All phenotypic data from longitudinal isolates were expressed as CFB and calculated as the ratios of post- to pre-PI treatment FC-IC50 values (data not shown). Based on similar phenotyping assays (eg, Monogram Biosciences PhenoSense Entry assay), CFB values ≤3 were considered within the “no-effect” range, and CFB values >3 were considered to be indicative of an effect on susceptibility.31 The relevance of this CFB cut-off in predicting GSK3532795 clinical efficacy is yet to be determined.
Phenotypic Susceptibilities of Highly Resistant PR Genes to GSK3532795: Nonlongitudinal Isolates
Consistent with their genotypic profiles, NLRepRluc proviruses expressing PR genes from a panel of 7 publicly available HIV-1 viruses containing multiple major and minor primary PI resistance-associated mutations (RAMs) were resistant to LPV (FC-IC50 range 15–442) and ATV (FC-IC50 range 11–415), but retained susceptibility to GSK3532795 and BMS MI B, with FC-IC50 values <1 (Supplemental Digital Content, Table 1 http://links.lww.com/QAI/A974). A virus with the A364V Gag substitution was used as a positive control for reduced GSK3532795 susceptibility.15 These data clearly indicate that highly PI-resistant viruses with PI RAMs in PR retain sensitivity to GSK3532795 and to a second structurally-related BMS MI (MI B).
Genotypic and Phenotypic Characteristics of Highly Resistant PR and Gag Genes: Longitudinal Isolates
Gag and PR genes from longitudinal isolates from 15 PI-resistant patients were cloned into recombinant virus vectors. Baseline (pre-PI treatment) samples, except those from patient 10 (Pt10), which contained a D30N mutation, contained no major PI RAMs. Conversely, all post-PI treatment samples had major PI RAMs in PR (Table 1) and 16/27 samples had PI-resistant mutations in Gag (at amino acid positions 128, 431, 436, 437, 449, 452, and 453)17–22 (Table 2, see also Supplemental Digital Content, Fig. 2, http://links.lww.com/QAI/A974 for full sequences of the entire Gag/PR region). In several patient samples (Pts06, 07, 09, and 15; Tables 2 and 3), changes were acquired in Gag at or near the purported site of action of MIs (near the CA/SP1 cleavage site).17,32 Ten of the 27 pre- or post-PI treatment samples had polymorphisms at Gag amino acids 362, 369, or 370, which are associated with BVM resistance.5,13,14,17,27,31,33–35 The phenotypic susceptibilities of these samples to 8 commonly used PIs [ATV, DRV, LPV, saquinavir (SQV), tipranavir (TPV), fosamprenavir (FPV), indinavir (IDV), and nelfinavir (NFV)] were predicted based on their PR genotype using the Stanford HIV Database Genotypic Resistance Interpretation Algorithm (Table 1). Pre-PI treatment samples from 14/15 patients were predicted to be either completely susceptible or exhibit only low resistance to all 8 PIs. Based on its D30N mutation, the sample from Pt10 was predicted to be susceptible to 7 PIs but highly resistant to NFV. All post-PI treatment samples were predicted to have intermediate/high resistance to ≥3 PIs.
Phenotypic Susceptibilities of All Longitudinal Isolates to GSK3532795
All pre- and post-PI treatment samples from 15 patients were analyzed by Monogram Biosciences for phenotypic susceptibility to GSK3532795 and DRV. The Monogram assay successfully reported results for at least 1 post-PI therapy time point from 11/15 patients (Pts02, 03, 04, 06, 07, 09, 10, 11, 12, 14, and 15). For Pt06, results were reported from the pre-PI treatment and only the second of 2 post-PI treatment samples. As shown in Figure 1, major PI RAMs were associated with a 1.9–4.37-fold increase in the median DRV CFB in post-PI treatment samples. The FC-IC50 median (range) was 1.19 (0.67–2.84) for pre-PI treatment samples and 2.72 (0.39–13.00) for post-PI treatment (FC-IC50 data not shown in Fig. 1). Although none of the samples had FC-IC50 >90, indicating clinically defined DRV resistance, the second post-PI treatment sample from Pt09 showed intermediate DRV resistance (FC-IC50 = 13). GSK3532795 susceptibility was observed in 9/11 pre-PI treatment samples, whereas low susceptibility to GSK3532795 was observed in the other 2 patients (FC-IC50 Pt11: 22.30; Pt14: 256.82). The determinants for reduced GSK3532795 susceptibility are currently being further examined in these samples and are not yet understood. However, the corresponding post-PI treatment samples had greatly reduced FC-IC50, indicating enhanced susceptibility. Overall, although no consistent change was observed in the distribution of the GSK3532795 FC-IC50s within this set, the distribution of the GSK3532795 FC-IC50s in post-PI treatment samples indicates that the presence of major PI RAMs did not reduce GSK3532795 susceptibility (Table 2 and Fig. 1).
