NEWLY-IDENTIFIED MUTATIONS IN HBV RT ARE ASSOCIATED WITH FAILURES OF - - PowerPoint PPT Presentation

NEWLY-IDENTIFIED MUTATIONS IN HBV RT ARE ASSOCIATED WITH FAILURES OF SELECTED ANTI-HBV TREATMENTS AND INDUCE ALTERATION OR STOP CODON FORMATION IN S ANTIGEN

• V. Cento1, F. Van Hemert2, V.C. Di Maio1, R. Salpini1, C. Mirabelli11, A. Bertoli3, C.

Gori4, V. Micheli5, G. Gubertini5, M. Bernassola6, S. Romano6, M. Visca6, C. Alteri1,

• G. Cappiello6, GM. De Sanctis7, N. Marino8, F. Mazzotta8, C. Sarrecchia9, M.

Andreoni9, A. Spanò6, M. Angelico10, F. Ceccherini-Silberstein1,

• C. F. Perno1,3, V. Svicher1

1 Department of Experimental Medicine and Biochemical Science, University of “Tor Vergata” Rome, Italy; 2 Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 3 Molecular Virology unit, “Tor Vergata” University Hospital, Rome, Italy; 4 Antiretroviral Drugs Monitoring unit, National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy; 5 Microbiology unit, “L. Sacco” Hospital, Milan, Italy; 6 Microbiology and Virology unit, “S. Pertini” Hospital, Rome, Italy; 7 Infectious and Tropical Diseases unit, University “La Sapienza”, Rome, Italy; 8 Infectious Diseases unit, “Santa Maria Annunziata” Hospital, Firenze, Italy; 9 Infectious Diseases unit, “Tor Vergata” University Hospital, Rome, Italy; 10 Hepatology unit, “Tor Vergata” University Hospital, Rome, Italy

Kwon, H. & Lok, A. S. Nat. Rev. Gastroenterol. Hepatol. 2011

HBV treatment and resistance

• However, there is the increasing evidence that

additional mutations can be involved in mechanisms underlying HBV drug resistance……

The rtL180M +A181C+M204V mutant decreased >1000-fold susceptibility to LAM, and 85.6 ± 10.8-fold susceptibility to ETV, but remained susceptible to ADV and tenofovir

HBV: an escape specialist …

IMMUNE ESCAPE HCC DEVELOPMENT

Torresi J, J Clin Virol 2002

6 - 21 yr 6 - 21 yr

Pre-S1 Terminal protein Spacer RT RNAse H S Pre- S2

HBsAg

Virion i interior Viri irion surface ce

RT

V173L M204V E164D I195M

The pattern of lamivudine resistance mutations M204V+L180M+V173L in RT correspond to I195M+E164D in the HBsAg, that strongly reduce the binding affinity with neutralizing antibodies including those induced by the vaccine

(Torresi et al. Virology 2002)

Hepatology 2008

• The adefovir resistance mutation rtA181T introduces a stop codon in

the HBsAg.

• This determines the production of a truncated HBsAg that is retained

and accumulates within the cell, thus impairing the production of viral particles.

• The impairment of viral production determines a decrease in viremia

that can mask the diagnosis of resistance if genotypic testing is not used.

Antivir Ther 2008

The accumulation of the truncated HBsAg has been demonstrated to favor the onset of hepatocellular carcinoma

A181T correlates with the onset of HCC even in HBV infected humans

Factors associated with occurrence of HCC. The cumulative incidence of HCC was depicted according to the presence of the rtA181T mutation (A), use of rescue therapy (B), the presence of liver cirrhosis (C), and age > 50 years (D).

2011

The goal of the study was to investigate:

• the correlation of novel mutations with anti-HBV treatment

and with the classical drug resistance mutations

• their impact on binding affinity between the RT and the drug
• their impact on HBsAg sequence and structure

Demographic and virological characteristics of the study population

Characteristic Drug-naive patients, N=197 Drug-treated patients, N=159 Male, N(%) 150 (76.1) 120 (75.5) Italian nationality, N(%) 91 (46.2) 140 (88.1) Age (years), Median (IQR) 45.0 (35.0-58.0) 49.5 (41.0-62.0) Plasma HBV-DNA (logIU/ml), Median (IQR) 4.6 (3.2-6.5) 4.1 (3.0-5.7) ALT (IU/ml), Median (IQR) 40 (27-77) 44 (30-77) AST (IU/ml), Median (IQR) 36 (26-54) 32 (23-50) Viral co-infections, N(%) Hepatitis C Virus 24 (12.2) 5 (3.1) Hepatitis D Virus 4 (2.0) 0 (0.0) Human Immunodeficiency Virus 72 (36.5) 31 (19.5) HBV-genotype, N(%) A 73 (37.1) 31 (19.5) D 124 (62.9) 128 (80.5) Type of anti-HBV treatment, N(%) Adefovir therapy

a

• 35 (22.0)

Entecavir mono-therapy

• 18 (11.3)

