TLR ligand functionalized nanocarriers to enhance immunogenicity of vaccines

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VHIR Seminar led by Gerrit Borchard, Section of Pharmaceutical Sciences University of Geneva, University of Lausanne Biopharmaceutical Sciences Geneva Switzerland. …

VHIR Seminar led by Gerrit Borchard, Section of Pharmaceutical Sciences University of Geneva, University of Lausanne Biopharmaceutical Sciences Geneva Switzerland.

Abstract: In order to enhance the efficacy of vaccines, antigen and adjuvants are combined in particulate carrier systems resembling pathogens in size, shape and surface properties. These novelnano- and microcarriervaccines strategies, using DNA or subunit vaccines as antigens and specific ligands of receptors of the innate immune system,offer several advantages, such as enhanced immune recognition, direction of immune response bias, and enhancement of vaccine stability. We are focusing on eliciting protective immune responses against M. tuberculosis, a pathogen transmitted through inhalation, bydeveloping vaccine delivery systems composed of different materialsand administered by the mucosal route.

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  • 1. TLR  ligand  func.onalized  nanocarriers  to  enhance   immunogenicity  of  vaccines   J.  Poecheim  &  G.  Borchard,  Ph.D.     Vall  d’Hebron,  Ins.ut  di  Recerca  VHIR   Barcelona,  Catalunya   5.11.2013  
  • 2. Adjuvants…   “…the  immunologist’s  dirty  liPle  secrets”   C.A.  Janeway,  Cold  Spring  Harb  Symp  Quant  Biol  1989      Adjuvant  
  • 3. What  makes  viruses  immunogenic?   If   drugs   are   similar   or   iden.cal   with   respect   to   structure   and   mechanism   of   ac.on   (MOA)   to   endogenous  substances…   …should   drug   delivery   systems   not   resemble   their  “natural”  counterparts,  as  well?      Adjuvant  
  • 4. What  makes  viruses  immunogenic?   Viruses   Nature’s  best  (and  worst)  delivery  systems  
  • 5. Viruses  are  par.cles   •  Uptake  by  an.gen-­‐presen.ng  cells  (APC)  depends  on  shape,   size  (10nm-­‐3µm),  surface  charge,  receptor  interac.ons,…   •  Uptake   triggers   matura.on   of   dendri.c   cells,   trafficking   to   lymph  nodes  and  T-­‐cell  ac.va.on   •  Viruses   interact   directly   with   B-­‐cells,   triggering   an.body   response   •  Uptake   of   par.culate   an.gen   leads   to   cross-­‐presenta.on,   which  is  absent  in  soluble  an.gens        
  • 6. Viruses  show  repe..ve  structures   •  Viruses  have  limited  gene.c  informa.on  for  proteins   •  Viral  surface  is  quasi-­‐crystalline,  of  repe..ve  subunits   •  Direct  ac.va.on  of  B-­‐cells,  breaking  tolerance     •  T-­‐cell  independent  IgM      
  • 7. Viruses  replicate   •  Sustained  an.gen  exposure   •  Induc.on  of  T-­‐cell  memory,  important  at  re-­‐infec.on   •  Size  of  T-­‐cell  memory  pool  is  dependent  on  dura.on  of   exposure  to  an.gen      
  • 8. Viruses  ac.vate  the  innate  immune  system   •  Interac.on  with  pathogenic  paPern-­‐recogni.on  receptors   (PRRs),  e.g.,  Toll-­‐like  receptors  (TLRs)   •  PRRs  are  expressed  on  many  cell  types,  including  APCs,   epithelial  and  B-­‐cells   •  First  line  of  defense  against  infec.on   •  Ac.va.on  of  adap.ve  immune  system        
  • 9. Adjuvants…   TLR,  Toll-­‐like  receptor   NLR,  nucleo.de-­‐binding   oligomeriza.on  domain  (NOD)-­‐like   receptor   RIG,  re.noic  acid-­‐inducible  gene   (RIG)-­‐1-­‐like  receptor      Adjuvant   Higgins  &  Mills,  Curr  Infect  Dis  Rep  2010  
  • 10. Toll-­‐like  receptors   C.  Nüsslein-­‐Volhard  
  • 11. 11
  • 12. Pathogenic  paPern  recogni.on  receptors  (PRR)      Adjuvant   NOD:  Nucleo2de  Oligomeriza2on  Domain   TLR:  Toll-­‐like  Receptors  
