Analysis of HBV resistant m utants by phenotypic assay - PowerPoint PPT Presentation

Analysis of HBV resistant m utants by phenotypic assay AREVIR-GenaFor-Meeting 22.04.2010 Corinna M. Bremer Institut für medizinische Virologie der JLU Gießen

HBV Developm ent of Resistance Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009; 137: 1593-1608

HBV Polym erase Secondary prim ary resistance /V resistance m utation m utation rtV173L ? adapted from Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009; 137: 1593-1608

Cloning Cloning polymerase-frame • HBV-replication-competent vector with HBV backbone • polymerase frame is replaced • cloning efficient >10 4 GE/ml • plasmid serves as wildtype (genotype D)

phenotypic assay Huh-7 cells transfection , 6h, 1% FCS/DMEM Fugene HD / 8µg DNA (thereof 1µg SEAP-plasmid)

phenotypic assay-SEAP • Standardization of the transfection by SEAP-test (secreted alkaline phosphatase) SEAP-Assay 96-Well 2 1,5 OD450 1 0,5 0 0,08 0,04 0,02 0,01 transfected µg plasmid/well 5 min 10 min

phenotypic assay Huh-7 cells 100 µl of nucleoside analoga in 96well Platte transfection , 6h, 1% FCS/DMEM Fugene HD / 8µg DNA (thereof 1µg SEAP-plasmid) 3 days wash 2x wash, detach cells, 3x wash by centrifugation HBV DNA in 25 ml DMEM / 1% FCS from supernatant + SEAP-test resuspend cells in 15 ml DMEM w/o phenol red

w ildtype inhibition IC50s: wildtype plasm id Lamivudin: 77.0 42.3 nM 1 4 0 ,0 0 1 2 0 ,0 0 1 0 0 ,0 0 % replicat ion 8 0 ,0 0 6 0 ,0 0 4 0 ,0 0 2 0 ,0 0 (average of 8 independent 0 ,0 0 experiments in triplicates) 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 Lam ivudin

w ildtype inhibition IC50s: wildtype plasm id Lamivudin: 77.0 42.3 nM 1 6 0 ,0 0 1 4 0 ,0 0 Adefovir: 1281.7 625.5 nM 1 2 0 ,0 0 % replicat ion 1 0 0 ,0 0 8 0 ,0 0 6 0 ,0 0 4 0 ,0 0 2 0 ,0 0 (average of 8 independent 0 ,0 0 experiments in triplicates) 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 Adefovir Lam ivudin

w ildtype inhibition IC50s: wildtype plasm id Lamivudin: 77.0 42.3 nM 1 6 0 ,0 0 1 4 0 ,0 0 Adefovir: 1281.7 625.5 nM 1 2 0 ,0 0 % replicat ion Entecavir: 1.1 0.6 nM 1 0 0 ,0 0 8 0 ,0 0 6 0 ,0 0 4 0 ,0 0 2 0 ,0 0 (average of 8 independent 0 ,0 0 experiments in triplicates) 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 Adefovir Entecavir Lam ivudin

w ildtype inhibition IC50s: wildtype plasm id Lamivudin: 77.0 42.3 nM 1 6 0 ,0 0 1 4 0 ,0 0 Adefovir: 1281.7 625.5 nM 1 2 0 ,0 0 % replicat ion Entecavir: 1.1 0.6 nM 1 0 0 ,0 0 Tenofovir: 1006.5 654.7 nM 8 0 ,0 0 6 0 ,0 0 4 0 ,0 0 2 0 ,0 0 (average of 8 independent 0 ,0 0 experiments in triplicates) 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 Adefovir Entecavir Lam ivudin Tenofovir

Relative replication yield of HBV m utants variability of different test systems and within the mutants 6.4 % of WT (n=5) n.d. (secretion deficient) 67 % of WT (n=1) 2.6 – 83.6 % of WT (n=6) 10.8 % of WT (n=4) 50 % of WT (n=2) 7.4 / 45.5 % of WT (n=2) 17-120 % of WT (n=5) 17-67 % of WT (n=3) R.Edwards, T.Shaw, V. Sozzi & S. Locarnini, 2005 HepDART

180M-184A-204V Zoulim & Locarnini, HBV resistance to nucleos(t)ide S: sensitive; R: resistance; I: intermediate analogues . Gastroenterology, 2009; 137: 1593-1608

L1 8 0 M-T1 8 4 A-M2 0 4 V 1 8 0 M-1 8 4 A-2 0 4 V - Adefovir ( viral fitness ~ 1 6 % of W T) 160 140 120 Adefovir % replication 100 resistance factor: 2.8 0.3 80 60 40 20 0 0,01 0,1 1 10 100 1000 10000 100000 Adefovir [ nM] 147- 1 155- 2 147- 3 WT 1 8 0 M-1 8 4 A-2 0 4 V - Tenofovir 140 resistance factor = 120 100 % replication mutant IC50 80 60 wildtype IC50 40 20 Tenofovir 0 0,01 0,1 1 10 100 1000 10000 100000 Tenofovir [ nM] resistance factor: 5.5 3.1 147- 1 155- 1 147- 3 WT

