Masters Degree in Medical Biotechnologies IMPACT OF THE HIV-1 - PowerPoint PPT Presentation

Department of Medical Biotechnologies Master’s Degree in Medical Biotechnologies IMPACT OF THE HIV-1 INTEGRASE NATURAL POLYMORPHISM E157Q ON SUSCEPTIBILITY TO INTEGRASE INHIBITORS Supervisor Prof. Maurizio Zazzi Advisor Candidate Dr. Francesco Saladini Diletta Tiezzi Academic Year 2015/2016

Intro  The resistance profile of HIV-1 integrase inhibitors (INI) has been not fully elucidated, particularly for the latest licensed compound dolutegravir (DTG)  While multiple substitutions at codons 138, 140 and 148 clearly have an impact on DTG activity, anecdotal cases have suggested a possible implication for few integrase polymorphisms including E157Q  This has been found to confer DTG resistance in the absence of major integrase mutations in a case report and to be occasionally selected under DTG treatment  The aim of this study was to evaluate the prevalence and role of E157Q on INI susceptibility

Per esclusivo uso interno di ViiV Anecdotal case in one patient with primary HIV infection 2 years after receiving a second  kidney transplant E157Q as a natural polymorphism  Both RAL and DTG fail without selection of additional IN mutations (DRV then suppresses VL)  The E157Q recombinant has stronger strand transfer activity which confers 9-fold  resistance to DTG in a biochemical assay (RAL and EVG not tested) Warrants confirmation in cell based assays  Danion, JAC 2015

Per esclusivo uso interno di ViiV The E157Q clone (NL4-3 backbone) is not resistant to DTG/RAL   The effect shown by Danion 2015 must have been strain specific E157Q increases DTG resistance by R263K  No cases of selection of E157Q and R263K have been reported in the  clinic Anstett, JAC 2016

O332 - French national survey of resistance to integrase inhibitors shows high differences of resistance selection rate in case of virological failure in a context of routine hospital care (ANRS-AC11 virology network) V Calvez et al Among the 96 patients failing to DTG, 49 received DTG as the first INI, neither INI resistance mutations among the major pathways (92, 118, 121, 140, 143, 148, 155) nor the R263K mutation were present at failur e. Conclusions: In this national survey, RAL and EVG are associated with 32–40% resistance at failure. However, in INI-naïve patients, failing to DTG when used as the first INI, no resistance to INI was detected whatever the antiretroviral associated to DTG .

Per esclusivo uso interno di ViiV 4/36 recently infected patients harbor E157Q IN virus  3 of these 4 started with DTG based therapy, all reached undetectable VL  Ambrosioni, JAC 2016

Role of E157Q in clinical isolates AIM OF THE THESIS Determination of phenotypic Determination of Relative susceptibility to INIs in a set Replication Capacity (RRC) of of 6 recombinant viruses recombinant viruses for defining carrying patient derived the effect of E157Q in viral integrase harboring E157Q fitness. polymorphism alone. Growth Phenotypic Assay Competition Assay

Prevalence of E157Q in the Italian HIV resistance ARCA database  70/2960 (2.4%) IN sequences harbour the E157Q polymorphism  E157Q is significantly more prevalent in CRF02_AG than B subtype ( P <0.0001, chi-square test)

Clinical features of the selected patients  Analysis of six samples of patients infected with HIV-1 and positive for E157Q polymorphism, available in HHML biobank.

MATERIALS AND METHODS Production of recombinant viruses RT-PCR PCR 293-LX cells Recombinant viruses PCR fragment Transfection pNL4- 3ΔIN Homologous Recombination

MATERIALS AND METHODS Expansion of recombinant viruses MT-2 cells Recombinant viruses Harvesting of Viral Titration through supernatants in presence measuring TCID 50 /ml of large syncytia.

