Five Antiretroviral Drug Class–Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection

Abstract

Transmitted HIV-1 exhibiting reduced susceptibility to protease and reverse transcriptase inhibitors is well documented but limited for integrase inhibitors and enfuvirtide. We describe here a case of transmitted 5 drug class–resistance in an antiretroviral (ARV)-naïve patient who was successfully treated based on the optimized selection of an active ARV drug regimen. The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report.

Background

Drug susceptibility can be a key determinant in choosing an initial antiretroviral (ARV) regimen for patients who are naive to ARV therapy. The selection of a regimen in which individual components have less than full susceptibility can result in virologic failure. Transmission of HIV-1 exhibiting resistance to protease (PR) and reverse transcriptase (RT) inhibitors is well documented ^{1 –3} and because of this, the US Department of Health and Human Services (DHHS) guidelines recommend baseline genotypic resistance testing to guide drug selection in patients who are ARV naïve. ⁴ However, given the relatively recent availability of the integrase (IN) inhibitor class and the limited use of the fusion inhibitor enfuvirtide (ENF), transmitted resistance for these drug classes is less well defined. ^{5 –7} To date, only 2 cases of raltegravir-resistant HIV-1 transmission have been reported in the literature. ^5,6

Although transmission of virus resistant to more than 1 ARV class occurs less frequently than a single class, ^2,3 when it occurs, the selection of a baseline regimen can be more challenging. Such was the case in 2005 when a New York City man acquired a dual tropic, multidrug-resistant HIV-1 strain, ⁸ during a time when few therapeutic options were available for multidrug class–resistant strains. Here, we describe the first documented multidrug-resistant HIV-1 strain containing variants that exhibit resistance to 5 ARV drug classes. This report not only demonstrates one of the earliest cases of transmitted resistance to the integrase strand transfer inhibitor (INSTI) class but also exemplifies the need to develop a detailed resistance profile prior to initiating therapy.

Case History

A man in his 40s was hospitalized in 2010 with severe flu-like illness. HIV-1 antibody testing during hospitalization was negative. HIV-1 RNA testing by polymerase chain reaction was not performed. Six months later, follow-up HIV-1 antibody testing was positive and infection was confirmed by Western blot. Initial CD4 count and viral load were 376 cells/mm³ and 211 540 RNA copies/mL, respectively. Baseline genotypic resistance testing demonstrated extensive resistance to nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) as well as protease inhibitors (PIs; Figure 1). Confirmatory testing was performed to verify the initial genotypic resistance profile. Additional genotypic testing for INSTI resistance and phenotypic testing for PI, NRTI, NNRTI, and INSTI susceptibility was conducted. Phenotypic coreceptor tropism testing was also performed to thoroughly explore potential ARV treatment options. Due to the complexity of the baseline resistance profile, ENF susceptibility was also assessed.

Figure 1.

The results of both genotypic and phenotypic drug resistance analyses are compiled here. The list of antiretroviral (ARV) drugs is arranged by drug class. The net assessment column considers both the genotype and phenotype test results and provides a final resistance call based on the cumulative data. † Single values represent either biological or clinical cutoffs, and ranges indicate lower and upper clinical cutoffs. ‡ Fold change is defined as the ratio of the measured half maximal inhibitory concentration (IC₅₀) for the patient-derived virus to that of the NL4-3 reference virus.

Methods

Resistance-associated mutations (RAMs) to inhibitors of HIV-1 PR, RT, and IN were identified by conventional DNA sequencing (GenoSure MG, GenoSure Integrase; Monogram Biosciences, South San Francisco, California, USA; and LabCorp, Burlington, North Carolina, USA). Phenotypic susceptibility to PR, RT, and IN inhibitors, ENF, and coreceptor tropism was also assessed using well-established pseudovirus infectivity assays (PhenoSense, PhenoSense GT, PhenoSense Integrase, PhenoSense Entry, and Trofile, respectively; Monogram Biosciences). Molecular clones of full-length envelope sequences were generated and evaluated for ENF susceptibility and coreceptor tropism (PhenoSense Entry and Trofile). The gp41 sequences of envelope clones were generated by conventional DNA sequencing. Phylogenetic analysis was conducted on clonal gp41 sequences to rule out coinfection.

