Mutations at codons 54 and 82 of HIV protease predict virological response of HIV-infected children on salvage lopinavir/ritonavir therapy

José Luis Jiménez{dagger}, Salvador Resino{dagger}, Alberto Martinez-Colom, José Ma Bellón, Ma Ángeles Muñoz-Fernández* on behalf of the Spanish Group of Paediatric HIV Infection{ddagger}

Laboratory of Immunobiología-Molecular, Hospital General Universitario ‘Gregorio Marañón’, Madrid, Spain


* Corresponding author. Tel: +34-91-586-8565; Fax: +34-91-586-8018; E-mail: mmunoz.hgugm{at}salud.madrid.org

Received 5 July 2005; returned 15 August 2005; revised 8 September 2005; accepted 9 September 2005


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
Background: Lopinavir/ritonavir is a protease inhibitor (PI) that has shown great effectiveness as salvage therapy in PI-experienced HIV-infected children.

Objectives: To study whether mutations in the HIV-1 protease gene can reliably predict virological responses to salvage therapy with lopinavir/ritonavir in HIV-infected children.

Patients and methods: We carried out a prospective study in 56 HIV-infected children. PI-associated resistance mutations were determined by genotypic testing and were scored according to the IAS-USA guidelines 2005.

Results: Children with a ‘lopinavir mutation score’ (LMS) ≥6 had a negative association for achieving viral load (VL) control [undetectable viral load (uVL) ≤400 copies/mL] and maintaining uVL for at least 6 months. Moreover, children with protease-associated mutations (PRAMs) ≥2 had a negative association for achieving VL control but not for maintaining uVL for at least 6 months. The relative proportion (RP) to uVL was 0.32 (CI95%: 0.16; 0.33; P = 0.002) in children with I54V (46% of total) and 0.48 (CI95%: 0.24; 0.97; P = 0.041) in children with V82A/F (52% of total). Children with I54V and V82A/F had higher prevalence of lopinavir-associated resistance mutations and showed RP of 0.36 (CI95%: 0.17; 0.76; P = 0.007) for achieving uVL.

Conclusions: LMS and PRAMs in HIV-infected children were associated with virological failure in pre-treated HIV-infected children on salvage therapy with lopinavir/ritonavir. Moreover, I54V and V82A/F led to the poorest virological response.

Keywords: HIV-1 , viral load , salvage therapy , resistance


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
Protease inhibitors (PIs) are very efficient in controlling human immunodeficiency virus (HIV)-1 viral replication, but the use of PIs as salvage therapy for heavily pre-treated patients has not fulfilled its expectations.1 However, lopinavir/ritonavir has shown great effectiveness as salvage therapy in PI-experienced HIV-infected children.24 Partial PI resistance accumulated during prior therapy can result in resistance to lopinavir.5 The exact pattern of lopinavir resistance is not yet known and may provide a prediction of virological response to lopinavir/ritonavir.610 Genotyping analyses have revealed that mutations in 16 amino acids of the HIV-1 protease gene (L10F/I/R/V, K20M/R, L24I, V32I, L33F, M46I/L, I47V/A, I50V, F53L, I54V/L/A/M/T/S/I, L63P, A71V/T, G73S, V82A/F/T/S, I84V and L90M), called ‘lopinavir mutation score’ (LMS) are likely to contribute to the reduced susceptibility to lopinavir,11 and provide a potential method as a baseline genotype to evaluate the hypothetical virological response to lopinavir/ritonavir. Other potential predictors include the protease-associated mutations (PRAMs) that evaluate mutations at codons L33 I/V or F, 82L/T, 84V or 90M which are associated with a high level of cross-resistance.12

To address the predictive value of PI mutations on viral load (VL) response to salvage therapy, we have carried out a study with PI-experienced HIV-infected children treated with lopinavir/ritonavir and followed up for a long period of time (24 months).


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
Patients and study design

The study involved a multicentre prospective cohort of 56 PI-experienced HIV-infected children on salvage therapy with lopinavir/ritonavir followed at 12 Spanish hospitals. The inclusion criteria were: (i) VL > 5000 copies/mL at baseline; (ii) at least 6 months of follow-up; (iii) older than 1 year of age; (iv) being previously treated with antiretroviral therapies (ART) and having records of virological failure with PI and/or non-nucleoside analogue (NNRTI); (v) starting salvage HAART (highly active antiretroviral therapy) with lopinavir/ritonavir; (vi) no CD4+ cell count restrictions. This study was conducted according to the declaration of Helsinki and was approved by the Ethics Committees of all hospitals involved.

