Received 15 February 1996/Accepted 23 May 1996

Transcription

1 JOURNAL OF VIROLOGY, Sept. 1996, p Vol. 70, No X/96/$ Copyright 1996, American Society for Microbiology Human Immunodeficiency Virus Type 1 Drug Susceptibility during Zidovudine (AZT) Monotherapy Compared with AZT plus 2,3 -Dideoxyinosine or AZT plus 2,3 -Dideoxycytidine Combination Therapy BRENDAN A. LARDER, 1 * ARINDER KOHLI, 1 STUART BLOOR, 1 SHARON D. KEMP, 1 P. RICHARD HARRIGAN, 1 ROBERT T. SCHOOLEY, 2 JOEP M. A. LANGE, 3 KEVIN N. PENNINGTON, 4 MARTY H. ST. CLAIR, 4 AND THE PROTOCOL 34, COLLABORATIVE GROUP Clinical Virology, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, United Kingdom 1 ; University of Colorado Health Sciences Center, Denver, Colorado 2 ; Academic Medical Centre, Amsterdam, The Netherlands 3 ; and Glaxo Wellcome Research Laboratories, Research Triangle Park, North Carolina 4 Received 15 February 1996/Accepted 23 May 1996 Human immunodeficiency virus type 1 (HIV-1) isolates obtained prior to and during a combination therapy trial comparing zidovudine (AZT; 3 -azidothymidine) monotherapy with AZT plus 2,3 -dideoxyinosine (ddi) or AZT plus 2,3 -dideoxycytidine (ddc) were assessed for the development of drug resistance. Drug susceptibility was measured by using two different phenotypic assays, one that requires infection of peripheral blood mononuclear cells with HIV-1 isolated from cocultures and a second based on infection of HeLa CD4 cells with recombinant virus containing the reverse transcriptase (RT) of the clinical isolate. In addition, genotypic assessment of resistance was obtained by DNA sequencing of the RT coding region. No difference in the development of AZT resistance was noted in isolates from individuals receiving AZT monotherapy or combination therapy. However, a low frequency of ddi or ddc resistance was seen in isolates from the combination arms, which may at least partially explain the enhanced efficacy observed with these drug combinations compared with monotherapy. It was noted from DNA sequencing that a relatively high frequency of the nonnucleoside RT inhibitor resistance mutation, codon 181 changed from encoding Tyr to encoding Cys, was present in some isolates both before and during nucleoside analog combination therapy. Since these patients were unlikely to have access to nonnucleoside RT inhibitors, it is probable that this mutation preexisted at a reasonable level in the wild-type virus population. Comparisons of the AZT susceptibility assays indicated a good correlation between the phenotypic and genotypic determinations. However, direct numerical comparisons between the phenotypic assays were not reliable, suggesting that valid comparisons of different resistance data sets will require the use of the same assay procedure. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) strains during antiretroviral therapy is perceived as an important factor contributing to the gradual loss of clinical benefit (4, 25, 34). Resistance was first reported to the reverse transcriptase (RT) inhibitor zidovudine (AZT) during the treatment of individuals with advanced HIV-1 disease (17, 29). The genetic basis of this resistance was elucidated by comparative DNA sequence analysis of HIV-1 isolates obtained before and after initiation of AZT therapy, together with susceptibility analysis of virus variants (constructed by site-directed mutagenesis) containing specific amino acid substitutions in RT (17, 20). Mutations at five codons in RT (Met41Leu [codon 41 changed from encoding Met to encoding Leu], Asp67Asn, Lys70Arg, Thr215Tyr or -Phe, and Lys219Gln) were shown to be responsible for the AZT-resistant phenotype (13, 20). These and subsequent studies identified specific combinations of mutations that conferred different degrees of AZT resistance (13). Mutations at codon 70 or 215 alone confer modest reductions in susceptibility (about 8- and 16-fold decreases in 50% inhibitory concentration [IC 50 ], respectively), while Leu41 and Tyr215 in the same genome confer about 60- to 70-fold decreases in susceptibility, * Corresponding author. Phone: (44) Fax: (44) and combinations of four (41, 67, 70, and 215 or 67, 70, 215, and 219) or all five mutations confer 100- to 200-fold increases in resistance (13, 14). Analysis of isolates collected sequentially during treatment of asymptomatic individuals revealed that the AZT resistance mutations tend to appear in a specific order (2, 14). The codon 70 mutation occurs first but is replaced by mutations at codons 41 and 215. Continued treatment can then lead to the appearance of the other three amino acid changes (2, 14). Resistance in clinical studies to a number of other nucleoside analog inhibitors and some nonnucleoside RT inhibitors (NNRTI) has been reported (for a summary, see reference 22). Treatment with 2,3 -dideoxyinosine (ddi) can result in the development of resistant isolates as a result of an RT mutation of Leu74Val (33). Of interest, it was found that when present in an AZT resistance background, this mutation was able to suppress the effects of Tyr215 (33), although the additional presence of Leu41 appears to abrogate this effect (19). In addition, it has been reported that two further RT mutations, Lys65Arg and Met184Val, can confer a modest decrease in ddi susceptibility (7, 8, 36). The first report of resistance to 2,3 dideoxycytidine (ddc) identified a novel mutation in RT (Thr69Asp) that results in about a fivefold decrease in susceptibility (6). The mutations at codons 65, 74, and 184 described above for ddi also appear to confer a modest decrease in ddc 5922