Protease and RT regions were also interrogated by massively parallel (“deep”) sequencing. A sequence library was generated using the Illumina Nextera XT library preparation kit and an Illumina MiSeq 2 × 250-bp paired-end run resulted in 1 017 032 reads with an average read depth of >15 000× (after alignment). Reads were trimmed using Cutadapt and FASTX toolkits and aligned to the NL4-3 reference genome (accession number AX032749.1) using Bowtie 2. ⁹

Results

Genotypic resistance analysis of the baseline virus identified mutations associated with resistance to PI (L10Y, I13V, K20I, E35D, M36I, K43T, I62I/V, and V82A), NRTI (M41L, D67N, L74V, and V118I), and NNRTI (K101E, Y181C, V189I, and G190S; Figure 1). Repeat genotypic resistance analysis from a second draw confirmed the initial findings, as well as mutations associated with INSTI resistance (G140S and Q148H). Reductions in susceptibility to PI, NRTI, NNRTI, and INSTI were confirmed by phenotypic testing, which demonstrated large reductions in susceptibility to efavirenz (EFV), nevirapine (NVP), and raltegravir. Notably, the NRTI resistance mutation M184V was not identified by genotypic assessment, although a phenotypic assessment revealed modest reductions in susceptibility to emtricitabine and lamivudine (3TC; half maximal inhibitory concentration IC₅₀ fold change [FC] = 7.16 and 5.25, respectively) that exceeded the biological cutoff (FC 3.5). Further analysis of the sample using deep sequencing failed to uncover minor variants harboring an M184V substitution. The results of both conventional and deep sequencing were consistent with a homogeneous virus population that contained the full constellation of resistance-associated substitutions reported previously, with the exception of PR position 62 where both wild type (I) and mutants (V) were detected. The phylogenetic analysis of the PR, RT, and IN regions, as well as the sequences of envelope clones, was consistent with a single group M, subtype B variant and yielded no evidence to suggest a coinfection.

Initial phenotypic analysis of ENF susceptibility (FC = 6.31) fell within 0.2 log₁₀ of the biological cutoff (FC = 6.48). This cutoff is based on a reference population of ENF-naive baseline isolates from the T-20 versus Optimized Regimen Only (TORO) clinical trials. ¹⁰ Given the proximity of the measured ENF susceptibility of the patient sample to the biological cutoff and considering both the broad distribution of the susceptibility of the reference population and the inherent variability of the phenotypic assay (± 3-fold), further analysis was warranted. Consequently, envelope gp41 sequencing was performed on 44 molecular clones generated from the virus population. Phenotypic analysis to determine ENF susceptibility was performed on 20 of the 44 clones that had unique gp41 sequences relative to the consensus sequence of the virus population. Two clones (#10 and #25) exhibited reduced ENF susceptibility (FC = 46 and FC > MAX, respectively), well above the biological cutoff. The gp41 sequence of these 2 clones revealed novel substitutions (Q40R and N43S) at amino acid positions previously associated with ENF resistance. ¹¹ Phenotypic coreceptor tropism testing indicated that 19 of the 20 selected clones exhibited R5 tropism, consistent with the phenotypic R5 tropism determination for the overall virus population.

Based on the compiled results of resistance and coreceptor tropism testing, the patient was placed on a regimen of ritonavir-boosted darunavir (DRV/r), tenofovir/emtricitabine, and maraviroc. Due to the presence of a DRV RAM, DRV/r was prescribed at 600 mg twice a day rather than 800 every day. This regimen successfully suppressed viral replication to <200 RNA copies/mL by month 2, which was maintained 1-year posttreatment initiation (Table 1). The CD4 count increased from 376 cells/mm³ at diagnosis to 614 cells/mm³ at 1 year of ARV treatment. After 3 years of ARV treatment, the patient’s virus remains fully suppressed at <75 RNA copies/mL and the CD4 count has risen to 865 cells/mm³ (Table 1).

