Axel Schubert Institut fr Virologie, Universittsklinikum Ulm - - PowerPoint PPT Presentation

AREVIR 2017

Emergence of drug-resistant cytomegalovirus (CMV) in immunosuppressed patients

Axel Schubert Institut für Virologie, Universitätsklinikum Ulm Consultant Laboratory for Cytomegalovirus

Clinical resistance

e.g. total loss of immune system/T-cell depletion

Virological resistance

drug resistant phenotype (drug resistance mutation)

Failure of therapy

WT

days

Virus load

5 10 15 5 5 5 10 5

anti-viral therapy

Mutant

time

Virus load

15 10 5

anti-viral therapy

(n ~ 1700 samples; ~ 17% also UL54 sequenced): 27% GCV (FOS/CDV) resistance 73 % wild-type

Failure of therapy

Clinical resistance

e.g. total loss of immune system/T-cell depletion

Virological resistance

drug resistant phenotype (drug resistance mutation)

months after transplantation

Tx 1 2 3

GCV-resistant HCMV in solid organ transplant recipients (n=97)

4 5 6 7 >7

GCV resistance can emerge early after transplantation

months after transplantation

Tx 1 2 3

GCV-resistant HCMV in solid organ transplant recipients (n=97)

4 5 6 7 >7

aa 595 aa 595

NTx ganciclovir resistance can emerge fast (L595S)

leucine

serine 60%

leucine 40%

10 days

aa 595 aa 595

NTx ganciclovir resistance can emerge fast (L595S)

leucine

serine 60%

leucine 40%

10 days

After ~ 14 days of antiviral therapy, genotypic resistance may be suspected

Molecular targets of the HCMV therapy

* Compassionate Use

* * **

** Phase III

*Leflunomide

*** Phase I

***

! regulatory approval only for Retinitis in AIDS patients *!

Leflunomide Anti-HCMV activity in vitro, many case reports, no “controlled” studies Brincidofovir Anti-HCMV (and anti-adenovirus) activity, lipid conjugated

CMX-001

prodrug of cidofovir, did not meet primary endpoint in two Phase III studies Letermovir Non-nucleoside HCMV inhibitor, inhibiting the viral terminase

MK-8228

complex, oral drug, Viral mutations described in UL56 codons 231 to 369 (V236M in a Phase 2 Prophylaxis Trial; (60 mg/day)*

“Waiting for regulatory approval” (End 2017? 2018)

Maribavir Anti-HCMV activity, UL97 protein kinase inhibitor, oral drug, effective in 2 Phase II studies, “no” efficacy in a Phase III study. Cyclopropavir Broad-spectrum anti-herpesvirus drug, anti-HCMV activity is comparable to GCV (cross-resistance), clinical trial study Phase Ib Artesunate Anti-CMV activity in vitro and in a rat model. Some case reports show effects on virus load.

"Alternatives" to the current HCMV therapy

*Lischka P 2016

Molecular targets of the HCMV therapy

* frequently used salvage therapy

* * **

** Phase III

*Leflunomide

*** Phase I

***

! regulatory approval only for Retinitis in AIDS patients *!

Mutations in UL97 and UL54 conferring reduced drug susceptibility

Exo I Exo II Exo III IV δ region II VI III I VII V

N C

pol regions

1242 AA

mutations associated with drug resistance

DNA polymerase (pUL54) Protein kinase (pUL97)

Chevillotte et al. 2010

460 590-607 301 987

I

N C

kinase regions

707 AA

mutations associated with drug resistance

VIB VII VIII IX

520

Genotyping UL97 AS 395-638 Genotyping UL54 AS 300-988

Mutations in UL97 and UL54 conferring reduced drug susceptibility

DNA polymerase (pUL54) Protein kinase (pUL97)

Chevillotte et al. 2010

Exo I Exo II Exo III IV δ region II VI III I VII V

N C

pol regions

1242 AA

mutations associated with drug resistance drug resistance phenotype

GCVr CDVr FOSr FOSr GCVr CDVr

I

N C

kinase regions

707 AA

mutations associated with drug resistance drug resistance phenotype

GCVr

VIB VII VIII IX

Genotyping UL97 AS 395-638 Genotyping UL54 AS 300-988

CMV-Polymerase Mutations Ratio of IC50 Strain/WT

Ratio = IC50 wild type IC50 mutant

1≤ sensitiv /potential resistance ≥ 2 ≤ potential/low resistance ≥ 3 ≤ low to high resistance

MRA - mutation resistance analyzer

Mutions found „more frequently“ in Ulm

Polymorphisms and resistance-associated mutations in human cytomegalovirus DNA polymerase

MRA - mutation resistance analyzer

Number of mutations at position “x" phenotype

MRA - mutation resistance analyzer

?