1 8 0 M-1 8 4 A-2 0 4 V - Lamivudin 1 8 0 M-1 8 4 A-2 0 4 V 160 viral fitness ~ 1 6 % of W T) 140 120 Lamivudin % replication 100 resistance factor: 6934.0 5310.4 80 60 40 20 0 0,01 0,1 1 10 100 1000 10000 100000 Lam ivudin [ nM] 147- 1 155- 1 147- 3 WT 1 8 0 M-1 8 4 A-2 0 4 V - Entecavir 160 140 120 % replication 100 80 60 40 20 Entecavir 0 0,01 0,1 1 10 100 1000 Entecavir [ nM] resistance factor: 200.0 145.3 147- 1 155- 1 147- 3 WT

cross-resistance M204I in combination with L80I/ V and/ or V173L Lamivudin / Entecavir 100 90 80 resistance factor 70 60 50 40 30 20 10 0 204I 204I 204I,80V 204I, 80I 204I,173L 204I, 80V,173L, Adefovir Ent ecavir Lamivudin Tenofovir Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009; 137: 1593-1608

Em ergence of m ultidrug resistance A181V in combination with N236T Adefovir / Tenofovir 20,00 18,00 16,00 resistance factor 14,00 12,00 10,00 8,00 6,00 4,00 2,00 0,00 181V 181V 181V 181V 236T 236T 236T 236T 181V,236T WT Adefovir Ent ecavir Lamivudin Tenofovir Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009; 137: 1593-1608 S: sensitive; R: resistance; I: intermediate

HBV overlapping ORFs rtA1 8 1 T Polym erase Surface proteins sW 1 7 2 * stop in some mutants  no genomes detectable in supernatant - replication deficient? - secretion deficient? - cloning? Warner & Locarnini, Hepatology , 2008; 48: 88-98

HBV overlapping ORFs – Antiviral resistance mutation rtA181T / sW172* stop mutant has a rtA181T dominant-negative effect on HBsAg secretion – Reduced amount of HBsAg and HBV in serum, although antiviral therapy was not successful sW172*stop – Misinterpretation of serological signs during treatment. – Treatm ent failure The hepatitis B virus rtA181T / sW172* stop mutant has oncogenic potential. (Lai and Yeh, Antiviral Therapy, 2009)  induction of hepatocellular carcinoma during antiviral therapy is possible

Conclusions • the developed phenotypic assay is an highly reproducible, reliable tool for the analysis of HBV resistance mutants with high throughput • yet unknown secondary mutations are important • 180M is not necessarily essential for Entecavir resistance • tenofovir shows a similar resistance pattern as adefovir in vitro (not noticed in therapy, because it can be used in higher concentrations)

open points • analysis of secondary mutations – variability of polymerase within different genotypes • criteria of viral fitness • secretion-deficient or replication-deficient mutants? – number of intracellular genomes must be determined

Acknow ledgem ent I nstitut für Med. Virologie Andreas Geipel Robert Schefter Dieter Glebe Wolfram H. Gerlich Universität zu Köln Maria Neumann-Fraune MPI Saarbrücken Bastian Beggel all other m em bers of the HOPE-project

W ildtype replication Resistance factors W T - Adefovir 0.84 2.2. 1 6 0 0.8 5.2. 1 4 0 0.84 12.2. 1 2 0 % replication 2.1 1 0 0 16.2. 8 0 1.7 23.2. 6 0 1.2 2.3. 4 0 4 9.3. 2 0 1.2 0 16.3. 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 1.1 9.4. Adefovir [ nM] IC50 ~ 1300 nM

W ildtype replication Resistance factors W T - Entecavir 0.75 2.2. 1 6 0 1.2 5.2. 1 4 0 1.3 12.2. 1 2 0 1 % replication 16.2. 1 0 0 1.1 8 0 23.2. 6 0 0.7 2.3. 4 0 1.1 9.3. 2 0 2.1 16.3. 0 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 0.6 9.4. Entecavir [ nM] IC50 ~ 80 nM

W ildtype replication Resistance factors W T - Lamivudin 2.2. - 1 6 0 5.2. 1 1 4 0 12.2. 0.72 1 2 0 % replication 16.2. 0.45 1 0 0 1.1 8 0 23.2. 6 0 1 2.3. 4 0 0.4 9.3. 2 0 1.3 16.3. 0 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 1.3 9.4. Lamivudin [ nM] IC50 ~ 77 nM

W ildtype replication Resistance factors W T - Tenofovir 2.2. 0.5 1 4 0 5.2. 0.87 1 2 0 12.2. 0.67 1 0 0 % replication 8 0 16.2. 0.46 6 0 23.2. 1.5 4 0 2.3. 1.1 2 0 9.3. 1.1 0 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 9.4. 1.1 Tenofovir [ nM] IC50 ~ 1000 nM