  • 13. Adjuvants:  Toll-­‐like  receptor  agonists   •  Insoluble  aluminum  salts  (alum)  and  uric  acid  crystals  poten.ally   ac.vate  the  NALP3  inflammasome,  as  does  chitosan  in  vitro     •  Muramyl  dipep.de  (MDP,  NOD2),  minimum  effec.ve  component  of   complete  Freund’s  adjuvant,  pyrogenic   •  Poly  I:C  (TLR3  and  RIG-­‐1),  synthe.c  analog  of  dsRNA,  Ampligen®,  in   clinical  trials   •  LPS  (TLR4),  1955,  too  toxic  for  use  in  human  vaccines   •  MPL  (TLR4),  modified  lipid  A  moiety  of  LPS,  included  in  Cervarix®  (HPV   vaccine)  as  AS04  (MPL  +  AlOH3)     •  E6020,  synthe.c  and  selec.ve  TLR4  ligand  based  on  lipid,  in   combina.on  with  MF59  (squalene,  Tween  80,  Span  85  in  citrate  buffer)   o/w  emulsion  
  • 14. Par.culate  carriers  for  mucosal  immuniza.on   •  TLRs   are   PaPern   Recogni.on   Receptors   present   on   diverse   cell  types  (epithelial,  immune  cells)   •  Recognize  specific  molecular  paPerns  present  in  pathogens   like  bacteria,  viruses  or  fungi   •  TLR   agonists   induce   matura.on   of   DC   and   ac.vate   the   immune  system   •  Pam3Cys   (TLR-­‐2),   bacterial   recogni.on,   favor   T H 2,   produc.on  of  Ab   •  IMQ  (TLR-­‐7),  viral  recogni.on,  favor  TH1,  cellular  IR   •  Synergy?    
  • 15. Mucosal  immuniza.on:  a  real  challenge   •  Protec.ve  mucosal  immune  responses  are  most  effec.vely   induced  by  mucosal  immuniza.on   •  Protec.ve  immunity  against  mucosal  pathogens  requires   novel  vaccine  strategies  ac.va.ng  mul.ple  arms  of  the   innate  and  adap.ve  immune  systems   Successes   Poliovirus   Influenza  virus   S.ll  pending…   HIV   Herpes  virus   Mycobacterium   Lehner,  J  infect  Dis,  1999    -­‐    De  Magistris,  Adv  Drug  Deliv  Rev,  2006   Belyakov  IM,  J.  Immunol  (2009)  
  • 16. Use  of  nanopar.cles  for  mucosal  vaccina.on   -­‐  Protec.on  of  the  an.gen  against  degrada.on   -­‐  Avoid  an.gen  dilu.on  on  mucosa   -­‐  Targe.ng  of  an.gen-­‐presen.ng  cells  (APC)   -­‐  Increase  an.gen  uptake  by  immune  cells   -­‐  Failed   aPempts   using   synthe.c   biodegradable   NPs   (PLGA/ PLA):  No  induc.on  of  dendri.c  cell  matura.on  in  vitro   -­‐  Strategy:  Addi.on  of  immunos.mulatory  molecules   -­‐  Combina.on  of  different  PRR  ligands:  synergis.c  effect?  
  • 17. 17 VACCINE ADJUVANTS “a  substance  used  to  stimulate  the  immune  system  to  provide  immunity  and  is  treated  to  act  as  an  antigen   without  inducing  the  disease”  Oxford dictionaries Latin vaccinus, from vacca 'cow‘ (Edward Jenner, 1796) "germ theory of disease“ (Louis Pasteur, 1880) Latin adjuvare, meaning "to help“ (G.Ramon, 1925) ↑  specific immune responses to the antigen special type of excipients
  • 18. 18 MODERN VACCINE STRATEGIES v Traditional vaccines: live-attenuated or whole-inactivated organisms.      →  Generally do not require adjuvants. v “Modern  vaccines”:   subunit vaccines Highly purified/ recombinant antigenic proteins/ epitopes DNA vaccines Plasmid encoding antigenic protein Safer, long-term protection, more specific BUT: far less immunogenic than traditional vaccines → Need for improved, safe, and more powerful adjuvants! www.niaid.nih.gov
  • 19. 19 New generation vaccine formulation Danger signals: Pathogen products (e.g. TLR ligands, NLR ligands) Delivery system: - Mineral salts (Alum) - Micro- and nanoparticles - Emulsions -  Liposomes -  Virosomes -  VLP Vaccine antigens: -  Recombinant proteins -  Gene delivered antigens Immune potentiators: MPL, MDP, CpG ODNs, Flagellin, Lipopeptides, Saponins, dsRNA, small molecule immune potentiators (Imiquimod)
  • 20. 3D  model  of  the  human  airway  barrier   PhD Defence 2010 / Heuking Blank,  et  al.,  Am.  J.  Respir.  Cell  Mol.  Biol  (2007)  36,  669-­‐677.  
