Open Access

The L76V mutation in HIV-1 protease is potentially associated with hypersusceptibility to protease inhibitors Atazanavir and Saquinavir: is there a clinical advantage?

  • Frank Wiesmann1Email author,
  • Jan Vachta1,
  • Robert Ehret1,
  • Hauke Walter2,
  • Rolf Kaiser3,
  • Martin Stürmer4,
  • André Tappe5,
  • Martin Däumer6,
  • Thomas Berg7,
  • Gudrun Naeth1,
  • Patrick Braun1 and
  • Heribert Knechten1
AIDS Research and Therapy20118:7

DOI: 10.1186/1742-6405-8-7

Received: 27 September 2010

Accepted: 13 February 2011

Published: 13 February 2011

Abstract

Background

Although being considered as a rarely observed HIV-1 protease mutation in clinical isolates, the L76V-prevalence increased 1998-2008 in some European countries most likely due to the approval of Lopinavir, Amprenavir and Darunavir which can select L76V. Beside an enhancement of resistance, L76V is also discussed to confer hypersusceptibility to the drugs Atazanavir and Saquinavir which might enable new treatment strategies by trying to take advantage of particular mutations.

Results

Based on a cohort of 47 L76V-positive patients, we examined if there might exist a clinical advantage for L76V-positive patients concerning long-term success of PI-containing regimens in patients with limited therapy options.

Genotypic- and phenotypic HIV-resistance tests from 47 mostly multi-resistant, L76V-positive patients throughout Germany were accomplished retrospectively 1999-2009. Five genotype-based drug-susceptibility predictions received from online interpretation-tools for Atazanavir, Saquinavir, Amprenavir and Lopinavir, were compared to phenotype-based predictions that were determined by using a recombinant virus assay along with a Virtual Phenotype™(Virco). The clinical outcome of the L76V-adapted follow-up therapy was determined by monitoring viral load for 96 weeks.

Conclusions

In this analysis, the mostly used interpretation systems overestimated the L76V-mutation concerning Atazanavir- and SQV resistance. In fact, a clear benefit in drug susceptibility for these drugs was observed in phenotype analysis after establishment of L76V. More importantly, long-term therapy success was significantly higher in patients receiving Atazanavir and/or Saquinavir plus one L76V-selecting drug compared to patients without L76V-selecting agents (p = 0.002).

In case of L76V-occurrence ATV and/or SQV may represent encouraging options for patients in deep salvage situations.

Background

The reduced susceptibility to certain antiretrovirals is often accompanied with a gradual loss of viral fitness, indicating that mutations with high fitness costs are less able to persist in the absence of drug pressure [1]. There have been recent reports about HIV strains with increased susceptibility to particular drugs when certain mutation patterns had developed under antiretroviral treatment [25]. This biological attribute enables new putative strategies for future treatment of HIV-infected patients with abundant resistance mutations by trying to take advantage of particular mutations [6].

As example, M184V/I, the most prevalent NRTI-mutations selected under 3TC or FTC in the reverse transcriptase, do for instance revert partially the effect of thymidine-analogue mutation- (TAM) on resistance [7]. K65R and L74V are further mutations which can confer hypersusceptibility or resensitization to AZT [8]. Beside these specific mutations in the reverse transcriptase, there are also reports about resensitizing mutations affecting the protease gene [9, 10].

Objectives

This article reports about possible clinical advantages of a valine substitution, instead of leucine, at position 76 in the HIV-1 protease. This mutation generally disappears quickly in replicating viruses in absence of selection pressure mediated by LPV, APV or DRV treatment. Thus, for deep salvage therapy situations in patients with strongly limited therapy options, it might be of advantage to maintain these drugs in treatment regimens to preserve L76V in the current replicating virus in combination with a "resensitized" drug ATV or SQV.

Results

Patients with protease gene mutation L76V show increased susceptibility for Atazanavir and Saquinavir

At first, the impact of L76V on ATV- and SQV-resistance characteristics was assessed before and after establishment of the mutation. Due to the manifestation of the L76V mutation as well as other minor mutations at resistance-relevant sites in the course of treatment, genotype-based interpretation tools predicted intermediate or mostly complete resistance against all PIs including ATV and SQV and the majority of NRTIs and NNRTIs resulting in an active drug score (ADS) of ≤ 1.0 for the failing regimen (Figure 1). Interestingly, in phenotypic analysis, the resistance factor (RF) for ATV and SQV remained at full susceptibility in both patients and even decreased for SQV from 31 to 1.1 (Figure 1A) and 1.1 to 0.6 (Figure 1B) and for ATV from 62 to 2.8 and 4.3 to 0.9, respectively.
https://static-content.springer.com/image/art%3A10.1186%2F1742-6405-8-7/MediaObjects/12981_2010_Article_183_Fig1_HTML.jpg
Figure 1

Resensitizing effects of the L76V mutation are visible in phenotype results: Phenotypic resistance analysis before and after manifestation of L76V in two representative patients (A+B). Although additional mutations developed in the progress of therapy (bold characters) the resistance factor for ATV and SQV decreased below the cut-off for full susceptibility in both patients compared to analyses one year before. Antiretroviral drugs are illustrated with corresponding resistance factors (cut-off: 0-3.5 = sensitive 3.6-9.5 (29 for LPV) = intermediate; >9.0 (29 for LPV) = resistant). Genotypic resistance interpretations derived from five common online tools showed considerable discrepancies in weighting of ATV and SQV resistance levels compared to each other and to phenotypic results (grey and white colour). A) One patient with failing APV containing therapy after week 72. B) Another patient with a failing IDV/LPV treatment before start of SQV containing therapy.

