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Cutting Edge New Chimeric NOD2 TLR2 Adjuvant Drastically
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Published November 12 2014 doi 10 4049 jimmunol 1402184. The Journal of, Cutting Edge Immunology, Cutting Edge New Chimeric NOD2 TLR2 Adjuvant. Drastically Increases Vaccine Immunogenicity, Vincent Pavot Nicolas Rochereau Julien Resse guier Alice Gutjahr. Christian Genin Gerard Tiraby Eric Perouzel Thierry Lioux. Fabienne Vernejoul Bernard Verrier and Ste phane Paul. TLR ligands are critical activators of innate immunity and Although TLR ligands are pyrogenic and crucial for the. are being developed as vaccine adjuvants However their induction of inflammatory cytokines NLR ligands have a poor. usefulness in conjunction with NOD like receptor agonists capacity to trigger these pathways without a synergistic boost. Downloaded from http www jimmunol org by Diane Razanajaona Doll on November 14 2014. remains poorly studied In this study we evaluated a new from TLR ligands in vitro 5 7 Despite these apparently. ligand that targets both TLR2 and NOD2 receptors We weak stimulatory properties the presence of NLR subfamily. assessed its ability to enhance dendritic cell maturation NOD1 and NOD2 ligands positively triggers the potential. in vitro in addition to improving systemic and mucosal im of some immune adjuvants including TLR ligands 8 10. mune responses in mice The chimeric NOD2 TLR2 ligand Therefore the interaction between NLRs and TLRs seems to. induced synergistic upregulation of dendritic cell matu be more crucial for adaptive immunity than previously ap. ration markers costimulatory molecules and secretion preciated An interesting new area in the field of adjuvants. could be the combination of immunostimulatory agents that. of proinflammatory cytokines compared with combina. target different pattern recognition receptor pathways 11. tions of separate ligands Furthermore when coadmi, Our purpose was to target both TLR2 and NOD2 aiming at. nistered with biodegradable nanoparticles carrying a inducing systemic and mucosal immune responses after par. model Ag the ligand was able to induce high Ag specific enteral immunization Indeed TLR2 appears to have a special. IgA and IgG titers at both systemic and mucosal sites after role in T cell polarization and differentiation especially TH17. parenteral immunizations These findings point out the 12 mucosal homing receptor expression and IgA produc. potential utility of chimeric molecules TLR NOD as tion by human B cells 13 whereas NOD2 has a role in the. adjuvants for vaccines to induce systemic and mucosal production of proinflammatory cytokines and autophagy 14. immune responses The Journal of Immunology 2014 15 and is highly expressed at the mucosal level 3 10. 193 000 000 In this study we evaluated the potential synergistic effects. of NOD2 and TLR2 stimulation by a chimeric ligand on the. induction of dendritic cell DC maturation and proinflam. ew directions in vaccine research concern the im, matory cytokine secretion in vitro as well as the induction. provement of Ag immunogenicity and the design of new. of systemic and mucosal immune responses after parenteral. adjuvants Recombinant protein subunits or synthetic. immunization in mice in the context of a nanoparticle NP. peptides are being tested in new vaccine strategies and offer many. based vaccine carrying the Gag p24 HIV 1 Ag, advantages such as a reduced toxicity compared with live.
