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

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

• analogues. Gastroenterology, 2009; 137: 1593-1608

HBV Developm ent of Resistance

HBV Polym erase

prim ary resistance m utation Secondary resistance m utation

/V

rtV173L ?

adapted from Zoulim & Locarnini, HBV resistance to nucleos(t)ide

• analogues. Gastroenterology, 2009; 137: 1593-1608

Cloning polymerase-frame

• HBV-replication-competent

vector with HBV backbone

• polymerase frame is replaced
• cloning efficient >104 GE/ml
• plasmid serves as wildtype

(genotype D)

Cloning

phenotypic assay

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

• Standardization of the transfection by SEAP-test

(secreted alkaline phosphatase)

SEAP-Assay 96-Well 0,5 1 1,5 2 0,08 0,04 0,02 0,01 transfected µg plasmid/well OD450 5 min 10 min

phenotypic assay-SEAP

phenotypic assay

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

w ildtype inhibition

wildtype plasm id 0 ,0 0 2 0 ,0 0 4 0 ,0 0 6 0 ,0 0 8 0 ,0 0 1 0 0 ,0 0 1 2 0 ,0 0 1 4 0 ,0 0 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 % replicat ion Lam ivudin

(average of 8 independent experiments in triplicates)

IC50s: Lamivudin: 77.0 42.3 nM

w ildtype inhibition

wildtype plasm id 0 ,0 0 2 0 ,0 0 4 0 ,0 0 6 0 ,0 0 8 0 ,0 0 1 0 0 ,0 0 1 2 0 ,0 0 1 4 0 ,0 0 1 6 0 ,0 0 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 % replicat ion Adefovir Lam ivudin

IC50s: Adefovir: 1281.7 625.5 nM Lamivudin: 77.0 42.3 nM

(average of 8 independent experiments in triplicates)

w ildtype inhibition

wildtype plasm id 0 ,0 0 2 0 ,0 0 4 0 ,0 0 6 0 ,0 0 8 0 ,0 0 1 0 0 ,0 0 1 2 0 ,0 0 1 4 0 ,0 0 1 6 0 ,0 0 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 % replicat ion Adefovir Entecavir Lam ivudin

IC50s: Adefovir: 1281.7 625.5 nM Entecavir: 1.1 0.6 nM Lamivudin: 77.0 42.3 nM

(average of 8 independent experiments in triplicates)

w ildtype inhibition

wildtype plasm id 0 ,0 0 2 0 ,0 0 4 0 ,0 0 6 0 ,0 0 8 0 ,0 0 1 0 0 ,0 0 1 2 0 ,0 0 1 4 0 ,0 0 1 6 0 ,0 0 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 % replicat ion Adefovir Entecavir Lam ivudin Tenofovir

IC50s: Adefovir: 1281.7 625.5 nM Entecavir: 1.1 0.6 nM Lamivudin: 77.0 42.3 nM Tenofovir: 1006.5 654.7 nM

(average of 8 independent experiments in triplicates)

Relative replication yield of HBV m utants

R.Edwards, T.Shaw, V. Sozzi & S. Locarnini, 2005 HepDART 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) 17-120 % of WT (n=5) 17-67 % of WT (n=3) 7.4 / 45.5 % of WT (n=2)

variability of different test systems and within the mutants

180M-184A-204V

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

• analogues. Gastroenterology, 2009; 137: 1593-1608

S: sensitive; R: resistance; I: intermediate

L1 8 0 M-T1 8 4 A-M2 0 4 V

( viral fitness ~ 1 6 % of W T)

1 8 0 M-1 8 4 A-2 0 4 V - Adefovir

20 40 60 80 100 120 140 160 0,01 0,1 1 10 100 1000 10000 100000 Adefovir [ nM] % replication

147- 1 155- 2 147- 3 WT

1 8 0 M-1 8 4 A-2 0 4 V - Tenofovir

20 40 60 80 100 120 140 0,01 0,1 1 10 100 1000 10000 100000 Tenofovir [ nM] % replication

147- 1 155- 1 147- 3 WT

resistance factor: 2.8 0.3 resistance factor: 5.5 3.1 resistance factor = mutant IC50 wildtype IC50 Adefovir Tenofovir

1 8 0 M-1 8 4 A-2 0 4 V - Entecavir

20 40 60 80 100 120 140 160 0,01 0,1 1 10 100 1000 Entecavir [ nM] % replication

147- 1 155- 1 147- 3 WT

resistance factor: 6934.0 5310.4 resistance factor: 200.0 145.3 1 8 0 M-1 8 4 A-2 0 4 V

viral fitness ~ 1 6 % of W T) 1 8 0 M-1 8 4 A-2 0 4 V - Lamivudin

20 40 60 80 100 120 140 160 0,01 0,1 1 10 100 1000 10000 100000 Lam ivudin [ nM] % replication

147- 1 155- 1 147- 3 WT

Lamivudin Entecavir

cross-resistance M204I in combination with L80I/ V and/ or V173L Lamivudin / Entecavir

10 20 30 40 50 60 70 80 90 100 204I 204I 204I,80V 204I, 80I 204I,173L 204I, 80V,173L, resistance factor 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

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

S: sensitive; R: resistance; I: intermediate 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 WT 181V 181V 181V 181V 236T 236T 236T 236T 181V,236T resistance factor Adefovir Ent ecavir Lamivudin Tenofovir

HBV overlapping ORFs

Polym erase Surface proteins

rtA1 8 1 T sW 1 7 2 * stop

Warner & Locarnini, Hepatology, 2008; 48: 88-98

in some mutants  no genomes detectable in supernatant

• replication deficient?
• secretion deficient?
• cloning?

– Antiviral resistance mutation rtA181T / sW172* stop mutant has a dominant-negative effect on HBsAg secretion – Reduced amount of HBsAg and HBV in serum, although antiviral therapy was not successful – 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

rtA181T sW172*stop

HBV overlapping ORFs

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)

• pen 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 all other m em bers of the HOPE-project MPI Saarbrücken Bastian Beggel

W ildtype replication

Resistance factors 0.84 0.8 0.84 2.1 1.7 1.2 4 1.2 1.1

IC50 ~ 1300 nM

W T - Adefovir

2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 Adefovir [ nM] % replication

2.2. 5.2. 12.2. 16.2. 23.2. 2.3. 9.3. 16.3. 9.4.

W ildtype replication

Resistance factors 0.75 1.2 1.3 1 1.1 0.7 1.1 2.1 0.6

IC50 ~ 80 nM

W T - Entecavir

2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 0 ,0 1 0 ,1 1 1 0 1 0 0 1 0 0 0 Entecavir [ nM] % replication

2.2. 5.2. 12.2. 16.2. 23.2. 2.3. 9.3. 16.3. 9.4.

W ildtype replication

Resistance factors

• 1

0.72 0.45 1.1 1 0.4 1.3 1.3

IC50 ~ 77 nM

W T - Lamivudin

2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 Lamivudin [ nM] % replication

2.2. 5.2. 12.2. 16.2. 23.2. 2.3. 9.3. 16.3. 9.4.

W ildtype replication

Resistance factors 0.5 0.87 0.67 0.46 1.5 1.1 1.1 1.1

IC50 ~ 1000 nM

W T - Tenofovir

2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 0 ,0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 Tenofovir [ nM] % replication

2.2. 5.2. 12.2. 16.2. 23.2. 2.3. 9.3. 9.4.