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Abstract Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells. Key messages • Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1. • The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells. • Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.

Adenovirus-based vectors comprise the most frequently used vector type in clinical studies to date. Both intense lab research and insights from the clinical trials reveal the importance of a comprehensive understanding of vector-host interactions. Especially for systemic intravenous adenovirus vector delivery, it is paramount to develop safe and efficacious vectors. Very early vector-host interactions that take place in blood long before the first cell is being transduced are phenomena triggered by the surface, shape, and size of the adenovirus vector particles. Not surprisingly, a multitude of different technologies ranging from genetics to chemistry has been developed to alter the adenovirus vector surface. In this review, we discuss the most important technologies and evaluate them for their suitability to overcome hurdles imposed by early vector-host interactions.

Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.

Background Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. Methods We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. Results FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10–35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3–5 days after injection of FAD vectors to the knee joints. Conclusions Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides. Copyright © 2013 John Wiley & Sons, Ltd.

Virotherapy is a unique modality for the treatment of cancer with oncolytic viruses (OVs) that selectively infect and lyse tumor cells, spread within tumors, and activate anti-tumor immunity. Various viruses are being developed as OVs preclinically and clinically, several of them engineered to encode therapeutic proteins for tumor-targeted gene therapy. Scientists and clinicians in German academia have made significant contributions to OV research and development, which are highlighted in this review paper. Innovative strategies for "shielding," entry or postentry targeting, and "arming" of OVs have been established, focusing on adenovirus, measles virus, parvovirus, and vaccinia virus platforms. Thereby, new-generation virotherapeutics have been derived. Moreover, immunotherapeutic properties of OVs and combination therapies with pharmacotherapy, radiotherapy, and especially immunotherapy have been investigated and optimized. German investigators are increasingly assessing their OV innovations in investigator-initiated and sponsored clinical trials. As a prototype, parvovirus has been tested as an OV from preclinical proof-of-concept up to first-in-human clinical studies. The approval of the first OV in the Western world, T-VEC (Imlygic), has further spurred the involvement of investigators in Germany in international multicenter studies. With the encouraging developments in funding, commercialization, and regulatory procedures, more German engineering will be translated into OV clinical trials in the near future.

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)β(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

Adenovirus vectors belong to the most frequently used vector type in gene therapy approaches. In addition, adenovirus vector particles and adenovirus subviral particles offer a great potential for the direct delivery of proteins into cells. In this review we discuss this potential and the technology of adenovirus as a protein delivery platform for applications ranging from vaccination to gene therapy.

Background The ability to induce cytotoxic T lymphocyte (CTL) responses that are multispecific is considered to comprise an essential feature for an efficacious genetic vaccine against many pathogens including HIV and hepatitis C virus. ΔE1Ad vectors are promising vectored vaccines but have been shown to induce antigen-specific CTLs with only limited multispecificity. In the present study, we investigated the applicability of gene-deleted high-capacity adenovirus (HC-Ad) vectors and focused on the induction of multispecific CTL responses. Methods We generated ΔE1 and HC-Ad vectors expressing hepatitis B virus small surface antigen (HBsAg). We comparatively analyzed the CTL profiles against various transgene product- and vector-derived epitopes in several mouse strains and HBsAg- and vector-directed antibody responses. Results HC-Ad vectors efficiently induced multispecific HBsAg-directed CTLs. By contrast, ΔE1Ad vectors mainly primed CTLs against one immunodominant epitope of HBsAg. This absence of multispecific CTL responses correlated with the induction of CTLs against viral epitopes generated by de novo expression of Ad genes from the ΔE1Ad vector. However, Ad-specific CTLs induced in trans did not impair HC-AdS-induced multispecific CTL responses against HBsAg. Finally, HC-Ad vectors also induced higher HBsAg antibody titers compared to ΔE1Ad vectors. Conclusions De novo expression of viral genes from ΔE1Ad vector genomes restricts the multispecificity of transgene product-specific CTLs by immunodominance effects. HC-Ad vectors devoid of Ad genes are favorable for the induction of both multispecific CD8 T-cell responses and high antibody responses. Our results suggest the deletion of Ad genes as an important means for developing potent Ad-based vectored vaccines. Copyright © 2011 John Wiley & Sons, Ltd.

