Exogenous LRRK2G2019S induces parkinsonian-like pathology in a nonhuman primate

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease among the elderly. To understand pathogenesis and to test therapies, animal models that faithfully reproduce key pathological PD hallmarks are needed. As a prelude to developing a model of PD, we tested the tropism, efficacy, biodistribution, and transcriptional impact of canine adenovirus type 2 (CAV-2) vectors in the brain of Microcebus murinus, a nonhuman primate that naturally develops neurodegenerative lesions. We show that introducing helper-dependent (HD) CAV-2 vectors results in long-term, neuron-specific expression at the injection site and in afferent nuclei. Although HD CAV-2 vector injection induced a modest transcriptional response, no significant adaptive immune response was generated. We then generated and tested HD CAV-2 vectors expressing LRRK2 (leucine-rich repeat kinase 2) and LRRK2 carrying a G2019S mutation (LRRK2G2019S), which is linked to sporadic and familial autosomal dominant forms of PD. We show that HD-LRRK2G2019S expression induced parkinsonian-like motor symptoms and histological features in less than 4 months

A review of 65 years of human adenovirus seroprevalence

International audienceIntroduction: Human adenovirus (HAdV)-derived vectors have been used in numerous pre-clinical and clinical trials during the last 40 years. Current research in HAdV-based vaccines focuses on improving transgene immunogenicity and safety. Because pre-existing humoral immunity against HAdV types correlate with reduced vaccine efficacy and safety, many groups are exploring the development of HAdV types vectors with lower seroprevalence. However, global seroepidemiological data are incomplete. Areas covered: The goal of this review is to centralize 65 years of research on (primarily) HAdV epidemiology. After briefly addressing adenovirus biology, we chronical HAdV seroprevalence studies and highlight major milestones. Finally, we analyze data from about 50 studies with respect to HAdVs types that are currently used in the clinic, or are in the developmental pipeline. Expert opinion: Vaccination is among the most efficient tools to prevent infectious disease. HAdV-based vaccines have undeniable potential, but optimization is needed and antivector immunity remains a challenge if the same vectors are to be administrated to different populations. Here, we identify gaps in our knowledge and the need for updated worldwide epidemiological data

IC-HAdV induce membrane damage and gain access to the cytosol.

<p>MoDCs were exposed to Alexa488-HAdV-C5, IC-HAd-488 or IVIg for 3 h and processed for confocal immunofluorescence analysis and stained with anti-p62 (n = 3) A) and galectin-3 (n = 3) B). Scale bar = 5 μm. C) Transmission electron microscopy of IC-HAdV in MoDCs at indicated times. HAdV-C5 can be seen due to their high electron density. White lines depict circumference of IC-HAdVs. White asterisks indicate artifacts.</p

IC-HAdV induce MoDC maturation through DNA sensors TLR9.

<p>TNF secretion in response to IC-HAdV was measured in MoDCs after lentivirus-mediated shRNA knockdown of TLR9, MyD88 and AP3B1. A) Immunoblotting demonstrating lentivirus-mediated shRNA knockdown of the TLR9 pathway in MoDC. MoDCs were exposed to LPS, HAdV-C5, IC-HAdV or IVIg for 3 and/or 6 h and B) TNF secretion was measured by ELISA. C) IL1B mRNA levels were assessed by qRT-PCR. D) Immunoblots of pro-IL-1β levels in MoDCs after incubation with HAdV-C5, IC-HAdV, IVIg or LPS at 6 h. β-tubulin levels were used as loading controls. The experiments were carried out in 2–3 donors with similar results.</p

IC-HAdV trafficking and cellular events leading to pyroptotic dendritic cell death.

<p>IC-HAdV are taken up via FcγR-mediated endocytosis and fuse with TLR9⁺ endolysosomal vesicles because of NAb-dependent capsid stabilization at endosomal pH. At the more acidic pH in lysosome-like vesicles, IC-HAdV begin to disassemble and to release viral DNA, membrane lytic protein VI, and cellular components. TLR9 engagement induces MyD88-NF-κB-dependent <i>de novo</i> expression of TNF and pro-IL1β. During vesicular DNA sensing, protein VI release from the capsid leads to endolysosomal membrane lysis and IC-HAdV gain access to the cytosol. The cytosolic inflammasome sensor AIM2 interacts with the HAdV-C5 genome and induces inflammasome assembly and activation of caspase 1, and downstream cleavage of pro-IL-1β and GSDMD. Together this leads to pyroptosis of MoDCs.</p

IC-HAdV induce loss of plasma membrane integrity and release of cytosolic proteins from MoDC.

