AREVIR 2017 Emergence of drug-resistant cytomegalovirus (CMV) in immunosuppressed patients Axel Schubert Institut für Virologie, Universitätsklinikum Ulm Consultant Laboratory for Cytomegalovirus
Failure of therapy Virological resistance drug resistant phenotype (drug resistance mutation) Virus Virus load load anti-viral therapy anti-viral therapy 5 10 15 time 5 5 5 5 5 10 10 15 days Mutant WT Clinical resistance e.g. total loss of immune system/T-cell depletion
Failure of therapy Virological resistance drug resistant phenotype (drug resistance mutation) (n ~ 1700 samples; ~ 17% also UL54 sequenced): 27% GCV (FOS/CDV) resistance 73 % wild-type Clinical resistance e.g. total loss of immune system/T-cell depletion
GCV-resistant HCMV in solid organ transplant recipients (n=97) Tx 1 2 3 4 5 6 7 >7 months after transplantation
GCV-resistant HCMV in solid organ transplant recipients (n=97) Tx 1 7 >7 2 3 4 5 6 months after transplantation GCV resistance can emerge early after transplantation
NTx ganciclovir resistance can emerge fast (L595S) aa 595 aa 595 10 days serine 60% leucine leucine 40%
NTx ganciclovir resistance can emerge fast (L595S) aa 595 aa 595 10 days serine 60% leucine leucine 40% After ~ 14 days of antiviral therapy, genotypic resistance may be suspected
Molecular targets of the HCMV therapy * Compassionate Use *** * *! *Leflunomide * ** ** Phase III ! regulatory approval only for Retinitis in AIDS patients *** Phase I
"Alternatives" to the current HCMV therapy Leflunomide Anti-HCMV activity in vitro , many case reports, no “controlled” studies Artesunate Anti-CMV activity in vitro and in a rat model. Some case reports show effects on virus load. Cyclopropavir Broad-spectrum anti-herpesvirus drug, anti-HCMV activity is comparable to GCV (cross-resistance), clinical trial study Phase Ib 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. *Lischka P 2016
Molecular targets of the HCMV therapy * frequently used salvage therapy *** * *! *Leflunomide * ** ** Phase III ! regulatory approval only for Retinitis in AIDS patients *** Phase I
Mutations in UL97 and UL54 conferring reduced drug susceptibility Protein kinase (pUL97) kinase regions I VIB VII VIII IX 707 AA Genotyping UL97 AS 395-638 N C mutations associated with drug resistance 460 520 590-607 DNA polymerase (pUL54) Exo I Exo II Exo III pol regions 1242 AA N δ region C IV II VI III I VII V Genotyping UL54 mutations associated AS 300-988 with drug resistance 987 301 Chevillotte et al. 2010
Mutations in UL97 and UL54 conferring reduced drug susceptibility Protein kinase (pUL97) kinase regions I VIB VII VIII IX 707 AA Genotyping UL97 AS 395-638 N C mutations associated with drug resistance drug resistance GCVr phenotype DNA polymerase (pUL54) Exo I Exo II Exo III pol regions 1242 AA N δ region C IV II VI III I VII V Genotyping UL54 mutations associated AS 300-988 with drug resistance drug resistance GCVr FOSr FOSr phenotype CDVr GCVr CDVr Chevillotte et al. 2010
CMV-Polymerase Mutations Ratio of IC 50 Strain/WT 1≤ sensitiv /potential resistance ≥ 2 ≤ potential/ low resistance ≥ 3 ≤ low to high resistance IC 50 mutant Ratio = IC 50 wild type Mutions found „more frequently“ in Ulm MRA - mutation resistance analyzer