In a further aspect, genotypic and phenotypic resistance data of 10 patients, all L76V positive, was assessed in order to analyze if these observed resensitizing effects represent ubiquitous drug resistance patterns. Figure 2 supports this hypothesis on a variety of other patients harbouring HIV populations with L76V mutation. The accuracy and concordance of predicted genotype-based interpretations were compared with obtained phenotypic resistance levels from recombinant virus assays and virtual phenotype analysis.
https://static-content.springer.com/image/art%3A10.1186%2F1742-6405-8-7/MediaObjects/12981_2010_Article_183_Fig2_HTML.jpg
Figure 2

The "resensitizing" effect of 76V could be observed in a variety of other patients before start of PI-containing therapy. Genotypic and phenotypic data of 10 representative PI-experienced patients were analysed by using five common resistance interpretation systems Stanford HIVdb 4.3.6; REGA v7.1.1, HIV-Grade 04/2008, ANRS 10/2007 and geno2pheno. Genotypic resistance results were compared to phenotypic resistance results derived from recombinant virus assay results and/or Virtual Phenotype™ analysis (Virco).

Despite a general concordance in genotype- and phenotype-based resistance predictions for LPV and APV, there were wide discrepancies in the weighting of resistance for ATV and SQV, mostly overestimation of resistance in genotype-based predictions (Figure 2). However, most phenotypic resistance interpretations uncovered full susceptibility for the drugs ATV and SQV. In most cases L76V appeared to be associated with a variety of other resistance relevant protease mutations without effecting the resensitizing effect. However, in particular, the copresence of the protease mutation L90 M was notably associated with high ATV and SQV resistance factors (Figure 2; #4, #26, #21).

Clinical outcome and follow-up in patients with L76V-adapted therapy

Considering the effect of L76V on susceptibility for ATV and SQV, the big question was obviously, how this mutation might affect the therapeutic option and strategy for patients with a narrow margin of remaining active drugs. A considerable issue remained to generalize data from a small cohort of patients with diverse optimized backbone therapies. Thus, this work focused on the amount of active drugs in the treatment of each patient. Table 1 shows the L76V-adjusted follow-up therapies that were administered after resistance prediction results.
Table 1

Active drug score (ADS) for the follow-up therapy

 

GROUP A

GROUP B

Pat-ID

#1

#2

#4

#12

#14

#17

#18

#21

#22

#46

#3

#5

#8

#9

#10

#11

#16

#19

#24

#25

#26

#27

#31

#33

#37

#39

#40

#41

Therapy

ATV SQV RTV FTC

ATV SQV

RTV 3TC

ATV SQV RTV

ATV/r SQV

3TC

T20

SQV

TPV/r

3TC

ddI

ddI

d4T

SQV

ATV

SQV

3TC

T20

ATV

EFV

TDF

AZT

3TC

MVC

ATV/r

SQV

AZT

3TC

TDF

SQV/r

TDF

FTC

LPV/r

SQV

3TC

d4T

TDF

LPV/r

ATV

AZT

3TC

LPV/r

SQV

d4T

LPV/r

SQV

ENF

LPV

ATV

FTC

TDF

LPV/r

ATV

EFV

LPV/r

SQV

LPV/r

SQV

APV

SQV

TDF

APV

SQV

ddC

d4T

APV/r

SQV

LPV/r

SQV

FTC

TDF

LPV/r

SQV

3TC

ETR

LPV/r

SQV

AZT

3TC

TDF

LPV/r

SQV

ddI

3TC

LPV/r

SQV

TDF

LPV/r

SQV

TDF

MVC

LPV/r

ATV

TDF

AZT

ABC

3TC

Rev.-Transcriptase mutations

41L

44D

67N

98G

103N

118I

210F

215Y

219R

41L

67ss

69S

188L

215Y

41L

118I

184V

215Y

41L

44A

67N

75I

103N

108I

118I

210W

215Y

41L

67N

69D

70R

74V

103N

181C

210W

215F

219Q

67N

70R

103NS

190A

184V

219Q

41L

67N

69E

75I

103N

108I

118I

178M

210W

215Y

41L

67N

74V

101Q

184V

215Y

41L

44D

67N

101E

103N

118I

184V

210W

215Y

219E

n.d.

