Evaluation of Primary Resistance to HIV Entry Inhibitors Among ...

AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 26, Number 12, 2010 ª Mary Ann Liebert, Inc DOI: 10.1089/aid.2010.0057

CLINICAL TRIALS/CLINICAL STUDIES

Evaluation of Primary Resistance to HIV Entry Inhibitors Among Brazilian Patients Failing Reverse Transcriptase/ Protease Inhibitors Treatment Reveal High Prevalence of Maraviroc Resistance-Related Mutations Cecı´lia S. Alencar,1 Anna S. Nishiya,2 Suzete Ferreira,2 Maria Teresa M. Giret,3 Ricardo S. Diaz,1 and Ester C. Sabino 2,4

Abstract

Entry inhibitor is a new class of drugs that target the viral envelope protein. This region is variable; hence resistance to these drugs may be present before treatment. The aim of this study was to analyze the frequency of patients failing treatment with transcriptase reverse and protease inhibitors that would respond to the entry inhibitors Enfuvirtide, Maraviroc, and BMS-806. The study included 100 HIV-1 positive patients from one outpatient clinic in the city of Sao Paulo, for whom a genotype test was requested due to treatment failure. Proviral DNA was amplified and sequenced for regions of gp120 and gp41. A total of 80 could be sequenced and from those, 73 (91.3%), 5 (6.3%) and 2 (2.5%) were classified as subtype B, F, and recombinants (B/ F and B/C), respectively. CXCR4 co-receptor use was predicted in 30% of the strains. Primary resistance to Enfuvirtide was found in 1.3%, following the AIDS Society consensus list, and 10% would be considered resistant if a broader criterion was used. Resistance to BMS-806 was higher; 6 (7.5%), and was associated to non-B strains. Strikingly, 27.5% of samples harbored one or more mutation among A316T, I323V, and S405A, which have been related to decreased susceptibility of Maraviroc; 15% of them among viruses predictive to be R5. A more common mutation was A316T, which was associated to the Brazilian B strain harboring the GWGR motif at the tip of V3 loop and their derivative sequences. These results may be impact guidelines for genotype testing and treatment in Brazil.

Introduction

T

he combined administration of nucleoside and nonreverse transcriptase inhibitors (NRTI and NNRTI), and protease inhibitors (IP) has resulted in a dramatic decrease of the morbidity and mortality of HIV-1 infection.1,2 However, after a few years of HAART, drug resistance may emerge with treatment failure. The number of AIDS patients caused by multi-resistant strains and the incidence of primary resistance have increased in many parts of the world.3 Therefore, the development of new drugs is essential to sustain the disease under control. One of the main areas of focus of this development is drugs that inhibit the interaction between the virus and its receptor

in the cell or the viral fusion process.4 A prominent example for such peptides, termed fusion inhibitors, is the peptide Enfuvirtide (ENF or T20) which is used in HIV/AIDS salvage therapy regimens.5 It is a synthetic peptide compounded of 36 amino acids, that binds to the gp41 protein in the viral envelope, therefore blocking the final fusion of the virus with the host cell.1,3,6,7 Salvage regimens that include ENF have demonstrated sustained efficacy in combination with more active drugs. However, the costs are very high for initiatives like the Brazilian Ministry of Health AIDS program that provide free treatment for all patients affected by the disease.8 Resistance mutations associated with ENF have been described9 and may be present before the introduction of the treatment.

1

Infectious Diseases Division, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil. Fundac¸a˜o Pro´-Sangue, Hemocentro, Sa˜o Paulo, Brazil. Divisions of 3Clinical Immunology and Allergy and 4Hematology and Hemotherapy, University of Sao Paulo, Brazil. This study was supported with funding from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (04/15856-9/Diaz, Sabino & Kallas). MTMG. was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (09/52381-2). 2