GSK3532795 and DRV susceptibilities of patient samples with reportable results from the Monogram assay were calculated as CFB (Table 2 and Fig. 1). Median CFB for DRV was generally >3 and increased in the 6 patients with multiple post-PI treatment samples from the first (median CFB = 1.92) to the second (median CFB = 4.37) sample, suggesting decreasing drug susceptibility with greater PI treatment experience (Fig. 1 and Table 2). These observations are consistent with the genotype data (Table 1), which show that in patients with multiple post-PI treatment samples, predicted resistance to one or more PIs increases from the first to the second sample. Median CFB for GSK3532795 was ∼1, suggesting minimal change for both the first (median CFB = 0.6) and second (median CFB = 1.33) sets of post-PI treatment samples. However, one of each of 2 time point samples from Pt04 and Pt09 had GSK3532795 CFB >3 (further analysis of these samples is presented in section “Further Analysis of Longitudinal Isolates With GSK3532795 CFB >3”). Conversely, samples from Pts11, 14, and 15 seemed to show increased susceptibility to GSK3532795 compared with pre-PI treatment (CFB <0.33) (Table 2). The genotype data indicate that these samples had intermediate or high predicted resistance to all PIs except DRV (Table 1).
Samples from patients with nonreportable results (Pts01, 05, 06, 08, and 16) using the Monogram assay were recloned and analyzed using the single-cycle assay for phenotypic susceptibilities to GSK3532795, BMS MI A and BMS MI B, and the clinically relevant PIs LPV and ATV. Pt10 samples were used as a control for cross-comparison purposes. The presence of major PI RAMs in 5 post-PI treatment samples from 4 of these patients was associated with ATV and/or LPV CFB values >3, indicative of PI resistance (Table 3, Supplemental Digital Content, Fig. 2, http://links.lww.com/QAI/A974). The exception was Pt08, whose major RAM was D30N (characteristic of NFV resistance) and thus still showed susceptibility to ATV and LPV. All post-PI treatment samples had CFB <3 for GSK3532795, BMS MI A, and MI B CFB (Table 3, Supplemental Digital Content, Fig. 2, http://links.lww.com/QAI/A974). In summary, analysis of these longitudinal samples demonstrates a lack of cross-resistance to GSK3532795 in the presence of high-level PI resistance and PI treatment-induced mutations in Gag. The observation of sensitivity to MIs A and B further generalizes this result of lack of cross-resistance of MIs to PR-resistant isolates (Table 3, Supplemental Digital Content, Fig. 2, http://links.lww.com/QAI/A974).
Further Analysis of Longitudinal Isolates With GSK3532795 CFB >3
The first and second post-PI treatment samples from Pts09 and 04, respectively, had GSK3532795 CFB values substantially >3, and were subsequently recloned and tested using both single- and multiple-cycle assays. The first post-PI treatment sample from Pt09 showed GSK3532795 CFB ∼1.5 in both assays. The second post-PI treatment sample from Pt04 reproduced a CFB >3 (4.17, n = 2 independent experiments) in the single-cycle assay and CFB <3 (2.1) in the multiple-cycle assay. Both were resistant to ATV and/or LPV in the single-cycle assay (Pt04 time point 2 FC-IC50: ATV = 402; LPV = 152; Pt09 time point 1 FC-IC50: ATV = 14.8; LPV = 9.6) and the multiple-cycle assay (Pt04 time point 2 FC-IC50: ATV = 217, LPV = 133; Pt09 time point 1 FC-IC50: ATV = 4.4, LPV = 5.1) (Supplemental Digital Content, Table 2, http://links.lww.com/QAI/A974). Thus, the data suggest that the post-PI treatment samples from Pt04 and Pt09 did not exhibit a significant CFB toward GSK3532795.
Impact of PI-Resistance Mutations in Gag Cleavage Sites on GSK3532795 Susceptibility
The most frequently observed mutations in the Gag polyprotein shown to affect PI susceptibility are in MA/CA (codon 128), NC/SP2 (codons 431, 436, and 437), and SP2/P6 (codons 449, 452, and 453).17–22 None of these mutations map to amino acids associated with BVM susceptibility.35 Among the samples analyzed using the Monogram assay, ≥1 Gag PI-resistance mutation (except for a change in codon 452) was present in ≥1 post-PI treatment sample from 10/11 patients. Although samples with Gag PI-resistance mutations had a wider range of GSK3532795 CFB values, median values were similar regardless of the presence of these mutations. In contrast, median DRV CFB values were higher when Gag PI-resistance mutations were present (median CFB = 2.94) than not (median CFB = 1.26) (Fig. 2). Samples analyzed using the single- or multiple-cycle assays had ≥2 Gag PI-resistance mutations in 4/5 patients (excluding Pt08) (Table 1). GSK3532795, BMS MI A, and BMS MI B CFB values for these samples were similar regardless of the presence of these mutations.
