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. Cento 1 , F. Van Hemert 2 , V.C. Di Maio 1 , R. Salpini 1 , C. Mirabelli 1 1, A. Bertoli 3 , C. Gori 4 , V. Micheli 5 , G. Gubertini 5 , M. Bernassola 6 , S. Romano 6 , M. Visca 6 , C. Alteri 1 , G. Cappiello 6 , GM. De Sanctis 7 , N. Marino 8 , F. Mazzotta 8 , C. Sarrecchia 9 , M. Andreoni 9 , A. Spanò 6 , M. Angelico 10 , F. Ceccherini-Silberstein 1 , C. F. Perno 1,3 , V. Svicher 1 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

HBV treatment and resistance Kwon, H. & Lok, A. S. Nat. Rev. Gastroenterol. Hepatol. 2011

• 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 Torresi J, J Clin Virol 2002 HCC DEVELOPMENT 6 - 21 yr 6 - 21 yr

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) V173L M204V RT Terminal protein Spacer RT RNAse H Pre- HBsAg Pre-S1 S S2 E164D I195M Viri irion surface ce Virion i interior

• 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. Hepatology 2008

Antivir Ther 2008 The accumulation of the truncated HBsAg has been demonstrated to favor the onset of hepatocellular carcinoma

2011 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).

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(%) 140 (88.1) 91 (46.2) 49.5 (41.0-62.0) Age (years), Median (IQR) 45.0 (35.0-58.0) Plasma HBV-DNA (logIU/ml), Median (IQR) 4.6 (3.2-6.5) 4.1 (3.0-5.7) 44 (30-77) ALT (IU/ml), Median (IQR) 40 (27-77) 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(%) a Adefovir therapy - 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

Novel RT mutations significantly correlated with virological failure to specific anti-HBV drugs p=0.039 18.0 Drug-Naive Patients, N=197 16.0 LMV-Treated Patients, N=106 p=0.007 14.0 ADF+LMV-Treated Patients, N=35 Prevelence (%) 12.0 ETV-Treated Patients, N=18 p=0.049 10.0 p=0.041 p=0.019 8.0 p=0.014 p=0.014 6.0 p=0.017 p=0.050 p=0.037 p=0.043 4.0 2.0 0.0 RT Mutation 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.

Some novel mutations localize in RT domains critical for its function LMV-treatment, rtL229V/F ADF-treatment rtK212Q rtA200V rtS85F rtS78T ETV-treatment rtA181I rtS135T rtN53T rtM309K Three-dimensional structure of HBV-RT. YMDD motif is reported in dark-grey. Novel RT-mutations are reported in space-filled format.

S85F is localized close (≤10 Å) to the classical drug-resistance related position 204, and to the 3 catalytically essential aspartic residues at positions 83, 205 and 206 SER 85 (C α ) LEU 80 (Cg) LEU 80 (C) distances (Å) distances (Å) distances (Å) LEU 80 16.323-15.384 - (C α -Cg) ASP 83 6.483-6.378 10.645 9.051 (C α -C β ) SER 85 15.384 15.127 - (C α ) 80 TYR 203 85 D83 13.851-14.931 12.286 (C α -Cg) D206 MET 204 10.266 13.485 (C) Y203 D205 M204 ASP 205 7.998-5.870 12.664-13.693 (C α -Cg) ASP 206 9.908-11.199 11.202 8.670 (C α -Cg)

The novel RT mutation rtS85F always occurs the classical rtM204I and never with the classical L80I/V Prevalence (%) a Combination prevalence (%) p-value b Mutation 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.

The novel RT mutation rtS85F always occurs the classical rtM204I and never with the classical L80I/V Prevalence (%) a Combination prevalence (%) p-value b Mutation 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. This suggests the existence of 2 distinct compensatory pathways underlying the rtM204I-mediated drug resistance M204I L80IV S85F

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

rtS78T occurs in around 12% of patients failing adefovir treatment either alone or…… p=0.039 18.0 Drug-Naive Patients, N=197 16.0 LMV-Treated Patients, N=106 p=0.007 14.0 ADF+LMV-Treated Patients, N=35 Prevelence (%) 12.0 ETV-Treated Patients, N=18 p=0.049 10.0 p=0.041 p=0.019 8.0 p=0.014 p=0.014 6.0 p=0.017 p=0.050 p=0.037 p=0.043 4.0 2.0 0.0 RT Mutation 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.

…..in combination with the classical adefovir resistance mutations -0.22 0.68 0.71 0.04 0.48 0.49 0.3 0.56 0.8 0.99 0.81 rtN236T rtA181T/V/ I rtL229V rtM204V rtL180M rtM204I rtL80I/V rtS78T ADF-resistance pattern LMV-resistance patterns 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