41L

67ss

69S

101Q

181C

190S

215Y

65R

70R

103N

108I

115F

151M

179E

184V

219E

67N

184V

210W

215Y

219Q

41L

44D

67N

75L

118I

181C

184V

190A

210S

215Y

41L

44D

67N

70R

190A

227L

184V

210W

215F

219R

41L

67N

74V

98G

118I

184V

210W

215Y

227L

41L

44D

103N

118I

184V

210W

215Y

no

data

no

data

no

data

67N

70R

103S

184V

190A

215F

219Q

41L

67N

75I

118I

210W

215F

41L

44A

67N

75M

101Q

118I

184V

210W

215F

41L

74V

101Q

103N

108I

181C

190A

210W

215Y

41L

44D

67G

103N

118I

184V

210W

215F

67N

70R

215I

219Q

67N

70R

215I

219Q

41L

67N

70R

184V

Protease mutations

10F

20I

36I

46I

50V

54I/V

76V

10V

46I

47V

71V

76V

77I

10V

20R

36I

46I

76V

90M

10I

33F

46L

76V

82F

90M

10V

20R

33F

36I

54V

73S

76V

90M

10F

20R

46I

54V

63P

76V

82F

10I

33F

36L

46L

76V

82F

84V

90M

10I

53L

54V

71V

76V

77I

82A

84V

90M

10I

33F

46L

54V

71I

76V

77L

82A

90M

10I

33V

60E

76V

10V

46L

54V

63P

71V

82A

93L

10I

20I

36I

46I

54L

76V

84V

20R

32I

46I

76V

82A

10I

24I

33F

46I

54V

63P

71V

76V

82A

10V

20I

36I

46I

47V

53L

76V

84V

90M

10F

33F

46L

54L

71V

76V

77I

84V

90M

10F

46I

54M

71V

76V

82A

10I

20R

24I

36I

46I

54V

76V

82C

10F

33F

54V

71V

76V

77I

82A

10I

20R

35D

46I

54V

76V

10I

20R

36I

46I

54V

71V

76V

82F

90M

10I

46I

47V

71V

76V

90M

10I

13V

32I

33F

36I

46I

76V

84V

90M

10F

46I

47V

76V

84V

10R

32I

33F

46I

47V

76V

84V

88S

90M

10F

20I

36I

46I

76V

84V

10V

13V

24I

33F

46I

54V

76V

82A

20I

36I

54V

76V

82A

Active Drug Scores

                            

   HIVdb 4.3.6

[1.5]

(2.25)

0.5

[1.75]

0.75

1,25

[1.5]

[1.75]

0.25

3.0

1,5

[0.5]

[0.75]

1.25

0.25

[0.75]

[0.5]

[0.5]

1.5

n.d.

0.5

[1.25]

[0.25]

[1.5]

0.0

[1.0]

[1.75]

[1.75]

   Rega V7.1.1

2.0

(2.75)

1.5

2.0

1.0

1.0

2.0

3.5

0.0

3.0

1.75

[1.0]

[1.5]

1.0

0.0

[0.25]

[0.75]

[0.75]

1.75

n.d.

0.75

[1.0]

[0.5]

2.75

0.5

2.0

2.25

4.25

   HIVGrade04/08

2.5

(3.0)

1.5

2.25

1.0

0.5

2.75

2.75

0.0

3.0

(2.0)

[1.0]

[1.0]

(2.0)

0.0

[1.0]

[1.0]

[1.0]

(2.0)

n.d.

1.0

[1.75]

[0.0]

[1.75]

0.0

[0.75]

2.0

2.0

   ANRS 10/2007

3.0

(2.0)

0.5

[1.0]

1.0

1.0

2.0

2.5

0.5

3.0

1.5

[0.0]

2.0

1.0

0.5

[1.0]

[1.0]

[0.5]

(2.0)

n.d.

0.5

2.0

[0.5]

2.5

1.5

[1.5]

2.5

3.0

   geno2pheno

2.0

(3.0)

1.0

2.5

(2.0)

n.d.

n.d.

2.0

n.d.

3.0

(2.0)

[0.5]

[0.5]

1.5

0.0

[1.0]

n.d.

n.d.

n.d.

n.d.

0.0

2.5

n.d.

2.0

0.0

1.0

2.0

4.0

   VircoType

2.25

(2.0)

(2.0)

2.5

1.5

n.d.

n.d.

3.5

n.d.

3.0

2.5

2.0

2.0

1.0

0.5

[0.5]

[1.0]

[1.0]

n.d.

n.d.

0.5

[1.0]

[1.0]

3.0

1.5

2.0

3.0

3.5

   Phenotype

2.5

n.d.

0.0

n.d.

n.d.

(3.0)

3.0

3.0*

n.d.

n.d.

n.d.

2.5

2.5

(2.0)

n.d.

n.d.

n.d.

[0.5]

n.d.

n.d.

