Mutation D30N Is Not Preferentially Selected by Human Immunodeficiency Virus Type 1 Subtype C in the Development of Resistance to Nelfinavir

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2004, p Vol. 48, No /04/$ DOI: /AAC Copyright 2004, American Society for Microbiology. All Rights Reserved. Mutation D30N Is Not Preferentially Selected by Human Immunodeficiency Virus Type 1 Subtype C in the Development of Resistance to Nelfinavir Zehava Grossman, 1 * Ellen E. Paxinos, 2 Diana Averbuch, 3 Shlomo Maayan, 3 Neil T. Parkin, 2 Dan Engelhard, 3 Margalit Lorber, 4 Valery Istomin, 5 Yael Shaked, 1 Ella Mendelson, 1 Daniela Ram, 1 Chris J. Petropoulos, 2 and Jonathan M. Schapiro 6 National HIV Reference Center, Central Virology Laboratory, Public Health Laboratories, Tel Hashomer, 1 Hadassah Medical Center, Jerusalem, 3 Rambam Medical Center, Haifa, 4 Hillel-Jaffe Medical Center, Hadera, 5 and National Hemophilia Center, Tel Hashomer, 6 Israel, and ViroLogic, South San Francisco, California 2 Received 14 October 2003/Returned for modification 18 December 2003/Accepted 3 February 2004 Differences in baseline polymorphisms between subtypes may result in development of diverse mutational pathways during antiretroviral treatment. We compared drug resistance in patients with human immunodeficiency virus subtype C (referred to herein as subtype-c-infected patients ) versus subtype-b-infected patients following protease inhibitor (PI) therapy. Genotype, phenotype, and replication capacity (Phenosense; Virologic) were determined. We evaluated 159 subtype-c- and 65 subtype-b-infected patients failing first PI treatment. Following nelfinavir treatment, the unique nelfinavir mutation D30N was substantially less frequent in C (7%) than in B (23%; P 0.03) while L90M was similar (P < 0.5). Significant differences were found in the rates of M36I (98 and 36%), L63P (35 and 59%), A71V (3 and 32%), V77I (0 and 36%), and I93L (91 and 32%) ( < P < 0.05) in C and B, respectively. Other mutations were L10I/V, K20R, M46I, V82A/I, I84V, N88D, and N88S. Subtype C samples with mutation D30N showed a 50% inhibitory concentration (IC 50 ) change in susceptibility to nelfinavir only. Other mutations increased IC 50 correlates to all PIs. Following accumulation of mutations, replication capacity of the C virus was reduced from 43% 22% to 22% 15% (P 0.04). We confirmed the selective nature of the D30N mutation in C, and the broader cross-resistance of other common protease inhibitor mutations. The rates at which these mutational pathways develop differ in C and subtype-b-infected patients failing therapy, possibly due to the differential impact of baseline polymorphisms. Because mutation D30N is not preferentially selected in nelfinavir-treated subtype-c-infected patients, as it is in those infected with subtype B, the consideration of using this drug initially to preserve future protease inhibitor options is less relevant for subtype-c-infected patients. Different subtypes of human immunodeficiency virus type 1 (HIV-1) possess distinct patterns of consensus amino acid sequences in viral proteins, including the protease, a highly polymorphic and flexible enzyme (10) Nearly 47% of the 99 protease amino acids can vary naturally in wild-type viruses (both within and between subtypes) (14). Mutations at 45 amino acid positions have been associated with resistance to one or more of the six presently used protease inhibitors (PIs) (8, 17, 18, 31). Mutations at nine amino acid positions have been commonly designated primary or major resistance mutations (D30N, V32I, M46I/L, G48V, I50V, I54L/M/V, V82A/F/S/T, I84A/V, N88S, and L90M) (11, 31). Other resistance mutations considered of a lesser significance are defined as secondary or minor. Although none of the primary mutations occur as polymorphisms in wild-type HIV-1, several secondary mutations contributing to reduced susceptibility (i.e., M36I and I93L) are found in nearly 100% of subtype C virus from drug-naive patients (5, 10). Upon antiretroviral treatment, such differences in baseline polymorphisms among subtypes may result in * Corresponding author. Mailing address: Central Virology Laboratory, Sheba Medical Center, Tel Hashomer 52621, Israel. Phone: Fax: lcgross@inter.net.il. the evolution of drug resistance along distinct mutational