attenuated vaccines However they often remain poorly immu. nogenic when administered alone particularly with regard to their Materials and Methods. NOD2 TLR2 chimeric NOD2 TLR2 ligands and p24 coated. capacity to induce mucosal immune responses The enhancement. polymeric NPs, of Ag specific immune responses through the development of. improved vaccine adjuvants remains an important challenge 1 CL429 is a novel chimeric compound that was designed to stimulate both. TLR2 and NOD2 This compound is composed of Murabutide NOD2. The discovery of the role of TLRs and NOD like receptors ligand covalently linked to Pam2C TLR2 ligand via a spacer Supplemental. NLRs in the action of certain adjuvants has provided new Fig 1A Murabutide Pam2C and CL429 were developed and manufactured. insights for vaccine development 2 3 These observations are by InvivoGen. particularly important with regard to the cross talk between The ligands specific activity was determined using the HEK Blue hTLR2. HEK Blue hNOD2 and RAW Blue reporter cell lines InvivoGen to monitor. NLRs and TLRs which has been the subject of numerous the activation of the NF kB and AP 1 pathways using a secreted embryonic. investigations in recent years 4 alkaline phosphatase SEAP Stimulation with ligands activates NF kB and. Centre National de la Recherche Scientifique Unite Mixte de Recherche 5305 Uni The online version of this article contains supplemental material. versite de Lyon Lyon F 69007 France Groupe Immunite des Muqueuses et Agents. Abbreviations used in this article DC dendritic cell MHC II MHC class II MoDC. Pathoge nes INSERM Centre d Investigation Clinique en Vaccinologie 1408 Universite de. monocyte derived DC NLR NOD like receptor NP nanoparticle PLA poly lactic. Lyon Saint Etienne F 42023 France and Cayla InvivoGen Toulouse F 31000 France. acid PLA p24 p24 adsorbed on PLA NP SEAP secreted embryonic alkaline phos. Received for publication September 11 2014 Accepted for publication October 13 phatase. Address correspondence and reprint requests to Dr Stephane Paul Faculte de Me decine Copyright 2014 by The American Association of Immunologists Inc 0022 1767 14 16 00. de Saint Etienne 15 rue Ambroise Pare Saint Etienne 42023 France E mail address. stephane paul chu st etienne fr, www jimmunol org cgi doi 10 4049 jimmunol 1402184. 2 CUTTING EDGE NOD2 TLR2 COSTIMULATION AS VACCINE ADJUVANT STRATEGY. AP 1 which induce the production of SEAP The level of SEAP can be easily. quantified with a detection medium that turns purple blue in the presence of. alkaline phosphatase HEK Blue Detection InvivoGen, HIV 1 p24 Ag was produced and purified by PX Therapeutics Pro. tein eXpert Grenoble France, Poly lactic acid PLA NPs 180 nm were prepared by nanoprecipitation. as previously described 16 and provided by Adjuvatis Lyon France P24. protein and PLA particles were diluted in PBS at 200 mg ml and 5 mg ml. respectively The two solutions were mixed volume to volume The adsorp. tion reaction occurred within 2 h at room temperature with moderate. overhead stirring Endotoxin contents were assessed using an Endosafe test. Charles River and were 5 EU ml, In vitro maturation of human monocyte derived DCs and cytokine.
Monocytes were purified from peripheral human blood and cultured in the. presence of IL 4 and GM CSF to differentiate into DCs monocyte derived. DCs MoDCs 17 After 6 d NOD2 TLR2 or chimeric NOD2 TLR2. Downloaded from http www jimmunol org by Diane Razanajaona Doll on November 14 2014. ligands 10 nmol ml were added to the MoDCs for 24 or 48 h LPS. 2 5 mg ml was used as a positive control Cells were then stained with FITC. labeled anti CD1a DC marker and DC maturation was assessed by cell im. munostaining using PE labeled mAbs against the maturation marker CD83. costimulatory molecules CD80 CD86 MHC class II MHC II BD Phar. mingen and the intracellular DC LAMP maturation marker Dendritics Ten. thousand events were acquired by FACS with a FACSCanto II BD and the. data were analyzed using FlowJo software MoDC culture supernatants were. collected after 24 or 48 h and expression of IL 12p70 TNF a IL 6 IL 1b and. IFN a was analyzed by Luminex using the Bio Plex Pro assay Bio Rad. Autophagy was analyzed by immunofluorescence analysis of LC3 After stimu. lation 8 h cells were fixed permeabilized incubated with LC3 specific Ab. NanoTools clone 5F10 and incubated with secondary Ab goat anti mouse. FITC An agent containing DAPI was added cells were observed using fluo. rescent microscopy and quantification of cells containing LC3 autophagosomes. was performed, Mouse study design, Eight to twelve week old female BALB c mice were purchased from Charles. River Laboratories Mice were divided into five groups of five animals and. immunized s c with 10 mg p24 adsorbed on PLA NPs PLA p24 suspended. in 100 ml PBS on days 0 14 and 28 A total of 20 nmol ligands NOD2. TLR2 both or chimeric was coadministered one group was immunized with. PLA p24 alone Sera vaginal lavage fluid and fecal pellets were collected at. days 0 14 28 and 34 and tested for the presence of p24 specific IgA IgG. IgG1 and IgG2a by ELISA, Serum samples were obtained from whole blood recovered by performing. a small incision at the retro orbital vein Vaginal secretions were collected from. mice with 