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  

Adjuvants…  

   Adjuvant  

“…the  immunologist’s  dirty  liPle  secrets”  C.A.  Janeway,  Cold  Spring  Harb  Symp  Quant  Biol  1989  

What  makes  viruses  immunogenic?  

   Adjuvant  

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?  

What  makes  viruses  immunogenic?  

Viruses  Nature’s  best  (and  worst)  delivery  systems  

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        

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      

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    

 

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        

Adjuvants…  

   Adjuvant  

Higgins  &  Mills,  Curr  Infect  Dis  Rep  2010  

TLR,  Toll-­‐like  receptor  NLR,  nucleo.de-­‐binding  

oligomeriza.on  domain  (NOD)-­‐like  receptor  

RIG,  re.noic  acid-­‐inducible  gene  (RIG)-­‐1-­‐like  receptor  

Toll-­‐like  receptors  

C.  Nüsslein-­‐Volhard  

11

Pathogenic  paPern  recogni.on  receptors  (PRR)  

   Adjuvant  

NOD:  Nucleo2de  Oligomeriza2on  Domain  TLR:  Toll-­‐like  Receptors  

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  

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   TH2,  produc.on  of  Ab  

•  IMQ  (TLR-­‐7),  viral  recogni.on,  favor  TH1,  cellular  IR  •  Synergy?    

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  

Lehner,  J  infect  Dis,  1999    -­‐    De  Magistris,  Adv  Drug  Deliv  Rev,  2006  Belyakov  IM,  J.  Immunol  (2009)  

Poliovirus   Influenza  virus   HIV   Herpes  virus   Mycobacterium  

Successes   S.ll  pending…  

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

VACCINE ADJUVANTS

Latin adjuvare, meaning "to help“ (G.Ramon, 1925)

↑  specific immune responses to the antigen

special type of excipients

Latin vaccinus, from vacca 'cow‘ (Edward Jenner, 1796)

"germ theory of disease“ (Louis Pasteur, 1880)

“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

MODERN VACCINE STRATEGIES

www.niaid.nih.gov

18

v Traditional vaccines: live-attenuated or whole-inactivated organisms.      →  Generally do not require adjuvants.

v “Modern  vaccines”:  

subunit vaccines DNA vaccines Highly purified/ recombinant antigenic Plasmid encoding antigenic protein proteins/ epitopes

Safer, long-term protection, more specific BUT: far less immunogenic than traditional vaccines

→ Need for improved, safe, and more powerful adjuvants!

19

New generation vaccine formulation

Immune potentiators: MPL, MDP, CpG ODNs, Flagellin, Lipopeptides, Saponins, dsRNA, small molecule immune potentiators (Imiquimod)

Vaccine antigens: -  Recombinant proteins -  Gene delivered antigens

Danger signals: Pathogen products (e.g. TLR ligands, NLR ligands)

Delivery system: - Mineral salts (Alum) - Micro- and nanoparticles - Emulsions -  Liposomes -  Virosomes -  VLP

PhD Defence 2010 / Heuking

3D  model  of  the  human  airway  barrier  

Blank,  et  al.,  Am.  J.  Respir.  Cell  Mol.  Biol  (2007)  36,  669-­‐677.  

Blank,  et  al.,  Am.  J.  Respir.  Cell  Mol.  Biol  (2007)  36,  669-­‐677.  

Study design

21  

Empty CTC NP CTC pGFP NP CTPPC pGFP NP

+  

+  

nm  scale   nm  scale   nm  scale  +

+ +

+

22  

Uptake  into  MDM:  CLSM  

     MDM        MDM  

     MDM  

 20  μm    20  μm    20  μm  

Empty CTC NP CTC pGFP NP CTPPC pGFP NP

+  

+  

nm  scale   nm  scale   nm  scale  

+

+

+

+

CTPPC pDNA NP (N/P 3:1)

 20  μm  

Uptake  into  MDDC:  CLSM  

 20  μm    20  μm    20  μm  

+  

+  

nm  scale   nm  scale   nm  scale  

Empty CTC NP CTC pDNA NP CTPPC pDNA NP

+

+ +

+

PhD Defence 2010 / Heuking 25

 20  μm  

CTC pGFP NP (N/P 3:1)

Uptake pattern

0,0

20,0

40,0

60,0

80,0

100,0

1  MDM                                                MDDC                                              EC

Uptake  [%

]

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.