MATERIALS AND METHODS «MonoCycle» Phenotypic Assay Single round infection of reporter cell line TZM-bl with wild type or recombinant viruses in presence of serial dilutions of RAL and DTG Viral Stock 48-hour incubation 2000 1500 RLU Luminescence 1000 + measurement 500 0 -2 0 2 4 6 log [c] drug TZM-bl cells IC 50 determination [c] RAL and DTG IC 50 and Fold Change (FC)* calculation *Fold Change values

Phenotypic assay results DTG RAL Sample ID IC 50 (95% CI) nM FC IC 50 (95% CI) nM FC 1.4 (0.6-3.3) 1 (ref) 5.1 (2.6-10.3) 1 (ref) NL4-3 3.2 (2.0-5.2) 2.3 7.9 (3.1-21.2) 1.5 142057 4.2 (3.3-7.4) 3.0 5.5 (2.5-12.3) 1.1 147829 2.2 (0.6-7.7) 1.6 3.9 (3.8-7.0) 0.8 147847 3.8 (1.1-13.4) 2.7 5.8 (1.4-24.3) 1.1 148505 4.2 (1.9-9.1) 3.0 2.4 (1.3-3.4) 0.5 148680 1.3 (0.9-1.8) 0.9 4.5 (0.8-24.3) 0.9 149029 1.7 (1.1-2.7) 1.2 3.6 (1.8-7.4) 0.7 NL4-3/IN_157Q Median FC values of 0.9 (range 0.7-1.5) for RAL and 2.3 (range 0.9-3) for DTG (p = 0.027, Wilcoxon Rank Sum test)

Phenotypic assay results DTG RAL Sample ID IC 50 (95% CI) nM FC IC 50 (95% CI) nM FC 1.4 (0.6-3.3) 1 (ref) 5.1 (2.6-10.3) 1 (ref) NL4-3 3.2 (2.0-5.2) 2.3 7.9 (3.1-21.2) 1.5 142057 4.2 (3.3-7.4) 3.0 5.5 (2.5-12.3) 1.1 147829 2.2 (0.6-7.7) 1.6 3.9 (3.8-7.0) 0.8 147847 3.8 (1.1-13.4) 2.7 5.8 (1.4-24.3) 1.1 148505 4.2 (1.9-9.1) 3.0 2.4 (1.3-3.4) 0.5 148680 1.3 (0.9-1.8) 0.9 4.5 (0.8-24.3) 0.9 149029 1.7 (1.1-2.7) 1.2 3.6 (1.8-7.4) 0.7 NL4-3/IN_157Q According to the Phenosense cut-off values (1.5 biological cut-off for RAL, 4-13 clinical cut-offs for DTG), 1/7 recombinant viruses had a borderline resistance to RAL (FC 1.5), while all the viruses were susceptible to DTG.

Agreement between an in- house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV- 1 protease, reverse transcriptase, and integrase inhibitors Assessment of the accuracy of Mono/BiCycle assays compared to Phenosense assay. (A) Correlation between log- transformed FC values obtained with the Mono/BiCycle assays and the reference Phenosense assay on a panel of drug- resistance viruses. (B) Bland- Altman plot showing the relationship between the average FC for the Phenosense and Mono/BiCycle assays and the ratio of the FC values obtained by the two systems Saladini, JCLA 2017

MATERIALS AND METHODS Growth competition assay 5 silent mutations have been introduced in the p17 Dual infection of MT-2 cell coding region of pNL4-3 through mutagenesis in order line culture with recombinant to be selectively recognized by a specific real time virus and NL4-3_RC. probe. After 5 days, viral RNA was extracted from cell culture supernatants and reverse transcribed. qPCR to quantify the amount of viral populations grown in each culture , interpolating Ct values with standard curves created with known amounts of pNL4-3 and pNL4-3_RC plasmids.

Results of growth competition assay 121,8 115 100 78,1 82,2 59,9 44,8

CONCLUSIONS  The E157Q integrase polymorphism is differently distributed across subtypes, particularly it is more frequent in CRF02_AG  The role of E157Q on INI susceptibility appears to be minimal in this limited case series, suggesting that INI resistance apparently driven by E157Q could depend on additional as yet unidentified and possibly rare polymorphism(s)  The relative impact of E157Q on RAL vs. DTG remains to be elucidated since the interpretation of the different FC values is currently dependent on two different types of cut-offs for these two drugs (biological vs. clinical).