Histidylated nanovectors for mRNA vaccine formulation: Induction of a strong anti-tumor T cell immunity combined with inflammatory state

These last years, we are witnessing the emergence of new class of biopharmaceuticals based-on transcribed mRNA. They emerged as an extremely tunable vaccination platform. Formulations made of mRNA and liposomes (lipoplexes) have yielded strong T cell responses, but require induction of cytokines identical to those that have plagued clinical development of siRNA therapeutics. We have developed histidylated Lipid Polymer mRNA nanocomplexes (LPR) that combine the beneficial properties of lipid based and polymer based nanoparticles, including lowered cellular toxicities and improved colloidal stabilities. Immunization with LPR instigated extremely potent T-cell responses and showed superior effectiveness in controlling tumor growth compared to intravenous immunization with antigen mRNA electroporated dendrictic cells. Early innate responses to LPR were characterized by a type I IFN signature in the spleen. Nonetheless, conversely to LR, LPR did not depend on type I IFN responses to generate cytolytic effectors. This unique behavior of LPR enabled the generation of a less pro-inflammatory yet equally potent systemic LPR vaccine by usage of N1-methylpseudo-uridine (N1mψ) modified mRNA required to improve mRNA translatability by avoiding mRNA sensors activation. Overall, our data indicate that LPR can combine excellent immunogenicity with improved inflammatory and they could be an interesting alternative to formulations that are currently explored in early phase clinical trials.. Please click Additional Files below to see the full abstract

Skin Immune Landscape: Inside and Outside the Organism

The skin is an essential organ to the human body protecting it from external aggressions and pathogens. Over the years, the skin was proven to have a crucial immunological role, not only being a passive protective barrier but a network of effector cells and molecular mediators that constitute a highly sophisticated compound known as the “skin immune system” (SIS). Studies of skin immune sentinels provided essential insights of a complex and dynamic immunity, which was achieved through interaction between the external and internal cutaneous compartments. In fact, the skin surface is cohabited by microorganisms recognized as skin microbiota that live in complete harmony with the immune sentinels and contribute to the epithelial barrier reinforcement. However, under stress, the symbiotic relationship changes into a dysbiotic one resulting in skin disorders. Hence, the skin microbiota may have either positive or negative influence on the immune system. This review aims at providing basic background information on the cutaneous immune system from major cellular and molecular players and the impact of its microbiota on the well-coordinated immune responses in host defense

Ultrasound sensitive O2O_{2} microbubbles radiosensitize murine breast cancer but lead to higher metastatic spread

The inadequate level of oxygenation in tumors has been shown to correlate not only with greater invasiveness of cancer cells, but also with a reduction in their sensitivity to anticancer therapies. Over the years, many attempts have been made to increase the oxygenation level of cancer, but most of them have been ineffective. We investigated the heterogeneous response of tumor tissue to phospholipid-coated oxygen microbubbles (OMB) in murine tumors in vivo using oxygen and hemoglobin saturation mapping and the influence of OMB treatment on microvasculature, perfusion, and radiotherapy effectiveness. Intravenous administration of OMB followed by ultrasound pulse leads to increased oxygenation of a tumor, found mainly in the vicinity of tumor vessels, while intratumoral delivery resulted in areas of increased $pO_{2}$ more evenly distributed within the tumor. Furthermore, hemoglobin contributes little to the increase in tumor oxygenation caused by oxygen microbubbles. Extensive vasculature disruption was observed in the groups treated with both oxygen/nitrogen microbubbles and ultrasound pulse. This therapy also led to a reduction in the coverage of the vessels by pericytes, while the density of the microvessels was unchanged. Radiotherapy with a single dose of 12Gy reduced tumor growth by 50% in all treated groups. Unfortunately, at the same time, the number of macroscopic metastases in the lungs increased significantly after intravenous administration of oxygen/nitrogen microbubbles and the application of an ultrasound pulse. In conclusion, ultrasound-sensitive oxygen microbubbles are effective in delivering oxygen to tumor tissue, thus increasing the effectiveness of radiotherapy. However, cavitation effects and destruction of the integrity of tumor vessels result in greater spread of cancer cells in the host organism

An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-regulation

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using a study model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5- mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We show that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking

In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.info:eu-repo/semantics/publishedVersio

The Flagella of an Atypical Enteropathogenic Escherichia coli Strain Are Required for Efficient Interaction with and Stimulation of Interleukin-8 Production by Enterocytes in Vitro

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. in this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. the role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. in addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Colegio Doutoral Franco BrasileiroInstitut PasteurFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Programa de Apoio a Nucleos de ExcelenciaPRONEXConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilInst Pasteur, Unite Pathogenie Bacterienne Muqueuses, F-75724 Paris 15, FranceInst Butantan, Bacteriol Lab, BR-05503900 São Paulo, BrazilInst Fleury Ensino & Pesquisa, BR-04344903 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilInstitut Pasteur: PTR165FAPESP: 05/59128-0Web of Scienc

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Abstract Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking

Regulation and novel action of thymidine phosphorylase in non-small cell lung cancer : crosstalk with Nrf2 and HO-1

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC

New histidylated cationic lipids for DNA and mRNA- based lipofection

Plasmid delivery into the cytosol remains one of the limiting factor to achieve efficient transfection. We have previously demonstrated that the presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers significantly enhances their gene delivery efficiencies. We designed and synthesized two novel non-glycerol and a cholesterol based histidylated cationic amphiphiles containing a single histidine head group. Physico-chemical characteristics of all the novel liposomes and lipoplexes including lipid:DNA interactions, global surface charge, sizes, etc. were evaluated. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-Histidine(N,N-di-n-hexadecylamine, N-methyl)ethylamide (lipid 2) in combination with cholesterol and Cholesteryl-L-Histidine-Ethylamide (lipid 3) in combinaison with DOPE gave efficient DNA and mRNA transfections into various cell lines. DNA transfection efficiency into A549, 293T7 and HeLa cells of Chol/lipid 1 lipoplexes was similar with that of FuGENE6 and DC-Chol lipoplexes but was two order of magnitude higher in HepG2 cells. Membrane fusion activity measurements using FRET technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. By using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter, we showed that the release of DNA from the endosomally trapped lipoplexes to the cytosol is acidic dependent and presumably mediated by the imidazole ring protonation of histidine head group of these cationic amphiphiles. A better efficiency was obtained with Chol/lipid 2 lipoplexes than with Chol/lipid 1 lipoplexes when using the cytosolic luciferase expression vector. As anticipated, transfection efficiency of Lipid 3 was greatly inhibited in the presence of Bafilomycin A1. By contrast, endosome escape of DNA with a new cholesterol based cationic lipid containing no histidine head-group (Alanine-Cholesteryl-Ethylamide; lipid 4) seemed to be independent of endosome acidification. However, transfection efficacy of lipids 3 & 4 was similar. In conclusion, we show that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds or cholesterol is sufficient to impart remarkable transfection properties on the resulting cationic amphiphile via endosome-disrupting characteristics of the histidine functionalities Figure 1