Background The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the causative agent of malaria tropica, is considered to be a promising vaccine candidate. Although its stable cloning and expression has been difficult in the past, adenoviral vectors expressing the complex protein are described in the present study. Methods Codon-optimized msp-1 was used to construct a set of first generation (ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral immune responses induced by the vectors were characterized in detail in mice. Results Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length MSP-1 and their production to high vector titers was found to be feasible. Epitope identification and analysis of frequencies of specific CD8 T-cells revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format, higher titers of MSP-1 specific antibodies were generated by Ad vectors expressing MSP-1 from a chicken β-actin (CAG) promoter comprising the cytomegalovirus early enhancer element and the chicken β-actin promoter. Conclusions The findings of the present study suggest that Ad vectors expressing full-length codon-optimized MSP-1 are promising candidate vaccines against P. falciparum infections. Use of the HC-Ad vector type for delivery, as well as the CAG promoter to control MSP-1 expression, may further increase the efficacy of this vaccine candidate. Copyright © 2011 John Wiley & Sons, Ltd.

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K(b)/S(190-197)-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

Adenoviral (Ad) vectors are widely used for gene therapy approaches. Because of the high abundance of the natural adenoviral receptors (coxsackievirus-adenovirus receptor and integrins) on a wide variety of cells, numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. Importantly, an increasing number of publications have shown that the success of targeting not only depends on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge about the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, the technologies currently available for fluorescent labeling of Ad vectors have significant limitations: (1) at present capsids are labeled all over the particle surface, and this imposes the risk of interference with particle infectivity; (2) capsomer-specific labeling requires extensive genetic modifications and has been demonstrated only at protein IX; and (3) two-color labeling approaches are not available. Here we present a novel, robust, and straightforward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomer's fiber, protein IX, or hexon and permits two-color labeling. We demonstrate the potential of this labeling technology by analyzing two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds are quickly hydrolyzed after uptake of the virions and are thus favorable for the generation of bioresponsive vectors.

The chemical modification of adenovirus (Ad) gene transfer vectors with synthetic polymers is a promising strategy for overcoming typical in vivo hurdles associated with Ad-mediated gene delivery. Polymer-modified Ad vectors induce significantly reduced innate immune responses, can evade pre-existing anti-Ad antibodies, allow for repeated vector delivery, and have been used for developing novel retargeting strategies. The most widely used polymers for covalent chemical capsid surface modification are poly-N-(2-hydroxypropyl)methacrylamide (poly-HPMA) and polyethylene glycol (PEG), and the latter is in wide clinical use for modifying protein biopharmaceuticals. In this review, we critically compare the properties of various polymers with respect to Ad vector shielding and retargeting, and identify areas for future research on polymer-modified viral vectors. We describe the potential technical pitfalls of polymer modification of Ad vectors and provide a technical guide for avoiding these while establishing polymer modification techniques in the laboratory.

