Download Quantitative assessment of resistance to fusion inhibitors in a

Quantitative Assessment of Resistance to Fusion Inhibitors (T20) in a Replicative Phenotyping Assay
POSTER
Institut für
medizinische
Mikrobiologie
Basel
Vincent BRONDANI, François
#
HAMY
2.22
and Thomas KLIMKAIT
Institute 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.
I. Infection experiment with Env-gp41 mutants
Literature describes several point mutations in the HIV-1 gene gp41 in patients treated with T-20 that are
3.50E+08
associated with decreased clinical activity. Accordingly, the motif –D35I -V- (found as GIV in reference virus HXB2)
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
3.00E+08
Viral RNA (copies)
was point mutated to GIV, GIA or DIA in pNL4-3. Mutated Env sequences were used to reconstitute infectious HIV-
gp 120
GIV
DIV
GIA
DIA
2.50E+08
2.00E+08
gp 41
-G-I-V-D-I-V-G-I-A-D-I-A-
1.50E+08
Location of mutants used in the study
1.00E+08
5.00E+07
Figure 1. Replication of Env gp41
mutant in infection assay.
1.00E+03
experiments (not shown).
0
2
4
6
8
10
12
14
Days of Infection
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 RTgenes 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
(nonreplicative; Figure 2).
The reporter system used in both assays allows to score viral replication as induced b-gal activity which is reported in figure 3 (results of triplicate experiments). Figure 4 depicts the relative percentage of readout 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.
III. Susceptibility to T-20 amongst engineered Env-gp41 mutants.
DIA
DIAmono
GIV
GIVmono
GIA
GIAmono
DIV
DIVmono
90
90
80
% inhibition
80
70
60
50
40
60
40
direct
comparison of IC50 values
Results
either
in
the
demonstrate
the
superiority of the replicative
10
10
a
PhenoTect format (red bars).
50
20
20
shows
single cycle (blue bars) or the
70
30
30
7
determined
100
100
Figure
8
Relative T-20 suceptibility
DIV Day 3 _ T20
DIV Day3 _ T20
% in h ib it io n
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.
0
0
-2
-1
0
1
2
7
non-replicative
replicative
6
5
4
3
2
1
0
-2
Log drug. Conc. microM
-1
0
Log drug. Conc. m icroM
Figure 5.
Figure 6.
1
2
format
in
discriminating
DIV
GIV
GIA
DIA
Env gp41 mutants
susceptibility to T-20 for the
Figure 7.
examined gp41-mutants.
IV. Micro-study on T-20 susceptibility amongst clinical Env-variants.
Patient#8
Patient#1
Patient#6
Patient#4
Patient#2
Patient#3
Patient#7
Patient#5
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
0.8
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”.
-2
-1
0
Log drug. Conc. microM
Figure 8.
CONCLUSIONS:
1
2
Patient 8
(GIV)
Patient 5
(GIV)
1
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
100
90
80
70
60
50
40
30
20
10
0
Patient 7
(GIV)
1.2
Relative T-20 suceptibility
% in h ib itio n
DIV Day3 _ T 20
0.6
Patient 3
(GIV)
0.4
Patient 4
(GIV)
Reference
clone
pNL4-3
GIV
0.2
0
-0.2
These findings rather indicate that the basis for resistance to the new drug T-20 certainly
-0.4
involves a more complex genetic picture, which is directly deciphered by replicative
-0.6
pNL4-3
Reference
DIV
Clone
DIV
Patient 6
(DIV)
Patient 2
(GIV)
Patient 1
(DIV)
phenotypic analysis, such as PhenoTect.
Figure 9.
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.
gp 120
gp 41
-G-I-V-D-I-V-G-I-A-D-I-A-
NON-REPLICATIVE
Env
Or
Mutated-Env
stable HeLa
expressing CD4/CXCR4/CCR5
and LTR-lacZ
(Reporter)
b-Gal
ONPG
HeLa
(production)
REPLICATIVE
Proviral cassette
(pNL4-3 background)
ONP
stable HeLa
expressing CD4/CXCR4/CCR5
and LTR-lacZ
(Reporter)
HeLa
(production)
b-Gal
ONPG
CEM-SS
Lymphocytes
(amplification)
ONP
0..750
0..500
GIV
DIV
GIA
DIA
Control
100 100 100
100
0..250
0..000
1
2
3
4
Days of co-culture
relative read-out
b -Gal activity (OD405nm)
1..000
Infection
non-replicative
replicative
80
60
63
60
50 49
43 41
40
20
15
11.2
9.6
b -Gal activity (OD405nm)
1..000
0..750
0..500
0
GIV
DIV
GIA
DIA
Control
GIV
GIA
DIA
Env gp41 mutant
Figure 4. Comparison of Virus
dynamics of Env mutants in
3 systems
0..250
0..000
1
Figure 2. Schematical representation
of PhenoTectTM (replicative) cellular assay and its non-replicative equivalent.
DIV
2
3
Days of co-culture
Figure 3.
4