MRA - mutation resistance analyzer

New!: HSV Thymidinkinase & Polymerase

Sequence

MRA - mutation resistence analyzer

http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/mra/app/index.php?plugin=form

Sequence

A594P A594L C603F A594S L595F

different Deletions

H520Q M460I C592G M460V C603W L595S A594V

Proportion of UL97 mutations detected by genotyping (n = 235)

A594G A594T C607Y Propotion >85%

A594P A594T A594L C603F A594S L595F

different Deletions

H520Q M460I C592G M460V C603W L595S A594V

Proportion of UL97 mutations detected by genotyping (n = 235)

A594G C607Y

A594P A594T A594L C603F A594S L595F

different Deletions

H520Q M460I C592G M460V C603W L595S A594V

Proportion of UL97 mutations detected by genotyping (n = 235)

A594G

High vs. low resistance?

C607Y

* Ratio IC50

1≤ sensitiv /potential resistance ≥ 2 ≤ potential/low resistance ≥ 3 ≤ low to high resistance

CMV UL97 Mutations

Ratio of IC50 Strain/WT

MRA - mutation resistance analyzer

* Ratio IC50

1≤ sensitiv /potential resistance ≥ 2 ≤ potential/low resistance ≥ 3 ≤ low to high resistance

CMV UL97 Mutations

Ratio of IC50 Strain/WT

Selection of drug resistance mutation: “Higher ratio is not better”

*Baldanti et al. 2002, and Ijichi et al. 2001a antiviral research, ** Schreiber et al. 2009 Expert Opin. Hanz et al. 2005b Antimicrob Agents Chemother.

Phosphorylation activity of HCMV-UL97 mutants Mutant ~ Occurence %

(n=221)**

% GCV phosph.

• f UL97 wt*

Mutant/wildtyp GCV IC50 ratio** M460I H520Q C592G del601b A594V L595S L595F G598S C607Y del590-593 ≤1 ≤0,1 15 5 35 10 18 16 28 52 32 11 8 5 10 3 16 9 16 1 13 ~10 8 7 8 25 20 3 1 ≤1 A594Pa 30 3 ≤1 M460V 13 9 8

*Baldanti et al. 2002, and Ijichi et al. 2001a antiviral research, ** Schreiber et al. 2009 Expert Opin. Hanz et al. 2005b Antimicrob Agents Chemother.

Mutant ~ Occurence %

(n=221)**

% GCV phosph.

• f UL97 wt*

Mutant/wildtyp GCV IC50 ratio** M460I H520Q C592G del601b A594V L595S L595F G598S C607Y del590-593 ≤1 ≤0,1 15 5 35 10 18 16 28 52 32 11 8 5 10 3 16 9 16 1 13 ~10 8 7 8 25 20 3 1 ≤1 A594Pa 30 3 ≤1 M460V 13 9 8 Phosphorylation activity of HCMV-UL97 mutants

Distribution of mutations in UL97 and/ or UL54 detected in the same sample (n = 290)

UL97GCVr UL54 % GCV* GCV/CDV FOS(±GCV) Mixed WT WT 65 MU WT 23 MUa MU 7 ~ 25% ~ 25% ~ 25% ~25% WT MU 5 #7 # 1 # 7b * Only UL54 a: ~12% of the samples with a resistance mutation in UL97 have an additional GCVr mutation in UL54 UL97~ 80% high resistance mutations (M460I/VT, C603W) UL97~ 20% low resistance mutations (e.g. C592G)

Distribution of mutations in UL97 and/ or UL54 detected in the same sample (n = 290)

UL97GCVr UL54 % GCV* GCV/CDV FOS(±GCV) Mixed WT WT 65 MU WT 23 MUa MU 7 ~ 25% ~ 25% ~ 25% ~25% WT MU 5 #7 # 1 # 7b * Only UL54 a: ~12% of the samples with a resistance mutation in UL97 have an additional GCVr mutation in UL54 UL97~ 80% high resistance mutations (M460I/VT, C603W) UL97~ 20% low resistance mutations (e.g. C592G) b: # 1 multi-resistance mutation (A834P) # 4 low level GCV resistance mutation (A809V) # 2 Foscarnet resistance mutation (L802M, V751M)