The clinical efficacy of adenovirus serotype 5-based vectors is limited by complex interactions of the vector with host blood components. These interactions trigger sequestration of systemically administered vector particles mainly by the reticuloendothelial system. In order to generate retargeted Ad vectors suitable for i.v. delivery it is mandatory to understand and manipulate these non-target interactions.In mice, it was shown that blood coagulation factor X bound to hexon reduces sequestration by shielding the virus from natural antibodies and macrophage uptake, but also mediates hepatocyte transduction. To generate Ad vectors deficient for hepatocyte transduction but shielded from natural antibodies, we employed a combination of point mutations ablating (i) CAR binding (Ad-ΔCAR), (ii) integrin binding, (iii) FX binding (Ad-ΔFX), and rendered the vectors amenable for position-specific PEGylation at hexon HVR1 (Ad-HVR1) to compensate the lack of a FX-mediated shielding. Surface plasmon resonance analysis confirmed complete ablation of FX binding by ΔFX mutation in combination with PEGylation. Upon i.v. delivery, unPEGylated Ad-HVR1-ΔCAR-ΔFX did not transduce hepatocytes in BALB/c mice, whereas robust transduction levels were observed in antibody-deficient JHD mice. In agreement with the literature, this suggested that the ΔFX vector particles became susceptible to natural antibodies. However and surprisingly, PEGylated Ad-HVR1-ΔCAR-ΔFX showed a strong FX-independent hepatocyte transduction in both BALB/c and JHD mice compared to unPEGylated Ad-HVR1-ΔCAR-ΔFX and wildtype capsid vectors. Preliminary results suggested that integrins were involved in this FX-independent transduction. Further, PEGylated Ad-HVR1-ΔCAR-ΔFX vectors displayed 20-fold increased blood persistence without being associated to blood cells in BALB/c mice.To analyze vector sequestration by macrophages more closely we performed in vitro assays measuring the uptake of vector particles by Raw264.7 cells in the presence of murine plasma. We found enhanced uptake of unPEGylated Ad-HVR1-ΔFX vectors compared to wildtype capsid vectors. Using antibody-deficient and heated plasma of Ad naive mice confirmed a natural antibody- and complement-dependent uptake mechanism. This uptake was completely prevented by PEGylation of the vector particles, suggesting that position-specific PEGylation of hexon HVR1 interfered with natural antibody binding. Moreover, using hirudinized human whole blood we could show that a complement-dependent natural antibody-mediated binding of unPEGylated Ad-HVR1-ΔCAR-ΔFX vectors to erythrocytes could be prevented by PEGylation of hexon HVR1 with large PEG moieties.Thus, we conclude that a PEG-mediated evasion from sequestration by macrophages by shielding from natural antibodies or by a yet unknown mechanism maintained high vector concentrations in blood followed by enhanced hepatocyte transduction.

Numerous attempts to target viral gene therapy vectors to specific cells have met with limited success. Here we describe a novel virus vector-targeting platform based on a unique combination of genetic and chemical vector particle modifications to overcome typical restrictions in virus vector targeting. We genetically introduced cysteines at solvent-exposed positions of the adenovirus capsid. The corresponding thiol groups were highly reactive, and we established procedures for controlled covalent coupling to them of protein and nonprotein ligands. After the coupling of transferrin, the particles were efficiently targeted to the transferrin receptor pathway. Depending on the chemistry used, ligands could be coupled under the formation of thioether or disulfide bonds, the latter allowing for separation of ligand and particle after cell entry in the endosome. Furthermore, this technology could be efficiently combined with vector shielding for true retargeting: after amino-PEGylation of the vector particles the genetically introduced thiols were still accessible for ligand coupling, and particles could be retargeted to the transferrin receptor. Since this platform is robust, can be scaled, is compatible with industrial standards, and can integrate chemically diverse molecules as ligands, it may be used for clinical gene therapy and, potentially, also for vaccination.

To confer adenovirus vectors (AdV), the feature of integration into the host cell genome hybrid vectors were characterized in vitro, which express vectors derived from the prototypic foamy virus (FV) in the backbone of a high-capacity AdV. FVs constitute a subfamily of retroviruses with a distinct replication pathway and no known pathogenicity. In the absence of envelope glycoprotein, the prototypic FV behaves like a retrotransposon, while it behaves like an exogenous retrovirus in its presence. Two principle types of vectors, which either allows the intracellular (HC-FAD-7) or, in addition, the extracellular (HC-FAD-2) pathway were constructed. In both chimeras the expression of the FV vector was controlled by the tetracycline-regulatable system. Hybrids were produced close to 10(10) infectious units/ml. By Southern blotting, the functionality of the hybrid vectors to generate host cell genomic integrants was shown. However, the efficiency of HC-FAD-7 to establish stable transgene expression was rather low, while around 70% of cells were stably transduced in secondary round following primary transduction with HC-FAD-2 at an MOI of 100. Given the benign characteristics of high-capacity adenovirus and FV vectors, hybrids based on HC-FAD-2 are probably suited for an in vivo application.

Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37 degrees C for up to 8 h. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.

