Novel mutations located outside the integrase gene confer HIV-1 integrasestrand-transfer inhibitors resistance.

Dr Olivier Delelis

Pr Anne-Geneviève Marcelin

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Resistance to Anti-Integrases

• Raltegravir(RAL) /Elvitegravir (FDA. 2007/2012) 1st generation molecules• Several pathways of resistance characterized (G148, N155, Y143)

• Dolutegravir (DTG) (2014) 2nd Generation• No pathways of resistance • Some patients have become non-responsive to DTG regimen

• Characterization achieved by: • in vitro recombinant Integrase assays • Virological assays mainly based on quantitative PCR

2

3

SELECTION OF VIRUS RESISTANT TO DTG ?

HIV-1

Dolutegravir

High concentration

Highly Resistant Mutant

4

Further selection demonstrates an increasing resistance and thereby anenrichment of a “selected” virus resistant to DTG.

EC50 5nM

EC50 nil

MUTANT IS INHIBITED BY RT INHIBITORS AS WELL AS THE WT

No mutation in the integrase

Mechanism of replication ?

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

4 5 6 7 10

Co

pie

s vi

ral D

NA

/ug

Days Post-Infection

Total Viral DNA PPT Mutant

Mutant+DTG

Mutant -DTG

Mutant+CBT

WT

-1

4

9

14

19

24

29

34

39

0 10 100 1000 10000

Rat

io S

ign

al A

LU

+/A

LU

-

Dilution in DEPC Treated Water

Increasing Specificity of Integrated Forms Upon

Dilution

Mutant DTG J11

Mutant DTG J13

WT 0 J5

Mechanism of replication ?

▪ qPCR data indicate the profile of a replicating mutant which bypasses integration

▪ No Integration

9

0

1

2

3

4

5

6

d3 d4 d5 d8 d11

WT

PPT

PPT+DTG

1-LTR circles ratio during WT and PPT infection 2-LTR circles: no accumulation

0

2

4

6

8

10

12

14

WT WT+DTG PPT PPT+DTG

d3

d4

d5

d8

d11

HIV-1 life cycle

INSTI

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Unintegrated Viral DNA forms are accumulated by INSTI treatment.

Function of episomes : synthesis of viral proteins.

Could they involved in HIV-1 replication ?

An alternative pathway of replication ?

1-LTR circle Accumulation

• 3’ PPT offers (+) strand synthesis and secondary LTR in viral DNA

• Augmented 1-LTR circle levels highlight the increased importance of this pathway

• PPT mutation leads to abnormal degradation 3’ PPT

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Conclusions:

- A virus has been obtained by DTG selection.- This virus is able to replicate.- This virus is resistant to DTG and all INSTIs.- No mutation in the integrase gene.

- No integrated viral DNA has been detected by 2 different methods.- Accumulation of 1-LTR circles.

Expression of Unintegrated viral DNA ?

Experiments are ongoing but expression from this virus seems to be stronger that the WT

Acknowledgements

LBPA, Eric Deprez and the Biophotonic Team

▪ Dr Fréderic Subra

▪ Dr Hervé Leh

▪ Béatrice Herrmann

Collaborators and Funding

- Dr Isabelle Malet

- Dr Gilles Collin

- Pr Vincent Calvez

- Pr Anne-Geneviève Marcelin

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- Dr Charlotte Charpentier

- Pr Diane DescampsFunding

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Sequencing of the integration sites ?

qPCR experiments : no integrated DNAConfirmed by integration sites sequencing?

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

S70 0 J5 S70 0 J5 10X S70 0 J5 100X S70 0 J5 1000X S70 0 J510000X

Dilution of WT integrated DNA

alu+

alu-

qPCR sensitivity

16

0

2

4

6

8

10

12

2 3 5 7 9 11 13

Ra

tio

alu

+/a

lu-

WT WT - 500nM DTG

Mutant Mutant - 500nM DTG

ratio

SamplesNumber of Insertions Gene Non-Gene

WT 0 DTG 8000 70 30

Mutant 0 DTG 10 p.i. 6000 71 29

WT 500nM DTG 5 p.i. nul

Mutant 500nM DTG 7 p.i. nul nul nul

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Unintegrated viral DNA expression

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Day 6 post-infection

Uninfected cells

WTWT+DTG

5% of GFP+ cells 0,035% of GFP+ cells

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INF BRU2 D116NBru

4,17% of GFP+ cells0,09% of GFP+ cells

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PPT MUTANT PPT MUTANT+DTG

8,7% of GFP+ cells 5,86% of GFP+ cells

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0

20

40

60

80

100

120

WT WT+DTG BRU BRU D116N PPT Mutant PPT mutant+ DTG

%o

f GF

P +

ce

llsn

orm

alis

edb

y th

e W

T (

wit

ho

ut

DT

G)

Escaping Dolutegravir

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WT PPT Mutant

Escaping Dolutegravir

23

WT PPT Mutant

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Conclusions :

- A replicative virus, without mutation in the integrase gene has been obtained.- « Stable mutations » were observed in the 3’-PPT.- The « selected » virus is resistant to all known INSTIs inhibitors.- No integrated viral DNA has been observed by 2 different approaches.

Relevance of this study : patient failing DTG treatment was identified with similarmutations in the 3’-PPT but no mutation in IN

Future directions :

-Cloning of the « selected virus ».-Site directed mutagenesis in the catalytic site of IN.-Check action of other IN inhibitors.-Localisation of viral DNA.

Acknowledgements

LBPA, Eric Deprez and the Biophotonic Team

▪ Dr Fréderic Subra

▪ Dr Hervé Leh

▪ Béatrice Herrmann

Collaborators and Funding

- Dr Isabelle Malet

- Pr Vincent Calvez

- Pr Anne-Geneviève Marcelin

- Dr Vincent Parissi

- Dr Zoltan Ivics

- Dr Marc Ruff

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