Institut für medizinische Mikrobiologie Basel Quantitative Assessment of Resistance to Fusion Inhibitors (T- 20) in a Replicative Phenotyping Assay Vincent

Institut für medizinischeMikrobiologieBasel

Quantitative Assessment of Resistance to Fusion Inhibitors (T-20) in a Replicative Phenotyping Assay

Vincent BRONDANI, François HAMY# and Thomas KLIMKAITInstitute of Medical Microbiology, University of Basel, Switzerland# InPheno AG, Basel, Switzerland

BACKGROUND: Fusion between HIV-env and target cells is now accepted as a valid target for therapeutic intervention. Nevertheless, like other drugs used in HAART, this new class of fusion inhibitors can

experience a rapid escape of HIV via rather stochastic mutations of the HIV genome with subsequent selection upon drug-pressure. We have developed the replicative phenotyping system “PhenoTect”, validated

as diagnostic resistance test platform for protease and RT inhibitors. We aimed at assessing the amenability of PhenoTect to analyse resistance against fusion inhibitors.

IV. Micro-study on T-20 susceptibility amongst clinical Env-variants.

The Env-gene from the viruses of 8 random patients, all T-20-naïve, was analyzed with the

replicative PhenoTect system. IC50 determinations for each one are compiled in the graph

shown in Figure 8. The susceptibility plot of Figure 9 compares the relative drug-sensitivity in

relation to clonal reference viruses (identical genomes except for the indicated change in

gp41). Bars below the line indicate a tendency towards hyper-susceptibility, whereas bars

above indicate a tendency towards T-20-resistance.

The results emphasize existing heterogeneities in susceptibility among viruses that cannot

be predicted from genomic analysis of the “GIV-motifs”.

These findings rather indicate that the basis for resistance to the new drug T-20 certainly

involves a more complex genetic picture, which is directly deciphered by replicative

phenotypic analysis, such as PhenoTect.Figure 9.

pNL4-3DIV

pNL4-3GIV

Patient 8 (GIV)

Patient 7 (GIV)

Patient 6 (DIV)

Patient 5 (GIV)

Patient 4 (GIV)

Patient 3 (GIV)

Patient 2 (GIV)

Patient 1 (DIV)-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

p N L 4 -3

D IV

p N L 4 -3

G IV

P a tie n t 1 (D IV ) P a tie n t 2 (G IV ) P a tie n t 3 (G IV ) P a tie n t 4 (G IV ) P a tie n t 5 (G IV ) P a tie n t 6 (D IV ) P a tie n t 7 (G IV ) P a tie n t 8 (G IV )

Re

lati

ve

T-2

0 s

uc

ep

tib

ilit

y

Reference clone

Reference CloneDIV

-2 -1 0 1 20

10

20

30

40

50

60

70

80

90

100

Patient#8

Patient#1

Patient#6

Patient#4

Patient#2

Patient#3

Patient#7

Patient#5

DIV Day3 _ T20

Log drug. Conc. microM

% in

hib

itio

n

Figure 8.

Figure 7 shows a direct

comparison of IC50 values

determined either in the

single cycle (blue bars) or the

PhenoTect format (red bars).

Results demonstrate the

superiority of the replicative

format in discriminating

susceptibility to T-20 for the

examined gp41-mutants.

Literature describes several point mutations in the HIV-1 gene gp41 in patients treated with T-20 that are

associated with decreased clinical activity. Accordingly, the motif –D35I -V- (found as GIV in reference virus HXB2)

was point mutated to GIV, GIA or DIA in pNL4-3. Mutated Env sequences were used to reconstitute infectious HIV-

1 clones, and expression was first quantitatively assessed (quantitative PCR) after in-vitro infection of the human

lymphocytic cell line CEM-SS. The results showing replication of the different engineered mutants depicted in

Figure 1 support the concept that discrete mutations do have an impact on replication capacity (i.e. Fitness). The

low replication capacities of mutants however did not allow to assess subtle differences in T-20 susceptibility

experiments (not shown).

I. Infection experiment with Env-gp41 mutants

1.00E+03

5.00E+07

1.00E+08

1.50E+08

2.00E+08

2.50E+08

3.00E+08

3.50E+08

0 2 4 6 8 10 12 14

Days of Infection

Vir

al R

NA

(co

pie

s)

GIV

DIV

GIA

DIA

Figure 1. Replication of Env gp41 mutant in infection assay.

gp 120 gp 41

-G-I-V--D-I-V--G-I-A--D-I-A-

Location of mutants used in the study

CONCLUSIONS: Both non-replicative and Replicative System (PhenoTect) are able to determine fitness features.