<p>MoDCs were challenged with IC-HAdV and plasma membrane integrity, LDH and GFP release was quantified. A) MoDCs were incubated with IVIg, HAdV-C5 and IC prepared with an escalating dose (100, 1,000, 2,000 5,000, 10,000 15,000, 20,000 pp/cell) of HAdV-C5 and cell membrane integrity was assessed by intercalation of propidium iodide (PI) into cellular DNA at 6 h. B) MoDCs were incubated with Alexa555-HAdV-C5, IC-HAdV-555 or IVIg and assayed by flow cytometry at 6 h. The quadrants were set for PI^- HAdV-C5^- (white), PI^- HAdV-C5⁺ (light grey), PI⁺ HAdV-C5⁺ (dark grey) and PI⁺ HAdV-C5^- (black). The percentage of each subpopulation at each condition is depicted in the histogram. These assays were performed in 3 donors with similar results. C) Loss of cytosolic content by MoDC exposed to IC-HAdV, IVIg, HAdV-C5 and LPS/nigericin was assessed by measuring LDH activity in the supernatant. D) Loss of cytosolic content by GFP-lentivirus transduced MoDC exposed to IC-HAdV, IVIg, HAdV-C5 and LPS/nigericin was assessed by flow cytometry measuring GFP fluorescent and plasma membrane integrity with PI.</p

IC-HAdV induced IL-1β expression, caspase-dependent IL-1β secretion and AIM2 inflammasome-dependent GSDMD cleavage in MoDCs.

<p>The involvement of the AIM2 inflammasome and caspases in the inflammatory response in IC-HAdV challenged MoDCs ± inhibitors was assessed by quantifying intra- and extracellular levels of IL-1β plasma membrane integrity and GSDMD cleavage. A) Effect of protein VI-mediated membrane lytic activity on IL-1β secretion was assessed by incubation of MoDCs with escalating doses of IC-HAdV or IC-AdL40Q. IL-1β secretion was quantified by ELISA (n = 5). P values were derived from two-way ANOVA with Bonferroni posttest. * and ** correspond to p < 0.05 and p < 0.01, respectively. B) IL-1β secretion in MoDCs pre-incubated with 20 μM YVAD and Z-VAD and stimulated as indicated. This experiment was performed in triplicate using 3 donors with similar results. C) Loss of cell membrane integrity was assessed by PI/flow cytometry for MoDCs pre-incubated with Z-VAD and exposure to HAdV-C5, IC-HAdV and IVIg at 6 h (n = 3). D) Caspase 1 activation was measured with FAM-YVAD-FMK FLICA by flow cytometry. MoDC were preincubated with YVAD-FLICA for 1 h and exposed to LPS, LPS/nigericin, IVIg HAdV-C5 and IC-HAdV for 3 h. Nigericin was added to MoDC at 2 h. This assay was performed in 3 donors with similar results E) GSDMD cleavage in IC-HAdV-challenged MoDC in presence of caspase inhibitors was monitored by western blot. MoDC were preincubated for 1 h with 20 or 100 μM Z-VAD, YVAD, WEHD or an equal volume of DMSO followed by exposure to LPS/nigericin (added at 4 h), IVIg, HAdV-C5 and IC-HAdV for 6 h. This assay was performed in 3 donors with similar results. F) GSDMD cleavage in IC-HAdV-challenged MoDC in presence of AIM2 inhibitors was monitored by immunoblotting. MoDC were pre-incubated for 2 h with 100 μM ODN A151 followed by exposure to IVIg, HAdV-C5 and IC-HAdV for 6 h This assay was performed in 3 donors with similar results.</p

Protein VI is indirectly linked to loss of plasma membrane integrity.

<p>MoDCs were challenged with IC prepared from HAdV-C5 and HAdV-C5 harboring mutations in protein VI or protease. A) Effect on cell membrane integrity was determined by incubating MoDCs with HAdV-C5, Ad2ts1 and HAdV-C5 empty capsid or IC of each virus, respectively (n = 3) for 6 h, then with PI followed by flow cytometry. B) Dose-dependent loss of cell membrane integrity was assessed for IC-HAdV and IC-AdL40Q at 6 h (n = 5) as in B. Statistical significance (<i>p</i> values) were derived from two-way ANOVA with Bonferonni post test: * and ** correspond to p < 0.05 and p < 0.01, respectively.</p

Immune-complexed HAdV-C5: role of IgGs, physical characteristics, and anti-hexon Abs.

<p>The effect of IgG-opsonization on HAdV-C5 particle size, stability, and aggregation, and how it affects MoDC maturation was assessed. A) TNF secretion by MoDCs incubated with LPS, HAdV-C5, IVIg and IC-HAdV formed using a fixed dose of 20,000 pp/cell HAdV-C5 and increasing volume of pooled human IgGs (IVIg). B) Size of HAdV-C5, IC-HAdV and IVIg was assayed by nanoparticle tracking. IC-HAdV were formed in PBS for 2 h at 37°C. C) Size of IC-HAdV-555 by flow cytometry. Fluorescent beads of 1 and 3 μm were used to determine the diameter of IC-HAdV based on side scatter (SSC). D) Fluorescent intensity used as a surrogate for capsid stability: the stability of HAdV-C5 or IC-HAdV was assessed at pH 6 and 7 at increasing temperatures by monitoring HAdV-C5 genome accessibility to Picogreen. E) Immune complexes were generated using either IVIg, mock-depleted IVIg (NAbs ΔCtrl), IgG recovered from mock-depleted IVIg (NAbs from ΔCtrl), from IVIg depleted for hexon-antibodies (NAbs Δα-hexon), or purified anti-hexon Abs (NAbs from Δα-hexon). MoDCs were incubated with HAdV-C5, IC-HAdV, IVIg for 3 or 6 h. TNF secretion was quantified by ELISA. All assays were repeated at least three times with similar results.</p