The HIV envelope glycoproteins, gp120 (surface) and gp41 (transmembrane), play an important role during viral infection and pathogenesis.1,2 gp120 recognizes and interacts with the CD4 receptor and with chemokine coreceptors (CCR-5 or CXCR-4).3,4 This interaction promotes conformational changes in gp120 and gp41, causing exposure of the gp41 fusion peptide, leading to fusion between the viral membrane and the host cell and release of viral genetic material into the cell cytoplasm.5,6 In its extracellular domain, gp41 has two N-terminal α-helical motifs, similar to leucine zippers, composed by heptad repeat regions, HR1 and HR2, which have an important role during the conformational changes that result in viral entry.7,8
Given the large genetic diversity of HIV-1 and the development of resistance mutations in patients undergoing highly active antiretroviral therapy, the use of novel drug classes for rescue therapy is necessary.9 Enfuvirtide (ENF, T20 or Fuzeon, Hoffmann-La Roche, Inc.) is a 36 amino acid synthetic peptide derived from the HR2 region of gp41.10 It blocks fusion with the host cell membrane through competitive binding with HR2.11 In 2005, Brazil included ENF in rescue therapy for patients resistant to protease and reverse transcriptase inhibitors.
Resistance can be quickly developed in patients receiving ENF rescue therapy and is often associated with mutations on the tripeptide motifs involving 36, 37, and 38 residues located on the HR1 domain of gp41.12 Secondary resistance usually involves mutations between the 36 and 45 residues,12 including G36D/E, I37V, V38A/M/E, Q40H, N42T, and N43D.13 Some compensatory mutations in the HR2 region such as N126K, E137K, and S138A were described in patients in ENF treatment and were involved in the establishment of HR1-HR2 interaction.14
Some Brazilian studies highlight the importance of gp41 genotyping for the evaluation of resistance mutations. Oliveira et al15 found a 7.69% prevalence of major mutations to ENF among multidrug-resistant ENF-untreated patients. Cardoso and Stefani16 identified resistance mutations in three samples from 14 multidrug-resistant patients, which included two in ENF treatment. DNA and RNA sequence analysis of ENF-untreated patients showed resistance mutations in 3.8% of DNA samples and 4.6% of RNA samples.17
A precise understanding of resistant strains and the impact of these mutations in HIV-1-infected individuals is important for the response to AIDS. This study compared genotypic differences of the gp41 HR1 region from isolates of patients undergoing treatment or untreated with ENF according to subtype, resistance profile, susceptibility-related mutations, and available clinical data (viral load).
The study analyzed 28 samples from HIV-positive patients, including 16 treated with ENF and 12 untreated with ENF (six ENF treatment-naive and six highly active antiretroviral therapy-naive). The samples were obtained from Rio de Janeiro, Rio de Janeiro state, and Porto Alegre, Rio Grande do Sul state, between 2006 and 2009.
Genomic DNA samples were extracted from peripheral blood mononuclear cells by “QIAmp DNA Blood Mini Kit” (QIAgen, Hilden, Germany) according to the manufacturer's protocol. Amplification of the gp41 gene (773 base pairs, HXB2 7638-8411) was done through nested polymerase chain reaction using specific primers to the HR1 and HR2 conserved domains. The polymerase chain reaction products were purified in a Microcon (Millipore Corp, Billerica, MA) microspin column and sequenced in an ABI 3100 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA) with BigDye Terminator Cycle Sequencing Reaction Kits (Version 3.1).
Electropherogram sequences obtained were analyzed and edited manually with SeqMan software (DNAStar, Madison, WI) using HXB2 as a reference sequence for HIV-1 subtype B. Fragments of 490 base pairs, corresponding to the fusion domain peptide and gp41 HR1, were aligned with reference sequences from the Los Alamos database (www.hiv.lanl.gov) using the ClustalW2 program.18 Sample subtypes were determined by phylogenetic analysis of the gp41 region (neighbor-joining with Kimura's two-parameter correction, Mega 3.1 package).19
The research project was approved by the Research Ethics Committee of the Evandro Chagas Clinical Research Institute-FIOCRUZ.
Figure 1 depicts the subtypes of the 28 patient samples analyzed for the gp41 gene: 23 were subtype B (82%), four were subtype C (14.3%), and one was subtype F1 (3.7%).
The HR1 region had diverse polymorphisms unrelated to resistance, as shown in Figure 2. Among these polymorphisms, L54M and K77Q/R/G were the most frequent (53.5% of total samples) (Fig 2). Of the 12 ENF-untreated patients, four (33%) belonged to subtype C and had the N42S polymorphism. Sample RS08188, from the untreated group of patients, had the G36D major resistance mutation (Table 1) in addition to diverse polymorphisms. Sample RJ09619, also from the untreated group, had the L44M accessory mutation. The other 16 isolates had major mutations between gp41 amino acid residues 36 and 45 with V38A as the most prevalent (three isolates from subtype B). Among these three cases, sample RS08084 had the V38A mutation in combination with N42D, whereas isolate RJ09479 had the G36D mutation. Sample RJ09357, with subtype F1, had a combination of G36E + N42S. The accessory mutations V38M and L44M were found in samples RS08074 and RJ09416, respectively, which belong to subtype B (Table 1). Table 2 corresponds to sample ID, its collection date, country of origin and GenBank accession number.
