transplantation-related viral infections Pier Giulio Conaldi Unit - - PowerPoint PPT Presentation
Fifth Annual Ri.MED Scientific Symposium Palermo 24.10.2011 PRIMING THE THERAPEUTIC PIPELINE: NEW STRATEGIES FOR DRUG DISCOVERY Immunotherapeutic strategies in the management of transplantation-related viral infections Pier Giulio Conaldi
Palermo 24.10.2011
Pier Giulio Conaldi
Unit of Regenerative Medicine and Biomedical Technologies Laboratory of Clinical Pathology, Microbiology and Virology
ISMETT
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Experimental Laboratory GMP Facility Pre-clinical Laboratory Immunobiology Molecular Medicine Regenerative Medicine Cell Therapy Preclinical animal models Experimental surgery
Adoptive immunotherapy
Pancreatic islet transplantation Fetal hepatocyte transplantation
Unit of Regenerative Medicine and Biomedical Technologies
(J. A. Fishman, N. Eng. J. Med., 357:2601-14, 2007)
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Ex vivo production and in vivo infusion of autologous (or heterologous) cytotoxic/helper T lymphocyte clones specific to
Prevention and treatment of PTLD Treatment of CMV infections caused by drug-resistant viral variants
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EBV normally infects naive B cells in Waldeyer’s ring, which differentiate into memory B cells, exit the cell cycle and are therefore not pathogenic. Normally, bystander B-cell blasts would be destroyed by CTLs, but if the CTL response is suppressed, the blasts can lead to PTLD
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lymphoma
incidence in children since PTLD is mainly related to post-transplant EBV primary infection)
mortality after solid organ transplantation (overall mortality is approximately 50%)
express all the latent viral genes (latency III)
bowel, lung, mediastinum, tonsil, bone marrow and CNS), including the organ allograft
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60 Gy irradiation
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10 20 30 40 50 60 70 80 auto allo auto allo auto allo auto allo auto allo auto allo 40:1 20:1 10:1 5:1 2,5:1 1,25:1 % of specific lysis
10 20 30 40 50 60 70 80 90 40:1 20:1 10:1 5:1 2,5:1 1,25:1 % of specific lysis control group SOT
Cytotoxic activity (Cr51 release assay)
10 20 30 40 50 60 70 80 CD4 CD8 NK % of positive cells
Immunophenotype of EBV-specific “CTLs”
Characterization of EBV-specific “CTLs” expanded in vitro with serial stimulations by autologous EBV-immortalized LCLs
Autologous LCLs Allogeneic LCLs High specific reactivity: >40% killing of autologous target cells at 20:1 effector/target ratio
Cytotoxic activity (Cr51 release assay)
Healthy controls Transplant patients
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COD age Transplantation Diagnosis ITA001 33 kidney High level EBV viral load ITA002 36 Kidney High level EBV viral load ITA003 24 Lung PRE-PTLD (lung) ITA005 5 Liver Polymorphic PTLD (tonsils, bowel) ITA006 9 Liver Polymorphic PTLD (bowel) ITA007 4 Liver Early lesions (tonsils) ITA008 4 Liver Early lesions (gut) ITA009 5 Liver Polymorphic PTLD (tonsils) ITA010 15 Liver Polymorphic PTLD (tonsils, bowel) ITA011 6 Liver Polymorphic PTLD (tonsils) ITA012 7 Liver Early lesions (liver) ITA014 7 Liver High level EBV viral load ITA016 7 Liver Polymorphic PTLD (tonsils, bowel) ITA017 5 Liver Early lesions (tonsils, bowel) ITA017 5 Liver Early lesions (tonsils, bowel) ITA018 6 Liver Early lesions (tonsils) ITA019 21 Liver Early lesions (tonsils) ITA020 4 Liver High level EBV viral load ITA021 9 Liver Early lesions (tonsils, bowel) ITA022 7 Liver Early lesions (tonsils, bowel) ITA023 9 Liver Early lesions (tonsils, bowel) ITA024 7 Liver Early lesions (tonsils, bowel) ITA025 20 Liver High level EBV viral load ITA026 6 Liver Polymorphic PTLD (bowel) ITA028 9 Liver Early lesions (tonsils, bowel)
