene expression cassette and whose fiber shaft and knob domains are from serotype Ad35. It was found that SG235-TRAIL preferentially replicated in gastric cancer cell lines, SGC-7901 and BGC-823 compared to in normal human fibroblast BJ cells. Also, when compared with a replication-deficient chimeric vector Ad5/35-TRAIL, SG235-TRAIL mediated a higher level of the transgene expression via viral replication in the cancer cells. Further, because of the more efficient cell-entry and infection, SG235-TRAIL induced stronger cell apoptosis than the Ad5 CRAD vector, ZD55-TRAIL. In addition, SG235-TRAIL in combination with the chemotherapeutic drug, taxol, produced a synergistic cytotoxic effect in cancer cells in vitro without causing significant toxicity to normal cells. In the gastric tumor xenograft mouse model, intratumoral SG235-TRAIL injection produced a significant antitumor effect 14 days after treatment. Pathological examination demonstrated TRAIL expression and associated apoptosis in majority of SG235-TRAIL-treated tumor cells. These results suggest that SG235-TRAIL is a potential novel, efficient anti-cancer agent, and in combination with taxol, it would be even more useful with considerably low toxic side effects.

841. Integrin-Binding RGD Insertion in Adenovirus-5 Fiber Shaft or Knob as a Selective Tumor-Targeting Strategy in Human Colorectal Cancer

841. Integrin-Binding RGD Insertion in Adenovirus-5 Fiber Shaft or Knob as a Selective Tumor-Targeting Strategy in Human Colorectal Cancer

Substitution of Hexon Hypervariable Region 5 of Adenovirus Serotype 5 Abrogates Blood Factor Binding and Limits Gene Transfer to Liver

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Interaction of Systemically Delivered Adenovirus Vectors with Kupffer Cells in Mouse Liver

When adenovirus (Ad) vectors are injected intravenously they are rapidly taken up by Kupffer cells (KCs) in the liver. This results in massive KC necrosis within minutes, followed by a more gradual disappearance of KCs from the liver. It is not known how KCs recognize Ad, or why Ad kills KCs. We used a variety of mutated and fiber-pseudotyped Ad vectors to evaluate how capsid proteins influence Ad uptake by KCs and to define the viral proteins that are involved in the destruction of KCs. We found that depletion of KCs from the liver was partially dependent on interactions between Ad and integrins, but was independent of the coxsackievirus and Ad receptor. The Ad5 fiber shaft was proven to be a particularly important contributory factor, because vectors with the shorter Ad35 shaft were not as effective at depleting KCs. In contrast, the fiber head played no discernible role. Variations in the ability of Ad vectors to deplete KCs could not be explained by differences in the amount of Ad that reached KCs, because all mutant Ads were accumulated by KCs at similar levels. Interestingly, we found that the Ad mutant ts1 did not cause KC death; this virus is known to bind and enter cells normally, but the capsid is unable to disassemble or lyse membranes. We conclude that Ad vectors kill KCs at a postbinding step and that this cell death can be mitigated if downstream events in viral entry are blocked.

Evaluation of Twenty-One Human Adenovirus Types and One Infectivity-Enhanced Adenovirus for the Treatment of Malignant Melanoma

Advanced melanoma is associated with poor prognosis warranting the development of new therapeutics, such as oncolytic adenoviruses for immunovirotherapy. Since this approach critically depends on efficient transduction of targeted tumor cells, we screened a panel of 22 different adenovirus types for their internalization efficiency in melanoma cells. We demonstrated that the virions of Ad35, Ad38, and Ad3 have significantly higher internalization efficiency in melanoma cells than Ad5, so far the only adenovirus type used in clinical trials for melanoma. Therefore, we developed a conditionally replication-competent Ad5-based vector with the Ad35 fiber shaft and knob domains (Ad5/35) and compared its therapeutic efficacy with the homologous vector carrying the native Ad5 fiber. To further enhance virotherapy, we combined the oncolytic adenovirus vectors with intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-H/F) and dacarbazine chemotherapy. In a human melanoma xenograft model, established from a short-term culture of primary melanoma cells, we demonstrated that the Ad5/35-based therapy had a significantly greater anti-neoplastic effect than the homologous Ad5-based therapy. Furthermore, the combination of virotherapy, intratumoral expression of MV-H/F, and chemotherapy was clearly superior to single- or double-agent therapy. In conclusion, Ad35-based vectors are promising for the treatment of melanoma.

Fiber Shaft-Chimeric Adenovirus Vectors Lacking the KKTK Motif Efficiently Infect Liver Cells In Vivo

The molecular mechanisms governing the infectivity of adenovirus (Ad) toward specific cell and tissue types in vivo remain poorly understood. The direct Ad binding to hepatic heparan sulfate proteoglycans via the KKTK motif within the fiber shaft domain was suggested to be the major mechanism of Ad liver cell infection in vivo. Here, we describe the generation and in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains, which lack the KKTK motif. We found that all the critical early steps of Ad infection, including attachment to the cellular receptor, internalization, and virus genome transfer into the nucleus, occurred with similar levels of efficiency for fiber shaft-chimeric vectors and unmodified Ad5. Upon intravenous delivery into mice, fiber shaft-chimeric vectors accumulated in liver tissue, transduced liver cells, and induced the production of proinflammatory cytokines (tumor necrosis factor alpha and interleukin-6) and the chemokine monocyte chemoattractant protein 1 at levels indistinguishable from those observed for Ad5. Thus, our data provide evidence that the Ad5 fiber shaft amino acid sequence does not play any substantial role in determining adenovirus infectivity toward hepatic cells in vivo. The data obtained contribute to improving our understanding of the molecular mechanisms determining Ad infectivity and biodistribution in vivo and may aid in designing novel Ad-based vectors for gene therapy applications.