0.0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

Follow-up

                            

   Baseline VL

1070

160000

26900

3231

47653

859000

7700

27000

35400

4248

6680

25900

4840

31400

39000

20163

8751

8800

88100

16100

310510

< 50

72100

14900

1300

744

2078

443

   Lowest VL (12-96 weeks)

< 50

3410

38700

< 50

4943

190

< 50

< 50

1020

< 50

1320

< 50

< 50

116

24000

< 50

< 50

< 50

559

< 50

18270

< 50

< 50

< 50

1300

< 50

< 50

< 50

   VL at week 96

13589

46570

152000

< 50

20626

LFU

LFU

LFU

16500

LFU

LFU

< 50

< 50

116

LFU

< 50

< 50

LFU

LFU

1777

LFU

391

60

< 50

LFU

< 50

< 50

< 50

Based on five genotypic resistance interpretation tools as well as two phenotypic resistance tests, an active drug score (ADS) for the follow-up therapy was calculated by adding the activity score for every active drug ranging from AS = 1.0 for complete sensitivity, AS = 0.75 for potential low-level resistance, AS = 0.5 for intermediate resistance, AS = 0.25 for possible resistance and AS = 0.0 for complete resistance. Their prediction on follow-up therapy was then compared with the virological response in a time frame of 96 weeks. Sucessfull therapies despite an active drug score prediction of <2 are displayed in bold and square brackets. Unsucessfull therapies despite an active drug score prediction of ≥2 are displayed in round brackets. LFU=loss of follow-up.

Sufficient virus suppression below the detection limit was initially observed in 50% of group A (ATV and/or SQV without L76V-selecting drug) and 67% of group B (ATV and/or SQV plus L76V-selecting drug LPV or APV) within the first weeks of follow-up therapy. Despite similar response rates at first, a sustained therapy success with virus suppression still below 50 copies/mL at week 96 and longer was predominantly achieved in group B patients where the selection pressure on L76V was constantly maintained by the drugs LPV or APV (Table 1, lower rows). While 66.7% of group B patients remained under detection levels at week 96, there was a significantly lower success rate in group A patients with 16.7% remaining <50 copies/mL in per-protocol analysis (p = 0.002; χ2 test) (Table 2 and Figure 3). Patients of group C did not show any virus suppression below detection limit.
Table 2

Comparison of results (group A and group B).

 

Virus Suppression

Therapy success

 

Group A

Median

Viral load

Group B

Median Viral load

Mann-Whitney

Group A

Success

Group B Success

χ2

Baseline

26,950

(N = 10)

8,800

(N = 17)

P = 0.12

0%

(N = 10)

5.9%

(N = 17)

P = 0.998

Week 12

370

(N = 10)

< 50

(N = 15)

P = 0.07

40.0%

(N = 10)

66.7%

(N = 15)

P = 0.035

Week 24

4650

(N = 8)

< 50

(N = 13)

P = 0.16

37.5%

(N = 8)

69.2%

(N = 13)

P = 0.166

Week 48

3410

(N = 7)

< 50

(N = 13)

P = 0.19

42.9%

(N = 7)

53.8%

(N = 13)

P = 0.425

Week 96

15,045

(N = 6)

< 50

(N = 9)

P = 0.044

16.7%

(N = 6)

54.5%

(N = 9)

P = 0.002

Comparison of results (group A and group B). Median viral loads and the therapy success rates illustrate a significantly better long-term suppression of HIV when SQV/ATV plus optimized backbone therapy is combined with a L76V-selecting drug. In bivariate analysis, these results were independent from slightly different baseline viral loads ( < 0,5log) between group A and B.

https://static-content.springer.com/image/art%3A10.1186%2F1742-6405-8-7/MediaObjects/12981_2010_Article_183_Fig3_HTML.jpg
Figure 3

Clinical outcome and suppression of viral load in a time frame of 96 weeks of follow-up therapy (illustrated as box plot figure). Median viral loads are illustrated in bold lines in between the upper- and lower quartile. Group B (ATV and/or SQV plus LPV or APV containing treatment) show a higher long-term success rate after 96 weeks of follow-up therapy in comparison to group A (ATV and or SQV without L76V-selecting drug).

Interestingly, despite a successful virus suppression <50 copies/mL, the majority of group B therapies were expected to have an ADS below 2.0 after genotypic resistance predictions, indicating a very likely event of therapy failure (bold numbers in square brackets) (Table 1).

Most of the cases where therapies were predicted to have active drug scores of ≥2.0 turned out to be successful. Only few experienced virological failure despite an ADS of more than 2.0 (indicated in round brackets).