International audience; BACKGROUND: The expression of Moloney murine leukemia virus (Mo-MLV) gag proteins is sufficient to generate retrovirus-like particles (retroVLPs) that can be used as antigen-display platforms by pseudotyping with heterologous envelope proteins or by insertion of epitopes in structural constituents. To circumvent the in vitro production of such retroVLPs, we used DNA plasmids generating recombinant retroVLPs (plasmo-retroVLPs) as immunogens. We previously demonstrated that plasmo-retroVLPs induce significantly better antigen-specific T cell responses and antiviral immune protection than plasmids bearing a single mutation preventing retroVLPs assembly. In the present study, we investigated the possibility of using such plasmo-retroVLPs in prime-boost immunization strategies for hepatitis C virus (HCV) vaccine development. METHODS: To define the best immunization regimen with plasmo-retroVLPs and serotype 5 recombinant adenovirus vectors (rAd5), we used standardized methodologies measuring immune responses to the GP(33-41) 'gold standard' antigen. The protective efficacy of these immunization schedules was also evaluated in mice after tumor challenge. We then applied the optimal prime-boost immunization strategy using vectors expressing HCV-E1/E2 envelope glycoproteins. RESULTS: Using vectors expressing the model antigen, we demonstrated that rAd5(GP33-41)/plasmo-retroVLP(GP33-41) regimen induced significantly higher cellular immune responses than plasmo-retroVLP(GP33-41)/rAd5(GP33-41). Consequently, HCV-specific plasmo-retroVLPs (plasmo-retroVLP(E1E2)) were used as boost in mice primed with rAd5(E1E2) and we observed that plasmo-retroVLP(E1E2) significantly increased E1/E2-specific interferon-gamma cellular responses and E2-specific antibody generation. By contrast, plasmids unable to form E1/E2-pseudotyped retroVLPs had no boosting effect, revealing the importance of presenting E1/E2 in a particulate form. CONCLUSIONS: Altogether, combining plasmo-retroVLPs that represent a new class of genetic vaccines in a heterologous prime-boost vaccination strategy appears to be a promising strategy for HCV vaccine development.

Background Chemical capsid modification of adenovirus vectors with synthetic polymers has been shown to aid in overcoming typical barriers for adenovirus vector-mediated gene transfer. Carbohydrate-based polymers for covalent modification of adenovirus vectors have been largely neglected so far. We utilized a reductive amination strategy to generate a novel class of adenovirus-based glycovectors with a mannan derivative. Methods Reductive amination to covalently couple polysaccharides to the capsid surface of adenovirus serotype 5-based vectors was investigated utilizing an oxidized derivative of mannan. After biochemical and physical characterization of mannanylated vectors, their performance was analysed in vitro in cell lines and primary human cells, and in vivo in mice after local and systemic vector injection. Results We describe the successful modification of adenovirus vectors with large polysaccharides by reductive amination. The particles were efficiently modified, physically intact and, importantly, detargeted from the natural Coxsackie and adenovirus receptor/integrin pathway in vitro. In addition, they exhibited significantly decreased transduction of muscle after local delivery and of liver after systemic delivery in mice. However, despite the modification of 60% of capsid surface amino groups, mannanylated particles were unable to evade neutralizing anti-Ad5 antibodies. Conclusions Mannanylated vectors are a paradigm for a novel class of glycoviruses modified with large polysaccharides. Vector promiscuity as one of the important hurdles for Ad-mediated gene transfer could be significantly decreased in vivo, whereas mannanylated vectors were unable to escape from anti-adenovirus antibodies. Our studies provide a detailed analysis of mannan-modified Ad vectors and suggest further improvements for this novel class of glycovectors. Copyright © 2008 John Wiley & Sons, Ltd.

Adenovirus (Ad) vector targeting requires presentation of specific ligands on the virion's surface. Geneti-chemical targeting is based on the genetic introduction of cysteine residues bearing reactive thiol groups into solvent-accessible capsomeres of the virion and subsequent chemical coupling of ligands. Here, we exploited this technology to modify the pIX capsomere with high-affinity ligands. Genetic introduction of C-terminal cysteines to pIX allowed for specific coupling of full-length proteins to the virion, while not affecting vector production. Direct comparison of the two high-affinity ligands receptor- associated protein (RAP) and transferrin (Tf) revealed that targeting after coupling of a high-affinity ligand to pIX presumably requires release of the ligand from its receptor after cell entry. In addition, data obtained by live cell imaging of labeled vector particles demonstrated that coupling of very large proteins to pIX can impair intracellular vector particle trafficking. Finally, we demonstrate that the geneti-chemical targeting technology is suitable for in vivo targeting to liver after intravenous injection. Our data provide significant insight into basic requirements for successful targeting of pIX-modified Ad vectors.