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1268 Therefore, understanding the level of primary resistance to this drug among patients failing therapy is important for the development of appropriate salvage therapy guidelines.1 Maraviroc is a CCR5 inhibitor also approved by the Food and Drug Administration (FDA) for salvage therapy. This drug is active against R5 with no net benefit against X4 or dual- or mixed-tropic HIV strains. The classification of coreceptor usage is done through a phenotypic commercially available technology (TrofileTM).10 Genotype classification based on the envelope gene sequence may also be applied for this purpose and may have similar sensitivity in predicting CCR5 antagonist virologic failure.11 Another drug called BMS-806, not approved yet by the FDA, is capable of inhibiting the interaction between gp120 subunits and the cell surface CD4 molecules, therefore blocking the entrance of the virus into the lymphocyte.2 Mutations related to the reduction of viral sensitivity to the drug have already been described.2,12,13 Despite the fact that ENF has been used in Brazil since 2005 and Maraviroc since 2007, there are scant data about prevalence of primary resistance to these drugs among patients failing therapy to RT and protease inhibitors. The aim of this study was to analyze the envelope gene of patients failing treatment with RT and protease inhibitors and infer the proportion of individuals that could take advantage of the new entry inhibitor drugs. Materials and Methods Study population. We enrolled 100 consecutive HIV-1 infected patients, from a public outpatient clinic in the city of Sao Paulo (Centro de Refereˆncia e Treinamento DST/AIDS de Sa˜o Paulo—CRT). Eligibility criteria included: viral load 5000 copies/ml, patients older than 18 years failing treatment with NRTIs, NNRTIs, or PIs, and no previous exposure to entry inhibitors. The subjects were invited to participate at the time of blood collection for a pol genotype test, which is routinely performed in Brazil for patients failing antiretroviral treatment. After informed consent, the patients were interviewed and an extra 10 ml of blood was collected. Medical charts were also reviewed to access previous antiretroviral exposure. DNA extraction, amplification for the gp 120 and gp 41 regions The DNA was obtained from 200 ml of blood samples with QIAamp DNA Blood Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer protocol. A fragment with approximately 1489 bp containing the gp120/gp41 region of the env gene was amplified. The primers 2142 (outer) 5’-GGTGAGTATCCCTGCCTAAC-3’ and ED5 (outer) 5’-ATG GGA TCA AAG CCT AAA GCC ATG TG-3’14 were used in the first round amplifications, and the primers ED31 (inner) 5’-CCT CAG CCA TTA CAC AGG CCT GTC CAA AG-3’14 and gp41/2as (inner) 5’-CTACCAAGCCTCCTAC TATC-3’15 were used in the second round amplifications. The nested PCR mixture was performed using 0.4 mM dNTPs (Amersham Pharmacia Biotec, Piscataway, NJ), 2.5 mM MgCL2 (Invitrogen, Carslbad, CA), 0.2 pmol/ml of each of the primer and 1.5 U of Taq-DNA polymerase (Invitrogen) for the final volume of 50 ml. Amplification for the first round was one

ALENCAR ET AL. cycle of 948C for 1 min., followed by 35 cycles of 948C for 45 sec, 558C for 45 sec, and 728C for 2 min, with a final extension of 728C for 10 min. The second round of amplification included an initial cycle of 948C for 1 min, followed by 35 cycles of 948C for 1 min, 558C for 1 min, and 728C for 1:30 min, and a final extension of 728C for 10 min. Sequencing, resistance analysis, HIV-1 subtype, and co-receptor use classification The PCR product of the second amplification was purified using QIAquick Kit (QIAGEN) according to the manufacturer protocol, and directly sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA) according to manufacturer procedure. We used the primers ED 31 50 -CCT CAG CCA TTA CAC AGG CCT GTC CAA-30 ; gp 41/2as: 50 -CTACCAAGCCTCCTACTATC30 . ED 33 50 -TTA CAG TAG AAA AAT TCC CCT -30 ; gp 41/1s: 50 -TGGAGGAGGAGATATGAGG-30 ; gp 41/6as 50 -CAGAT GYTGTTGMGCCTC-30 14–16 and the sequences were edited using the SEQUENCHER program (Gene Codes Corporation Ann Arbor, MI). The sequences were aligned to HXB2 using the Software Bioedit Sequence Alignment Editor. HIV-1 subtype was performed using Rega HIV Subtyping Tool Version 2.0 available at http://www.Bioafrica.net/subtypetool/html. Co-receptor usage was defined using the algorithm Geno2pheno Version 1.1 available at http://coreceptor.bionf.mpiinf.mpg.de/. Drug resistance analysis To define resistance to ENF, we have considered the following classification: The International AIDS Society (IAS) consensus list: Q36D/S; I37V; V38A/M/E; Q39R; Q:40H, N42T e N43D,17 and the expanded IAS list, including previous mutations plus: Q32H/Q, Q39H, Q40R, N42D/Q/H, N43S/Q/K/, L44M, L45M, R46M e V69I.15,18,19 For BMS-806, we have considered the following mutations: M426L, M475I, and M434I.12,13,20 For Maraviroc, we have considered the mutations A316T, I323V, and S405A (HXB2 positions) that are selected in vitro and related to the decrease of susceptibility to this inhibitor among R5 strain.21 Statistical analysis Frequencies, medians of main variables, and statistical significance were assessed by the Mann–Whitney test for comparison between two groups ( p < 0.05) using the program Epi-info (version 6.04). To evaluate the correlations between patients failing treatment with HAART and selection of mutations in gp120 and gp41 was done using the Chi-square (with and without Yate’s correction) test and Fisher’s exact test. Results Of the 100 samples collected, 87 yielded a PCR product and a sequence was obtained for 80 of them. Table 1 compares the demographic characteristics of the enrolled population versus the studied population; no difference could be identified between the sequenced versus the nonsequenced samples. Of the 80 samples, 73 (91%) were classified as subtype B, five (6%) as subtype F, and two (2.5%) were mosaic (1 B/F and 1 B/C). The frequency of the samples presenting primary resistance to ENF varied according to the criteria applied. When the IAS