The final viral load levels of patients treated or untreated with ENF showed no difference between the two groups (P = 0.675). Nonetheless, a statistically significant difference was encountered between ENF-treated patients and the presence or not of resistance mutations (P = 0.051) (data not shown).
Subtype analysis showed a high frequency of subtype B (82%), confirming it as the main strain circulating in Brazilian states.15,20,21 All subtype C samples (RS08053, RS08170, RS08171, and RS08224) (14.3% of the total) were from the city of Porto Alegre, Rio Grande do Sul. According to Brindeiro et al,21 the prevalence of subtype C reaches 30% in the south and southeast regions of Brazil. According to Soares et al,20 the prevalence is 37% in Porto Alegre, where subtype C was first detected in Brazil. Only one sample belonging to subtype F1 (RJ09357) was obtained from Rio de Janeiro state, where a 10% subtype frequency has been observed.15,21
Genotypic analysis also revealed a high prevalence of polymorphisms unrelated to ENF resistance in the gp41 region. The L54M polymorphism was the most common (53.5%) followed by K77Q/R/G. The R46M and V69I polymorphisms were unrelated to increased drug susceptibility in a study of patients on ENF monotherapy.22 The N42S polymorphism related to ENF hypersusceptibility23,24 was found in approximately 16% of subtype B virus untreated with ENF23; this polymorphism was observed in 33% of isolates from untreated patients (17% of the total) in subtype C. The N42S mutation is considered a natural polymorphism of gp41 in HIV-1 subtype C, and its actual impact in relation to ENF susceptibility is still unknown. The G36D major resistance mutation was observed in the group of untreated patients (RS08188), and just this substitution causes a 10-fold decreased susceptibility to ENF.25 Sample RJ08619 had a L44M mutation, also identified in samples by Teixeira et al.17
Half of isolates from the patient group in ENF treatment showed primary and secondary mutations. Nonetheless, the appearance of resistance mutations was unrelated to duration of drug use, because average duration of ENF use did not differ between resistant and nonresistant patients. Interestingly, half of the study patients who experienced ENF treatment failure did not show any resistance mutations. According to Cabrera et al,26 resistance mutations can be detected within 4 weeks of treatment initiation. Mutations in the V38 locus were the most frequent. The V38A mutation was observed in three of eight isolates, and V38M was identified in sample RS08074. V38A is strongly associated with susceptibility to ENF.25,27 In sample RS08084, the V38A mutation appeared in combination with N42D. According to Greenberg et al,25 this combination experiences a 140-fold reduction in drug susceptibility, although when N42D appears as a sole mutation (as seen in sample RJ09202), there is no relationship to resistance. Mutations in samples from subtype B such as in G36D (RJ09479) and L45M (RJ09416) do not confer resistance as single base substitutions, according to Menzo et al.27 Sample RJ09357, from subtype F1, had a combination of G36E + N42S. The N42S polymorphism is most frequently detected in non-B subtypes,28 and, in our study, it was found in untreated samples from subtype C, which is the most prevalent non-B subtype in Brazil. Therefore, it is very important to analyze the actual impact of ENF resistance mutations on drug susceptibility, already described for subtype B and present in circulating non-B virus in Brazil. This need can be achieved through in vitro phenotypic testing that can provide a quick and precise answer. No statistically significant difference was observed in the viral load (RNA copies/mL) of ENF-treated and untreated patients. This unexpected finding may be explained by low adherence to ENF treatment, because administration is by subcutaneous injection twice per day. This is inconvenient for the patient, in addition to eventual adverse events, principally injection site reaction.
The number of Brazilian patients using ENF reached 21,946 in 2008 and 23,035 in 2009 (www.aids.gov.br). With the introduction of integrase inhibitors and darunavir, ENF is being discontinued and patients experiencing ENF treatment failure are changing their therapeutic regimen. Our data show that ENF failure may also be associated with low adherence for a large part of cases.
In conclusion, the study results show that phenotypic analysis for viral resistance is necessary to understand the role of single and combined substitutions as well as to investigate the level of resistance conferred by mutations.
The authors thank the technicians of the Laboratory of Molecular Virology of Rio de Janeiro Federal University, Mônica Arruda and Lidia Boullosa, for their help with DNA sequencing and José Boullosa for assistance with data analysis.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
HIV-1; gp41; enfuvirtide; antiviral drug resistance