Heat shock proteins (Hsp) of the Hsp70/90 families facilitate cellular immune responses to antigenic peptides or proteins bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70, Hsp73) or bacterial (DnaK) stress proteins and accumulate to high steady-state levels. J domains from different species (viruses/SV40, bacteria/Chlamydia trachomatis or plants/Arabidopsis thaliana) efficiently capture murine or human stress proteins in this system, thus making different J domains available for vaccine production. A novel expression and purification method was developed to produce native Hsp/antigen complexes in transfectants. These purified Hsp/antigen complexes efficiently elicited antigen-specific CD8 T cell responses in mice when delivered as vaccines without adjuvants. In situ complex formation of antigen with Hsp was critical for CD8 T cell priming. Because the described expression system supports the flexible design of multivalent vaccines, it is an attractive strategy to elicit CD8 T cell responses either to recombinant proteins or to selected antigenic domains of these molecules.

El pdf del artículo es la versión de autor.-- et al., Drug-inducible systems allow modulation of the duration and intensity of cytokine expression in liver immuno-based gene therapy protocols. However, the biological activity of the transgene may influence their function. We have analyzed the kinetics of interleukin-12 (IL-12) expression controlled by the doxycycline (Dox)- and the mifepristone (Mif)-dependent systems using two long-term expressing vectors directed to liver: a plasmid administered by hydrodynamic injection and a high-capacity adenoviral vector. Daily administration of Dox or Mif was associated with a progressive loss of inducibility and a decrease of murine IL-12 production. This inhibition occurred at the transcriptional level and was probably caused by an interferon (IFN)-γ-mediated downmodulation of liver-specific promoters that control the expression of transactivators in these systems. Genome-wide expression microarrays studies revealed a parallel downregulation of liver-specific genes in mice overexpressing murine IL-12. However, a promoter naturally induced by IL-12 was also inhibited by this cytokine when placed in a plasmid vector. Interestingly, treatment with sodium butyrate, a class I/II histone deacetylase inhibitor, was able to rescue liver-specific promoter activity solely in the vector. We conclude that biologically active IL-12 can transiently inhibit the function of drug-inducible systems in non-integrative DNA vectors by reducing promoter activity, probably through IFN-γ and protein deacetylation-dependent mechanisms., This project was founded through the UTE project CIMA, DIGNA Biotech and Grants from Education Department, Gobierno de Navarra and Fondo de Investigación Sanitaria (FIS) and CIBERehd. RHA is a recipient of Ramón y Cajal research contract. MGK is a recipient of an FIS research contract. IM is a recipient of a Torres Quevedo contract.

Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product–specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.

Vectors based on Adenovirus type 5 (Ad5) are well suited for the purpose of genetic vaccination due to their high immunogenicity. However, it is known that immune complexes formed upon contact with vector-specific antibodies (Abs) can impede gene transfer and influence vector immune profiles. To model, analyze, and understand the effects of immune complex formation on in vivo biodistribution and vector immunogenicity, we established a novel model based on chemical tagging of Ad capsomers with a carbohydrate epitope. Of note, this model allows to direct antibodies to specific capsid regions and thus to generate highly defined Ad immune complexes independent of species and MHC restrictions.As an artificial immunogenic epitope, we chemically coupled alpha-Gal (Galactose-a1,3-Galactose-b1,4-N-Acetylglucosamine) to specific sites of Ad capsomers. We generated ΔE1 Ad5 vectors being alpha-Galylated at hexon HVR1, hexon HVR5, penton or fiber, and an unmodified control vector.We demonstrated that on account of the alpha-Gal epitope, anti-Gal IgG or IgM bound to selected tagged capsomers. Thus, we analyzed biodistribution and vector-induced immune responses in alpha-1,3-Galactosyltransferase knockout (a1,3-GT KO) mice, which harbor large amounts of anti-Gal Abs.First, to compare the effect of epitope tagging on vector biodistribution, we analyzed hepatic and splenic transgene expression after i.v. injection of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. We observed that transgene expression from fiber-modified vectors was mildly decreased, while expression from hexon-modified vectors decreased drastically, in a way resembling neutralization. These data suggested that, in fact, our model is suitable to precisely analyze the role of capsomer-specific Ab binding on in vivo vector biodistribution and activity.Furthermore, we analyzed the effect of immune complexation on the induction of vector- and transgene product-directed immune responses after repetitive i.m. delivery of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. Here, we found that the Ad immune complexes induced transgene product-directed immune responses, even upon repeated vector administration. In particular, the extents of hepatic and splenic transgene expression, and transgene-directed immune responses were inversely correlated. For hexon-modified vectors, in spite of weakest transgene expression, transgene-directed cellular immune responses were strongest.In summary, we showed that alpha-Galylation of Ad capsomers markedly influenced specific immune responses directed to the vector as well as the transgene product. Our concept shall be used as an in vivo model to characterize whether directing or diverting immune complexation to or from specific capsomer sites can improve future strategies for vaccination regimens using Ad vectors.