A major question remained obviously, why patients of group A display earlier therapy failures than patients of group B. After two years of follow-up therapy, only one patient of group A, who additionally received a fusion-inhibitor containing treatment, showed a viral load still below 50 copies/mL. The median viral load increased after 24 weeks of follow-up in group A (Figure 3). More interestingly, due to a loss of selection pressure on the L76V mutation, it was then undetectable in those patients who failed therapy, resulting in a decrease of the ADS below <2.0 (Table 3). While L76V was undetectable in patients where no L76V-selecting drug was applied, it persisted in group B and group C where selection pressure on mutation L76V was maintained (Table 3). In these patients the therapy failure had other reasons (e.g. acquisition of L90M).
Table 3

Compensatory changes in virus genotypes within 96 weeks of follow-up therapy.

Group

Patient ID

Protease mutations at start of therapy

Time of therapy failure

Time of 2nd genotype

Protease mutations after therapy failure

A

#1

L10FL, K20I, M36I, M46I, I50V, I54IV, L63P, L76V

Week 48

Week 144

L10F, V11I, I13V, K20R, V32I, L33F, M36I, M46I, I47V, I54M, L63P, A71V, G73S, I84V, L90M

 

#2

L10V, M46I, I47V, L63P, A71V, L76V, V77I

Week 12

Week 48

L63P, V77I

(therapy interruption)

 

#4

L10V, K20RK, M36I, M46I, L63P, L76V, L90M

Week 12

Week 24

L10V, K20R, M36I, M46I, F53L, L63P, I84IV, L90M

 

#22

L10I, L33F, M46L, I54V, L63P, A71I, L76V, V77I, V82A, L90M

Week 12

Week 48

L10I, L33F, M46L, F53L, I54V, L63P, A71T, G73S, V77I, V82A, L90M

B

#8

K20R, V32I, M46I, L76V, V82A

Week 48

Week 48

K20R, V32I, M36I, M46I, F53FL, L76V, V82A, L90LM

 

#9

L10I, L24I, L33F, M46I, I54V, L63P, A71V, L76V, V82A

Week 12

Week 24

L10I, L24I, L33F, M46I, F53L, I54V, L63P, A71V, L76V, V82A, I84V

 

#10

L10IV, K20I, M36I, M46I, I47V, F53L, L63P, A71V, G73 D, L76V, I84V, L90M

Week 12

compliance

Week 48

L10V, K20I, L33I, M36I, M46I, I47V, F53L, L63P, A71V, G73 D, L76V, I84V, L90M

 

#27

L10I, M46I, I47V, L63P, A71V, L76V, L90M

Week 48

Week 48

L10I, M46I, I47V, L63P, A71V, L76V, I84V, L90M

C

#6

L10V, L33F, M46L, I54V, A71V, L63P, A71V, L76V, V82A

Week 12

Week 96

L10V, K20R, L33F, M36I, M46L, I54V, A71V, L76V, V82A

 

#23

L10FIRV, L33F, I54MV, D60E, L63P, A71V, L76V, V82F

Week 12

Week 24

L10FIRV, L33F, I54MV, D60E, L63P, A71T/V, L76V, V82F

Compensatory changes in virus genotypes within 96 weeks of follow-up therapy. Patients with failing therapies within the 96 weeks received a second resistance testing. While L76V was still present in patients receiving L76V-selecting drugs, it was then absent in patients without these drugs (new detected mutation are underlined). Therapy failure in group B was noticeable associated with an additional establishment of the protease mutation L90 M.

These results additionally indicate benefits for patients with L76V-selecting drugs in combination with L76V-"resensitized" drugs. A major issue remains the establishment of additional protease gene mutations i.e. L90 M and further compensatory changes over the time (Figure 2; #4, #21, #26), making it crucial to suppress the virus completely and monitor viral load in close intervals.

Discussion

Little is known about the impact of drug-resensitizing mutations on antiretroviral therapy. Most works mainly describe the effects of resistance mutations on reductions in drug susceptibility. However, selective pressure of drug therapy may also lead to shifts in the quasispecies distribution and fitness of those mutants with decreased sensitivity to the respective antiretrovirals [11, 12]. This loss in replication fitness may be even larger for a multi-drug resistant virus and might lead to a better starting point for particular antiretroviral regimens [13]. Nevertheless, it is not always applicable that the acquisition of drug-resistance mutations inevitably result in loss of viral fitness. Even in case a loss is apparent, the virus may select compensatory changes over time [12, 14]. This may explain why current treatment guidelines still advocate a switch to antiretroviral treatment regimens following the emergence of drug resistance mutations and possibly prior to selection of compensatory changes [11]. In summary, drug hypersusceptibility mutations which reduce viral fitness are difficult to maintain in the predominant virus population in multiple pretreated patient.

In this article we provide insights into a possibility how to maintain efficient selection pressure on the protease mutation at position L76V by combining one drug, which selects L76V (in this case LPV, APV, DRV) and another drug which gains efficiency when L76V develops. This article reports about a significant clinical benefit of the protease mutation L76V on drug susceptibility to ATV and SQV due to resensitizing effects in multi-resistant patients resulting in a significantly higher long-term therapy success. These results may be in line with explanations from molecular dynamics- and free energy studies recently reported by Alcaro et al. who found that in the presence of the L76V substitution, ATV reveals a more productive binding affinity, in agreement with hypersusceptibily data [15].