pathways, or in differences in the incidence of these specific pathways (1, 4, 9, 12, 16, 25, 28, 29). These genetic differences may be clinically relevant when considering long-term treatment strategies for patients infected with different subtypes. In this study, we document the emergence of drug resistance following PI therapy in a large cohort of patients infected with HIV-1 subtype C (referred to herein as subtype-c-infected patients ) and compare these patients with a cohort of subtype- B-infected patients treated in the same clinics and to the large Stanford public database (13, 24). MATERIALS AND METHODS Clinical specimens and database. Genotype testing was performed in accordance to treating-physicians requests. Samples were collected by seven HIV treatment centers located throughout Israel. Levels of HIV RNA in plasma and CD4 -T-cell counts were determined by the local treatment center laboratory. Demographic information, detailed antiretroviral treatment history, current and past CD4 counts, and HIV RNA viral load measurements were provided along with the samples on standardized forms and were stored in an anonymous database at the National HIV Reference Center. Samples were genotypically characterized at the National HIV Reference Center. Patients were considered to be failing treatment when plasma HIV RNA concentration was higher than 1,000 copies per ml while on antiretroviral treatment for at least 6 months, including at least 1 month on PI. All successfully analyzed subtype B and C 2159

2 2160 GROSSMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Summary of patients failing their first PI treatment (at genotype testing) Subtype No. (%) of patients failing treatment with first PI Nelfinavir Indinavir Saquinavir Ritonavir Lopinavir Amprenavir B(n 65) 22 (34) 24 (37) 6 (9) 2 (3) 10 (15) 1 (3) C(n 159) 80 (50) 56 (34) 11 (7) 5 (3) 6 (4) 1 (1) Total (n 224) 102 (46) 80 (36) 17 (8) 7 (3) 16 (7) 2 (1) samples from PI failing patients submitted between August 1999 and June 2003 were included in the analyses. HIV-1 RNA extraction, sequencing and subtyping. Viral RNA was isolated from patient plasma samples using the QIAamp kit (Qiagen, Hilden, Germany) according to manufacturer s instructions. The protease gene was sequenced (codons 4 to 99) using the Open Gene system and the TruGene HIV-1 Genotyping kit and prototype 1.5 RT-PCR Primers (research use only; Visible Genetics Inc., Toronto, Canada) as previously described (10). Sequences from 26 of 330 subtype C and 2 of 154 subtype B viruses were also independently verified using ViroLogic s GeneSeq HIV assay. Classification of samples into subtypes was done by peptide subtyping of the envelope region (6) and/or by comparing the polymerase sequence to consensus sequences in the Stanford database (13, 24). Drug susceptibility and replication capacity determination. Drug susceptibility and replication capacity were measured using a cell based, single replication cycle assay (PhenoSense HIV; ViroLogic). In this assay, drug susceptibility is measured using HIV resistance test-vector libraries (23). The PhenoSense drug susceptibility test has also been adapted to measure the replication capacity (RC) of recombinant test viruses. In this RC assay, the total amount of luciferase activity expressed in infected cells in the absence of drug represents a measure of the RC of the virus. Viruses with high RC generate high levels of luciferase activity, while viruses with low RC generate low levels of luciferase activity. Relative measures of RC can be derived by comparing the total amount of luciferase generated by two or more viruses. Replication capacity measurements are reported as a percentage of the mean RC of wild-type viruses, that is, those that possess no genotypic or phenotypic drug resistance. Replication capacity measurements are normalized for differences in transfection efficiencies by monitoring the luciferase activity generated in transfected cells, which is independent of viral enzyme function. Statistical analysis. Clinical data and differences in mutation frequencies were compared and analyzed using the chi-square test, Fisher s two-tailed exact tests, the Mann-Whitney test, and logistic regression. Nucleotide sequence accession numbers. GenBank accession numbers for protease sequences