CTL dose 6 1.5 x 106 cells/kg 3 1.5 x 106 cells/kg 3 2.0 x 106 cells/kg
PTLD histology EBV-DNA (copies x105 PBMC) EBV-DNA (copies/ml blood) Follow up (wks)
ITA005
From Polymorphic to Early Lesion From 23 to undetectable From 408 to undetectable 32
ITA010
From Polymorphic to Early Lesion From 141 to 20 From 6700 to 1700 21
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ITA 005 colon - treatment with anti-EBV-CTLs before treatment after treatment
CD20+ B cells CD20+ B cells
EBER EBER EBER
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ITA 010 tonsil - treatment with anti-EBV-CTLs before treatment after treatment
CD20+ B cells CD20+ B cells
EBER EBER
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Pulsed with pp65 and IE-1 peptide-mix X ray Co-colture with lymphocites IL-2
CMV-specific T cells
PBMC
Evaluation for cell therapy purpose: specifity, phenotype and sterility
CMV pp65 and IE-1: a mix of 15-mers peptides overlapping by 11 amino acids spanning the entire IE-1 and pp65 protein sequences
IL-4 +GM-CSF
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Ganciclovir therapy (GC) GC GC
Severe leucopenia
Pneumonitis
Treatment of a SOT recipient with severe and persistent CMV infection with allogenic CMV-specific T cells
Male, 16 yrs old, affected by primary sclerosing cholangitis Liver transplantation: D+/R- for anti-CMV Abs Primary CMV infection after transplantation with serious clinical manifestations Severe leucopenia, CMV infecton of bone marrow
Cut-off of CMV-DNA level in blood for pre-emptive therapy
bone marrow
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Cytotoxic activity (Cr51 release assay)
10 20 30 40 50 60 70 80 90 100 40:1 20:1 10:1 5:1 2,5:1 Effector/target ratio % of specific lysis T1 autologous PHA-blasts +pept T2 autologous PHA-blasts T3 recipient PHA-blasts +pept) T3 recipient PHA-blasts
infusion of CMV-specific T cells (1x106cells/kg) normal level of blood leucocytes
* * evaluation of CMV infection in
the bone marrow
In vitro production of CMV-specific T cells using the PBMC of the mother of the patient
Immunophenotype
Follow-up after CMV-specific T cell infusions
1 w after 1° inf.: significant reduction of CMV-DNA level in bone marrow 2 w after 1° inf.: normalization of blood leucocyte number (still now) detectable CMV-specific T cells in the peripheral blood (ELISPOT) until 2 months after 1° inf. Undetectable level of CMV-DNA emia (still after 1 year) No further treatment with ganciclovir CMV-DNAemia
GC No GC treatment
> 6000/ml <2000/ml
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Funded by
With a global burden of 180 million infected people HCV is a major public health challenge. Of the infected patients 60-80% develop chronic hepatitis, leading cause of liver cirrhosis and hepatocellular carcinoma. HCV persistence is mainly due to virus capability to evade host immune response. Particularly, in the early phase of the infection HCV attenuates innate immune responses (including NK cells functions), weakening the development of specific T cell responses, and impairs the production of anti-HCV neutralizing Abs. Chronic hepatitis C is the most common (almost 50%) indication of liver transplantation, but viral recurrence of the graft is universal and the damage of the new livers occurs usually and is associated with accelerated progression to cirrhosis, graft loss and death. A prophylactic strategy for prevention of HCV recurrence is lacking and drug-based antiviral therapies have limited efficacy and tolerability in liver transplantation recipients.
