Strong foreign promoters contribute to innate inflammatory responses induced by adenovirus transducing vectors

E1-deleted adenovirus (FG Ad) transducing vectors are limited for use in vivo by their induction of strong innate and adaptive inflammatory responses. We have examined the contribution of the transgene cassette, particularly the foreign promoter driving transgene expression, in the induction of innate inflammation using a mouse ear model in which swelling is measured as a sensitive surrogate marker of the total innate inflammatory response. The commonly used cytomegalovirus major immediate early (CMV) promoter led to high-level swelling that was independent of transgene expression, while the Rous sarcoma virus and human ubiquitin C promoters led to intermediate levels of swelling and the Ad E1A promoter or no promoter led to equally low levels of swelling. Significant swelling was induced by a virus in which the E1A promoter directed pIX expression, supporting the possibility that activation of expression of Ad genes retained in the vector plays an important role in the inflammatory response. Taken together, our findings support the idea that strong foreign promoters likely play the limiting role in the induction of innate and adaptive immune responses that limit the duration of transgene expression after transduction by FG Ad vectors.     

Current strategies and future directions for eluding adenoviral vector immunity

Adenoviral (Ad) vectors can efficiently transduce a broad range of cell types and have been used extensively in preclinical and clinical studies for gene delivery applications. The presence of preexisting Ad immunity in the majority of human population and a rapid development of immune response against the Ad vector backbone following the first inoculation with the vector have impeded clinical use of these vectors. In addition, a number of animal inoculation studies have demonstrated that high systemic doses of Ad vectors invariably lead to initiation of acute inflammatory responses. This is mainly due to activation of innate immunity by vector particles. In general, vector and innate immune responses drastically limit the vector transduction efficiency and the duration of transgene expression. In order to have a predictable response with Ad vectors for gene therapy applications, the above limitations must be overcome. Strategies that are being examined to circumvent these drawbacks of Ad vectors include immunosuppression, immunomodulation, serotype switching, use of targeted Ad vectors, microencapsulation of Ad vectors, use of helper-dependent (HD) Ad vectors, and development of nonhuman Ad vectors. Here we review the current understanding of immune responses to Ad vectors, and recent advances in the strategies for immune evasion to improve the vector transduction efficiency and the duration of transgene expression. Development of novel strategies for targeting specific cell types would further boost the utility of Ad vectors by enhancing the safety, efficacy and duration of transgene expression.     

Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors

Hexon modification of adenovirus type 5 (Ad5) vectors with the hypervariable regions (HVRs) of Ad48 has been shown to allow Ad5HVR48 vectors to circumvent the majority of the preexisting Ad5-neutralizing antibodies. However, it remains unclear whether modifying hexon HVRs impacts innate or adaptive immune responses elicited by this vector. In this study, we investigated the influence of the HVR substitution of Ad5 on innate and adaptive immune responses following vaccination. Ad5HVR48 displayed an intermediate level of innate immune cytokines and chemokines relative to those of Ad5 and Ad48, consistent with its chimeric nature. Hepatotoxicity was observed after Ad5 immunization but not after Ad5HVR48 or Ad48 immunization. However, the CD8⁺ T-cell responses elicited by Ad5HVR48 vectors displayed a partially exhausted phenotype, as evidenced by the sustained expression of programmed death 1 (PD-1), decreased effector-to-central memory conversion, and reduced memory recall responses, similar to those elicited by Ad5 vectors and in contrast to those induced by Ad48 vectors. Taken together, these results indicate that although Ad5HVR48 largely bypasses preexisting Ad5 neutralizing antibodies and shows reduced hepatotoxicity compared to that of Ad5, it induces adaptive immune phenotypes that are functionally exhausted similar to those elicited by Ad5.     

Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells

An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells.     

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p''HR.cppt.3''1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.     

Adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications

Extensively characterized, modified, and employed for a variety of purposes, adenovirus (Ad) vectors are generally regarded as having great potential by many applied virologists who wish to manipulate and use viral biology to achieve beneficial clinical outcomes. Despite widespread functional prominence and utility (i.e., Ad-based clinical trials have begun to progress to critical Phase III levels, it has recently become apparent that investigations regarding the innate immune response to Ads may reveal not only reasons behind previous failures, but also reveal novel insights that will allow for safer, more efficacious uses of this important gene transfer platform. Insights gained by the exploration of Ad induced innate immune responses will likely be most important to the fields of vaccine development, since Ad-based vaccines are regarded as one of the more promising vaccine platforms in development today. Adenovirus is currently known to interact with several different extracellular, intracellular, and membrane-bound innate immune sensing systems. Past and recent studies involving manipulation of the Ad infectious cycle as well as use of different mutants have shed light on some of the initiation mechanisms underlying Ad induced immune responses. More recent studies using microarray-based analyses, genetically modified cell lines and/or mouse mutants, and advanced generation Ad vectors have revealed important new insights into the scope and mechanism of this cellular defensive response. This review is an attempt to synthesize these studies, update Ad biologists to the current knowledge surrounding these increasingly important issues, as well as highlight areas where future research should be directed. It should also serve as a sobering reality to researchers exploring the use of any gene transfer vector, as to the complexities potentially involved when contemplating use of such vectors for human applications.     

High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.     

