Airi Harui

Helper-Dependent Adenoviral Vectors Efficiently Express Transgenes in Human Dendritic Cells but Still Stimulate Antiviral Immune Responses

Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd-AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275-fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV.

Redirecting Lentiviral Vectors Pseudotyped with Sindbis Virus-Derived Envelope Proteins to DC-SIGN by Modification of N-Linked Glycans of Envelope Proteins

ABSTRACT Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.

Modifying Adenoviral Vectors for Use as Gene-Based Cancer Vaccines

The past decade has produced significant advances in our understanding of antigen-presenting cells, tumor antigens, and other components of the immune response to cancer. Gene-based vaccination is emerging as one of the more promising approaches for loading dendritic cells (DC) with tumor-associated antigens. In this respect, it is proposed that adenoviral (AdV) vectors can deliver high antigen concentrations, promote effective processing and MHC expression, and stimulate potent cell-mediated immunity. While AdV vectors have performed well in pre-clinical vaccine models, their application to patient care has limitations. The in vivo administration of AdV vectors is associated with both innate and adaptive host responses that result in tissue inflammation and injury, viral neutralization, and premature clearance of AdV-transduced cells. A variety of strategies have been developed to address these limitations. The ideal vaccine would avoid vector-related immune responses, have relative specificity for transducing DC, and induce high levels of transgene expression. This review describes the range of host responses to AdV vaccines, identifies strategies to reduce viral recognition and enhance transgene antigen expression, and suggests future approaches to vector development and administration. There is every reason to believe that safer and more effective forms of AdV-based vaccines can be developed and applied to patient therapy.

Vaccination with helper-dependent adenovirus enhances the generation of transgene-specific CTL

Recombinant adenoviral vectors (AdV) have been used experimentally as vaccines to present antigenic transgenes in vivo. However, administration of first-generation vectors (FG-AdV) is often limited by their induction of antiviral immunity. To address this limitation, helper-dependent vectors (HD-AdV) were developed that lack viral coding regions. While the administration of HD-AdV results in long-term gene expression in vivo, their utility as immunogens has never been examined. Direct vaccination with 108 blue-forming units (BFU) of HD-AdV injected into C57BL/6 mice lead to superior transgene-specific CTL and antibody responses when compared to the same amount of a FG-AdV. The antibody responses to viral antigens were high in response to both the vectors. As a mechanism to reduce viral exposure, dendritic cells (DC) were transduced with HD-AdV in vitro and then used as a cell-based vaccine. DC transduced with HD-AdV expressed higher levels of transgene-specific mRNA and up to 1200-fold higher levels of transgene protein than did DC transduced with a FG-AdV. In addition, HD-AdV-transduced DC stimulated superior transgene-specific CTL responses when administered in vivo, an effect that was further enhanced by maturing the DC with LPS prior to administration. In contrast to direct immunization with HD-AdV, vaccination with HD-AdV-transduced DC was associated with limited antibody responses against the AdV. We conclude that HD-AdV stimulates superior transgene-specific immune responses when compared to a FG-AdV, and that immunization with a DC-based vaccine maintains this efficacy while limiting antiviral reactivity.

Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

AbstractmRNA encoding for the CB2 cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB2 and GFP, we developed a flow cytometry assay for characterizing CB2 protein expression. A monoclonal antibody directed against human CB2 selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB2 corresponded closely with the level of GFP expression. When exposed to Δ9-tetrahydrocannabinol, CB2-expressing cells internalized cell surface CB2 receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB2 protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB2 expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB2 mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB2 protein but that its distribution is predominantly intracellular with only B cells expressing CB2 protein at the extracellular membrane. The differential role of intracellular and extracellular CB2 receptors in mediating ligand signaling and immune function remains to be determined.

Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model.

BackgroundThe complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and the systemic immune system are likely to define critical factors in the host response to cancer. While conventional animal models have identified an array of potential anti-tumor therapies, mouse models often fail to translate into effective human treatments. Our goal is to establish a humanized tumor model as a more effective pre-clinical platform for understanding and manipulating TIL.MethodsThe immune system in NOD/SCID/IL-2Rγnull (NSG) mice was reconstituted by the co-administration of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells (PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating cytokines and chemokines were also assessed.ResultsA tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG animals closely matched the features of TIL recovered from primary human prostate cancers. Human cytokines were readily detectible in the serum and exhibited a different profile in animals implanted with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but could not eliminate tumor growth and this effect required the presence of CD4+ T cell help.ConclusionsSimultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that recapitulates the development of human TIL and allows an assessment of tumor and immune system interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual features and cell populations provides an opportunity for hypothesis testing and outcome monitoring in a humanized system that may be more relevant than conventional mouse models.

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of αvβ3 integrins

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)‐based gene therapy. However, DC lack the coxsackie‐Ad receptor, and little is known about the process by which they acquire and present Ad‐encoded antigens. We examined the expression of ανβ3 integrins (CD51/CD61) on mouse bone marrow‐derived DC (BM‐DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM‐DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage‐colony stimulating factor (GM‐CSF)/interleukin (IL)‐4. After 7 days, 28 ± 1.7% of CD11c+ DC expressed high levels of CD51 (CD51hi), and the remaining DC expressed low levels of CD51 (CD51lo). CD51hi CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first‐generation Ad vector, transgene expression was restricted to the CD51hi DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD‐binding sequence in its fiber knob (Ad‐RGD) transduced the CD51hi DC subset with significantly higher efficiency. When BM‐DC were transduced with an Ad‐expressing ovalbumin (Ad‐OVA), the CD51hi subset proved superior in activating OT‐I (T cell receptor‐OVA) T cells. Similar to in vitro effects, systemic administration of GM‐CSF/IL‐4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of ανβ3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad‐encoded antigens.

Clarifying CB2 Receptor-Dependent and Independent Effects of THC on Human Lung Epithelial Cells

Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that Delta(9)-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p<0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p<0.05). THC decreased ATP level and mitochondrial membrane potential (Psi(m)) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against ATP loss, did not increase cell migration. Moreover, CB2R-transduced cells displayed higher Psi(m) than did control cells. Since both Psi(m) and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of IkappaB-alpha and JNK was regulated in a CB2R- and THC-dependent manner. We conclude that airway epithelial cells are sensitive to both CB2R-dependent and independent effects mediated by THC.