Masters Degree in Medical Biotechnologies IMPACT OF THE HIV-1 - - PowerPoint PPT Presentation
Department of Medical Biotechnologies Masters 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.
Department of Medical Biotechnologies Master’s Degree in Medical Biotechnologies
Supervisor
Advisor Candidate
Diletta Tiezzi
Academic Year 2015/2016
been not fully elucidated, particularly for the latest licensed compound dolutegravir (DTG)
clearly have an impact on DTG activity, anecdotal cases have suggested a possible implication for few integrase polymorphisms including E157Q
absence of major integrase mutations in a case report and to be occasionally selected under DTG treatment
role of E157Q on INI susceptibility
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
E157Q increases DTG resistance by R263K
No cases of selection of E157Q and R263K have been reported in the clinic
Anstett, JAC 2016
Per esclusivo uso interno di ViiV
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 failure. 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.
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 Per esclusivo uso interno di ViiV
Determination of phenotypic susceptibility to INIs in a set
carrying patient derived integrase harboring E157Q polymorphism alone.
Phenotypic Assay
Determination of Relative Replication Capacity (RRC) of recombinant viruses for defining the effect of E157Q in viral fitness.
Growth Competition Assay
Recombinant viruses PCR fragment
pNL4-3ΔIN Transfection
293-LX cells
Homologous Recombination PCR RT-PCR
Harvesting of supernatants in presence
Recombinant viruses MT-2 cells Viral Titration through measuring TCID50/ml
[c] RAL and DTG
+
Viral Stock TZM-bl cells
Luminescence measurement
2 4 6 500 1000 1500 2000
log [c] drug RLU
IC50 determination
48-hour incubation
*Fold Change values
Sample ID DTG RAL IC50 (95% CI) nM FC IC50 (95% CI) nM FC NL4-3 1.4 (0.6-3.3) 1 (ref) 5.1 (2.6-10.3) 1 (ref) 142057 3.2 (2.0-5.2) 2.3 7.9 (3.1-21.2) 1.5 147829 4.2 (3.3-7.4) 3.0 5.5 (2.5-12.3) 1.1 147847 2.2 (0.6-7.7) 1.6 3.9 (3.8-7.0) 0.8 148505 3.8 (1.1-13.4) 2.7 5.8 (1.4-24.3) 1.1 148680 4.2 (1.9-9.1) 3.0 2.4 (1.3-3.4) 0.5 149029 1.3 (0.9-1.8) 0.9 4.5 (0.8-24.3) 0.9 NL4-3/IN_157Q 1.7 (1.1-2.7) 1.2 3.6 (1.8-7.4) 0.7
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)
Sample ID DTG RAL IC50 (95% CI) nM FC IC50 (95% CI) nM FC NL4-3 1.4 (0.6-3.3) 1 (ref) 5.1 (2.6-10.3) 1 (ref) 142057 3.2 (2.0-5.2) 2.3 7.9 (3.1-21.2) 1.5 147829 4.2 (3.3-7.4) 3.0 5.5 (2.5-12.3) 1.1 147847 2.2 (0.6-7.7) 1.6 3.9 (3.8-7.0) 0.8 148505 3.8 (1.1-13.4) 2.7 5.8 (1.4-24.3) 1.1 148680 4.2 (1.9-9.1) 3.0 2.4 (1.3-3.4) 0.5 149029 1.3 (0.9-1.8) 0.9 4.5 (0.8-24.3) 0.9 NL4-3/IN_157Q 1.7 (1.1-2.7) 1.2 3.6 (1.8-7.4) 0.7
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
log- transformed FC values
assays and the reference Phenosense assay on a panel
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
Dual infection of MT-2 cell line culture with recombinant virus and NL4-3_RC. 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.
5 silent mutations have been introduced in the p17 coding region of pNL4-3 through mutagenesis in order to be selectively recognized by a specific real time probe.
78,1 121,8 115 82,2 44,8 59,9 100