PRIMARY RESISTANCE TO ENTRY INHIBITOR

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Table 1. Demographies and Social-Epidemiological Characteristics of the Study Population from Sa˜o Paulo Sequenced (80)

Non-sequenced (20)

Characteristics Gender Education Intravenous drugs usage Exposure

N (%) Male Basic education (8 years less) School graduates (11 years) Graduated (>11 years) Yes MSMa Heterosexual

Age (mean) Time of treatment (mean in years) a

64 30 30 22 6 42 38 45 11

(80) (37.5) (37.5) (27.5) (7.5) (52.5) (47.5) (7.6) (7.6)

18 2 12 4 3 9 11 45 11

(90) (10) (60) (20) (15) (45) (55) (7.6) (7.6)

Men who have sex with men.

mutation list was used, only one sample was resistant due to the presence of the N42T mutation.9 However, if the criteria were expended, seven more samples would be classified as resistant due to the mutations: N42D, N42H, L44M, and R46M.15,18,19 In addition, other polymorphisms nonrelated to resistance were observed in the gp41 region studied as shown in Table 2. When analyzing the region related to BMS-806 resistance, we could detect six (7.5%) resistant strains due to the presence of mutation M426L in three, and M434I in three.12,13,20 Table 2 also summarizes the resistance mutation and polymorphisms for this drug. In relation to CCR5 antagonists susceptibility, 24 (30%) of the samples were classified as capable of using CXCR4. Mutations A316T, I323V, and S405A are related to the decrease of Maraviroc.21 Susceptibility among R5 strains was detected in 22 individuals, 12 of those samples harboring these above mutations were found among virus genotypically classified as R5 (Table 2). We found 48 out of 80 samples harboring the Proline at the GPGR motif at the tip of V3 loop and 32 typical Brazilian clade B strains harboring tryptophan or its derivative amino acids, including R, V, G, L, E, F, A, and S (Table 3). Mutation A316T, which was the more prevalent substitution among the above, was found in 4 (8.3%) out of GPGR sequences and in 10 (31.2%) of the GWGR or GWGR related sequences (Fischer exact test p < 0.01). When five sequences from non-B clade are excluded of this analysis, in the GPGR group, the association between A316T substitution and GWGR-related sequences is maintained ( p ¼ 0.02). The BMS-806 mutations were more prevalent among non-B strains ( p ¼ 0.03), but no association was found between resistance-related mutations to ENF and subtype, or BMS-806 and ENF resistance and co-receptor usage. There was no association between viral load or HIV subtype and co-receptor usage. Table 3 summarizes the results of subtype and coreceptor usage versus the presence of BMS-806 and ENF mutation resistance. Discussion In this study, we have analyzed the frequency of individuals sensitive to three HIV entry inhibitors among patients failing treatment in an outpatient clinic in Sao Paulo city. There are few studies in Brazil that have comprehensively analyzed the envelope region to understand the rate of primary resistance to new envelope inhibitors. Sa´ Ferreira et al. evaluated 74 drug naive blood donors in Brazil, and found 1(1.3%) patient resis-