Conclusion

The strategy of combining mutation-selecting drugs with "resensitized" drugs has already been discussed for the reverse transcriptase mutation M184V in NRTI-containing therapies [6, 13, 16] and has also been shown to be an adequate option for a couple of other mutations including the mutation N88 S [10].

Despite initially adequate therapy response rates in 50% (group A) - 67.7% (group B) of cases, it remains a major issue that virological failure under these therapies often occur due to compensatory changes in the virus genotype over the time mostly due to an additional establishment of further mutations in the respective gene [12, 14]. As shown in Table 3, failure of therapy in L76V-positive patients with ATV and/or SQV containing therapy was noticeable associated with an additional establishment of the protease gene mutation at position L90 M which resulted in resistance against all available PIs [17]. Six of eight patients who received a second genotypic resistance test following therapy failure were diagnosed positive for L90 M. The remaining two were therapy non-compliant. Thus, it might be questionable if SQV, which primarily selects L90 M should be replaced in favour of ATV [1820]. In addition, due to the approval of new drug classes over the past years one might err on the side of caution to supplement therapy regimens with new drugs. Due to the low potency of the present cohort and varying amounts of HIV non-B infected individuals in the examined patient groups, caution should be additionally advised, since these limitations might have effect on clinical outcomes.

Nevertheless, since there are still distinct discrepancies, mostly overestimation of resistance, in the prediction of the resistance level for Atazanavir and Saquinavir in five of the most common genotypic interpretation systems, there is still a need for further evaluation in the case of L76V occurrence.

Methods

Clinical material

HIV strains of 46 intensely pretreated (34 showed NRTI/NNRTI/PI resistance/12 showed NRTI/PI resistance) and one naïve with transmitted mutation, L76V-positive patients derived from 24 centres throughout Germany between 1999-2009 were retrospectively analysed for HIV-resistance patterns and success of follow-up therapy. The inclusion criterion is provided in Figure 4. Descriptional statistics concerning person-to-person variations of virological and immunological parameters were assessed at baseline before switch of therapy and are provided in Table 4.
https://static-content.springer.com/image/art%3A10.1186%2F1742-6405-8-7/MediaObjects/12981_2010_Article_183_Fig4_HTML.jpg
Figure 4

Inclusion criterion for the retrospective analysis of 47 L76V-positive patients.

Table 4

Patient characteristics and parameters.

Parameter

Total

Group A (N = 10)

Group B (N = 18)

Group C (N = 19)

Gender

    

Male

84%

100%

100%

68,7%

Female

16%

0%

0%

31,3%

HIV-1 subtype

    

Patients with subtype B

69%

80%

64%

67%

Patients with non-B subtype

31%

20%

36%

33%

Treatment history

    

Mean duration under ART in months (mean)

66

80

88

54

Current active drug score (mean)

-

1.5

1.3

0.5

HIV-1 RNA [copies/ml; median]

    

Baseline

20,163

26,950

8,800

42,600

CD4 cell counts [cells/μl; median]

    

Baseline

260

291

307

246

Patient characteristics and parameters. All patients were categorized in three groups as described throughout the article: Group A (ATV and or SQV), group B (ATV and or SQV plus L76V selecting drug LPV, APV or DRV) and group C (L76V selecting drug plus optimized backbone therapy).

All patient data was categorized in three groups concerning the follow-up therapy:

Group A: ATV and/or SQV (no selection pressure on L76V)

Group B: LPV or APV plus ATV or SQV (maintained selection pressure on L76V)

Group C: LPV or APV plus other drugs (maintained selection pressure on L76V)

All patients received an optimized backbone therapy.

HIV-1 RNA Quantification

Plasma of patients was analysed at baseline and week 12, 24, 48 until end of investigation at week 96 to monitor efficiency of therapy. Plasma RNA was measured by using the COBAS AMPLICOR HIV-1 Monitor system and the Abbott m2000sp/rt system according to the manufacturer's recommendations.

Genotypic resistance testing

Plasma samples of all 47 patients were collected and stored at -20°C until time of RNA extraction. All specimens were processed by using the FDA-approved Siemens HIV TruGene system as well as the Abbott HIV-1 Genotyping System on the Applied Biosystems' 3100 capillary electrophoresis platform according to the manufacturer's recommendations. HIV-1 genotypes were processed and analyzed by using the wildtype LAV-1 sequence as reference. The sensitivity for detecting minor quasispecies variants was 15%. In this cohort, only patients with major L76V positive population were included. Minor wildtype variants were not detected at this position.

Phenotypic resistance testing

Phenotypic resistance analysis of the complete protease gene and the first 900bp of the RT were performed according to an earlier described recombinant virus assay by determining a virus specific resistance factor [21]. In addition, the Virtual Phenotype™(based on 53,000 paired genotypes and phenotypes) from Virco was assessed for those patient samples where no recombinant virus assay was realizable.