Changes in Gag at or near the site of MI action, near CA/SP1, were observed in Pts06, 07, 09, and 15 (Tables 2 and 3). Despite the presence of these Gag changes, some of which have been associated with BVM resistance, and could thus be associated with resistance to other MIs, post-PI treatment samples from these patients remained susceptible to GSK3532795 and other structurally related-MIs.
MIs inhibit the final PR-mediated cleavage event in Gag, between the CA protein and SP1, whereas PIs inhibit all the PR-mediated cleavage steps required for virus maturation. Given the related mechanisms of action of these agents, there is potential for emergent PI RAMS to reduce MI susceptibility. This is the first comprehensive study to examine in detail the potential for cross-resistance between MI and PI ARVs. Using both nonlongitudinal (containing only the PR genes and mutations within) and longitudinal (containing PR and Gag genes and mutations within) clinical isolates from patients with acquired PI resistance, we found no definitive examples of viruses exhibiting reduced susceptibility to GSK3532795 in the presence of baseline or progressive PI resistance. Larger sample sizes will be helpful to support these findings.
Analysis of 7 nonlongitudinal HIV-1 viruses containing highly PI-resistant PR genes with multiple major and minor PI RAMs showed that these mutations were not associated with reduced sensitivity to GSK3532795. In a converse analysis, PI susceptibility was studied in viral isolates that exhibited reduced GSK3532795 sensitivity. As expected, viruses with reduced GSK3532795 susceptibility (FC-IC50 3.3–67) retained susceptibility to DRV, LPV, ATV, and NFV (unpublished data, Bristol-Myers Squibb). These data suggest that previous use of PIs will not affect subsequent use of GSK3532795 in PI–treatment-experienced patients and vice versa.
Pre- and post-PI treatment samples of all longitudinal clinical isolates were genotyped and predictions performed on their susceptibility to 8 PIs. As the patients were PI-naive at baseline, major PI RAMs were not present in most of the pre-PI treatment samples, but were present in all the post-PI treatment samples and associated with a predicted reduction in susceptibility to DRV, ATV, LPV, and a number of other less commonly used PIs. Phenotypic susceptibilities to PIs (DRV, ATV, and LPV) and MIs (GSK3532795, BMS MI A, and BMS MI B) were determined using a combination of Monogram and BMS single- and multiple-cycle susceptibility assays. The Monogram assay reported that longitudinal clinical isolates from 9/11 patients, except the first and second post-PI treatment samples for Pts09 and 04, respectively, retained susceptibility to GSK3532795 even in the presence of major PI RAMs. Pre- and/or post-PI treatment samples from Pts01, 05, 06, 08, and 16 yielded a nonreportable result from the Monogram assay and were thus re-analyzed (BMS single- and multiple-cycle assays). As for the set analyzed by Monogram, despite the high-level PI resistance mediated by PR and Gag RAMs, these samples remained susceptible to GSK3532795.
As the predicted PI-resistance profiles of samples from Pts04 and 09 were similar to others within the same set, further phenotypic analyses were performed to verify the Monogram GSK3532795 results. However, the single- and multiple-cycle assays showed that the first post-PI treatment sample for Pt09 had no significant change in susceptibility (CFB <3) to GSK3532795 and BMS MI A and BMS MI B. The second post-PI treatment sample for Pt04 had variable results: GSK3532795 CFB was >3 using the single-cycle assay but <3 using the multiple-cycle assay. The Monogram and BMS single- and multiple-cycle assays used the same primary PCR product for cloning and, additionally, positive control samples from Pt10 produced the same results from both the Monogram and BMS single-cycle assays. Thus, we speculate that differences in results between the assays might be attributable to small differences in PCR reamplification before cloning or small differences in assay conditions, although this was not formally tested. In summary, this detailed analysis generally showed that samples from Pt04 and Pt09 remained susceptible to GSK3532795.
The impact of PI-resistance mutations near the C-terminus of Gag on GSK3532795 susceptibility was also tested. Median GSK3532795 CFB values were similar in the presence or absence of such Gag PI-RAMs. In addition, 4 PI-resistant, post-PI therapy samples contained changes near the CA/SP1 site, but retained susceptibility to GSK3532795.
The results of this study indicate that GSK3532795, a potent, once-daily, second-generation MI, maintains activity toward clinical isolates from PI-treated patients harboring baseline and/or progressive genotypic and phenotypic PI resistance. Emergent mutations in PR and Gag were not linked to reduced viral susceptibility to GSK3532795. A lack of cross-resistance of GSK3532795 to PI-resistant isolates with primary PI resistance supports the use of PIs and MIs simultaneously or in succession, and supports the continued development of GSK3532795 in treatment-experienced patients with previous PI treatment exposure.
The authors thank Matthew Healy for sharing unpublished studies assessing the prevalence of MI-resistance mutations in PI-resistant viruses from the Los Alamos National Laboratory Database. Editorial support was provided by Sharmin Naaz at MediTech Media and funded by Bristol-Myers Squibb.
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HIV-1; maturation inhibitor; GSK3532795; protease inhibitor; cross-resistance; in vitro
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