High-capacity adenoviral (HC-Ad) vectors are devoid of all viral genes. Therefore, these vectors feature reduced toxicity, immunogenicity, and increased capacity for foreign DNA. HC-Ad vectors are produced in E1-transformed cell lines in the presence of an E1-deleted helper virus that provides in trans all viral functions necessary for vector production. By cre/loxP- or FLPe/Frt-mediated recombination the packaging signal of the helper virus is excised during vector production resulting in nonpackagable helper virus genomes. Although recombinase-mediated excision of the packaging signal from the helper virus genome is highly efficient, a small number of helper virus genomes with retained packaging signals are still packaged into capsids. For clinical trials, HC-Ad vector preparations have to be characterized accurately with respect to the number of (1) total HC-Ad vector particles, (2) infectious HC-Ad vector particles, and (3) the number of contaminating helper virus particles. We describe a fast and versatile DNA-based biologic assay for determination of these three parameters by standard laboratory methods. This assay is a useful tool for determining bioactivity data of adenoviral vector preparations and, importantly, allows their comparison among different studies.

Multiple forms of muscular dystrophy are due to the absence of cytoskeletal muscle proteins that normally protect the integrity of muscle cells. The lack of any adequate treatments for these devastating diseases propels research toward the development of strategies for gene delivery to skeletal muscle. High-capacity adenoviral vectors (HC-AdV) devoid of all viral coding sequences have been developed to avoid expression of viral proteins by the gene therapy vector. However, the capsid proteins that are an essential component of the input viral vector and any residual helper virus in the vector preparation could induce an immune response. Furthermore, the therapeutic protein provided by a gene transfer vector presents the potential to induce an immune response in a patient who does not express a normal cellular protein due to genetic mutation. Therefore, we hypothesize that some immune suppression will be required with therapeutic gene delivery designed for the treatment of patients with inherited muscle diseases. In this study, we constructed and rescued three HC-AdVs expressing murine CTLA4Ig, murine CD40Ig, or both. The backbone vector without a gene insert was rescued as a negative control vector. The production of relevant proteins from each vector was determined in vitro. In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP)-expressing first-generation adenoviral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice. Higher levels of muscle EGFP expression were observed in animals receiving an immunosuppressant vector. Furthermore, the production of total anti-AdV and anti-EGFP antibodies was reduced in mice treated with each of the three immunosuppressant vectors. A second intramuscular administration of AdEGFP alone 4 weeks after the initial co-injection was successful in all immunosuppressant vector-treated groups, but not in the negative control vector-treated group. All groups had a high antibody response to adenoviral proteins after the second injection of AdEGFP alone, indicating that the initial co-injection did not tolerize against vector capsid antigens.