50 ml PBS two times placed in the vagina of the animal using an. adapted pipette Pipette M100 and tips CP100 both from Gilson A total. of five microliters of 253 Halt Protease Inhibitor Cocktail Thermo Scien. tific was added to the vaginal lavage fluids to protect Igs from degradation. Feces were collected and diluted at 100 mg ml with 13 Halt Protease In. hibitor Cocktail to allow normalization, To study acute cytokine production new groups of mice were immunized. twice one week apart Sera and vaginal and intestinal tissues were collected 24 h. after the boost The vaginal cell and intestine cell lysates were prepared with the. Bio Plex Cell Lysis Kit Bio Rad as specified by the manufacturer Evaluation. of multiple cytokines chemokines was performed with a Luminex 100 in. strument in combination with the Bio Plex mouse cytokine panels Bio Rad. Cytokine concentrations were determined as the mean of three replicates time. Statistical analysis, FIGURE 1 Maturation related patterns expressed by human MoDCs. Analyses were performed using SigmaPlot software version 11 0 Statistical upon exposure to NOD2 TLR2 or chimeric NOD2 TLR2 ligands A. data analyses were performed using one way ANOVA with Bonferroni post Phenotypes of MoDCs were determined after 24 h of stimulation and. hoc test for comparison of pairs Statistical significance is indicated in the mean expression of maturation markers CD83 and DC LAMP costim. figures ulatory molecules CD80 and CD86 and MHC II were analyzed B. Quantification of cytokine secretion pattern of MoDCs by a Bio Plex Pro. Results and Discussion assay The supernatants were collected and the concentrations of IL 6. The chimeric ligand CL429 activates both TLR2 and NOD2 TNF a IL 1b IL 12p70 and IFN a were analyzed Data are means SD. The specific activity of each ligand at different concentrations n 3 C Chimera molecule NOD2 TLR2 improves autophagy induc. tion in DCs DCs were incubated in the presence of the ligands for 8 h. was monitored in vitro using cell lines expressing only TLR2 Cells were stained with Ab to LC3 and cells containing LC3 autopha. NOD2 or both As shown in Supplemental Fig 1B HEK cell gosomes were quantified Data are mean 6 SD from eight independent. line activation was dose dependent and the TLR2 ligand counts Data are representative of more than three independent experi. Pam2C was able to stimulate only HEK cells expressing ments p 0 05 p 0 01 p 0 001. The Journal of Immunology 3, TLR2 whereas the NOD2 ligand Murabutide was able to This strong adjuvant effect could be explained by improved.
stimulate only HEK cells expressing NOD2 Both cell lines NOD2 agonist uptake by DCs Although TLR2 receptors are. were efficiently stimulated with the chimeric ligand CL429 expressed on the cell surface and so widely exposed to ligands. meaning that the chimeric ligand can activate both TLR2 and NOD2 receptors are within the cytoplasm and are exposed. NOD2 and induce NF kB and or AP 1 signaling Moreover to few diffusing NOD2 agonists We hypothesized that the. stimulation of the RAW Blue cell line expressing both recep chimera is taken up through its TLR2 agonist domain in lipid. tors showed that the chimeric ligand was significantly more rafts and internalized within endosomes 19 20 enabling. efficient than separated ligands more NOD2 ligands to enter the cells How the NOD2 ag. onist domain of the chimera interacts with the NOD2 re. The chimeric ligand NOD2 TLR2 induces synergistic human DC ceptor after endosomal internalization is still beyond our. maturation in vitro comprehension and requires further study but involvement of. Adjuvants and vaccine delivery systems address the interface. between innate and adaptive immune responses where the. extent and direction of innate immune activation determine. the quality and magnitude of the resulting adaptive response. Downloaded from http www jimmunol org by Diane Razanajaona Doll on November 14 2014. DCs play a major role at this interface their maturation is. correlated with the presentation of Ag fragments by MHC. molecules as well as with the expression of specific cell mark. ers To determine the adjuvant potential of NOD2 and or. TLR2 ligands to stimulate human DCs in vitro expression. of the cell markers of maturation was assessed by flow cytometry. 24 h after equimolar ligand addition As shown in Fig 1A. a statistically significant increase in the expression of CD80. CD86 and DC LAMP was observed in MoDCs incubated, with NOD2 or TLR2 ligand alone p 0 05 CD83 ex. pression was enhanced to a statistically significant extent by the. NOD2 ligand p 0 001 whereas the amount of MHC II, was not modified by the NOD2 or TLR2 ligand The com. bination of the two ligands significantly increased the expres. sion of CD86 CD80 and DC LAMP markers but not, CD83 compared with NOD2 alone suggesting a cumulative. Cutting Edge New Chimeric NOD2 TLR2 Adjuvant Drastically Increases Vaccine Immunogenicity Endotoxin contents were assessed using an Endosafe test Charles River and were 5EU ml In vitro maturation of human monocyte derived DCs and cytokine secretion Monocytes were puri ed from peripheral human blood and cultured in the presence of IL 4 and GM CSF to differentiate into DCs monocyte

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