TLR	
  ligand	
  func.onalized	
  nanocarriers	
  to	
  enhance	
  
immunogenicity	
  of	
  vaccines	
  
J.	
  Poecheim	
 ...
Adjuvants…	
  

“…the	
  immunologist’s	
  dirty	
  liPle	
  secrets”	
  
C.A.	
  Janeway,	
  Cold	
  Spring	
  Harb	
  Sy...
What	
  makes	
  viruses	
  immunogenic?	
  
If	
   drugs	
   are	
   similar	
   or	
   iden.cal	
   with	
   respect	
  ...
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...
Viruses	
  show	
  repe..ve	
  structures	
  
•  Viruses	
  have	
  limited	
  gene.c	
  informa.on	
  for	
  proteins	
  ...
Viruses	
  replicate	
  
•  Sustained	
  an.gen	
  exposure	
  
•  Induc.on	
  of	
  T-­‐cell	
  memory,	
  important	
  a...
Viruses	
  ac.vate	
  the	
  innate	
  immune	
  system	
  
•  Interac.on	
  with	
  pathogenic	
  paPern-­‐recogni.on	
  ...
Adjuvants…	
  

TLR,	
  Toll-­‐like	
  receptor	
  
NLR,	
  nucleo.de-­‐binding	
  
oligomeriza.on	
  domain	
  (NOD)-­‐li...
Toll-­‐like	
  receptors	
  

C.	
  Nüsslein-­‐Volhard	
  
11
Pathogenic	
  paPern	
  recogni.on	
  receptors	
  (PRR)	
  

	
  	
  Adjuvant	
  
NOD:	
  Nucleo2de	
  Oligomeriza2on	
  ...
Adjuvants:	
  Toll-­‐like	
  receptor	
  agonists	
  
•  Insoluble	
  aluminum	
  salts	
  (alum)	
  and	
  uric	
  acid	
...
Par.culate	
  carriers	
  for	
  mucosal	
  immuniza.on	
  
•  TLRs	
   are	
   PaPern	
   Recogni.on	
   Receptors	
   pr...
Mucosal	
  immuniza.on:	
  a	
  real	
  challenge	
  
•  Protec.ve	
  mucosal	
  immune	
  responses	
  are	
  most	
  effe...
Use	
  of	
  nanopar.cles	
  for	
  mucosal	
  vaccina.on	
  
-­‐  Protec.on	
  of	
  the	
  an.gen	
  against	
  degrada....
17

VACCINE ADJUVANTS
“a	
  substance	
  used	
  to	
  stimulate	
  the	
  immune	
  system	
  to	
  provide	
  immunity	
...
18

MODERN VACCINE STRATEGIES
v Traditional vaccines: live-attenuated or whole-inactivated organisms. 	
  	
  
	
  →	
  G...
19

New generation vaccine formulation
Danger signals:
Pathogen products (e.g.
TLR ligands, NLR ligands)

Delivery system:...
3D	
  model	
  of	
  the	
  human	
  airway	
  barrier	
  

PhD Defence 2010 / Heuking
Blank,	
  et	
  al.,	
  Am.	
  J.	
...
Study design
Empty CTC NP
pGFP NP

CTC pGFP NP

+

+

nm	
  scale	
  

CTPPC

+

+

nm	
  scale	
  

Blank,	
  et	
  al.,	...
Uptake	
  into	
  MDM:	
  CLSM	
  
	
  	
  	
  MDM	
  

	
  	
  	
  MDM	
  

	
  	
  	
  MDM	
  

	
  20	
  μm	
  

	
  20...
 20	
  μm	
  

CTPPC pDNA NP (N/P 3:1)
Uptake	
  into	
  MDDC:	
  CLSM	
  

	
  20	
  μm	
  

	
  20	
  μm	
  

Empty CTC NP

CTC pDNA NP

+

+

nm	
  scale	
  
...
 20	
  μm	
  
PhD Defence 2010 / Heuking

CTC pGFP NP (N/P 3:1)