included in this study are AY to AY RESULTS Sample analysis. Sequences were obtained from a total of 542 patients failing a PI containing regimen. These included 168 subtype-b-infected and 374 subtype-c-infected patients. Of these, 65 subtype-b-infected and 159 subtype-c-infected samples were patients failing their first PI regimen. The common PIs used as first regimen for this cohort of patients were nelfinavir and indinavir (Table 1). All subtype C and B samples from patients failing nelfinavir were compared to those failing indinavir as their only PI (total of 46 and 136 samples, for subtypes B and C, respectively; Table 1). Patients and treatments. Subtype-B and subtype-c-infected patients were treated at the same clinics and by the same physicians. Overall usage of antiretroviral drugs was similar in the two groups. Nelfinavir and indinavir were commonly used as part of combination regimens, and one of these two drugs was often introduced as the first PI (Table 1). Several demographic and clinical characteristics varied between subtype-band subtype-c-infected patients. 76% of patients infected with subtype B were male, compared to 52.2% of patients infected with subtype C. Additionally, there were only five children (4.3%) in the group of subtype-b-infected patients, while 38 subtype-c-infected patients (17%) were younger than 13. Further, the primary risk factors for HIV infection differed between the two groups. Specifically, risk factors for HIV subtype B transmission included men having sex with men (42.7%), intravenous drug users (12.8%), heterosexual relations (26.5%), and receipt of blood transfusion (10.3%). In contrast, subtype-c-infected individuals acquired HIV infection either through heterosexual relations (83%) or mother to child transmission (17%). While adults received nelfinavir and indinavir in similar proportions (P 0.4), all children but one were treated with nelfinavir as the first PI (P ). Consequently, we genotyped a higher percentage of subtype-c-infected patients failing nelfinavir as their first PI (21%) than subtype B-infected patients failing nelfinavir as their first PI (13%; P 0.044; Table 1). The specific data concerning first PI failure patients after receiving nelfinavir or indinavir are summarized in Table 2. Mutations and polymorphisms. The frequency at which specific mutations emerged following initial PI therapy with nelfinavir or with indinavir varied in subtype-b-infected patients versus subtype-c-infected patients. As the number of samples from subtype-b-infected patients failing first regimens was relatively small, we compared our data from subtype-c-infected patients also to the large public Stanford database (13, 24), having first verified that there were no major differences in the frequencies of resistance-related mutations among the Israeli subtype-b-infected patients and those documented in the Stanford pool (Table 3). In the comparison between subtypes B and C, some of the differences that were evident but not statistically significant when the subtype C data were compared with the small set of Israeli subtype B data became significant on comparing with the larger set (Table 3). Most notably, among patients failing initial nelfinavir therapy, the D30N mutation emerged significantly less frequently in subtype-c-infected patients than in subtype-b-infected patients (P 0.03). This was also the case for L10I/V (P 0.03), L63P (P 0.05), A71V (P 0.001), and V77I (P 0.005). The L10I/V, I54V, and V82A mutations were more common in subtype-b-infected patients failing first indinavir therapy than in subtype-c-infected patients only when compared to the large Stanford database (P 0.03, 0.03, and 0.004, respectively). Further, as M36I and I93L are the consensus amino acid for HIV subtype C viruses, they were more common in C than in B, irrespective of PI therapy (P 0.001). The frequencies of I84V, N88S, and L90M were not significantly different between subtypes in patients failing either treatment (chi-square test and Fisher s two-tailed exact tests). Subtype-B- and subtype-c-infected patients were compared

