The HIV1 reverse transcriptase E138A polymorphism decreases the genetic

barrier to resistance to etravirine in vitro

Saladini Francesco, Giannini Alessia, Omesuh Franklin, Boccuto Adele, Zazzi Maurizio

ETR resistance associated mutations and predicted genotypic susceptibility of RT 138A carrying viruses to NNRTI

Source: HIVdb Stanford

Wensing et al. 2014

Prevalence of 138 ETR RAMS in untreated vs. treated patients

Source: HIVdb Stanford

UNTREATED PATIENTS

AA NumSeq NumMut % Mutant

A

52533

1404 2.60 Q 16 0.03 K 69 0.10 G 141 0.20

TREATED WITH >=1 NRTI

AA NumSeq NumMut % Mutant p Ratio treated/untreated

A

5833

147 2.50 0.519 0.96 Q 9 0.10 0.000 3.33 K 23 0.30 0.000 3.00 G 10 0.10 0.212 5.00

TREATED WITH >=1 NNRTI

AA NNRTI NumSeq NumMut % Mutant p Ratio treated/untreated

A >=1 21684 704 3.20 0.000 1.23 NVP 4605 138 2.90 0.210 1.12 EFV 4380 191 4.30 0.000 1.65

Q >=1 21684 245 1.10 0.000 36.66 NVP 4605 76 1.60 0.000 53.33 EFV 4380 29 0.60 0.000 20.00

K >=1 21684 83 0.30 0.000 3.00 NVP 4605 10 0.20 0.195 2.00 EFV 4380 29 0.60 0.000 6.00

G >=1 21684 147 0.60 0.000 3.00 NVP 4605 25 0.50 0.001 2.50 EFV 4380 23 0.50 0.004 2.50

No data available for ETR and RPV

Median (IQR) FC of thirteen isolates: 2.4 (1.6-2.9)

Phenotypic susceptibility of RT 138A carrying viruses to etravirine

Source: HIVdb Stanford

Tambuyzer et al., JAIDS 2011

QUESTION

• Does the natural polymorphism E138A have any impact on the genetic barrier to resistance to ETR?

Workflow

- Creation of recombinant viruses with RT-RNaseH region from untreated patients with only 138E (wild-type) or A. - Creation of pNL4-3 + 138A through site directed mutagenesis

Determination of IC50 of ETR to recombinant viruses

In vitro resistance selection with 50 nM ETR (around 3 times higher than IC50 of wild-type strain)

Creation of recombinant viruses (RVs)

Clinically derived RT-RNAseH PCR fragment

pNL4-3ΔRT-RNAseH (Linearized)

PRECIPITATION

TRANSFECTION 293LX cells

HOMOLOGOUS RECOMBINATION Recombinant Virus

AFTER 60 HOURS

MT-2 INFECTION MONITORING OF

CYTOPATHIC EFFECTS (SYNCYTIA)

HARVESTING RV TITRATION RV RT-RNAseH SEQUENCING

Confirmation of RVs RT-RNaseH viral sequences

SN Major NNRTI Polymorphisms compared to HXB2

NL4-3 None K102Q, S162C, K277R, I293V, R358K, V435I, S468P

473 None K20R, D123E, I135T, T165I, T200A, I202V, F214L, V245E, A272P, V276I, L283I, A288T, V292I, I293V, E297A, R356K, K366R, A376T, T386I, K390R, E399EG, A400T, V435I, R461KR, S468PS, T470N, H483Y, L491S, L517I, I522V, V548I, A554D

474 None K64KR, A98S, D123E, K166R, I180V, T200AT, R211K, A272S, P294Q, K311KR, E312T, M357S, K366R, A376V, T386A, A400T, V435A, D460N, R461K, K476Q, H483Y, L517I, S519NS, K527GR