The children were monitored at least every 3 months with physical examinations and blood sample collection for serial measurements as previously described.13 Laboratory markers of HIV-1 infection (T cells subsets, VL and genotypic HIV-1 drug resistance) were measured as previously described.13 There was not a uniform approach regarding antiretroviral treatment in the background regimen given together with lopinavir/ritonavir. Instead, each paediatrician administered the appropriate antiretroviral therapy (ART) regimen and changed the drugs according to his/her interpretation of the data and international guidelines.13

HIV-1 VL

VL was measured in plasma using the Amplicon Monitor assay (Amplicon Monitor; Roche Diagnostic Systems, Brandenburg, NJ, USA). The limit of quantification was 400 copies/mL.

Genotypic analysis of HIV-1 isolates

Baseline plasma samples for viral genotype were collected between day –60 (60 days prior to initiation of lopinavir/ritonavir treatment) and day 1. Genotypic HIV-1 drug resistance was determined from plasma-associated HIV-1 RNA using the TruGene HIV-1 Resistance Kit (Visible Genetics, Toronto, Canada). The complete HIV-1 protease gene was analysed using Gene Objects software (Visible Genetics). Drug resistances were defined according to the IAS-USA consensus statement.14

Statistical analysis

The ANOVA test was used to compare between the means of two groups. The Fisher exact test was used for all other comparisons between groups. Differences in the characteristics among number of mutations in children were analysed using a non-parametric test (Mann–Whitney). All P values were two-tailed, and the threshold of significance was set at 0.05.

The outcome variables examined were the time necessary for achieving VL ≤ 400 copies/mL and for maintaining VL ≤ 400 copies/mL for at least 6 months. These variables were analysed by the Kaplan–Meier method. Cox regression analyses were performed to assess the relative proportion (RP) achieving VL ≤ 400 copies/mL according to the presence of the PI mutation, and adjusted by baseline characteristics (%CD4+, VL and age at baseline, and additional new drugs in the salvage regimen at baseline). Also, we performed a logistic regression analysis to determine the odds ratio (OR) of VL control (to maintain VL ≤ 400 copies/mL for at least 6 months).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
Characteristics of HIV-1-infected children at baseline

Table 1 shows the baseline characteristics of HIV-infected children enrolled in this study. Approximately 50% of children had an AIDS diagnosis. Also, 42/56 of children had CD4+ T cells ≥ 15% whereas 25/56 had VL ≤ 50 000 copies/mL. Throughout the follow-up period, no child progressed to a new AIDS-defining condition or death. Twenty-nine percent of HIV-infected children did not show any PRAMs whereas 59% had <6 LMS.


View this table:
[in this window]
[in a new window]
 
Table 1. Demographics and clinical, immunological and virological characteristics of vertically HIV-1-infected children at baseline

 
Table 2 shows the ART regimens and drugs used with lopinavir/ritonavir in the salvage regimen. Sixty-six percent of children had >3 ART switches prior to baseline. Stavudine (NRTI) and efavirenz (NNRTI) were the drugs most frequently used. In addition, about 50% of children took 2 NRTI + 1 PI. One year after salvage therapy with lopinavir/ritonavir, 47/56 of children continued with the same regimen they had after initiation of HAART.


View this table:
[in this window]
[in a new window]
 
Table 2. Characteristics of antiretroviral treatments of vertically HIV-1-infected children

 
Prevalence of genotypic resistances at baseline and its impact on virological response

At baseline, children had a median of 5 lopinavir mutations (range: 0; 10). The most frequent PI mutations were L63A/P, A71T/V, L10I and L90M. In addition, all children had a median of 4.5 (range: 0; 8) NRTI mutations and 0 (range: 0; 3) NNRTI mutations. The most frequent reverse transcriptase (RT) mutations were M41L, D67N, Q151M, G190A and T215F.