tant to ENF.22 Oliveira et al. evaluated 65 patients failing HAART therapy, but not exposed to ENF, and found a high prevalence of ENF resistance (7.6%), due to the presence of five samples harboring the G36D mutation.23 More recently, Teixeira et al. found 3.8% of samples with similar characteristics harboring either L44M or N43K mutations.24 In contrast to what has been detected in Oliveira et al., the frequency of ENF resistance was low in our study (1%), and G36D mutation was not found. This inconsistency may represent regional differences or study design. Our study was focused on one clinic in Sao Paulo and all patients were interviewed to assure no previous ENF exposure. In the Oliveira study, the sample inclusion criteria and geographic distribution are not clearly described. The previous treatment history seems to be based on the medical summary sent to the laboratory for routine pol genotype test, so previous exposure to ENF in the study population may be underestimated. Results are also contradictory in other areas of the world. For instance, Razollini et al. have not found any cases of Enfuvirtide resistance among patients undergoing pol genotyping test and naı¨ve to entry inhibitors in Italy.25 In contrast, Leung et al. found a high level of G36D mutation in naı¨ve patients from Hong Kong.26 Thus, understanding the discrepancies between the studies is important to have a clear view of primary resistance to this antiretroviral. Polymorphisms in the HR1 genomic region are common, and some of them were previously described to be associated with resistance.15,18,19 If those mutations prove to decrease the drug response and were taken into consideration, our prevalence of Enfuvirtide resistance would reach 8 (10%). Therefore, studies may be of importance to evaluate the impact of these polymorphisms in the efficacy of the drug; we may be underestimating sensitivity when using IAS consensus list criteria. The BMS-806 was discontinued in phase 2 clinical development due to low genetic barrier to resistance, but it may have potential use as a topical microbicide.20 No other study has evaluated the sensitivity of Brazilian strains to this drug. We could detect a clear association between non-B strain and drug resistance to BMS-806, and it would be wise to take this feature into consideration in case of approval of this drug. Most striking was the high prevalence of mutations A316T, I323V, and S405A in our set of samples. These mutations have been selected by Maraviroc in cell culture leading to a decrease of susceptibility to this drug.21 The impact of these mutations have been confirmed upon reverse mutation which

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ALENCAR ET AL. Table 2. Frequency of Mutation and Polymorphisms Detected in gp120 and gp41 of HIV-1

Mutation associated to ENF N (%) Mutation related to IAS N42T Other mutation N42D/H L44M R46M Other polymorphisms A30T R31K Q32L L34M N42R/S R46K E49D A50V Q52H H53Q/R L54M Q56K I62V Q66R I69L

1(1)

Mutation associated to BMS-806 Mutations M426 M434I

3 (4) 3 (4)

3 1 13 2 6 7 1 1 1 3 33 1 2 1 2

(4) (1) (16) (3) (8) (9) (1) (1) (1) (4) (41) (1) (3) (1) (3)

Other polymorphisms G410E/I/K/L/N/P/S/T/V/W S411A/D/E/G/K/L/N/P/T/V D412A/E/G/K/N/S/T/V T413I/L/N/P/Y I414V T415I/R/S/V L416I P417L/Q/S R419K/Q K421 R Q422R I423F I424V M426R/T/K Q428W K429E/G/Q V430T K432Q A433S/R A436N P437A/N I439S/V S440A/D/E/G/K/P/Q/R G441E K442E/H/I/K/L/M/N/P/R/S/V/Y I443L/V R444I/K/L/N/S/T/Y S446A/L/N/T/V N448I/K/S T450S

Table 3. Number of Samples with ENF, BMS-806, and Maraviroc Resistance ENF

a

p < 0.05.

N (%)

Mutations A316T I323V S405A

14 (17.5) 8 (10) 4 (5)

Other polymorphisms G312A P313W/R/V/L/F R315A/S/R F317M/I/L/W V318Y/W/H T319A I320T/E/A G321 E K322I/V

3 24 12 9 72 56 74 4 73

2 (3) 2 (3) 3 (4)

restored the wild type susceptibility to Maraviroc. Authors pointed out that mutations A316T and I323V are exceedingly rare in clade B viruses, accounting for only two out of 23,000 sequences in the (Los Alamos HIV sequence database).21

Co receptor CCR5 (n ¼ 56) CXCR4 (n ¼ 24) Subtypes Subtype B (n ¼ 73) Subtype Non-B (n ¼ 7) GPGR (n ¼ 48) GWGR and others (n ¼ 32)

N (%) Mutation associated to Maraviroc

BMS-806 Maraviroc

N (%) N (%) 5 (8.9) 4 (7.1) 3 (12.5) 2 (8.3) 8 (11) 0

A316T

N (%) N (%) 12 (21.4) 9 (16.7) 10 (41.67) 5 (20.8)

4 (5.5)a 20 (27.3) 13 (17.8) 2 (28.6)a 2 (28.5) 1 (14.3)

5 (10.4) 4 (8.33) 11 (22.92) 4 (8.3)a 3 (9.38) 2 (6.25) 11 (34.38) 10 (31.25)a

33 (41) 40 (50) 48 (60) 26 (33) 1 (1) 9 (11) 1 (1) 12 (15) 5 (6) 5 (6) 1 (1) 2 (3) 16 (20) 7 (9) 32 (40) 74 (93) 1 (1) 1 (1) 11 (14) 1 (1) 5 (6) 3 (4) 43 (54) 2 (3) 31(39) 2 (3) 31 (39) 13 (16) 3 (4) 1 (1)