Top of pageAbstract Safe and successful genetic vaccination with adenovirus gene transfer vectors requires specific and efficient transduction of antigen- presenting cells (APCs) in vivo. Due to the high promiscuity of adenovirus and the presence of its natural receptor CAR on a wide variety of cells there is a strong demand for vectors which on the one hand are efficiently detargeted from their natural receptor and on the other hand possess specificity for endocytic receptors on antigen- presenting cells like dendritic cells. However, current genetic and chemical de- and retargeting strategies suffer from significant disadvantages. For example, genetically detargeted vectors are often difficult to produce and only a very limited number of peptidic ligands can be incorporated into the particles. Chemical detargeting strategies based on polymers like PEG and HPMA frequently fail to produce retargeted particles. Here we propose to use high molecular weight polysaccharide mannan as a polymer for chemical detargeting. We coupled mannan from S. cerevisiae onto the Ad vector particle surface by reductive amination. Mannan was partially oxidized and the resulting carbonyl groups were coupled to the amine groups on the vector particle surface under formation of Schiff bases. Subsequently, the Schiff bases were reduced to amine bonds which covalently linked the polysaccaride moiety to the vector particle surface. We carefully analyzed each step of the amination process for interference with physical or biological vector particle integrity and we established a protocol that allows for reductive amination maintaining particle integrity. SDS-PAGE analysis of the mannan- modified vector particles suggested highly efficient mannanylation of the vector surface. Photon correlation spectroscopy revealed that no particle aggregates had formed during the amination process. The mannanylated particles were tested for infectivity in vitro on the high and low CAR-expressing cell lines A549 and K562, respectively. Flow cytometric analysis of transgene expression (EGFP) after transduction with different MOIs revealed that, compared to unmodified controls, efficient detargeting of the mannanylated vectors was achieved. At the vector genome level this finding was supported by a DNA-based slot-blot assay. The detargeting efficiency was correlated with the extent of surface mannanylation. Using a fluorescamine-based assay we could show that modification of 25% of the amine groups on the vector particle surface was sufficient to detarget the vector particles 30-fold. To analyze whether mannan-detargeted particles were still capable to induce immune responses against the transgene in vivo we subcutaneously injected 5E+08 particles of a mannanylated vector coding for EGFP in Balb/c mice. Four weeks after injection a surprisingly strong cellular immune response against the transgene was detected. In summary, we could efficiently couple mannan to Ad vectors by reductive amination resulting in an efficient detargeting. Furthermore, the detargeted particles retained their potential to induce cytotoxic T cell responses against the transgene in vivo. This renders them potential candidates for vaccination.

Modifying the tropism of adenoviral vectors to obtain retargeted particles usually involves modification of the vector genome to genetically incorporate retargeting epitopes. In many cases impaired vector production and limitations with respect to the epitope size have been the consequence. We developed a novel platform for specific covalent capsid modification by combining a genetic approach with cysteine-based chemical capsid modification techniques.

Modifying the tropism of adenoviral vectors to obtain retargeted particles usually involves genetic modification of the vector genome to incorporate retargeting epitopes. In many cases impaired vector production and low vector yield has been the consequence frequently requiring even the generation of new production cell lines. Furthermore, the incorporation of large retargeting epitopes has been found to be difficult. We developed a novel platform for specific covalent capsid modification by combining a genetic approach with cysteine-based chemical capsid modification techniques.

Top of pageAbstract Foamy virus (FV) infections are known to take a non-pathogenic course and the application of high-capacity (“gutless”) adenovirus (AdV) vectors has also been shown to be superior to first and second generation AdV vectors. To confer AdV vectors the feature of integration into the host cell genome hybrid vectors were characterized in vitro, which express vectors derived from the prototypic FV in the backbone of a high-capacity AdV vector. FV constitute a subfamily of retroviruses with a distinct replication pathway. In the absence of envelope glycoprotein the prototypic FV behaves like an retrotransposon, while it behaves like an exogenous retrovirus in its presence. Two principle types of vectors, which either allow the intra-cellular (HC-FAD-7) or, in addition, the extra-cellular (HC-FAD-2) pathway were constructed. In both chimeras expression of the FV vector was controlled by the tetracycline regulatable system. Hybrids were produced to close to 10E10 infectious units per ml. By Southern blotting the functionality of the hybrid vectors to generate host cell genomic integrants was shown. However, the efficiency of HC-FAD-7 to establish stable transgene expression was rather low, while around 70% of cells were stably transduced in secondary round following primary transduction with HC-FAD-2 at an MOI of 100. Given the benign characteristics of high-capacity AdV and FV vectors, hybrids based on HC-FAD-2 are probably suited for an in vivo application.