  • 21. Study design Empty CTC NP pGFP NP CTC pGFP NP + + nm  scale   CTPPC + + nm  scale   Blank,  et  al.,  Am.  J.  Respir.  Cell  Mol.  Biol  (2007)  36,  669-­‐677.   +   nm  scale   +   21  
  • 22. Uptake  into  MDM:  CLSM        MDM        MDM        MDM    20  μm    20  μm    20  μm   Empty CTC NP pGFP NP CTC pGFP NP + + nm  scale   CTPPC + + nm  scale   +   nm  scale   +   22  
  • 23.  20  μm   CTPPC pDNA NP (N/P 3:1)
  • 24. Uptake  into  MDDC:  CLSM    20  μm    20  μm   Empty CTC NP CTC pDNA NP + + nm  scale    20  μm   CTPPC pDNA NP + + nm  scale   +   nm  scale   +  
  • 25.  20  μm   PhD Defence 2010 / Heuking CTC pGFP NP (N/P 3:1) 25
  • 26. Uptake pattern 100,0 Uptake  [%] 80,0 60,0 40,0 20,0 0,0 1  MDM                                                MDDC                                              EC Uptake of pDNA NP into MDM, MDDC or epithelial cells (EC): unloaded CTC NP (white bar), CTC pGFP NP (sheded bar) and CTPPC pGFP NP (dotted bar). Presented data are the mean ± standard error of the mean of three independent experiments. Differences were considered significant for * p<0.05.
  • 27. Immune response: IL-8            *   IL-­‐8  [ng/ml] 20.0          *   15.0      NS   10.0                  *   +   +   +   5.0 +   0.0 Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP ELISA: IL-8 release in the basolateral compartment from co-culture model due to pDNA NP exposure. Differences were considered significant for * (p<0.05); NS, not significant. Heuking,  et  al.  Nanobiotech.  11  (2013)  29  
  • 28. Immune  response:  TNF-­‐α              *   3.0 TNF-­‐alpha  [ng/ml]          *        NS   2.0        NS   +   +   1.0 +   +   0.0 Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP ELISA: TNF-α release in the basolateral compartment from co-culture model due to pDNA NP exposure. Differences were considered significant for * (p<0.05); NS, not significant. Heuking,  et  al.  Nanobiotech.  11  (2013)  29   28  
  • 29. Summary  (I)        +   +                      +        +   +   +   nm  scale   *** IL-8 (ng/mL) 20 *** 10 *** ** 0 Medium pDNA NP CM25-TMC35 NP Conjugate q  Chemistry:       Successful   synthesis   of   TLR-­‐1/2   (Pam3Cys)   agonist   functionalized  chitosan  derivatives.     q  Formulation:      Ability  of  Pam3Cys  decorated  pDNA  nanoparticles:   i)  to  complex  DNA  (~400  nm,  ~15-­‐20  mV),  by  forming   stable  particles  (release  study,  heparin  challenge),     ii)  to   protect   the   plasmid   against   DNase   degradation   and  to  transfect  A549  and  HBE  cells.     q  Immunogenicity  in  THP-­‐1  Φ:       Due   to   Pam3Cys   decoration   pDNA   nanoparticles   induced   higher  IL-­‐8  secretions  from  by  mTHP-­‐1  macrophages  and   3DCC.  
  • 30. Summary  (II)     IL-­‐8  [ng/ml] 20.0 15.0 10.0 5.0 0.0 Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP q For  pulmonary/bronchial  pDNA  vaccination,   the  use  of  CTTPC  versus  pDNA  alone   contributes  to  an  overall  higher  adjuvanticity:         q  protection  against  enzymatic  degradation   q  transfection  in  vitro   q  transport  of  DNA  into  the  most  immune   competent  APC  type,  namely  dendritic   cells;     q  increasing  the  overall  immune  response   (IL-­‐8,  TNF-­‐α).  