3 VOL. 48, 2004 MUTATION D30N IS SELDOM SELECTED BY HIV-1 SUBTYPE C 2161 d treatment duration until failure (test time). a Demographic and clinical data of patients treated with nelfinavir or indinavir as their only PI. Age and viral load data were taken at test time; CD4 counts were determined at test time or within 3 months prior to the test. Risk groups: blood, receipt of blood transfusion; heterosexual, heterosexual relations; IVDU, intravenous drug user; MSM, men having sex with men; MTCT, mother-to-child transfer. b P given in the column for males applies to genders taken together. c P given in the column for MSM applies to risk groups taken together. P for: Subtype B vs subtype C Nelfinavir b < c Indinavir Nelfinavir vs indinavir Subtype B Subtype C Subtype C Nelfinavir (n 80) 33 (2 72) , ( ) 344 (12 1,188) 18.5 (3 52) Indinavir (n 56) 37 (5 67) , ( ) 189 (29 872) 23.8 (1 69) Subtype B Nelfinavir (n 22) 36 (9 55) , ( ) 284 (31 596) 19.1 (4 47) Indinavir (n 24) 40 (28 63) , ( ) 267 (5 900) 34.4 (4 83) Patient group and treatment Median age (yr) (range) Male Female MSM Heterosexual Blood MTCT IVDU Median (copies/ml) Log 10 (range) Median no. of CD4 cells/ l range Median mo on PI d (range) % Gender % Risk group Viral load TABLE 2. Clinical data at first failure a for incidence of the D30N mutation using logistic regression to adjust for differences in viral load, CD4 count, time of treatment with nelfinavir, age and sex. A significant difference between subtype-b- and subtype-c-infected patients was found in the incidence of the D30N mutation (P 0.03). Drug susceptibility and replication capacity. Subsets of samples from both drug-naive and treated subtype-c-infected patients were examined for PI drug susceptibility (change in IC 50 ) and RC (Table 4). In samples containing the D30N mutation, large reductions in susceptibility to nelfinavir but not to other PIs were seen (e.g., patients and [Table 4]). Samples containing L90M, when it was present in combination with additional mutations (e.g., M46I, I54V, A71V, V82A, and/or I84V; Table 4), demonstrated reduced susceptibility to several PIs. RC relative to a subtype B reference strain (NL4-3) was reduced in drug-experienced patients, although variability within each group was high (22% 15% versus 43% 22% for drug-naive patients; P 0.04; Table 4). We have compared the RC of the subtype C viruses harboring mutation D30N or L90M with or without additional mutations to genotypically similar subtype B samples from the ViroLogic database. All the raw RC values were adjusted to account for the difference between the reference virus (NL4-3) and an average wild-type clade B virus (using the same correction factor for clade C). We did not find obvious differences in relative RC between the D30N and L90M groups either in clade B or in clade C. Both groups appear to possess significantly lower RC compared to wild type (data not shown). DISCUSSION We have compared the frequencies at which individual drug resistance mutations were selected in subtype-b- and subtype- C-infected patients failing their first PI regimen containing nelfinavir or indinavir. We found that mutation D30N was selected at a much lower rate in subtype-c-infected patients relative to subtype-b-infected patients, while other major mutations were selected at a similar rate in both subtypes. The frequency of other known PI mutations also varied between the subtypes. Similar findings regarding the pattern of mutations in persons failing nelfinavir therapy have been observed in a smaller number of patients infected with subtypes A (30), AE (1), C (5), and G (P. Gomes, I. Diogo, M. F. Gonçalves, P. Carvalho, J. Cabanas, et al., 9th Conf. Retroviruses and Opportun. Infect., abstr. 46, 2002) viruses. These studies strengthen one another because none is corrected for multiple comparisons (examining each residue in protease). Subtype-Cinfected patients harboring mutation D30N showed reduced susceptibility to nelfinavir but not to other PIs, whereas other primary mutations (i.e., L90M) showed broader cross-resistance. A broad range of RC values was found in subtype-cinfected patients, and the presence of a major PI mutation was associated with reduced RC. This study has a number of limitations. Patients were not randomized into the different treatments. Additionally, subtype-c- and -B-infected populations in Israel differ in a number of socioeconomic characteristics that may impact drug taking behavior. Nevertheless, this does not appear to be a significant confounding factor here for several reasons. (i) Data for this study were collected from multiple centers treat-