Children with LMS ≥ 6 presented a negative association with achieving VL ≤ 400 copies/mL and with maintaining virological suppression (VL ≥ 400 copies/mL) for at least 6 months (Table 3). Moreover, children with PRAMs ≥ 2 had a negative association with achieving VL ≤ 400 copies/mL but not with maintaining virological suppression (VL ≤ 400 copies/mL) for at least 6 months (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. Crude and adjusted risk of virological control (VL < 400 copies/mL) in HIV-infected children

 
On the other hand, I54V (24/56 of children) and V82A/F (23/56 of children) were individually associated with virological failure. HIV-infected children with PI mutations at positions 82 and 54 had the lowest probability for achieving VL ≤ 400 copies/mL (Table 3). There was a significant negative association between the presence of I54V and V82A/F at baseline and the probability of achieving a VL < 400 copies/mL. Also, we have shown that PI mutations at positions 82 and 54 were inversely associated with maintaining virological suppression (VL ≤ 400 copies/mL) for at least 6 months (Table 3).

Association of I54V and V82A/F mutations and its impact on virological response

Twenty-four children showed I54V and 20 of these showed V82A/F. When we analysed the virological impact of the I54V and V82A/F mutations combined, we have shown that children with I54V and V82A/F had a high prevalence of other lopinavir mutations (L10I, K20R, L24I, V32I, L33F, M46L, L63P, A71V/T, G73S, V82A/F, I84V and L90M) (Figure 1a). Children with both mutations had a median of 7 lopinavir mutations (range: 4; 10) in contrast to a median of 4 (range: 0; 8) in children without the two mutations (P < 0.001). Moreover, children with I54V and V82A/F mutations also showed a high prevalence of RT mutations (Figure 1b and c). Thus, they had a median of 5 NRTI mutations (range: 2; 8) in contrast to a median of 4 (range: 0; 9) in children without I54V and V82A/F (P = 0.532). Also, HIV-infected children with I54V and V82A/F showed a median of 2 (range: 0; 2) in contrast to a median of 0 (range: 0; 3) NNRTI mutations in children without the two mutations (P = 0.018).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 1. Summary of viral load (VL) evolution of pre-treated vertically HIV-1-infected children according to baseline HIV-protease mutations at positions 82 and 54. (a) Prevalence of HIV-protease mutations. (b) Prevalence of HIV-RT mutations. (c) Kaplan–Meier estimates for undetectable VL (VL ≤ 400 copies/mL). (d) Mean of log10 VL during the first 12 months of follow-up. Differences between groups (P < 0.05). NRTI, nucleoside analogue; NNRTI, non-nucleoside analogue. Only the genotypic HIV-1 mutations with absolute frequency ≥3 are shown.

 
I54V and V82A/F were associated with virological failure; so, the median time to achieve VL ≤ 400 copies/mL was higher with I54V and V82A/F (P = 0.005) (Figure 1c). In addition, children with I54V and V82A/F had VL higher than children without the two mutations (P < 0.05) for the first 12 months of follow-up (Figure 1d).

Children with I54V and V82A/F had the lowest probability of achieving VL ≤ 400 copies/mL (Table 3). I54V and V82A/F PI had a negative association with achieving undetectable VL and with having virological suppression (VL ≤ 400 copies/mL) for at least 6 months.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
Lopinavir/ritonavir is relatively well-tolerated, provides potent antiviral activity3 and may be used as an effective option for the treatment of children in combination with another antiretroviral as part of salvage therapy regimens.15 Kempf et al.9 found that lopinavir showed a low cross-resistance pattern to other PIs in HIV-infected patients. However, we have demonstrated that a quite significant resistance can be observed in PI-experienced patients. In addition, patients who fail lopinavir may develop mutations associated with significant resistance to other PIs. Predictive factors of virological response to lopinavir/ritonavir in children are unknown, especially in children who have been pre-treated with PIs. In this study, we have shown that some PI mutations present at baseline have a predictive value for virological response.

Previous studies have shown that the 16-mutation LMS was inversely associated with virological suppression.2,7,9,13,16,17 Algorithms for interpreting viral genotypes have suggested that the efficacy of lopinavir/ritonavir treatment is significantly reduced by the presence of six or more mutations (www.hivdb.stanford.edu). However, one other study found that this scoring system may lead to under-appreciation of lopinavir-associated resistance.18 Mutations at positions 46, 54 and 82 showed a great resistance to lopinavir. The threshold number of lopinavir mutations found by Delaugerre et al.16 was ~4, but we found a threshold of 6 as determinant of the virological outcome in agreement with other studies in children13 and adults.6,7,9,19 The differences could be because the children in the Delaugerre et al. report were less heavily treated than the children in our study.

The number of PRAMs necessary to confer resistance to lopinavir seems to be quite low (2 or more).20 We have found that only PRAMs ≥ 2 in plasma of children had a negative association with virological response to salvage therapy with lopinavir/ritonavir. However, this association was not found for maintaining virological suppression for at least 6 months.