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Resistance of infected hepatocytes to the effect of IFN HCV antigen uptake Modulation of DCs function Inhibition of NK cells by HCV E2 Poly- & monoclonal B cell expansions Late and limited humoral immune responses Reduced T cell priming HCV mutations and quasispecies escape from antibody and T cell responses Inhibiti
Treg Delayed trafficking to the liver Impaired effector functions Impaired differentiation & maturation Reduced proliferation
DC NK
Innate immune response Adaptive immune response Adapted from Rehermann B at el. Nat Rev Immunol Mar 2005: Vol 5; 215-229
CD4+ CD8+
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The incubation phase: viral attenuation of the innate immune responses (hepatocytes, DC, NK cells)
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HCV escapes the anti-viral immune response :
Natural Killer cells (NK) - upregulation of inhibitory receptors Dendritic Cells (DCs) - stimulation of anti-inflammatory phenotype CD4+ and CD8+ T lymphocytes - insufficient expansion & maturation B cells - impairment of anti-viral Ab production
Why does acute HCV infection successfully turns into chronicity?
20 1. Broad and vigorous T cells responses are observed in resolving HCV patients In chronic infected patients the T cells response to HCV is weak and oligospecific
increased numbers of CD56+ NK cells with anti-viral function This population is only scarcely represented in HCV chronic individuals 3. Resolving HCV infection may be associated with rapid induction of virus neutralizing. Viral entry end escape from Ab-mediated neutralization influence HCV recurrence after liver transplantation 20
HCV kinetics before, during and after liver transplantation
P: Pretransplant A: Anhepatic phase R: Reperfusion
(Garcia-Retortillo, Hepatology 35:680-687, 2002)
AIM : Prevention of post-transplant HCV recurrence by infusion of
activated NKs, not affected by the immunosuppressive regimen, and anti-HCV broadly neutralizing Abs early after liver transplantation 1) To produce NKs with strong anti-HCV activity, comparing the benefits of using cytotoxic NKs killing HCV+ hepatocytes versus NKs blocking HCV replication through the release of IFNg without lysing the infected cells. 2) To standardize NK purification, activation, expansion and cryopreservation conforming to GMP-standards for advanced therapy purpose. 3) To produce HCV-specific nAbs from immortalized B cells. To establish HCV neutralization assay in vitro and in vivo
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Isolation of Buffy coat from liver perfusate by apheresis B cell B cell HCV-specific Neutralizing Antibodies Hepatic Lymph nodes from HCV chronic patients undergoing liver transplant PBMC from HCV chronic patients and healthy donors B cell B cell B cell EBV- Immortalized HCV-specific B cells IV infusion activated NK Low viremic Recipient (anhepatic phase) Depletion of CD3+ cells with CliniMACS
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Sample 1 - 7,2 Lt
WBC (106/ml) HCT (%) Lymphocytes (% of WBC) Neutrophils (%WBC) Liver perfusate centrifuged (1,2 Lt)
26 54 Out of scale Out of scale
Waste (1,5 Lt)
16 46 8.6 90
Buffy Coat post-Ficoll (100ml)
80 2 20 ND
8 billion TOTAL cells from Buffy Coat Volume reduction Buffy coat isolation Purification of NK cells In vitro activation/expansion of NK cells
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phenotype ex vivo 60%
500 1000 1500 day 0 day 14 day 17 day 21 day 26
>1 x 109 cells in 3 weeks
2.5x106 Cell number x106
Expansion with IL2 and irradiated feeders
Isolation and expansion CD16+56+CD3- NK 100%
Day 0
100%
Day 14
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hepatic NK O/ IL-2 +/- IFNa
10 20 30 40 50 60 70 80 40:1 20:1 10:1 5:1 2.5:1 1:1 Effector:target ratio % Chromium release
Huh7.5 HCV-infected Huh7.5 Chromium release assay
100 101 102 103 104 CD56 APC-A 100 101 102 103 104 CD56 APC-A
16% 30%
NK + Huh7.5 NK + HCV-Huh7.5
IFN-g CD56 IFN-g CD56
NK cells are indeed ACTIVATED by HCV
Virus inhibits NK cells?