Interpretation of drug resistance

Several algorithms are available worldwide, both in public and private domains. The concordance of resistance predictions was analysed between the five most commonly used algorithms [REGA v7.1.1 [22] HIVGrade ver.12/2008 [23] ANRS ver.10/2007 [24] Stanford HIVdb ver.4.3.6 [25] and the geno2pheno online tool [26]] for the drugs ATV, SQV, LPV and/or APV. Multiple resistance tests in treatment history were cumulatively documented. In addition, an active drug score (ADS) was determined in order to analyze the amount of remaining active drugs in follow-up therapies of each patient (susceptible = +1/low intermediate = +0.75/intermediate = +0.5/high intermediate = +0.25/resistant = +0.0). This ADS allowed a statement concerning the prediction of follow-up therapy. It is generally accepted that a successful therapy should contain at least two active drugs, preferably three (ADS ≥2.0) [27, 28].

Declarations

Authors’ Affiliations

(1)
HIV&Hepatitis Research Group, PZB Aachen
(2)
University of Erlangen, Institute for Clinical and Molecular Virology, Schloßgarten
(3)
University of Cologne, Institute for Virology
(4)
University of Frankfurt, Institute for Virology
(5)
Roche Pharma, Clinical Project Management
(6)
Laboratories Thiele, Institute for Immunology and Genetics
(7)
Medical Laboratories Berg, HIV Research