Lamivudine mono-therapy

• 106 (66.7)

Time to virological failure (years), Median (IQR)b

• 2.4 (1.3-4.5)

a Among the 35 patients included in the “ADF-treated” group, 22 were under ADF monotherapy (all after LMV-failure), while 13

received ADF+LMV combination therapy. b Failure was defined as detectable plasma HBV-DNA after at least six month of therapy. IQR, interquartile range

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

Prevelence (%) RT Mutation

p=0.017 p=0.050 p=0.037 p=0.043 p=0.019 p=0.041 p=0.007 p=0.014 p=0.049 p=0.039 p=0.014

Histogram representing the prevalence of RT mutations in drug-naïve patients and in drug-treated patients. P-values were calculated by Fisher exact test, applying the Benjamini-Hochberg correction for multiple comparison.

Novel RT mutations significantly correlated with virological failure to specific anti-HBV drugs

Drug-Naive Patients, N=197 LMV-Treated Patients, N=106 ADF+LMV-Treated Patients, N=35 ETV-Treated Patients, N=18

rtS78T rtK212Q rtL229V/F rtA181I rtN53T rtS85F rtS135T rtM309K rtA200V

Three-dimensional structure of HBV-RT. YMDD motif is reported in dark-grey. Novel RT-mutations are reported in space-filled format.

LMV-treatment, ADF-treatment ETV-treatment

Some novel mutations localize in RT domains critical for its function

D83 Y203 M204 D205 D206 80 85

SER 85 (Cα) distances (Å) LEU 80 (Cg) distances (Å) LEU 80 (C) distances (Å) LEU 80 (Cα-Cg) 16.323-15.384

• ASP 83

(Cα-Cβ) 6.483-6.378 10.645 9.051 SER 85 (Cα)

• 15.384

15.127 TYR 203 (Cα-Cg) 13.851-14.931 12.286 MET 204 (C) 10.266 13.485 ASP 205 (Cα-Cg) 7.998-5.870 12.664-13.693 ASP 206 (Cα-Cg) 9.908-11.199 11.202 8.670

S85F is localized close (≤10Å) to the classical drug-resistance related position 204, and to the 3 catalytically essential aspartic residues at positions 83, 205 and 206

Mutation Prevalence (%)a Combination prevalence (%) p-valueb Phy rtM204I resistance pattern rtM204I 44.8 rtL80I/V 14.7 14.7 4.05E-05 0.410 rtS85F 3.9 3.9 0.037 0.221

The novel RT mutation rtS85F always occurs the classical rtM204I and never with the classical L80I/V

a The prevalence was determined in 106 LMV-treated patients. b P values were determined by Fisher exact test.

The novel RT mutation rtS85F always occurs the classical rtM204I and never with the classical L80I/V

This suggests the existence of 2 distinct compensatory pathways underlying the rtM204I-mediated drug resistance M204I L80IV S85F

Mutation Prevalence (%)a Combination prevalence (%) p-valueb Phy rtM204I resistance pattern rtM204I 44.8 rtL80I/V 14.7 14.7 4.05E-05 0.410 rtS85F 3.9 3.9 0.037 0.221

a The prevalence was determined in 106 LMV-treated patients. b P values were determined by Fisher exact test.

A different scenario is observed for the novel rtS78T……..

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

Prevelence (%) RT Mutation

p=0.017 p=0.050 p=0.037 p=0.043 p=0.019 p=0.041 p=0.007 p=0.014 p=0.049 p=0.039 p=0.014

Histogram representing the prevalence of RT mutations in drug-naïve patients and in drug-treated patients. P-values were calculated by Fisher exact test, applying the Benjamini-Hochberg correction for multiple comparison.

Drug-Naive Patients, N=197 LMV-Treated Patients, N=106 ADF+LMV-Treated Patients, N=35 ETV-Treated Patients, N=18

rtS78T occurs in around 12% of patients failing adefovir treatment either alone or……

…..in combination with the classical adefovir resistance mutations

0.71 rtS78T 0.49 rtN236T rtA181T/V/I 0.68 rtL229V 0.48 0.99 rtM204V rtL180M 0.8 rtM204I rtL80I/V 0.81 0.56 0.3 0.04

• 0.22

LMV-resistance patterns ADF-resistance pattern

Dendrogram obtained from average linkage hierarchical agglomerative clustering. The analysis was performed on 35 ADF-treated patients: 22 were under ADF monotherapy (all after LMV-failure), while 13 received ADF+LMV combination therapy. The length of branches reflects distances between genotypes in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes.