Our data clearly indicate that I54V and V82A/F mutations were associated with virological failure. These data differ from another study in children that found an association of the PI mutations L10I/F, M46I, I54V/L, A71V/I, V82A/F/T/S and L90M with virological failure.16 Thus, in our study undetectable VL was observed more commonly in children without I54V and V82A/F PI mutations; and VL values were stable between 3 and 15 months in most of these children. In contrast, viral replication was not well controlled in children with I54V and V82A/F as indicated by the low OR for achieving undetectable VL for at least 6 months of follow-up. Children with I54V and V82A/F had been heavily pre-treated and had a high overall number of mutations against lopinavir, NRTIs and NNRTIs. We also found that HIV-infected children with I54V and V82A/F also had a high prevalence of other lopinavir mutations.11

Moreover, we did not find any influence of previous ART at baseline and additional new drugs in the salvage regimen at baseline on the virological response because we did not find statistical significance in Cox regression analysis for achieving undetectable VL and for maintaining undetectable VL for at least 6 months (data not shown). Besides, with regard to the mutations in the HIV-1 protease gene, only VL at baseline showed statistical significance in Cox regression analysis.

In conclusion, our study indicates that both LMS and PRAMs in plasma of children were associated with virological failure to salvage therapy with lopinavir/ritonavir. Despite there being several studies, the exact pattern of lopinavir/ritonavir resistance is not yet known and the beneficial role for lopinavir/ritonavir in salvage antiretroviral therapy remains under debate. However, we have shown that I54V and V82A/F led to the worst virological response. Further studies are necessary on specific mutation patterns associated with a low response to lopinavir/ritonavir in HIV-infected children.


    Transparency declarations
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
We do not have commercial or other associations that might pose a conflict of interest.


    Footnotes
 
{dagger} Both authors contributed equally to this work. Back

{ddagger} Participants are listed in the Acknowledgements section. Back


    Acknowledgements
 
Financial support: Fundación para la Investigación y la Prevención del SIDA en España, FIPSE (grant 12456/03, 36365/02), the Red Temática Cooperativa de investigación en SIDA and the Red Temática Cooperativa de investigación en Genética of FIS (grants RIS G03/173 and RIG C03/07, respectively), Plan Nacional de Salud (grant SAF 2003-09209, SAF-2004-06778), and Abbott Laboratories. J. L. J., S. R. and J. M. B. are supported by a grant from FIS (grant CP03/00140; CP04/00090, 01/A016, respectively) FIS04/0883.

Participating hospitals and personnel staff of the Spanish Group of Paediatric HIV Infection. MADRID: Hospital 12 Octubre: J.T. Ramos. Hospital Gregorio Marañón: J.L. Jiménez, A. Martinez-Colom, J.M. Bellón, M.D. Gurbindo, M.L. Navarro, S. Resino, M. Gonzalez-Rivera, L. Diaz, M.A. Muñoz-Fernández. Hospital La Paz: M.I. Isabel de José, B. Larru. Hospital Carlos III: P. Martín-Fontelos, M.J. Mellado. SEVILLA: Hospital Virgen del Rocio: J.A. León Leal. BARCELONA: Hospital S.Juan de Dios: C. Fortuny. Hospital Valle de Hebrón: J.M. Bertrán, L. García. Hospital del Mar: A. Mur. ALICANTE: Hospital S Juan: R. González-Montero. BILBAO: Hospital de Cruces: I. Pocheville, C. Gutierrez. PALMA DE MALLORCA: Hospital Son Dureta: J. Dueñas. VALENCIA: Hospital La Fe: A. Orti, M.C. Otero, F. Asensi. ZARAGOZA: Hospital Clínico: M. Gracia, L. Ciria.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Transparency declarations
 References
 
1. Walmsley S. Individualized therapy for the treatment-experienced patient. AIDS Reader 2003; 13: S11–15.[Medline]

2. Resino S, Bellón JM, Ramos JT et al. Salvage antiretroviral therapy in HIV-infected children: advantages of lopinavir-ritonavir. Pediatr Infect Dis J 2004; 23: 923–30.[ISI][Medline]

3. Saez-Llorens X, Violari A, Deetz CO et al. Forty-eight-week evaluation of lopinavir/ritonavir, a new protease inhibitor, in human immunodeficiency virus-infected children. Pediatr Infect Dis J 2003; 22: 216–24.[CrossRef][ISI][Medline]