0.0

5.0

10.0

15.0

20.0

Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP

IL-­‐8  [ng/ml]

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.

     NS                    *  

         *  

Heuking,  et  al.  Nanobiotech.  11  (2013)  29  

           *  

+  

+  +  

+  

Immune response: IL-8

0.0

1.0

2.0

3.0

Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP

TNF-­‐alpha  [ng/m

l]

28  

Immune  response:  TNF-­‐α    

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.

     NS          NS  

         *  

         *  

+  

+   +  

+  

Heuking,  et  al.  Nanobiotech.  11  (2013)  29  

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.  

Summary  (I)    

0

10

20

Medium pDNA NP CM25-TMC35 NP Conjugate

IL-8

(ng/

mL)

***

***

**

***

nm  scale  

+  

+  

           

           

   +  +  +    +  

 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).  

Summary  (II)  

0.0

5.0

10.0

15.0

20.0

Medium  control CTC  NP CTC  pGFP  NP CTPPC  pGFP  NP

IL-­‐8  [ng/ml]

31

TLR 9 ligand pDNA with CpG sequence

encoding antigen 85A

NOD 2 ligand MDP

Muramyl dipeptide

Nanocarrier

TLR and NLR signaling pathways Vaccine formulation

Enhancing cellular immune responses

Proinflammatory cytokines Nucleus

Presentation of the project

32

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 2: Squalene in water emulsion nanodroplets

Vector 1: Trimethyl chitosan nanoparticles

Antigen: Ag85A (Mycobacterium tuberculosis)

Immunostimulator #1: unmethyl. CpG sequence (TLR 9 ligand)

Immunostimulator #2: MDP (NOD 2 ligand)

Vector 3: Cationorm ®

33

TMC +

CS -

Complex coacervation method: Positively charged TMC 0.5% + negatively charged CS 0.1%

Nanoparticle preparation techniques 1) Trimethyl chitosan (TMC)/Chondroitin sulfate (CS) nanoparticles

500 nm

Mean size: 283.3 nm ±  4.3

34

2) O/W emulsion preparation

Nanoparticle preparation techniques

5 % squalene

0.5% Span 85

Distilled water

0.5% Tween 80

DOTAP

1)  homogenized for 1 min, 10 000 rpm

2) High shear processing (Microfluidizer M110S)

500 nm

Mean size: 129.5 nm ±  3.3

35

500 nm

3) Cationorm ® cationic O/W emulsion

Cationorm ®  Oil   Mineral oil  Cationic agent   Cetalkonium chloride  Surfactants   Poloxamer, tyloxapol  Water   Water for injection  

Size, zeta potential of nanocarriers

36

Carrier Size [nm] Zeta potential [mV]

Poly- dispersity

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

Toxicity profile of nanocarriers

37

•  Cell  line:  RAW264.7  murine  macrophages  •  Dilu.on:  1:10  •  Incuba.on  .me:  24  h  •  Evalua.on:  XTT  prolifera.on  assay  

*** ***

***

38

Immunogenicity of functionalized pDNA nanocarriers in vitro

Values are means of 3 experiments; *** p <0.001

Synergistic expression of TNF- α!

•  Cell  line:  RAW264.7  murine  macrophages  •  Dilu.on:  1:10  •  Incuba.on  .me:  24  h  •  Evalua.on:  ELISA  mTNF-­‐α  

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  

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.

40

Perspectives

41

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? 

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

Acknowledgements

• UNIGE • VFL •  IBCP

43

Prof. Gerrit Borchard Dr. Christoph Bauer Emmanuelle Sublet Dr. Annasara Hansson Dr. Leonardo Lauciello Christian Reichert Shqipe Kelmendi Najoua Bennani

Dr. Charlotte Primard

Dr. Nicolas Colin Dr. Simon Heuking Dr. Livia Brunner