Polymorphisms and resistance-associated mutations in human cytomegalovirus DNA polymerase Number of mutations at position “x" phenotype MRA - mutation resistance analyzer
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
Proportion of UL97 mutations detected by genotyping (n = 235) C607Y A594G A594T A594P A594L C603F A594S L595F A594V different Deletions H520Q M460I C592G L595S M460V C603W Propotion >85%
Proportion of UL97 mutations detected by genotyping (n = 235) C607Y A594G A594T A594L C603F A594P A594S L595F A594V different Deletions H520Q M460I C592G L595S M460V C603W
Proportion of UL97 mutations detected by genotyping (n = 235) High vs. low C607Y A594G resistance? A594T A594L C603F A594P A594S L595F A594V different Deletions H520Q M460I C592G L595S M460V C603W
CMV UL97 Mutations Ratio of IC 50 Strain/WT Ratio IC50 * 1≤ sensitiv /potential resistance ≥ 2 ≤ potential/ low resistance ≥ 3 ≤ low to high resistance MRA - mutation resistance analyzer
CMV UL97 Mutations Ratio of IC 50 Strain/WT Ratio IC50 * 1≤ sensitiv /potential resistance ≥ 2 ≤ potential/ low resistance ≥ 3 ≤ low to high resistance Selection of drug resistance mutation: “Higher ratio is not better”
Phosphorylation activity of HCMV-UL97 mutants % GCV phosph. Mutant/wildtyp ~ Occurence % Mutant of UL97 wt* GCV IC50 ratio** (n=221)** 8 25 A594V 18 L595S 9 16 20 9 M460V 13 8 8 15 5 M460I 3 8 C592G 35 7 H520Q 5 10 L595F 3 28 16 C607Y 1 32 13 ≤ 1 A594P a 30 3 ≤ 1 ~10 del590-593 11 ≤ 1 16 del601 b 10 ≤ 0,1 G598S 52 1 *Baldanti et al. 2002, and Ijichi et al. 2001 a antiviral research, ** Schreiber et al. 2009 Expert Opin. Hanz et al. 2005 b Antimicrob Agents Chemother.
Phosphorylation activity of HCMV-UL97 mutants % GCV phosph. Mutant/wildtyp ~ Occurence % Mutant of UL97 wt* GCV IC50 ratio** (n=221)** 8 25 A594V 18 9 L595S 16 20 9 M460V 13 8 8 15 5 M460I 8 3 C592G 35 7 10 H520Q 5 L595F 3 28 16 1 C607Y 32 13 ≤ 1 A594P a 30 3 ≤ 1 ~10 del590-593 11 ≤ 1 10 16 del601 b ≤ 0,1 G598S 52 1 *Baldanti et al. 2002, and Ijichi et al. 2001 a antiviral research, ** Schreiber et al. 2009 Expert Opin. Hanz et al. 2005 b Antimicrob Agents Chemother.
Distribution of mutations in UL97 and/ or UL54 detected in the same sample (n = 290) UL97 GCVr UL54 % GCV* GCV/CDV FOS(±GCV) Mixed WT WT 65 MU WT 23 MU a MU 7 ~ 25% ~ 25% ~ 25% ~25% WT MU 5 #7 # 1 # 7 b * 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) UL97 GCVr UL54 % GCV* GCV/CDV FOS(±GCV) Mixed WT WT 65 MU WT 23 MU a MU 7 ~ 25% ~ 25% ~ 25% ~25% WT MU 5 #7 # 1 # 7 b * 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)
UL97- From phenotype to genotype to consequences for therapy A478V T601M V466M C603S C603del A497T C592G C603W A594E A594V D605E M460V H520Q L592S L600I G598S C607F Switch to FOS N597D E596G H469Y K599E A591V May permit GCV therapy using higher doses Maintain therapy
UL97- From phenotype to genotype to consequences for therapy A478V T601M V466M C603S C603del A497T C592G C603W A594E A594V D605E M460V H520Q L592S L600I G598S C607F Switch to FOS N597D E596G H469Y K599E A591V May permit GCV therapy using higher doses Maintain Genotyping of UL54 should be considered therapy
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.
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