High Therapeutic Efficacy of a New Survivin LSP-Cancer Vaccine Containing CD4+ and CD8+ T-Cell Epitopes

The efficacy of an antitumoral vaccine relies both on the choice of the antigen targeted and on its design. The tumor antigen survivin is an attractive target to develop therapeutic cancer vaccines because of its restricted over-expression and vital functions in most human tumors. Accordingly, several clinical trials targeting survivin in various cancer indications have been conducted. Most of them relied on short peptide-based vaccines and showed promising, but limited clinical results. In this study, we investigated the immunogenicity and therapeutic efficacy of a new long synthetic peptide (LSP)-based cancer vaccine targeting the tumor antigen survivin (SVX). This SVX vaccine is composed of three long synthetic peptides containing several CD4+ and CD8+ T-cell epitopes, which bind to various HLA class II and class I molecules. Studies in healthy individuals showed CD4+ and CD8+ T-cell immunogenicity of SVX peptides in human, irrespective of the individual's HLA types. Importantly, high frequencies of spontaneous T-cell precursors specific to SVX peptides were also detected in the blood of various cancer patients, demonstrating the absence of tolerance against these peptides. We then demonstrated SVX vaccine's high therapeutic efficacy against four different established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses. When tumors were eradicated, generated memory T-cell responses protected against rechallenge allowing long-term protection against relapses. Treatment with SVX vaccine was also found to reshape the tumor microenvironment by increasing the tumor infiltration of both CD4+ and CD8+ T cells but not Treg cells therefore tipping the balance toward a highly efficient immune response. These results highlight that this LSP-based SVX vaccine appears as a promising cancer vaccine and warrants its further clinical development

Kallikrein-related peptidase 5 contributes to H3N2 influenza virus infection in human lungs

Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype in vitro, but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans

Recognition of pleural mesothelioma by mucin-1(950-958)/human leukocyte antigen A*0201-specific CD8+ T-cells

International audienceRecent clinical investigations have demonstrated that T-cell-based immunotherapy of malignant pleural mesothelioma (MPM) could represent an alternative to the other therapeutic strategies. However, its development suffers from the lack of identified tumour antigenic targets. Mucin (MUC)1, which is expressed and recognised by cytotoxic T-cells in numerous cancer types, has not been investigated as a potential immune target in MPM. Thus, the objective of this study was to analyse MUC1 expression by MPM cells and to determine whether this antigen can be the target of cytotoxic CD8+ T-cells (cytotoxic T-lymphocytes (CTLs)). We first evaluated the expression and glycosylation of MUC1 by MPM cell lines using different MUC1-specific monoclonal antibodies. We then obtained a CTL clone specific for a MUC1 peptide (residues 950-958) presented by human leukocyte antigen (HLA)-A*0201 and studied its interferon-c and cytotoxic response to MPM cell lines. We found that all MPM cell lines expressed MUC1 protein at the cell surface with different glycosylation profiles. We also observed that HLA-A*0201+ MPM cell lines are recognised and lysed by a HLA-A*0201/MUC1(950-958)-specific CTL clone independently of the MUC1 glycosylation profile. Thus, MUC1 expression and antigen presentation by MPM cells may represent an attractive target for immunotherapeutic treatment of MPM despite its hyperglycosylated profile

Dehydroepiandrosterone up-regulates the Adrenoleukodystrophy-related gene (ABCD2) independently of PPAR alpha in rodents

International audienceX-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC transporter, ALDP, supposed to participate in the transport of very long chain fatty acids (VLCFA). The adrenoleukodystrophyrelated protein (ALDRP), which is encoded by the ABCD2 gene, is the closest homolog of ALDP and is considered as a potential therapeutic target since functional redundancy has been demonstrated between the two proteins. Pharmacological induction of Abcd2 by fibrates through the activation of PPARa has been demonstrated in rodent liver. DHEA, the most abundant steroid in human, is described as a PPARa activator and also as a prohormone able to mediate induction of several genes. Here, we explored the in vitro and in vivo effects of DHEA on the expression of peroxisomal ABC transporters. We show that Abcd2 and Abcd3 but not Abcd4 are induced in primary culture of rat hepatocytes by DHEA-S. We also demonstrate that Abcd2 and Abcd3 but not Abcd4 are inducible by an 11-day treatment with DHEA in the liver of male rodents but not in brain, testes and adrenals. Finally and contrary to Abcd3, we show that the mechanism of induction of Abcd2 is independent of PPARa

