ReplicacióndelCitomegalovirus(CMV)y*dinámicadela ... · Quinnan et al. N Engl J Med, 1982 Reusser et al. Blood, 1991 Riddell et al. Science, 1992 Li et al.Blood, 1994 Walter

SEIMC, Bilbao, 2012

Replicación del Citomegalovirus (CMV) y dinámica de la respuesta inmunitaria frente al virus en el paciente trasplantado

David Navarro Servicio de Microbiología Hospital Clínico Universitario. Departamento de

Microbiología, Facultad de Medicina, Valencia.

Replicación del CMV y dinámica de la respuesta inmunitaria frente al virus en el paciente trasplantado

SEIMC, Bilbao, 2012

• Implicaciones patogénicas de la ciné@ca replica@va, variabilidad y complejidad genómicas del CMV en el paciente trasplantado • Marcadores inmunológicos de protección frente al CMV en el paciente trasplantado • Ciné@ca de las respuestas T y NK frente al CMV durante los episodios de replicación viral en el Alo-‐TPH

• Rate of increase in CMV load in the blood of 18 bone marrow transplant recipients after allogeneic transplantation

J Exp Med 1999;190:177-‐182

Ciné@ca replica@va del CMV en el paciente trasplantado

dt: median 1.5 days (0.38-‐4.7)

SEIMC, Bilbao, 2012

TPH


SEIMC, Bilbao, 2012

dt: median 1.72 days (0.60-‐12.8)

Type of episode0 Doubling @me (dt)

Treated episodes 2.18 (1.04-‐8.65)

Type A episodes (rapid response) 4.45 (1.20-‐8.76)

Type B episodes (delayed response) 2.01 (1.04-‐3.10)

M/DCs

Fibroblasts Endothelial cells Epithelial cells


Virus Dissemina@on Doubling @me (dt)

Intracellular delay

R0

CMV Cell-‐associated virions 1-‐3 d 18 h 0.83-‐1.5

HCV Cell-‐free virions 0.4-‐1.5 d 6-‐12 h >2.2

Low-‐level non-‐ly@c replica@on

High-‐level ly@c replica@on Long intracellular-‐delay

High-‐level ly@c replica@on Short intracellular-‐delay

Funk et al., Lancet Infect Dis, 2007


SEIMC, Bilbao, 2012

Factores dependientes del virus

Amplia variabilidad en la ciné@ca replica@va del CMV

• ¿Virulencia específica de cepa? • Complejidad genómica viral

Factores dependientes del hospedador

• Ciné@ca de la respuesta inmunitaria

Variabilidad del CMV en el paciente trasplantado

SEIMC, Bilbao, 2012

gps (UL55,UL73,UL74, UL139) TNF-‐α receptor (UL144) α-‐Chemokines (UL33, UL 146-‐147) Puta@ve gps (UL4-‐UL11)

235 Kb 165 ORF 45 ORF esenciales

SEIMC, Bilbao, 2012

•  No existe una asociación consistente entre geno@pos y patogenicidad

•  Geno@pos gB, gN, gH, UL144//ciné@ca replica@va del CMV y riesgo de enfermedad orgánica en TPX

• Geno@pificación no es@ma con precisión la diversidad del CMV • No hay estudios de segregación de polimorfismos (polymorphisms linkage)

Secuenciación del genoma completo necesaria en estudios de patogenicidad

Variabilidad del CMV en el paciente trasplantado

High throughput or ultra-‐deep sequencing methods

Trans-‐complementa@on within co-‐infected cells increases virus fitness and pathogenic poten@al

SEIMC, Bilbao, 2012

Mixed infec@on (genotypes) as a marker for pathogenicity

Do mixed infec@ons “challenge” the immune system?

Complejidad genómica del CMV en el paciente trasplantado

SEIMC, Bilbao, 2012

D+/R+ > D+/R-‐


Kidney: 186 Heart: 19 Liver: 19 Lung: 15

SEIMC, Bilbao, 2012

• Complejidad genómica no inferible por métodos convencionales de geno@pificación


Detectan variantes presentes en frecuencias entre 0,1-‐5%

Detectan variantes presentes en frecuencias >5-‐10%

Quinnan et al. N Engl J Med, 1982 Reusser et al. Blood, 1991 Riddell et al. Science, 1992 Li et al. Blood, 1994 Walter et al. N Engl J Med,1995

Evidences based on studies employing CTL assays

Func@onal CD8+ T-‐cell responses protect against CMV viremia and CMV end-‐organ disease Adop@ve transfer of CMV-‐specific CD8+ T cells protects from CMV end-‐organ disease and mediates resolu@on of episodes of CMV replica@on refractory to an@viral therapy