A594V L592S C603W C592G A594E C603S H469Y V466M C607F M460V H520Q A497T G598S E596G A591V L600I

UL97- From phenotype to genotype to consequences for therapy

D605E A478V Switch to FOS May permit GCV therapy using higher doses Maintain therapy C603del N597D K599E T601M

A594V L592S C603W C592G A594E C603S H469Y V466M C607F M460V H520Q A497T G598S E596G A591V L600I

UL97- From phenotype to genotype to consequences for therapy

D605E A478V Switch to FOS May permit GCV therapy using higher doses Maintain therapy C603del N597D K599E T601M Genotyping of UL54 should be considered

Observations during HCMV therapy

• Multiple resistance conferring UL97 mutations are encoded on

different viruses (subpopulations).

• Over time some mutations (subpopulations) may „disappear“ under

therapy while others are maintained.

• During GCV-therapy pauses (or changes e.g. FOS) „repopulation“ with

wildtype can occur. (Cave: Resistant strains may persist as “latent” infection and may reappear after restarting GCV-therapy. Even after several (>12) Months).

• GCV-resistant variants have a selection advantage if GCV-therapy is

continued.

• qPCR may overestimate “hole genome” copy numbers
• >“fragmented DNA in plasma”
• > we prefer EDTA-Blood for genotyping (“cell associated Virus”).
• Frameshift or stop codon mutations in UL97 or UL54 are sequencing

errors.

Observations during HCMV therapy

• Multiple resistance conferring UL97 mutations are encoded on

different viruses (subpopulations).

• Over time some mutations (subpopulations) may „disappear“ under

therapy while others are maintained.

Observations during HCMV therapy

• Multiple resistance conferring UL97 mutations are encoded on

different viruses (subpopulations).

• Over time some mutations (subpopulations) may „disappear“ under

therapy while others are maintained.

• During GCV-therapy pauses (or changes e.g. FOS) „repopulation“ with

wildtype can occur. (Cave: Resistant strains may persist as “latent” infection and may reappear after restarting GCV-therapy. Even after several (>12) Months).

• GCV-resistant variants have a selection advantage if GCV-therapy is

continued.

• qPCR may overestimate “hole genome” copy numbers
• >“fragmented DNA in plasma”
• > we prefer EDTA-Blood for genotyping (“cell associated Virus”).
• Frameshift or stop codon mutations in are sequencing errors.

Institut für Virologie Diagnostic

• Prof. Dr. Mertens
• Prof. Dr. Michel
• Dr. Schubert

Universitätsklinikum Ulm

Page 34

A G T C purine R pyrimidine Transition ~ 77% SNP

1. 2. 3. A594 R Y R GCG NCG GNG GCN T,S,P V,E,G A ACG GTG GCA TCG GAG GCT CCG GGG GCC

Molecular aspects of drug resistance mutation

codon AA ratio IC50 Frequency in % GTG V 8 25 ACG T 2,7 ~ 1 GGG G 14 <1 TCG S 4,6 <1 GAG E 3 <1 CCG P 2,8 <1

Page 36

Molecular aspects of drug resistance mutation „Possible “ mutants Mutant(s) Selection

Viremia shell vial culture 1 to 3 days Real-time PCR DNA 2 to 4 h Antigenemia (pp65) 5 to 6 h Phenotyping after virus isolation, 1-2 weeks

(GCV, FOS, CDV)

UL97 (GCV, “Maribavir”) UL54 (Polymerase) (GCV, FOS, CDV) UL56 (“Letermovir”)

HCMV - diagnostics

Serology IgG, IgM, (IgG avidity) 6 h to 24 h Virus culture isolate 1 to 6 weeks Quantiferon-CMV ELISA (interferon-γ) 24 h Genotyping 2-3 days

Page 38

Fast selection of maribavir resistant cytomegalovirus in a bone marrow transplant recipient

409 411 MBV r MBV r MBV r M V T V H R Y H411Y T409M

direct sequencing from EDTA-blood cloning in

• E. coli

clone 2

Cloning proofs accumulation of different Maribavir- resistant strains under therapy