4. Fraaij PL, Neubert J, Bergshoeff AS et al. Safety and efficacy of a NRTI-sparing HAART regimen of efavirenz and lopinavir/ritonavir in HIV-1-infected children. Antivir Ther 2004; 9: 297–9.[ISI][Medline]

5. Mo H, King MS, King K et al. Selection of resistance in protease inhibitor-experienced, human immunodeficiency virus type 1-infected subjects failing lopinavir- and ritonavir-based therapy: mutation patterns and baseline correlates. J Virol 2005; 79: 3329–38.[Abstract/Free Full Text]

6. Kempf DJ, Isaacson JD, King MS et al. Identification of genotypic changes in human immunodeficiency virus protease that correlate with reduced susceptibility to the protease inhibitor lopinavir among viral isolates from protease inhibitor-experienced patients. J Virol 2001; 75: 7462–9.[Abstract/Free Full Text]

7. Monno L, Saracino A, Scudeller L et al. HIV-1 phenotypic susceptibility to lopinavir (LPV) and genotypic analysis in LPV/r-naive subjects with prior protease inhibitor experience. J Acquir Immune Defic Syndr 2003; 33: 439–47.[CrossRef][Medline]

8. Prado JG, Wrin T, Beauchaine J et al. Amprenavir-resistant HIV-1 exhibits lopinavir cross-resistance and reduced replication capacity. AIDS 2002; 16: 1009–17.[CrossRef][ISI][Medline]

9. Kempf DJ, Isaacson JD, King MS et al. Analysis of the virological response with respect to baseline viral phenotype and genotype in protease inhibitor-experienced HIV-1-infected patients receiving lopinavir/ritonavir therapy. Antivir Ther 2002; 7: 165–74.[ISI][Medline]

10. Tsuchiya K, Matsuoka S, Hachiya A et al. Accumulation of lopinavir resistance-associated mutations over 3 years follow-up of patients on highly active antiretroviral therapy: implication in salvage therapy. AIDS 2001; 15: 1183–4.[CrossRef][ISI][Medline]

11. Johnson VA, Brun-Vézinet F, Clotet B et al. Update of the drug resistance mutations in HIV-1: 2005. Top HIV Med 2005; 13: 51–7.[Medline]

12. Perez-Elias MJ, Garcia-Arota I, Munoz V et al. Phenotype or virtual phenotype for choosing antiretroviral therapy after failure: a prospective, randomized study. Antivir Ther 2003; 8: 577–84.[Medline]

13. Resino S, Bellón JM, Ramos JT et al. Positive virologic outcome after lopinavir/ritonavir salvage therapy in protease inhibitor-experienced HIV-1-infected children. A prospective cohort study. J Antimicrob Chemother 2004; 54: 921–31.[Abstract/Free Full Text]

14. Hirsch MS, Brun-Vezinet F, D'Aquila RT et al. Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel. JAMA 2000; 283: 2417–26.[Abstract/Free Full Text]

15. Sharland M, Blanche S, Castelli G et al. PENTA guidelines for the use of antiretroviral therapy, 2004. HIV Med 2004; 5 Suppl 2: 61–86.[CrossRef][ISI][Medline]

16. Delaugerre C, Teglas JP, Treluyer JM et al. Predictive factors of virologic success in HIV-1-infected children treated with lopinavir/ritonavir. J Acquir Immune Defic Syndr 2004; 37: 1269–75.[Medline]

17. Loutfy MR, Raboud JM, Walmsley SL et al. Predictive value of HIV-1 protease genotype and virtual phenotype on the virological response to lopinavir/ritonavir-containing salvage regimens. Antivir Ther 2004; 9: 595–602.[ISI][Medline]

18. Parkin NT, Chappey C, Petropoulos CJ. Improving lopinavir genotype algorithm through phenotype correlations: novel mutation patterns and amprenavir cross-resistance. AIDS 2003; 17: 955–61.[CrossRef][ISI][Medline]

19. Bongiovanni M, Bini T, Adorni F et al. Virological success of lopinavir/ritonavir salvage regimen is affected by an increasing number of lopinavir/ritonavir-related mutations. Antivir Ther 2003; 8: 209–14.[Medline]

20. Plosker GL, Figgitt DP. Tipranavir. Drugs 2003; 63: 1611–8.[ISI][Medline]