Evidences based on studies using Tetramer /ELISPOT/ICS assays

Cwynarski et al. Blood, 2001 Aubert et al. J Infect Dis 2001 Hebart et al. Blood, 2002 Ohnishi et al. Br J Hematol, 2005 Lacey et al. J Infect Dis 2006 Lilleri et al Haematologica, 2008

Evidences based on adop@ve T-‐cell transfer treatments

Koehne et al. Blood, 2000 Szmania et al. Blood, 2001 Kondo et al. J Immunol, 2002 Kleihauer et al Br J Haematol, 2001 Vannucchi et al. Br J Haematol, 2001 Peggs et al, Blood, 2001 Keenan et al. Br J Haematol, 2001 Rauser et al. Blood, 2004 Cobbold et al J Exp Med, 2005 Micklehwaite et al. Biol Blood Marrow Transplant, 2007 Zhong et al. Plos ONE, 2008

SEIMC, Bilbao, 2012

Marcadores inmunológicos de protección frente al CMV

CD8+ T cells are cri@cal in the control of CMV infec@on in the TPX semng

RLLQTGIHV

NLVPMVATV LMNGQQIFL RIFAELEGV ILARNLVPM

IGDQYVKVY TVQGQKLEY

YSEHPTFTSQY

40 epitopes restricted by HLA-‐ A (1,2,24,11,68) HLA-‐B (7,15,40,44,51,35,38,51,52) HLA-‐Cw (1,4,8,12,15)

pp65

HLA-‐ A2

HLA-‐ A1

SLLSEFCRV VLAELVKQI VLEETSVML

YILEETSVM CLQNALDIL YIGADPLRV

IKEHMLKKY DEEEAIVAY CVETMCNEY

15 epitopes restricted by HLA –A (1,2,23,24,) HLA-‐B (7,8,27)

IE-‐1

CD8+ T-‐cell responses to pp65 (UL83) and IE-‐1 (UL123) are immunodominant and confer protec@on against CMV infec@on

McLaughlin-Taylor et al. J Med Virol ,1994 Wills et al. J Virol ,1996 Kern et al. J Virol ,1999 Kern et al. J Infect Dis, 2002 Sylwester et al. J Exp Med, 2005

SEIMC, Bilbao, 2012


Indirect: crucial for adequate ac@va@on and expansion of CD8+ T cells

Direct: lysis of CMV-‐infected cells expressing HLA-‐II molecules

Li et al. Blood ,1994 Walter et al. N Engl J Med, 1995 Krause et al. Bone Marrow Transplant, 1997 Aubert et al. J Infect Dis, 2001 Hebart et al. Blood, 2002 Einsele et al. Blood, 2002

IFN-‐γ

At least, 60% of the CMV proteome is targeted by CD4+ T cells Sylwester et al. J Exp Med, 2005

IE (10%)/E (20%)/L (70%)

Frec

uenc

y (%

)

SEIMC, Bilbao, 2012


gB pp65

CD4+ T cells are cri@cal in the control of CMV infec@on in the TPX semng

Autores Recuento/feno@po Ensayo Anpgeno Protección

Cwynarski et al. Blood, 2001 >10/µL Tetrámeros pp65 495-‐503 (A02) pp65 417-‐426 (B07)

DNAemia/ Andgenemia/E.O.

Aubert et al. JID, 2001 Hassan-‐Walker et al. JID, 2001

≥20/µL Tetrámeros pp65 495-‐503 (A02) DNAemia

Hebart et al. Blood ,2002 >5/µL IFN γELISPOT /ICS

pp65 varios pépddos (A01/A02/B07)

DNAemia

Ohnishi et al. BJH , 2005 >10/µL

>1/µL Tetrámeros IFN γELISPOT

pp65 495-‐503 (A02) pp65 varios pépddos

Andgenemia

Lilleri et al. Haematologica, 2008

>3/µL IFNγ/TNFα ICS Células dendrídcas Infectadas VR1814

DNAemia/ Andgenemia

Solano et al Haematologica, 2008 Tormo et al. BMT, 2009 Tormo et al. BBMT,2009 Tormo et al. J Med Virol, 2010 Tormo et al., BMT, 2011

>1/µL

IFNγ ICS Pepmix pp65+IE-‐1 DNAemia/

Andgenemia

Moins-‐Teisserenc et al. JID , 2008

>1/µL T CD8+/ CD27-‐/CD28-‐/CD45RA+

Tetrámeros+IFNγ ICS/fenodpo membrana

pp65 495-‐503 (A02) pp65 417-‐426 (B07)

DNAemia

Solano y Navarro, Future Virology, 2010 SEIMC, Bilbao, 2012


Valores umbral T CD8+ que confieren protección frente al CMV en TPH

PPV:100% NPV:65%

Solano y Navarro, Future Virology, 2010

Autores Recuento/ feno@po

Ensayo Anpgeno Protección

Lilleri et al. Haematologica, 2008

>1/µL

IFNγ/TNFα ICS

Células dendrídcas Infectadas VR1814

DNAemia/ Andgenemia

Solano et al. Haematologica, 2008 Tormo et al. BMT, 2009 Tormo et al. J Med Virol, 2010 Tormo et al., BMT, 2011