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No NK
100 101 102 103 104 100 101 102 103 104 CFSE FITC-A 100 101 102 103 104
80 8
CFSE 7ADD
100 101 102 103 104 100 101 102 103 104
no NK 37 + NK 1:1 9
HCV-GFP 7ADD
100 101 102 103 104
76 + NK 5:1 50 30 30 37 5 17
Not infected Huh7.5 HCV-infected Huh7.5
+ NK 1:1 + NK 5:1
Dead cells infected cells Dead cells
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3.5% 30% reduction of total Venus+ cells (HCV replicating cells) Full depletion of Venusbright cells IL2 IL2/IFNa HCV HCV-Huh7.5 Huh7.5
Venus-HCV infected Huh7.5
+ Activated NK
54%
no NK
Core 400x
HCV-infected Huh7.5
HCV-GFP HCV-GFP
Issue: HCV escapes NK-mediated antiviral effect due to low expression levels in host cell? Repeat experiment by extending the time of exposure of HCV replicating cell population to NK cell-derived soluble factors
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Stegmann KA et al Gastroenterology 2010; Feb 2 Ohira M, et al. JCI 2009; Vol 119:11:3226-3235
Ohira M, et al. JCI 2009; Vol 119:11:3226-3235 Crotta S et al J Hepatol 2010;52:183-19
CD56+
Killing of HCV+ hepatocytes Tissue destruction? Block of HCV replication with preservation of liver integrity?
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– Test IL12, IFNa, IL2, IL15, TNFa as growth factors – ELISA/ELISPOT/FC/RT-PCR for cytokine profiling
– Permanent/transient effect of NK cells on HCV-infected cell population (on/off co-cultures with transwell) – Human hepatocyte 3D primary cultures on matrigel – Comparison of anti-viral outcome of TRAIL+NK vs IFNg+NK
– uPA/SCID chimeric mice
in vitro
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Potential targets for Abs capable to neutralize HCV infectivity
Isolation of B cells from hepatic lymph nodes (among which HCV-specific B cells)
EBV immortalization
Hepatic Lymph Nodes taken from HCV+ patients undergoing L-Tx
B cell lines releasing high levels of Ab Screening for B cell clones producing anti-HCV Abs by ELISA
Experimental approach
PBMC from HCV chronic patients 30
47 66.8
a) ex vivo
Gated on B cells 80 78
b) Day 30 of Culture with CD40L, CpG & IL-4, IL21
Gated on B cells 96
Ab secreting B cells
1.8
Ab secreting B cells
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ELISA to identify B cells producing HCV-specific Antibodies
0.5 1 1.5 2 2.5 3
+VE K A1 B1 C1 D1 E1 F1 A2 B2 C2 D2 E2 F2 A3 B3 C3 D3 E3 F3 A4 B4 C4 D4 E4 well ID Abs OD 492 nm
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Neutralization assay
20 40 60 80 100 120
No Antibody Polyclonal Antibody #1 % Infection
neat 1:2 1:10 1:50 1:100 1:1000 1:10,000 Dilution Factor
Neutralization assay
20 40 60 80 100 120 No Ab #1 #2 #3 #4 #5 #6
Neat polyclonal Ab % infection Luc-Jc1 infected Huh7.5 +/- Polyclonal Abs HCV expressing luciferase reporter gene Neutralizing Abs
Neutralization
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Main Expected Results and Impact:
the anhepatic phase might prevent HCV recurrence or limit disease progression with an invaluable clinical impact.
IFNg+NKs and TRAIL+NKs concerning HCV clearance and liver damage will allow to establish optimized immunotherapeutic strategies, valuable also for the treatment of chronic hepatitis C.
could be applied to other agents of infectious diseases
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Anti-EBV/CMV adoptive immunotherapy:
Mariangela Di Bella Anti-HCV adoptive immunotherapy:
Giovanna Russelli Paola Pizzillo
Acknowledgments Giandomenico Amico Chiara Di Bartolo Floriana Barbera Massimiliano Gaetani Cinzia Chinnici Maria Grupillo Nicola Cuscino Salvatore Pasqua Danilo D’Apolito Francesca Timoneri
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