Docking analysis reporting free binding energy between RT and adefovir for the wild-type and mutated RT

Total Ligand-Receptor Interaction Energy Kcal/mole Adefovir wtRT

• 9.63

S78T

• 7.37

A181T

• 9.30

A181V

• 7.96

S78T+A181T

• 4.33

S78T+A181V

• 5.43

rtS78T decreases the RT binding affinity for adefovir at a level that is comparable or stronger to that observed to the classical mutations at position 181

Total Ligand-Receptor Interaction Energy Kcal/mole Adefovir wtRT

• 9.63

S78T

• 7.37

A181T

• 9.30

A181V

• 7.96

S78T+A181T

• 4.33

S78T+A181V

• 5.43

The co-presence of the novel rtS78T and the classical rtA181T/V further exacerbates the decrease in the RT binding affinity for adefovir

Total Ligand-Receptor Interaction Energy Kcal/mole Adefovir wtRT

• 9.63

S78T

• 7.37

A181T

• 9.30

A181V

• 7.96

S78T+A181T

• 4.33

S78T+A181V

• 5.43

• These results suggest that the novel mutation rtS78T

could contribute to adefovir resistance by itself or could increase the level of resistance when present with the classical mutations at position 181.

• Further studies are necessary to investigate the role of

this mutation in adefovir resistance and in modulating virological response to tenofovir

Terminal protein Spacer RT RNAse H Pre-S1 S Pre- S2

HBsAg RT

rtA181T

Virion interior Virion surface Virion interior Virion surface

rtS78T sC69stop sW172stop

Similarly to A181T, rtS78T introduces a stop codon in the HBsAg and in particular at the HBsAg position 69 known to be critical for HBsAg secretion (Mangold et al., J

Virol 1993, Mangold et al., Virology 1995)

The issue of drug-resistance related stop codons is clinically relevant Indeed……

The currently available anti-HBV drugs can suppress the synthesis of viral genome and viral particles production, but can not suppress at all HBsAg production

Nguyen, T. and Locarnini, S. (2009) Monitoring drug therapy for hepatitis B—a global challenge?

• Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2009.160

• In presence of HBsAg stop codons, the production of truncated HBsAg can still go on

even if the patient receives a fully suppressive therapy

• Thus, in presence of HBsAg stop codons, the risk of liver cancer can persist

even if the patient has undetectable viremia

As final step of the study…

HBsAg mutations associated with virological failure to NUCs-therapy and….

Histogram representing the relative prevalence of HBsAg mutations found to be statistically associated with anti-HBV treatment. The analysis was performed on 197 drug-naïve patients and on 159 drug-failing patients. All p-values were <0.05 after Benjamini-Hochberg correction for multiple comparison.

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 sS154L sP217L sC69stop sL192F sL173F sL175F sI195M sW196L sW196S Prevalence (%) Drug-Naive Patients (N=197) Drug-Treated Patients (N=159)

p=0.039 p=0.049 p=0.050 p=0.039 p=0.001 p=0.008 P<0.001 p=0.008 P<0.001

Dependent upon classical drug-resistance development Silent in RT- protein rtA200V rtS78T

……..Clustering with the classical drug resistance mutations

0.93

C69stop L173F

0.3 0.58

P217L

0.45

W196S/L S154L

0.47

L192F

0.95

I195M L175F

0.21 0.14 0.07

• 0.07

rtA181V rtM204I rtM204V

Dendrogram obtained from average linkage hierarchical agglomerative clustering. The analysis was performed on 158 drug-failing patients. The length of branches reflects distances between genotypes in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes.

These HBsAg mutations are localized in the a- determinant or in a HBsAg domain critical for viral assembly

S154 P217

L192 C69

Three-dimensional ab initio structure of HBsAg protein. HBsAg residues interested by the development of newly- identified mutations associated with failure to anti-HBV treatment are represented in space-filled format. The “a”- determinant (amino acids 122-158) is colored in blue.

IN CONCLUSION …

Beside classical resistance-mutations, anti-HBV treatments correlates with the appearance of novel RT-mutations.

• To some extent, these mutations seem to be drug-specific.
• These can, at the same time, affect drug-binding affinity of

mutated HBV-RT, and alter HBsAg structure and/or functionality. HBV drug resistance is a highly complex phenomenon that can compromise not only the efficacy of the drugs but also increase HBV pathogenetic potential.

ACKNOWLEDGEMENTS

University of Rome “Tor Vergata”

• C.F. Perno
• V. Svicher
• F. Ceccherini-Silberstein
• M.M. Santoro
• R. Salpini
• C. Mirabelli
• V. C. Di Maio
• L. Fabeni
• D. Armenia
• C. Alteri
• F. De Luca
• M. Pollicita
• D. Di Pinto
• A. Bertoli
• C. Sarrecchia
• M. Andreoni
• M. Angelico

INMI “L. Spallanzani” Rome



• C. Gori
• L. Sacco University Hospital,

Milan



• V. Micheli



• G. Gubertini

“Pertini” Hospital, Rome



• G. Cappiello



• A. Spanò



• S. Romano



• M. Visca



• M. Bernassola

University “La Sapienza”, Rome



G.M. De Sanctis Hospital “Santa Maria Annunziata”, Firenze



• N. Marino



• F. Mazzotta

Center for Infection and Immunity, Amsterdam



• F. Van Hemert