  • 31. 31 Enhancing cellular immune responses Vaccine formulation TLR and NLR signaling pathways Nanocarrier TLR 9 ligand pDNA with CpG sequence encoding antigen 85A NOD 2 ligand MDP Muramyl dipeptide Proinflammatory cytokines Nucleus
  • 32. 32 Presentation of the project The aim is the preparation, characterization and in vitro testing of particulate carrier systems that are able to target and stimulate immune cells by combinations of PRR ligands incorporated and/or decorated on the particle surface. Vector 1: Trimethyl chitosan nanoparticles Vector 2: Squalene in water emulsion nanodroplets Vector 3: Cationorm ® Antigen: Ag85A (Mycobacterium tuberculosis) Immunostimulator #1: unmethyl. CpG sequence (TLR 9 ligand) Immunostimulator #2: MDP (NOD 2 ligand)
  • 33. 33 Nanoparticle preparation techniques 1) Trimethyl chitosan (TMC)/Chondroitin sulfate (CS) nanoparticles Complex coacervation method: Positively charged TMC 0.5% + negatively charged CS 0.1% CS Mean size: 283.3 nm ±  4.3 TMC + 500 nm
  • 34. 34 Nanoparticle preparation techniques 2) O/W emulsion preparation 1)  homogenized for 1 min, rpm 2) High shear processing (Microfluidizer M110S) DOTAP Mean size: 129.5 nm ±  3.3 Distilled water 0.5% Span 85 0.5% Tween 80 5 % squalene 500 nm 10 000
  • 35. 35 3) Cationorm ® cationic O/W emulsion Cationorm ®   Oil   Mineral oil   Cationic agent   Cetalkonium chloride   Surfactants   Poloxamer, tyloxapol   Water   Water for injection   500 nm
  • 36. 36 Size, zeta potential of nanocarriers Carrier Size [nm] Zeta potential [mV] Polydispersity TMC/CS 283.3 ±  4.3 33.0 ± 0.7 0.27 TMC/CS-pDNA 356.8 ± 33.4 16.9 ± 3.8 0.41 DOTAP-SWE 129.5 ±  3.3 22.8 ±  0.1 0.09 DOTSP-SWE-pDNA 165.8 ±  4.3 -17.5 ±  1.0 0.14 Cationorm ® 158.17 ± 2.28 14.53 ± 0.38 0.24 Cationorm ®-pDNA 216.62 ±  1.48 -35.65 ±  4.17 0.34
  • 37. 37 Toxicity profile of nanocarriers •  •  •  •  Cell  line:  RAW264.7  murine  macrophages   Dilu.on:  1:10   Incuba.on  .me:  24  h   Evalua.on:  XTT  prolifera.on  assay  
  • 38. 38 Immunogenicity of functionalized pDNA nanocarriers in vitro •  •  •  •  Cell  line:  RAW264.7  murine  macrophages   Dilu.on:  1:10   Incuba.on  .me:  24  h   Evalua.on:  ELISA  mTNF-­‐α   *** *** *** Synergistic expression of TNF- α! Values are means of 3 experiments; *** p <0.001
  • 39. 39 Uptake of functionalized pDNA nanocarriers in vitro •  •  •  •  Cell  line:  A549  human  alveolar  basal  epithelial  cells   Dilu.on:  1:10   Incuba.on  .me:  over  night   Evalua.on:  Confocal  microscopy   -­‐  Vectashield  moun.ng  media  containing  DAPI   -­‐  GFP-­‐pDNA   -­‐   MDP-­‐Rhodamine  
  • 40. 40 Summary •  The DNA vaccine formulations have been shown to be safe •  Both resulted in an increased pro-inflammatory cytokine release by targeting TLR-9 and NLR-2. •  They elicited a synergistic enhancement as a result of delivering two innate immune receptor ligands at the same time. •  Uptake and protein expression has been confirmed.
  • 41. 41 Perspectives a) In vitro: 2 questions to answer: Repetition of synergistic studies: additive or synergistic effect How do the ligands get into the cell/ into the nucleus? → Investigation of uptake mechanisms b) In vivo: - in Balb/c mice as immunological model for Th1 response - Nod2 knock out mice: Synergistic effect NOD2-receptor dependent? 
  • 42. Immune response studied §  Increase of anti-Ag85A antibodies by ELISA on serum: Total IgG  §  Cellular responses : isolation of spleen b1) ex-vivo protein stimulation : IFN-γ, IL-2, TNF-α, IL-4 (ELISA) b2) Lymphocyte proliferation (XTT reagent) b3) FACS – IFN-gamma, CD4+/CD8+ b4) ELISPOT Restimulation of splenocytes with recombinant protein Ag85A in vitro, ev. with CD8+ specific peptide
  • 43. 43 Acknowledgements •  UNIGE •  VFL •  IBCP Dr. Nicolas Colin Dr. Simon Heuking Dr. Livia Brunner Dr. Charlotte Primard Prof. Gerrit Borchard Dr. Christoph Bauer Emmanuelle Sublet Dr. Annasara Hansson Dr. Leonardo Lauciello Christian Reichert Shqipe Kelmendi Najoua Bennani