References

  1. Cong ME, Heneine W, Garcia-Lerma JG: Mutational interactions modulate the fitness cost of drug-resistant HIV-1. J Virol. 2007, 81 (6): 3037-41. 10.1128/JVI.02712-06PubMed CentralView ArticlePubMed
  2. Hu Z, Giguel F, Hatano H, Reid P, Lu J, Kuritzkes DR: Fitness comparison of thymidine analog resistance pathways in human immunodeficiency virus type 1. J Virol. 2006, 80 (14): 7020-7. 10.1128/JVI.02747-05PubMed CentralView ArticlePubMed
  3. Catucci M, Venturi G, Romano L, Riccio ML, De Milito A, Valensin PE, Zazzi M: Development and significance of the HIV-1 reverse transcriptase M184V mutation during combination therapy with lamivudine, zidovudine, and protease inhibitors. J Acquir Immune Defic Syndr. 1999, 21 (3): 203-8.View ArticlePubMed
  4. Masquelier B, Descamps D, Carriere I, Ferchal F, Collin G, Denayrolles M, Ruffault A, Chanzy B, Izopet J, Buffet-Janvresse C, Schmitt MP, Race E, Fleury HJ, Aboulker JP, Yeni P, Brun-Vézinet F: Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study. Antivir Ther. 1999, 4 (2): 69-77.PubMed
  5. Matamoros T, Franco S, Vazquez-Alvarez BM, Mas A, Martinez MA, Mendez-Arias L: Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers. J Biol Chem. 2004, 279 (23): 24569-77. 10.1074/jbc.M312658200View ArticlePubMed
  6. Gallant JE: The M184V mutation: what it does, how to prevent it, and what to do with it when it's there. AIDS Read. 2006, 16 (10): 556-9.PubMed
  7. Wolf K, Walter H, Beerenwinkel N, Keulen W, Kaiser R, Hoffmann D, Lengauer T, Selbig J, Vandamme AM, Korn K, Schmidt B: Tenofovir Resistance and Resensitization. Antimicrob Agents Chemother. 2003, 47 (11): 3478-84. 10.1128/AAC.47.11.3478-3484.2003PubMed CentralView ArticlePubMed
  8. Götte M, Weinberg MA: Biochemical mechanisms involved in overcoming HIV resistance to nucleoside inhibitors of reverse transcriptase. Drug Resist Updat. 2000, 3 (1): 30-38.View ArticlePubMed
  9. de Mendoza C, Valer L, Bacheler L, Pattery T, Corral A, Soriano V: Prevalence of the HIV-1 protease mutation I47A in clinical practice and association with lopinavir resistance. AIDS. 2006, 20 (7): 1071-4. 10.1097/01.aids.0000222084.44411.ccView ArticlePubMed
  10. Ziermann R, Limoli K, Das K, Arnold E, Petropoulos J, Parkin NT: A mutation in human immunodeficiency virus type 1 protease, N88 S, that causes in vitro hypersusceptibility to amprenavir. J Virol. 74: 4414-4419.
  11. Andreoni M: Viral phenotype and fitness. New Microbiol. 2004, 27 (2 Suppl 1): 71-6.PubMed
  12. Svarovskaia ES, Feng JY, Margot NA, Myrick F, Goodman D, Ly JK, White KL, Kutty N, Wang R, Borroto-Esoda K, Miller MD: The A62V and S68G mutations in HIV-1 reverse transcriptase partially restore the replication defect associated with the K65R mutation. J Acquir Immune Defic Syndr. 2008, 48 (4): 428-36. 10.1097/QAI.0b013e31817bbe93View ArticlePubMed
  13. Averbuch D, Schapiro JM, Lanier ER, Gradstein S, Gottesman G, Kedem E, Einhorn M, Grisaru-Soen G, Ofir M, Engelhard D, Grossman Z: Diminished selection for thymidine-analog mutations associated with the presence of M184V in Ethiopian children infected with HIV subtype C receiving lamivudine-containing therapy. Pediatr Infect Dis J. 2006, 25 (11): 1049-56. 10.1097/01.inf.0000243211.36690.d5View ArticlePubMed
  14. Nijhuis M, Wensing AM, Bierman WF, de Jong D, Kagan R, Fun A, Jaspers CA, Schurink KA, van Agtmael MA, Boucher CA: Failure of Treatment with First-Line Lopinavir Boosted with Ritonavir Can Be Explained by Novel Resistance Pathways with Protease Mutations 76V. J Infect Dis. 2009, 200 (5): 698-709. 10.1086/605329View ArticlePubMed
  15. Alcaro S, Artese A, Ceccherini-Silberstein F, Ortuso F, Perno CF, Sing T, Svicher V: Molecular Dynamics and Free Energy Studies on the Wild-Type and Mutated HIV-1 Protease Complexed with Four Approved Drugs: Mechanism of Binding and Drug Resistance. J Chem Inf Model. 2009, 49 (7): , 1751-1761. 10.1021/ci900012kView ArticlePubMed
  16. Zaccarelli M, Tozzi V, Perno CF, Antinori A: The challenge of antiretroviral-drug-resistant HIV: is there any possible clinical advantage?. Curr HIV Res. 2004, 2 (3): 283-92. 10.2174/1570162043351192View ArticlePubMed
  17. Rhee SY, Taylor J, Wadhera G, Ben-Hur A, Brutlag DL, Shafer RW: Genotypic predictors of human immunodeficiency virus type 1 drug resistance. Proc Natl Acad Sci USA. 2006, 103: 17355-17360. 10.1073/pnas.0607274103PubMed CentralView ArticlePubMed
  18. Zolopa AR, Shafer RW, Warford A, Montoya JG, Hsu P, Katzenstein D, Merigan TC, Efron B: HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed. Ann Intern Med. 1999, 131: 813-821.PubMed CentralView ArticlePubMed
  19. Dragsted UB, Gerstoft J, Youle M, Fox Z, Losso M, Benetucci J, Jayaweera DT, Rieger A, Bruun JN, Castagna A, Gazzard B, Walmsley S, Hill A, Lundgren JD: A randomized trial to evaluate lopinavir/ritonavir versus saquinavir/ritonavir in HIV-1-infected patients: the MaxCmin2 trial. Antivir Ther. 2005, 10: 735-743.PubMed
  20. Clotet B, Bellos N, Molina JM, Cooper D, Goffard JC, Lazzarin A, Wohrmann A, Katlama C, Wilkin T, Haubrich R, Cohen C, Farthing C, Jayaweera D, Markowitz M, Ruane P, Spinosa-Guzman S, Lefebvre E: Efficacy and safety of darunavir-ritonavir at week 48 in treatment-experienced patients with HIV-1 infection in POWER 1 and 2: a pooled subgroup analysis of data from two randomised trials. Lancet. 2007, 369: 1169-1178. 10.1016/S0140-6736(07)60497-8View ArticlePubMed
  21. Walter H, Schmidt B, Korn K, Vandamme AM, Harrer T, Uberla K: Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors. J Clin Virol. 1999, 13 (1-2): 71-80. 10.1016/S1386-6532(99)00010-4View ArticlePubMed
  22. REGAv.7.1.1.http://bioafrica.net/rega-genotype/html/subtypinghiv.html
  23. HIV-Grade ver 12/2008.http://www.hiv-grade.de
  24. ANRS ver. 10/2007 Agence Nationale de Recherche sur le SIDA (ANRS).http://www.hivfrenchresistance.org
  25. Stanford HIVdb ver.4.3.6.http://hivdb.stanford.edu
  26. geno2pheno.http://www.geno2pheno.org
  27. Raltegravir Treatment in Patients Failing Highly Active Antiretroviral Therapy (HAART) in Denmark.http://clinicaltrials.gov/ct2/show/NCT01061957
  28. Thompson MA, Aberg JA, Cahn P, Julio S, Montaner G, Rizzardini G, Telenti A, Gatell JM, Günthard HF, Hammer SM, Hirsch MS, Jacobsen DM, Reiss P, Richman DD, Volberding PA, Yeni P, Schooley RT, : Antiretroviral Treatment of Adult HIV Infection - 2010 Recommendations of the International AIDS Society-USA Panel. JAMA. 2010, 304 (3): 321-333. 10.1001/jama.2010.1004View ArticlePubMed

Copyright

© Wiesmann et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement