The capacity of recombinant adenoviruses (rAd) to induce immunization against their transgene products has been well documented. In the present study, we evaluated the vaccinal adjuvant role of rAd independently of its vector function. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along with AdE1°(10 9 particles), a first-generation rAd empty vector. Although coinjected with a suboptimal dose of lipopeptides, AdE1°significantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected with AdE1°plus LP6 developed both a polyspecific T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epitopes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted virions. When mice were immunized with LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the results obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen. Our results thus have some important implications not only in vaccinology but also for gene therapy using rAd vectors.

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.Viruses have developed natural strategies to infect and grow in specific tissues of particular hosts, and their specificity and restricted host range make them theoretically the best-adapted vectors to transfer therapeutic genes into recipient cells that are their natural targets. However, when desired cell targets are not part of the natural host range of the virus, genetic modifications are required to adapt the virus and its derived vectors to these novel hosts. Since viral tropism is, in large part, under the control of primary virus-cell interaction occurring at the cell surface, the most obvious modification consists of...

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the nontelomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

Here we describe the development of a two-step chromatography process based on the use of ion-exchange resins for the purification of recombinant adeno-associated virus (rAAV) serotypes-2 and-5. In vitro and in vivo results demonstrate that this method, which does not require any prepurification step of the cell lysate, can be applied to obtain highly pure rAAV2 and rAAV5 stocks. As such,this procedure can be easily transferred in vector cores and also scaled up, allowing the direct comparison of these two, and potentially other, AAV serotypes in large animal models.

Band shift assays were used to study proteins from the fission yeast that bind double-stranded telomeric repeat sequences. We also examine general DNA binding properties of the telobox domain, which characterizes telomere-binding proteins from a range of species. We demonstrate that Taz1p has a high affinity for the fission yeast telomeric repeat, consistent with genetic results implicating this protein in telomere maintenance. A second Schizosaccharomyces pombe telobox protein, Teb1p, is shown to bind with high affinity to the vertebrate repeat and with low affinity to the fission yeast telomeric DNA. When tested on G-rich single-stranded telomeric DNA, all these proteins bind with very low affinity, much like the human telomere-binding protein TRF1. Recombinant proteins containing just the telobox domains reproduce the specificity of binding demonstrated for the corresponding full-length proteins, indicating that the telobox domain is indeed responsible for specific DNA recognition. The presence of possible Teb1p-binding sites upstream of many genes suggests a role for this protein as a general transcription factor. Finally, band shift experiments with whole cell extracts from wild-type and taz1 (-)strains suggest that in addition to Taz1p, S.pombe has another major telomere-binding activity.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.