David Escors

Lentiviral vectors in gene therapy: their current status and future potential

The concept of gene therapy originated in the mid twentieth century and was perceived as a revolutionary technology with the promise to cure almost any disease of which the molecular basis was understood. Since then, several gene vectors have been developed and the feasibility of gene therapy has been shown in many animal models of human disease. However, clinical efficacy could not be demonstrated until the beginning of the new century in a small-scale clinical trial curing an otherwise fatal immunodeficiency disorder in children. This first success, achieved after retroviral therapy, was later overshadowed by the occurrence of vector-related leukemia in a significant number of the treated children, demonstrating that the future success of gene therapy depends on our understanding of vector biology. This has led to the development of later-generation vectors with improved efficiency, specificity, and safety. Amongst these are HIV-1 lentivirus-based vectors (lentivectors), which are being increasingly used in basic and applied research. Human gene therapy clinical trials are currently underway using lentivectors in a wide range of human diseases. The intention of this review is to describe the main scientific steps leading to the engineering of HIV-1 lentiviral vectors and place them in the context of current human gene therapy.

Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

ABSTRACT Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Δ3abΔE) was successfully rescued in these cell lines. rTGEV-Δ3abΔE reached high titers (107 PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 × 105 PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Δ3abΔE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E+ packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.

HIV-1 Lentiviral Vector Immunogenicity Is Mediated by Toll-Like Receptor 3 (TLR3) and TLR7

ABSTRACT Lentiviral vectors are promising vaccine vector candidates that have been tested extensively in preclinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunization. The activation of DC cultures was independent of the lentiviral pseudotype but dependent on cell entry and reverse transcription. In vivo-transduced DC also displayed a mature phenotype, produced tumor necrosis factor alpha (TNF-α), and stimulated naive CD8+ T cells. The lentiviral activation of DC was Toll-like receptor (TLR) dependent, as it was inhibited in TRIF/MyD88 knockout (TRIF/MyD88−/−) DC. TLR3−/− or TLR7−/− DC were less activated, and reverse transcription was important for the activation of TLR7−/− DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8+ T-cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the hosts immune response to the transgene.

Targeting dendritic cell signaling to regulate the response to immunization

Dendritic cells (DCs) are key regulators of the immune system; they capture antigens and then can either stimulate an immune response or induce tolerance. Our aim was to activate individual DC signaling pathways to regulate the immune response. We therefore expressed constitutive activators of mitogen-activated protein kinase (MAPK) pathways or the interferon pathway, together with tumor antigens, using lentivectors. Triggering of p38 activated DCs substantially enhanced the antitumor immune response and prolonged survival of tumor-bearing mice. Activation of extracellular signal-regulated kinase (ERK) increased TGF-beta expression while expression of a constitutively activated interferon regulatory factor-3 (IRF3) stimulated IL-10 secretion by DCs. ERK and IRF3 suppressed the immune response and stimulated expansion of regulatory T cells. These results provide a toolkit to regulate immune responses to viral vector or DC immunization; vaccine responses to foreign or tumor antigens can be enhanced and harmful responses to self-antigens or introduced transgenes can be reduced.

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

PD-L1 co-stimulation contributes to ligand-induced T cell receptor down-modulation on CD8(+) T cells

T cell receptor (TCR) down‐modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down‐modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD‐L1) on dendritic cells (DCs) and programmed death 1 (PD‐1) on CD8 T cells contributes to ligand‐induced TCR down‐modulation. We provide evidence that this occurs via Casitas B‐lymphoma (Cbl)‐b E3 ubiquitin ligase up‐regulation in CD8 T cells. Interference with PD‐L1/PD‐1 signalling markedly inhibits TCR down‐modulation leading to hyper‐activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD‐L1 silencing accelerates anti‐tumour immune responses and strongly potentiates DC anti‐tumour capacities, when combined with mitogen‐activated kinase (MAPK) modulators that promote DC activation.

The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

ABSTRACT The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg2+ ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.

The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells

In T lymphocytes, p38 MAP kinase (MAPK) regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative T cell receptor (TCR) activation pathway. Here we show that senescent human T cells lack the canonical and alternative pathways of p38 activation, but spontaneously engage the metabolic master regulator AMPK to trigger p38 recruitment to the scaffold TAB1 causing p38 auto-phosphorylation. Signaling via this pathway inhibits telomerase activity, T cell proliferation and expression of key components of the TCR signalosome. Our findings identify an unrecognized mode of p38 activation in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (‘intra-sensory’ pathway). The proliferative defect of senescent T cells is reversed by blocking AMPK-TAB1-dependent p38 activation.

Nonintegrating Lentivector Vaccines Stimulate Prolonged T-Cell and Antibody Responses and Are Effective in Tumor Therapy

ABSTRACT Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8+ T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8+ T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-κB activator from the same vector. Second, we demonstrated systemic CD8+ T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Organization of Two Transmissible Gastroenteritis Coronavirus Membrane Protein Topologies within the Virion and Core

ABSTRACT The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of 35S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.