(3.7) (30) (15) (11) (90) (70) (92.5) (5) (91.2)

Especially interesting was the association of A316T substitution and the Brazilian specific clade B strains. The presence of the tryptophan at the tip of the V3 loop is highly prevalent in Brazilian samples mainly detected during the first years of infection in a given individual.27 As disease progresses, TGG nucleotide triplet that encodes the tryptophan gives raise to other related amino acids, being more prevalent among individuals with low CD4 T cell counts.27 Conceivably, hypermutation processes may be related to the Tryptophan substitution by other related amino acids over time, since any G to A substitutions will incur in stop codons in the TGG triplet. Therefore, we grouped in our analysis sequences from the so-called GWGR lineage, which is extremely common in Brazil and rare elsewhere. Therefore, the phenotypical impact of these mutations detected here in Maraviroc susceptibility needs to be confirmed, and a resistance test may be required together with the tropism determination before initiating therapy with this drug. In conclusion, we analyzed the envelope gene of individuals failing antiretroviral therapy in Brazil and these results may be of impact to guidelines for genotyping test and treatment in Brazil.

PRIMARY RESISTANCE TO ENTRY INHIBITOR Sequence Data The HIV-1 env sequences used in this study were deposited in GenBank under accession numbers GU980491-GU980570. Author Disclosure Statement The authors have no competing conflicts of interest to declare. References 1. Van Laethem K, Schrooten Y, Lemey P, et al.: A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene. J Virol Methods 2005;123:25–34. 2. Wang T, Zhang Z, Wallace OB, et al.: Discovery of 4-benzoyl1-[(4-methoxy-1H- pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)methylpiperazine (BMS-378806): A novel HIV-1 attachment inhibitor that interferes with CD4-gp120 interactions. J Med Chem 2003;46:4236–4239. 3. Chinnadurai R, Rajan D, Munch J, Kirchhoff F: Human immunodeficiency virus type 1 variants resistant to first- and second-version fusion inhibitors and cytopathic in ex vivo human lymphoid tissue. J Virol 2007;81:6563–6572. 4. Bar S, Alizon M: Role of the ectodomain of the gp41 transmembrane envelope protein of human immunodeficiency virus type 1 in late steps of the membrane fusion process. J Virol 2004;78:811–820. 5. Steffen I, Pohlmann S: Peptide-based inhibitors of the HIV envelope protein and other Class I viral fusion proteins. Curr Pharm Des 2010;16:1143–1158. 6. Lu J, Sista P, Giguel F, Greenberg M, Kuritzkes DR: Relative replicative fitness of human immunodeficiency virus type 1 mutants resistant to enfuvirtide (T-20). J Virol 2004;78:4628– 4637. 7. Menzo S, Castagna A, Monachetti A, et al.: Genotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term treatment. Antimicrob Agents Chemother 2004;48:3253–3259. 8. Nunn AS, Fonseca EM, Bastos FI, Gruskin S, Salomon JA: Evolution of antiretroviral drug costs in Brazil in the context of free and universal access to AIDS treatment. PLoS Med Nov 13 2007;4:e305. 9. Johnson VA, Brun–Vezinet F, Clotet B, et al.: Update of the drug resistance mutations in HIV-1: Spring 2008. Top HIV Med 2008;16:62–68. 10. Hughes A, Nelson M: HIV entry: New insights and implications for patient management. Curr Opin Infect Dis 2009;22:35–42. 11. Harrigan PR MR, Dong W, et al.: Screening for HIV tropism using population-based V3 genotypic analysis: A retrospective virological outcome analysis using stored plasma screening samples from MOTIVATE-1. XVIII International Drug Resistance Fort Myers, Florida.; 2009:June 9–13, 2009. 12. Guo Q, Ho HT, Dicker I, et al.: Biochemical and genetic characterizations of a novel human immunodeficiency virus type 1 inhibitor that blocks gp120-CD4 interactions. J Virol 2003;77:10528–10536. 13. Madani N, Perdigoto AL, Srinivasan K, et al.: Localized changes in the gp120 envelope glycoprotein confer resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155. J Virol 2004;78:3742–3752. 14. Delwart EL, Shpaer EG, Louwagie J, et al.: Genetic relationships determined by a DNA heteroduplex mobility assay: Analysis of HIV-1 env genes. Science 1993;262:1257–1261.

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Address correspondence to: Ester Cerdeira Sabino, M.D., Ph.D. Fundac¸a˜o Pro´-Sangue Hemocentro Sa˜o Paulo Brazil Sa˜o Paulo–SP 05403-000 E-mail: [email protected]