Detection of Hepatitis C Mutants With Natural Resistance to NS3/4A Protease Inhibitors in HIV/HCV-Coinfected Individuals Treated With Antiretroviral Therapy

To the Editor: A number of hepatitis C virus (HCV) protease inhibitors (PIs) are in the pipeline and available data1-5 seem encouraging for Telaprevir (WX-950), Bocepravir (SCH-503034) SCH-6, and ITMN-191. Combination therapy of specific HCV NS3/NS4 PIs with pegylated interferon alpha and ribavirin can synergically inhibit HCV RNA replication, thus providing a rationale for future combination therapy in HCV-monoinfected persons as in HIV/HCV-double infected persons. Developing newer anti-HCV antivirals is urgent in HIV/HCV-infected individuals because these patients show a high rate of resistance to standard treatment with interferon and ribavirin,6 and a faster progression of the HCV-related liver disease.7 We Evaluated the frequency of NS3 protease mutations in the natural strains of 37 HIV/HCV-coinfected individuals (harboring HCV genotype 1) that were compared with 250 sequences (all assigned to HCV genotype 1) retrieved from the GenBank database. Among the HIV-infected patients, 31 were males and 6 were females, the median age was 40.3 years, interquartile range 37.1-46.2. The main risk factor for HIV infection was the intravenous drug use in 24 individuals and the sexual contact in 2 patients, whereas in 11 subjects the risk factor was unknown. Twenty-eight patients were infected with HCV genotype 1a, 8 with genotype 1b, and the remaining patient with genotype 1c. Eighteen patients were under antiretroviral therapy (ART) including PI (fosamprenavir boosted by ritonavir in 3 patients, nelfinavir in 2 patients, lopinavir boosted by ritonavir in 9 patients, saquinavir in 3 patients, and indinavir in 1 patient) and 19 under ART regimen sparing PI, since at least 6 months before HCV protease analysis was performed in plasma samples. All patients were naive to standard treatment for chronic hepatitis C. The NS3 protease domain was amplified by reverse nested polymerase chain reaction. Polymerase chain reaction products were directly sequenced and multiple alignment of nucleotides and deduced amino acid sequences was inferred by Clustal_X, version 1.64b. Fisher exact test was performed to compare the frequency of mutations in the sequences obtained from HIV-infected persons and GenBank control group. Wilcoxon rank sum test was used to evaluate the distribution of clinical and virologic variables between HIV-infected individuals with or without amino acid mutations in the protease domain. Six of 37 sequences obtained from HIV-infected individuals and 2 of 250 sequences retrieved from GenBank database showed natural amino acid polymorphism at positions associated with anti-HCV PIs resistance. The proportion of mutations between these 2 groups was statistically significant, P < 0.0001. Interestingly, amino acid mutations detected in HIV-infected persons were not invariably the same as those identified by in vitro and clinical studies with HCV NS3-4A PIs.8 Three of 18 patients taking ART including PI showed amino acid mutation at positions that confer resistance to PIs: patient 1 infected with HCV genotype 1a (under ART including PI, indinavir) exhibited a natural polymorphism at 3 different positions, R155K, A156T, and V170F; patient 2 infected with genotype 1a (under ART including PI, lopinavir boosted by ritonavir) carried R155K mutation that is associated with decreased sensitivity to PIs; and patient 3 infected with genotype 1b (under ART including PI, saquinavir) had amino acid substitution A156T. Three of 19 subjects under ART without PI and infected with HCV genotype 1a showed amino acid variation at position 170 (V170E, V170Y, and V170T, respectively) of the protease domain. The proportion of mutations was not different between patients under ART including or not PI, P = 0.340. However, amino acid substitution in the sequences of 2 individuals (patient 1 and patient 3) taking ART including PI was located in a site of primary resistance to boceprevir and telaprevir, whereas in the individuals taking ART sparing PI, amino acid mutation was in a site (V170) with low-level resistance to these 2 compounds. The NS3 catalytic triad was highly conserved among all the sequences analyzed. Additional amino acid mutations that were not located in sites associated with compensatory mutations or enhanced replication capacity were found along the protease domain of some HIV-infected patients. Comparison between 6 patients with HCV protease mutations and 31 without amino acid variation in sites of resistance to HCV inhibitors did not reveal any difference concerning the variables included in the analysis (age, CD4+ cells count, alanine amino transferase, aspartate amino transferase, HCV RNA and HIV RNA load, Table 1).TABLE 1: Clinical and Immunovirological Characteristics of HIV-Infected Individuals Showing or not a Variations in the HCV NS3 Protease Associated With Resistance to Investigative HCV NS3/4 PIsRecently, Lopez-Labrador et al9 performed an interesting study on the natural polymorphism of the protease domain in isolates probably belonging to HIV-negative individuals and retrieved from the European and US HCV sequence databases, showing a high degree of natural polymorphism in genotype 1 proteases, and few major variations associated with primary resistance to anti-HCV PIs. Accordingly, we found in the GenBank database only 2 of 250 sequences exhibiting amino acid mutation at position 156 that is a site of primary resistance. An other recent study by Halfon et al10 did not show any mutation at positions associated with anti-HCV PIs in the natural strains of 17 HIV-infected persons under ART including PI, as in a small control group of HIV-negative individuals. Conversely, we found that 6 of 37 natural protease sequences of HIV-infected individuals under ART including or not PI exhibited inhibitor-resistant mutants. We studied a similar group of individuals with PI-based ART, in term of proportion of HCV infecting genotype (13 genotype 1a, 4 genotype 1b, and 1 genotype 1c in our study), of those analyzed by Halfon et al.10 Indeed, we evaluated a group of patients taking ART without PI and compared sequences of HIV-infected patients with 250 sequences retrieved from GenBank database. Furthermore, the full NS3 protease domain was analyzed in the present study. The identification of naturally occurring variations that may modify inhibitor binding is an important issue in the design of broad-spectrum PIs. Thus, further studies will be important to determine the prevalence of resistant strains as dominant or minor viral populations in HIV/HCV-coinfected individuals because HIV/HCV coinfection, at least in patients under ART, seems to be more prone to induce HCV protease polymorphisms with respect to HCV monoinfection. Giulia Morsica, MD* Sabrina Bagaglio, PhD* Caterina Uberti-Foppa, MD* Laura Galli, MsC* Adriano Lazzarin, MD*† *Infectious Diseases Department, San Raffaele Scientific Institute, Milan, Italy †Vita-Salute San Raffaele University, Milan, Italy

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