Table 1.

Patient Clinical Parameters.^a

Month/Year	Viral Load, copies/mL	CD4, cells/mm3	CD8, cells/mm3	CD4/CD8 Ratio
7/11	211540	376	1571	0.24
12/11	60	381	966	0.38
3/12	80	385	785	0.49
7/12	40	614	1000	0.61
11/12	<75	704	934	0.75
2/13	<75	619	808	0.77
6/13	<75	774	834	0.93
12/13	<75	725	707	1.03
5/14	<75	865	888	0.97

^aViral loads, CD4, and CD8 counts, and CD4/CD8 ratios for this patient are listed over the treatment period. The initial viral load measurements were obtained using the Roche COBAS TaqMan version 2.0 (Roche Molecular Systems, Inc, Branchburg, New Jersey, USA) until July 2012. Subsequent values were obtained using the Siemens Versant HIV-1 Branched DNA assay (Siemens Healthcare GmbH, Erlangen, Germany).

Discussion

To our knowledge, this case represents the first confirmed report of the transmission of an HIV-1 variant exhibiting resistance to 5 antiretroviral drug classes as well as the third confirmed report of transmitted INSTI-resistant HIV-1. The selection of tenofovir/emtricitabine in the treatment regimen was based upon an assumption that an M184V variant might have been present below the limit of detection for population sequencing. Often, 3TC or emtricitabine is incorporated into ARV treatment regimens to exploit the impaired replication of M184V variants, despite limited evidence to support this approach. Detectable reductions in phenotypic susceptibility due to M184V variants require a viral subpopulation approximating 40% of the total viral population. ¹² Here, the absence of an M184V-containing subpopulation below the limit of detection of genotypic assays was confirmed by deep sequencing. Thus, the observed reduction in phenotypic susceptibility to emtricitabine and 3TC was likely due to the combination of L74V and V118I substitutions along with the thymidine analog selected substitutions M41L and D67N. Despite the presence of M41L and D67N substitutions, this virus retained susceptibility to zidovudine (ZDV) and stavudine (d4T), which is likely attributed to several substitutions in RT (L74V, W88S, and Y181C) that increase susceptibility to thymidine analogs.

This case further demonstrates the clinical utility of coreceptor tropism testing to guide maraviroc prescription. Antiretroviral treatment-experienced patients have a lower prevalence of R5 tropic virus compared with ARV treatment-naïve patients. Laboratory studies demonstrate preferential transmission of R5 virus (reviewed in ¹³ ), and data from clinical cohorts demonstrate that over 70% of ARV-naïve patients harbor R5 tropic virus. ¹⁴ In ARV treatment-naïve patients, there is no genetic linkage data to suggest that ARV-resistant profiles in pol influence envelope coreceptor tropism. Despite the extensive ARV resistance profile identified within the pol gene, the case patient was recently infected and treatment naive and thus more likely to harbor R5 tropic virus.

Envelope substitutions associated with ENF resistance include Q40H and the N43D. Clonal analysis of env in this case virus led to the identification of 2 novel substitutions Q40R and N43S that conferred high-level phenotypic resistance to ENF when tested in a pseudovirus reporter assay.

The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report. Current DHHS guidelines recommend supplemental genotypic INSTI resistance testing when transmitted INSTI resistance may be a concern. ⁴ There is evidence that transmitted INSTI resistance is following the same temporal course previously observed for NRTIs, NNRTIs, and PIs. ¹⁵ With the recent approval of a third INSTI, more widespread INSTI use, overlapping INSTI cross-resistance profiles, and documentation of this third case of transmitted INSTI resistance, baseline testing for INSTI resistance may become prudent.