25
Uptake pattern
100,0

Uptake	
  [%]

80,0
60,0
40,0
20,0
0,0
1
	
  MDM	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  ...
Immune response: IL-8
	
  	
  	
  	
  	
  	
  *	
  

IL-­‐8	
  [ng/ml]

20.0

	
  	
  	
  	
  	
  *	
  

15.0

	
  	
  	
 ...
Immune	
  response:	
  TNF-­‐α	
  	
  
	
  	
  	
  	
  	
  *	
  
3.0

TNF-­‐alpha	
  [ng/ml]

	
  	
  	
  	
  	
  *	
  
	
...
Summary	
  (I)	
  	
  
	
  	
  +	
  

+	
   	
  	
  	
  	
  
	
  	
  	
  	
  	
  	
  +	
   	
  	
   	
  +	
  

+	
  

+	
 ...
Summary	
  (II)	
  
	
  

IL-­‐8	
  [ng/ml]

20.0

15.0

10.0

5.0

0.0
Medium	
  control

CTC	
  NP

CTC	
  pGFP	
  NP

C...
31

Enhancing cellular immune responses
Vaccine formulation

TLR and NLR signaling pathways

Nanocarrier

TLR 9 ligand
pDN...
32

Presentation of the project
The aim is the preparation, characterization and in vitro testing of particulate carrier s...
33

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

Complex coacer...
34

Nanoparticle preparation techniques
2) O/W emulsion preparation
1)  homogenized for 1 min,
rpm
2) High shear processin...
35

3) Cationorm ® cationic O/W emulsion
Cationorm ®	
  
Oil	
  

Mineral oil	
  

Cationic agent	
  

Cetalkonium chlorid...
36

Size, zeta potential of nanocarriers
Carrier

Size [nm]

Zeta potential
[mV]

Polydispersity

TMC/CS

283.3 ±	
  4.3

...
37

Toxicity profile of nanocarriers
• 
• 
• 
• 

Cell	
  line:	
  RAW264.7	
  murine	
  macrophages	
  
Dilu.on:	
  1:10	...
38

Immunogenicity of functionalized pDNA nanocarriers in vitro
• 
• 
• 
• 

Cell	
  line:	
  RAW264.7	
  murine	
  macrop...
39

Uptake of functionalized pDNA nanocarriers in vitro
• 
• 
• 
• 

Cell	
  line:	
  A549	
  human	
  alveolar	
  basal	
...
40

Summary
•  The DNA vaccine formulations have been shown to be safe
•  Both resulted in an increased pro-inflammatory c...
41

Perspectives
a) In vitro: 2 questions to answer:
Repetition of synergistic studies: additive or synergistic effect
How...
Immune response studied
§  Increase of anti-Ag85A antibodies by ELISA on serum:
Total IgG 
§  Cellular responses : isola...
43

Acknowledgements
•  UNIGE
•  VFL
•  IBCP

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

Dr. Charlotte Primard...
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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.

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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Transcript of "TLR ligand functionalized nanocarriers to enhance immunogenicity of vaccines"

  1. 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. 2. Adjuvants…   “…the  immunologist’s  dirty  liPle  secrets”   C.A.  Janeway,  Cold  Spring  Harb  Symp  Quant  Biol  1989      Adjuvant  
  3. 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. 4. What  makes  viruses  immunogenic?   Viruses   Nature’s  best  (and  worst)  delivery  systems  
  5. 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. 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. 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. 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. 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. 10. Toll-­‐like  receptors   C.  Nüsslein-­‐Volhard  
  11. 11. 11
  12. 12. Pathogenic  paPern  recogni.on  receptors  (PRR)      Adjuvant   NOD:  Nucleo2de  Oligomeriza2on  Domain   TLR:  Toll-­‐like  Receptors  
  13. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23.  20  μm   CTPPC pDNA NP (N/P 3:1)
  24. 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. 25.  20  μm   PhD Defence 2010 / Heuking CTC pGFP NP (N/P 3:1) 25
  26. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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

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