>1,2/µL IFNγ ICS Pepmix pp65+IE-‐1

DNAemia/ Andgenemia

Valores umbral T CD4+ que confieren protección frente al CMV en TPH

SEIMC, Bilbao, 2012


PPV:100% NPV:60%

T r a s p l a n t e / C M V receptor

Método Población T Anpgeno Protección frente

Referencia

Corazón/Pulmón/+ ICS CD8+ IFNγ p é p d d o s solapados IE-‐1

Enfermedad orgánica

Bunde et al., 2005

C o r a z ó n / P u lm ó n / Riñón/+

ICS CD8+ IFNγ 0.4 cel/µL

C é l u l a s d e n d r í d c a s i n f e c t a d a s CMV-‐VR1814

I n f e c c i ó n acdva

Gerna, et al., 2006; Lilleri et

al., 2007

Hígado/ + o -‐ ICS CD8+ IFNγ P é p d d o s solapados pp65


Nebbia et al., 2008

Riñón/+ o -‐ ICS TET

CD8+IFNγ CD8+

P é p d d o s solapados pp65 Pépddo NLV


Maoes et al., 2008

Pulmón/+ Quan@ferónCMV CD8+IFNγ P é p d d o s (pp65/IE-‐1/gB/pp50)


Westall et al., 2008

Pulmón/Riñón/Hígado/ Páncreas/+ o -‐

Quan@ferón CMV CD8+IFNγ P é p d d o s (pp65/IE-‐1/gB/pp50)

Enfermedad orgánica

Kumar et al., 2009

Riñón/Corazón/ Pulmón/+ o -‐

ELISPOT CD8+IFNγ P é p d d o s solapados pp65

S í n d r o m e C M V / Enfermedad orgánica

Crough et al., 2007

SEIMC, Bilbao, 2012

Marcadores inmunológicos de protección frente al CMV en TOS

UCL, March 2012

Kinedcs of CMV-‐specific T cells during episodes of acdve CMV infecdon in TPH

Dinámica de la respuesta inmunitaria frente al CMV

pepmix pp65 and IE-‐1 (1 µg/mL/pep@de) + CD28/CD49d

Staining (CD3+/CD4+ or CD8+/CD69+/IFNγ+)

ICS

UCL, March 2012

• La dinámica de expansión de T CD8+ y T CD4+ frente a pp65/IE-1 determina la cinética replicativa del CMV en el Alo-TPH


• 24 episodes (14 A and 10 B) •  Median, 46 days (24-‐310) • 16 Inidal and 6 relapsing

9 days 10.5 days

Type A Type B

16.5 days 13.5 days

12-fold increase 6.8-fold increase

Tormo et al., BMT, 2010

Immunological monitoring

SEIMC, Bilbao, 2012


Cor@costeroid therapy (2 mg/Kg/day) Lack of control during the study period

Eventual control of ac@ve CMV infec@on

Eventual control of ac@ve CMV infec@on

Type B episodes

CMV DNAemia clearance

CMV DNAemia clearance

Persistent CMV DNAemia

T-‐cell expansion

T-‐cell expansion

Tormo et al., BMT, 2010

SEIMC, Bilbao, 2012


Lack of consistent T-‐cell expansion

Clearance of CMV DNAemia without therapeu@c interven@on in Allo-‐SCT depends on the expansion of func@onal CMV-‐specific CD8+ T cells above the protec@ve threshold (1 cell/μL)

UCL, March 2012 Solano et al., Transplantation, 2011

Recurrent

Recurrent

Initial

Initial

+14

+22

+15

+12


SEIMC, Bilbao, 2012

Clearance of CMV DNAemia with therapeu@c interven@on (pre-‐emp@ve therapy)


Rapid expansion above protec@ve thresholds (>1 cell/μL) within the first week aver ini@a@on of pre-‐emp@ve an@viral therapy

0 7 32 61 78 85 92

0

5

10

15

20

25

30

35

40

45

50

55

0

100

200

300

400

500

600

700

800

900

Day aqer initadon of pre-‐empdve therapy

cells

/µL

Interrrupdon P-‐T

Early and late episodes

First detected response

SEIMC, Bilbao, 2012


Delayed expansion above protec@ve thresholds (>1 cell/μL) associated with resolu@on of ac@ve CMV infec@on

Day after initation of pre-emptive therapy

cells

/µL

0 7 14 21 28 33 47 51 56 64 70 74 78 83 90 94 101

0,00,51,01,52,02,53,03,54,04,55,05,56,06,57,07,5

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

Interruption P-T

CM

V D

Aem

ia (copies/mL)