4 2162 GROSSMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Protease resistance mutations at first failure a Nelfinavir Indinavir Mutation BSt (n 297) % Resistance in subtype P % Resistance in subtype P (n 22) (n 80) vs BSt (n 381) (n 24) (n 56) Primary and major D30N M46I I54V V82A I84V N88D N88S L90M vs Minor L10I/V K20R L63P A71V V77I V82I Subtype C specific M36I I93L a Protease resistance mutations at first failure following nelfinavir or indinavir treatment. Primary and major mutations are indicated. In order to strengthen the comparison, Israeli subtype C data (Is) were compared also to subtype B data available from the public Stanford (St) database (16, 27). ing patients infected with either subtype who live in different geographic locations throughout the country. Local effects of adherence would thus be minimized. (ii) Despite years of nelfinavir therapy in the non-subtype-b populations, there have been no reports of specific adherence problems or unique toxicity issues with nelfinavir in these populations. (iii) Preliminary reports of similar findings in other populations (Japanese and Portuguese) (1; Gomes et al., 9th Conf. Retroviruses Opportun. Infect) that are very different socially from Ethiopian Israelis and are unlikely to share adherence patterns suggest an effect of the common baseline mutations rather than of drug taking behavior. (iv) Such an effect of baseline non-subtype-b mutations on efficiency and stability of the enzyme, and hence on resistance development, is supported by biochemical studies in these patients (28, 29). Susceptibility assays and RC assays were not designed specifically for subtype C viruses and used a standard subtype-b-based vector. Incorporating a subtype C virus sequence into a subtype B vector backbone may influence viral replication and impact the results. Additionally, as replication capacity assays were applied to a fragment of pol including both PR and RT regions, RT mutations may also affect RC measurements. Although these potential pitfalls must be kept in mind, our preliminary studies and those of others (1, 5, 30; Gomes et al., 9th Conf. Retroviruses Opportun. Infect.) showing an impact of viral subtype on nelfinavir resistance pathways suggest that such issues of subtype dependency should be formally addressed in controlled clinical trials. Resistance is one of several factors to consider in selecting the optimal drug regimen for an individual patient. Mutation D30N is a primary nelfinavir resistance mutation and appears to be specific to this inhibitor (19). In contrast, L90M, like other major protease mutations (e.g., V82A and I84V) is a primary mutation involved in resistance to several PIs including nelfinavir, indinavir, and saquinavir (3, 11, 19, 27). These two mutations rarely appear together on the same isolate (26). In the majority of subtype-b-infected patients developing resistance after failing nelfinavir as their first PI, mutation D30N is selected, while mutation L90M or other primary mutations are selected in a significantly smaller proportion (7, 19). Since mutation D30N does not engender resistance to PIs other than nelfinavir, patients failing nelfinavir will commonly retain susceptibility to other drugs of this class. This is to be taken into consideration when choosing the initial PI. In our study, we found that the D30N mutation was selected far less frequently in subtype-c-infected than in subtype-subtype-b-infected patients who failed nelfinavir as the first PI, while other key PI mutations such as L90M were seen more frequently. Susceptibility assays confirmed the selective resistance pattern of mutation D30N-containing viruses and the broader cross-resistance of those containing other key PI mutations in subtype C. These findings are consistent with other studies from distinct geographic regions showing that mutation D30N is selected at low frequency in non-subtype-b-infected patients (1; Gomes et al., 9th Conf. Retroviruses Opportun. Infect.). As the majority of HIV-infected individuals worldwide harbor a variety of nonsubtype-b viruses, it is important to determine the significance of subtype classification in making treatment choices. Our findings suggest that the D30N pathway will be selected in a small percentage of subtype-c-infected patients and that alternative mutations, including those implicated in the development of PI cross-resistance, will be selected in a high proportion of pa-