A replicative System is superior in discerning susceptibilities to fusion inhibitors hence is amenable to be used in diagnosing

resistance/susceptibility to fusion inhibitors.

Individual mutations may insufficiently predict phenotypic susceptibility to fusion inhibitors.

Even drug-naïve patients may need phenotypic analysis for susceptibility to fusion inhibitors.

III. Susceptibility to T-20 amongst engineered Env-gp41 mutants.

We then evaluated the T-20- susceptibilty of the

Env-gp41-mutants: GIV, DIV, GIA and DIA in

the two reporter systems. Triplicate experiments

were performed using either the non-replicative

(Figure 5) or the replicative (PhenoTect, Figure

6) format. Results from reporter read-outs were

averaged, normalized and curve- fitted.

Percentage of virus inhibiton is expressed as a

function of T-20 concentration. IC50 values were

extrapolated for the two methods as shown if

Figures 5 and 6.

-2 -1 0 1 20

10

20

30

40

50

60

70

80

90

100

DIA

GIV

GIA

DIV

DIV Day3 _ T20


% in

hib

itio

n

Figure 6. Figure 7.

0

1

2

3

4

5

6

7

8

DIV GIV GIA DIA

Env gp41 mutants

Rel

ativ

e T-

20 s

ucep

tibili

ty

non-replicative

replicative

Figure 5.

-2 -1 0 1 20

10

20

30

40

50

60

70

80

90

100

DIAmono

GIVmono

GIAmono

DIVmono

DIV Day3 _ T20


% in

hib

itio

n

POSTER

2.22

II. Comparison of features of replicative vs. non-replicative assay format.

We next wanted to check

whether the replicative system

PhenoTectTM (depicted in Figure

2) that is routinely used for

phenotypic diagnosis of HIV

resistance of Protease and RT-

genes was directly adaptable to

assess variations on Env gene

by transfection of the four

constructs described above.

PhenoTect was performed in

comparison with a cellular

system in which fusion is

directly scored without

amplification step (non-

replicative; Figure 2).

The reporter system used in both assays allows to score viral replication as induced -gal activity which is reported in figure 3 (results of triplicate experiments). Figure 4 depicts the relative percentage of read-

out amongst the different variant at day 3 of cultivation and compared to the infection experiment (grey bars). The histogram shows that both replicative and non-replicative methods produce comparable results

where dynamics of the virus is less affected than in infection but still reflects the lower fitness of Env-gp41 mutants.

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control

REPLICATIVEProviral cassette(pNL4-3 background)

HeLa(production)

-Gal

ONP

ONPG

CEM-SSLymphocytes(amplification)

stable HeLaexpressingCD4/CXCR4/CCR5

andLTR-lacZ (Reporter)

HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env

Figure 2. Schematical representation of PhenoTectTM(replicative) cellular assay and its non-replicative equivalent. Figure 3.

Figure 4. Comparison of Virusdynamics of Env mutants in3 systems

100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env



100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative


100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative


HeLa(production)

-Gal

ONP

ONPG


stable HeLaexpressing CD4/CXCR4/CCR5

and LTR-lacZ (Reporter)

HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env

gp 120 gp 41

-G-I-V--D-I-V--G-I-A--D-I-A-

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

cti

vit

y (

OD

40

5n

m)

GIV

DIV

GIA

DIA

Control

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(OD

405n

m) GIV

DIV

GIA

DIA

Control


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env

Figure 2. Schematical representation of PhenoTectTM (replicative) cellular assay and its non-replicative equivalent. Figure 3.

Figure 4. Comparison of Virus dynamics of Env mutants in 3 systems

100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

rela

tive

rea

d-o

ut

Infection

non-replicative

replicative

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env



100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control

0..000

0..250

0..500

0..750

1..000

1 2 3 4

Days of co-culture

-G

al a

ctiv

ity

(O

D4

05

nm

)

GIV

DIV

GIA

DIA

Control


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env


HeLa(production)

-Gal

ONP

ONPG




HeLa(production)

-Gal

ONP

ONPG



NON-REPLICATIVE

EnvOr

Mutated-Env



100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative


100

9.615

11.2

100

60

5043

100

63

49

41

0

20

40

60

80

100

GIV DIV GIA DIA

Env gp41 mutant

re

la

tiv

e re

ad

-o

ut

Infection

non-replicative

replicative

Documents

Institut für medizinische Mikrobiologie Basel Quantitative Assessment of Resistance to Fusion Inhibitors (T- 20) in a Replicative Phenotyping Assay Vincent