GvHD

GvHD

Early episodes

SEIMC, Bilbao, 2012


Inconsistent or lack of expansion above protec@ve levels thresholds (>1 cell/μL) are associated with persistent CMV DNAemia

Day aqer initadon of pre-‐empdve therapy

cells

/µL

CM

V D

Aem

ia (copies/mL)

0 7 14 20 25 32 50 80 90 97

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

0

500

1000

1500

2000

2500

3000

3500

4000

GvHD

GvHD

GvHD

NO emergence of Ganciclovir –resistant strains during therapy

Early and late episodes

SEIMC, Bilbao, 2012


SEIMC, Bilbao, 2012


Lacey et al., JID, 2006 Zhou et al.., Blood, 2009 Makadonas et al., 2010

Analysis of kine@cs of polyfunc@onal CD8+ T cells during episodes of ac@ve CMV infec@on

Monofunc@onal IFN-‐γ TNF-‐α CD107a

Bifunc@onal IFN-‐γ/ TNF-‐α IFN-‐γ/CD107a TNF-‐α/CD107a

Trifunc@onal IFN-‐γ/ TNF-‐α//CD107a

IFN-‐γ CD4 T cells

pp65/IE-‐1 Ga@ng strategy

SEIMC, Bilbao, 2012


*Within 72 h. following detecdon of CMV DNAemia

*

*

*

*

*

Self-‐resolved

SEIMC, Bilbao, 2012


No consistent expansion of any subset was observed in unresolved episodes

Resolved aqer treatment

SEIMC, Bilbao, 2012


c

NCR (NKp30,44,46)

KIR

2D S

3D L

ILT (LIR,CD85)

NKG/CD94

CD3/FCRIγ ITIM ITAM

ITIM

ITIM ITAM

ACT ACT/INH INH ACT:NKG2C/D INH:NKG2A

SEIMC, Bilbao, 2012

NK cells


CD3- CD16- / CD56+++ NKG2C+

CD3- CD16+ CD56+/- NKG2C+

An@viral cytokines (IFN-‐γ)

Cytotoxic poten@al

SEIMC, Bilbao, 2012


≥4-fold increase

Self-resolving

Treated

No expansion in non-‐resolved episodes

Muñoz-‐Cobo et al. J. Med. Virol., 2012 SEIMC, Bilbao, 2012


1

2

3

UCL, March 2012

Demonstrates expansion of NK cells during episodes of CMV DNAemia

Expansion of NKG2C+ NK cells producing IFN-γ and expressing self-KIR inhibitory receptors


DC

IFN-γ

IL-12

Robbins et al., 2007

IFN-α/β

Th1

DC/MΦ

TCD8 pDC

NK

DC/NK “crosstalk” Correladon between NKG2C+ NK cells and IFN-‐γ/TNF-‐α/CD107a CD8+ T cells (Muñoz-‐Cobo et al., J Med Virol, 2012)

SEIMC, Bilbao, 2012


Conclusions

• Definidve control of episodes of CMV replicadon either treated with andvirals or untreated is uldmately dependent on the consistent expansion (above 1.0 cell/µL) of pp65 and IE-‐1 IFN-‐γ-‐ producing CD8+ and CD4+ T cells

SEIMC, Bilbao, 2012


• Resoludon of early and late episodes of CMV DNAemia in the absence of andviral treatment is associated with a consistent expansion of both mono and polyfuncdonal CD8 + T cells

• Resoludon of episodes of CMV DNAemia treated with andvirals is associated with disdnct funcdonal paoerns of expanding CD8+ T cells

• Expansion of muldfuncdonal CD8+ T cells depends on adequate expansion of cognate andgen CD4 + T cells

• Expansion of NKG2C cells is associated with resoludon of either treated or untreated episodes of acdve CMV infecdon and may promote the expansion of polyfuncdonal CMV-‐specific CD8+ T cells

TPH

Nuria Tormo M. Aª Clari Isana Benet Carlos Solano Mª José Remigia Paula Amat Elisa Costa Dayana Bravo Beatriz Muñoz-‐Cobo

Rafael de la Cámara Ana García-‐Noblejas Laura Cardeñoso

Javier López

José B. Nieto

SEIMC, Bilbao, 2012

Documents

Replicación*del*Citomegalovirus*(CMV)*y*dinámica*de*la ... · Quinnan et al. N Engl J Med, 1982 Reusser et al. Blood, 1991 Riddell et al. Science, 1992 Li et al.Blood, 1994 Walter

ReplicacióndelCitomegalovirus(CMV)y*dinámicadela ... · Quinnan et al. N Engl J Med, 1982 Reusser et al. Blood, 1991 Riddell et al. Science, 1992 Li et al.Blood, 1994 Walter