5 VOL. 48, 2004 MUTATION D30N IS SELDOM SELECTED BY HIV-1 SUBTYPE C 2163 TABLE 4. Change in IC 50 s and replication capacities of subtype C viruses a Status Sample no. Replication capacity (%) Fold change in IC 50 Protease mutation at position: APV IDV LPV NFV RTV SQV Naive K20R M36I Naive K20R M36I L63P Naive K20R M36I Naive K20R M36I L63P Naive K20R M36I Naive K20R M36I L63P Naive K20R M36I L63P Treated M36I Treated K20R M36I L63P Ave SEM Treated K20R D30N M36I A71V N88D Treated D30N M36I L63P N88D Treated M36I L90M Treated NA L10V K20R M36I M46I L63P V82A I84V L90M Treated K20R M36I L63P L90M Treated L10V K20R M36I L90M Treated M36I M46I L63P A71V I84V L90M Treated K20R M36I L63P A71V I84V L90M Treated M36I M46I V82A L90M Treated L10I K20R M36I M46I I54V L63P A71V V82A L90M Treated K20R M36I A71V L90M Treated NA K20R M36I L63P A71V I84V L90M Treated L10V K20R M36I I54V A71V L90M Treated L10V K20R M36I I54V L63P A71V V82A L90M Ave SEM a Changes in IC50 s and replication capacities of subtype C viruses from drug-naive and drug-experienced patients compared to a subtype B reference database (NL4-3). Key resistance mutations present in selected drug-naive and drug-treated patients are specified. For clarity, the mutations D30N and L90M are shown in boldface type. Abbreviations: APV, amprenavir; IDV, indinavir; LPV, lopinavir; NA, Not Available; NFV, nelfinavir; SQV, saquinavir; RTV, ritonavir.

6 2164 GROSSMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. tients. Therefore, the patient s viral subtype is an important factor to be considered when selecting an initial PI regimen. The susceptibility testing results from these clinical samples confirmed the drug-specific nature of the resistance associated with the mutation D30N in HIV subtype C, which is consistent with what was found in subtype B (19, 22). Similarly, the cross-resistance conferred by L90M or multiple other PI mutations was also confirmed. Although preliminary, our RC results suggest that major PI mutations have a detrimental effect in subtype C consistent with previous findings in subtype B (2, 21). When mutation D30N is present it causes high resistance to nelfinavir, specifically, and not to other PI. Thus, the reason for not finding it in subtype-c-infected patients treated with nelfinavir is not that the mutation fails to confer resistance to the drug but that the virus finds other routes to propagate in the presence of the drug. This in turn has important implications. While nelfinavir is very often the first-line drug of choice for subtype-b-infected patients, because the frequent emergence of the non-cross-resistant D30N mutants would not bar the use of alternative drug combinations, it has no such advantage over other PI in the treatment of subtype-c-infected patients. It seems that HIV subtypes can take different pathways in evolving resistance to antiretroviral drugs. Our present findings pertain to one specific PI. Wainberg and colleagues found that the V106M mutation confers cross-resistance to nonnucleoside reverse transferase inhibitors (4). This mutation appears frequently in subtype-c-infected patients treated with efavirenz and rarely in subtype-b-infected patients or in subtype-c-infected patients treated with nevirapine (4, 9, 20). Moreover, in vitro studies of this group showed that the final drug concentration required for the development of resistance mutations conferring nonnucleoside reverse transferase inhibitors resistance was significantly lower for subtype C than for subtype B viruses and that resistant variants were fully selected more rapidly with the subtype C isolates than with the subtype B control (15, 16). All these studies highlight the importance of further characterization of the development of resistance in non-subtype B viruses. For some drugs, the differences seen in baseline polymorphisms between subtypes may influence which mutational patterns develop. Subtle effects of such polymorphisms on susceptibility and RC may underlie such changes. 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