476 None S48T, E53D, D123E, A158AS, D177E, R211G, A272P, V276IV, K277KR, K281R, E297K, I326T, G333E, Q334N, A360T, A376T, E399D, A400T, E404D, R461K, H483Y, E492Q, Q520H, K527N, A554N

469 138A P52PR, T69IT, K102Q, D123E, I135T, I142V, K173E, Q174R, D177E, V245I, A272P, K277R, Q278H, R356K, G359S, A360T, S379G, T386I, K390R, L452I, L469I, H483Y, L517Q, A554N

470 138A K122EK, K166R, F171Y, I202V, R211K, V245T, K277R, L283I, E297K, E312Q, I329V, S379G, T386I, K390R, T403M, K431T, V435A, T477AT, H483HY, L491P, I495V, Q524L, V531NT

471 138A K102Q, D123E, I135IT, I142IV, K173E, D177E, V245I, A272P, K277KR, Q278H, E297AE, R356K, G359S, A360AT, S379G, V381I, T386I, K390R, L452I, H483Y, L517Q, A554N

NL4-3 + 138A 138A K102Q, S162C, K277R, I293V, R358K, V435I, S468P

Determination of IC50 of ETR with RVs

- Recombinant viruses at M.O.I 0.01 (200-500 TCID50/well) - Five-fold triplicate dilutions of ETR from 10 µM to 0.005 nM - RLU measurement at 48h

RV ID 138 AA IC50 (nM) 95% CI (nM) FC NL4-3 E 17.1 8.2 – 35.8 1 473 E 8.4 3.5 – 20.2 0.5 474 E 17.8 8.5 – 37.0 1.1 476 E 15.8 8.2 – 30.4 0.9 469 A 31.0 13.8 – 69.6 1.8 470 A 29.5 13.2 – 65.8 1.7 471 A 38.5 17.2 – 86.3 2.3

NL4-3 + 138A A 21.3 12.0 – 37.4 1.2

Clinically derived recombinant viruses Median FC 138E = 0.8 Median FC 138A = 1.9

In vitro resistance selection experiments

INFECTION OF MT-2 CELLS WITH RVs AND 50 nM ETR

MONITORING OF VIRAL REPLICATION EVERY 48-72 h

PRESENCE OF LARGE SYNCYTIA?

NO

YES VIRUS

TITRATION AND DETERMINATION

OF ETR IC50

HARVESTING OF SUPERNATANT (SN)

VIRAL RNA EXTRACTION

AND RT-RNAseH SEQUENCING

Virus 138 AA

IC50 T0

(nM)

FCA T0

(nM)

Day of harvesting

(T1)

IC50 (T1) (nM) FCA (T1) Acquired

mutations

NL4-3 E 17.1 1 / /B 473 E 8.4 0.5 / /B 474 E 17.7 1.0 / /B 476 E 15.8 0.9 / /B

469 A 30.9 1.8 14 70.5 4.12 R174Q, E177D, V179E, A371T

470 A 30 1.8 / /B

471 A 38.5 2.6 28 NAC /C V108I, Q174R, Y181C, K385R

NL4-3 +138A A 21.3 1.2 / /B

A) Fold change (FC) values are referred to the HIV-1 reference strain NL4-3 (virus 206) B) No replication at 50 nM ETR C) Not assayed due to poor replication capacity

In vitro resistance selection experiments Viral growth after 45 days of culture with ETR 50 nM

CONCLUSIONS

• All 138A carrying recombinant viruses showed a minimal increase (around 2-fold) in ETR resistance compared to 138E viruses at baseline.

• During in vitro selection experiments, 2/4 138A viruses and 0/4

138E viruses replicated at 50nM ETR, suggesting a role of 138A mutation in the development of resistance to ETR.

• Viruses 469 and 471 acquired mutations A371T and K385R,

respectively. The potential role in ETR resistance of mutations in RT region not considered in certified genotypic assays should be investigated.

• To confirm these results, a larger panel of 138E and 138A carrying recombinant viruses is being analyzed.