Differentially imprinted innate immunity by mucosal boost vaccination determines antituberculosis immune protective outcomes,independent of T-cell immunity

Homologous and heterologous parenteral prime–mucosal boost immunizations have shown great promise in combating mucosal infections such as tuberculosis and AIDS. However, their immune mechanisms remain poorly defined. In particular, it is still unclear whether T-cell and innate immunity may be independently affected by these immunization modalities and how it impacts immune protective outcome. Using two virus-based tuberculosis vaccines (adenovirus (Ad) and vesicular stomatitis virus (VSV) vectors), we found that while both homologous (Ad/Ad) and heterologous (Ad/VSV) respiratory mucosal boost immunizations elicited similar T-cell responses in the lung, they led to drastically different immune protective outcomes. Compared with Ad-based boosting, VSV-based boosting resulted in poorly enhanced protection against tuberculosis. Such inferior protection was associated with differentially imprinted innate phagocytes, particularly the CD11c⁺CD11b^+/− cells, in the lung. We identified heightened type 1 interferon (IFN) responses to be the triggering mechanism. Thus, increased IFN-β severely blunted interleukin-12 responses in infected phagocytes, which in turn impaired their nitric oxide production and antimycobacterial activities. Our study reveals that vaccine vectors may differentially imprint innate cells at the mucosal site of immunization, which can impact immune-protective outcome, independent of T-cell immunity, and it is of importance to determine both T-cell and innate cell immunity in vaccine studies.     

T-cell-biased immune responses generated by a mucosally targeted adenovirus-σ1 vaccine

As most pathogens enter through the mucosa, it is important to develop vaccines that induce mucosal immunity. To this end, we generated a novel adenovirus (Ad) vaccine that displays the σ1 protein from reovirus to target junctional adhesion molecule 1 and sialic acid. Replication-defective Ad5 vectors were modified by replacement of the Ad fiber protein with σ1 (T3Dσ1) protein of reovirus T3D in previous work. Ad5 and Ad5-σ1 were compared in mouse models for gene delivery and vaccination to monitor cytokine, antibody, and T-cell responses. The viruses were also tested for the ability to transduce and mature dendritic cells. Ad5-σ1 was 40-fold less efficient at gene delivery in vivo, yet it was capable of inducing equal or greater cellular immune responses and systemic interferon-γ levels than Ad5 after intranasal administration. Despite weaker gross transduction, intranasal administration of Ad5-σ1 produced more green fluorescent protein-positive (GFP+) major histocompatibility complex class II (MHC II) cells in the draining lymph nodes, less GFP+/MHC II+ cells in the lungs, and mediated modestly better maturation of dendritic cells in vitro. These data suggest that targeting gene-based vaccination via the σ1 protein may enhance the T-cell immune response, perhaps by skewing immune responses to encoded antigens.     

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.     

Centrifugation enhances integrin-mediated transduction of dendritic cells by conventional and RGD-modified adenoviral vectors

The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(-)/E3Delta Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine-Glycine-Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9-20-fold at each MOI. The addition of centrifugal force (2000xg) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.     

Enhancing the therapeutic efficacy of adenovirus in combination with biomaterials

With the reason that systemically administered adenovirus (Ad) is rapidly extinguished by innate/adaptive immune responses and accumulation in liver, in vivo application of the Ad vector is strictly restricted. For achieving to develop successful Ad vector systems for cancer therapy, the chemical or physical modification of Ad vectors with polymers has been generally used as a promising strategy to overcome the obstacles. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of therapeutic Ad vectors and well accomplished to extend circulation time in blood and reduce liver toxicity. However, although polymer-coated Ads can successfully evacuate from a series of guarding systems in vivo and locate within tumors by enhanced permeability and retention (EPR) effect, the possibility to entering into the target cell is few and far between. To endow targeting moiety to polymer-coated Ad vectors, a diversity of ligands such as tumor-homing peptides, growth factors or antibodies, have been introduced with avoiding unwanted transduction and enhancing therapeutic efficacy. Here, we will describe and classify the characteristics of the published polymers with respect to Ad vectors. Furthermore, we will also compare the properties of variable targeting ligands, which are being utilized for addressing polymer-coated Ad vectors actively.     

Cytotoxic T lymphocyte antigen 4 decreases humoral and cellular immunity by adenovirus to enhance target GFP gene transfer in C57BL/6 mice

Abstract

Adenoviruses (Ad) are once potential and promising vectors for gene delivery, but the immunogenicity attenuates its transfer efficiency. Cytotoxic T lymphocyte antigen 4 (CTLA-4) can inhibit T cell immunity. Thus, we aimed to study the effect of CTLA-4 in the process of Ad-mediated gene transfer. The C57BL/6 mice were injected by Ad vectors at twice, and CTLA-4 was administrated after the first Ad injection. Then, the CD3⁺CD4⁺ T cells and circulating levels of IL-2, IL-4, and anti-Ad IgG were decreased by CTLA-4, while Ad generated immune responses. The green fluorescence protein (GFP) expressions of tissues were enhanced by CTLA-4 till injection of Ad at twice. Our results indicate that CTLA-4 can inhibit humoral and cellular immunity by adenovirus generation to enhance GFP delivery, and provide a potential way to assist in Ad-mediated gene transfer.     

Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.