Nanoparticles Produced by Ring-Opening Metathesis Polymerization Using Norbornenyl-poly(ethylene oxide) as a Ligand-Free Generic Platform for Highly Selective In Vivo Tumor Targeting

We described a norbornenyl-poly(ethylene oxide) nanoparticles ligand-free generic platform, made fluorescent with straightforward preparation by ring-opening metathesis polymerization (ROMP). Our method allowed to easily obtain a drug delivery system (DDS) with facilitated functionalization by means of azide-alkyne click chemistry and with a high selectivity for the tumor in vivo, while cellular internalization is obtained without cell targeting strategy. We demonstrated that our nanoparticles are internalized by endocytosis and colocalized with acidic intracellular compartments in two models of aggressive tumoral cell lines with low prognostic and limited therapeutic treatments. Our nanoparticles could be of real interest to limit the toxicity and to increase the clinical benefit of drugs suffering rapid clearance and side effects and an alternative for cancers with poorly efficient therapeutic solutions by associating the drug delivery in the tumor tissue with an acid-sensitive release system

Tissue kallikrein regulates alveolar macrophage apoptosis early in influenza virus infection

International audienceHost cell proteases are involved in influenza pathogenesis. We examined the role of tissue kallikrein 1 (KLK1) by comparing wild-type (WT) and KLK1-deficient mice infected with influenza H3N2 virus. The levels of KLK1 in lung tissue and in bronchoalveolar lavage (BAL) fluid increased substantially during infection. KLK1 did not promote virus infectivity despite its trypsin-like activity, but it did decrease the initial virus load. We examined two cell types involved in the early control of pathogen infections, alveolar macrophages (AMs) and natural killer (NK) cells to learn more about the antiviral action of KLK1. Inactivating the Klk1 gene or treating WT mice with an anti-KLK1 monoclonal antibody to remove KLK1 activity accelerated the initial virus-induced apoptotic depletion of AMs. Intranasal instillation of deficient mice with recombinant KLK1 (rKLK1) reversed the phenotype. The levels of granulocyte-macrophage colony-stimulating factor in infected BAL fluid were significantly lower in KLK1-deficient mice than in WT mice. Treating lung epithelial cells with rKLK1 increased secretion of this factor known to enhance AM resistance to pathogen-induced apoptosis. The recruitment of NK cells to the air spaces peaked 3 days after infection in WT mice but not in KLK1-deficient mice, as did increases in several NK-attracting chemokines (CCL2, CCL3, CCL5, and CXCL10) in BAL. Chronic obstructive pulmonary disease (COPD) patients are highly susceptible to viral infection, and we observed that the KLK1 mRNA levels decreased with increasing COPD severity. Our findings indicate that KLK1 intervenes early in the antiviral defense modulating the severity of influenza infection. Decreased KLK1 expression in COPD patients could contribute to the worsening of influenza

Histone deacetylase inhibitor-polymer conjugate nanoparticles for acid-responsive drug delivery

We report the synthesis of acid responsive polymeric nanoparticles (NPs) consisting of a polymerhistone deacetylase inhibitor conjugate. An innovative aspect of this drug delivery particle lies in the NP conjugation of a histone deacetylase (HDAC) inhibitor, CI-994 (Tacedinaline), introduced with a clickable acid-responsive prodrug during monomer synthesis, prior to polymerization. Another novelty lies in the selected norbornene (NB)-polyethylene oxide (PEO) macromonomer allowing standardization of the polymerization process by Ring-Opening Metathesis Polymerization (ROMP) and functionalization through azide-allcyne click chemistry. Herein we demonstrate that the synthesized polymer gave 300 nm core-shell spherical nanoparticles with low dispersity (0.04), high water dispersability thanks to the PEO shell and well controlled HDAC inhibitor prodrug loading. Bioluminescence Resonance Energy Transfer (BRET) assay in living cells and viability experiments demonstrated efficient cellular internalization without additional chemistry, drug release inside cells with restoration of the HDAC inhibition and induction of apoptosis. Such NPs should minimize drug release in vivo during blood circulation and trigger intracellular delivery after endocytosis, holding promises for improved efficacy of this class of epigenetic inhibitors. This standardized synthesis paves the way for multifunctional nanoparticles synthesis

Vorinostat-Polymer Conjugate Nanoparticles for Acid-Responsive Delivery and Passive Tumor Targeting

International audienc

Implication de KLK12 dans le remodelage de la matrice extracellulaire (MEC) et la migration des cellules endothéliales pulmonaires

National audienc

A novel cell model to study the function of the adrenoleukodystrophy-related protein

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycycline dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural contex