2 VOL. 70, 1996 HIV-1 SUSCEPTIBILITY DURING COMBINATION THERAPY 5923 susceptibility (6, 7, 33, 36). A large number of RT mutations that confer resistance to NNRTIs (1, 3, 24), including amino acid substitutions in the residue and regions, have been identified. However, a frequently observed NNRTI resistance mutation, arising both in vitro and in vivo, is Tyr181Cys (26, 28). Two major clinical endpoint trials, ACTG 175 and DELTA, have recently shown a benefit of AZT-ddC or AZT-ddI combination therapy over AZT monotherapy (9, 35). It has been reasoned that combination therapy with antiretroviral agents might slow or even prevent the development of drug resistance. There have been a number of small combination therapy studies in which limited assessment of HIV-1 drug susceptibility was performed (27). Although there did not appear to be a delay in AZT-resistant isolates emerging, these studies were not specifically designed to address this issue (27). Recent reports have suggested that a set of novel amino acid changes in RT (including substitution at codon 151) seen in isolates from a few individuals may confer cross-resistance to AZT, ddi, and ddc (10, 31, 32). However, most individuals receiving AZT and ddi or ddc in these studies had isolates that remained susceptible to ddi and ddc (31, 32). We have recently completed a combination therapy trial which compared AZT monotherapy with the combination of AZT and ddi or AZT and ddc (30). This was a double-blind, placebo-controlled study that enrolled 180 individuals. It was specifically designed to assess the potential effect of combination therapy on the development of antiretroviral resistance. Both combination therapy arms proved superior to AZT monotherapy with respect to changes in CD4 cell numbers and suppression of HIV-1 RNA copies in the serum. However, assessment of AZT susceptibility by either a peripheral blood mononuclear cell (PBMC)-based assay or PCR analysis of RT codon 215 showed that AZT resistance developed to similar degrees in all treatment arms (30). In the present study, we performed extensive phenotypic and genotypic analysis of multiple HIV-1 samples from individuals enrolled in this trial. This analysis has enabled comparisons of different techniques used to assess drug resistance and offers insight into the likely reason for the superior activity of combination therapy. MATERIALS AND METHODS Study participants and treatment regimens. HIV-positive individuals with fewer than 300 CD4 cells per mm 3 were enrolled in the trial. In addition, only participants with less than 4 weeks of AZT therapy and no prior therapy with any other antiretroviral agent were allowed to enroll. Individuals were randomized to receive either (i) AZT (200 mg three times per day) plus ddi (100 mg twice a day) or ddi placebo or (ii) AZT plus ddc (0.75 mg three times per day) or ddc placebo. Complete details of subject characteristics at entry have been described elsewhere (30). All virological assessments were made during the first 48 weeks of study, which corresponded to the initial randomization period. Isolation of HIV-1 from PBMCs. Virus isolates were obtained by coculture of each study subject s PBMCs with phytohemagglutinin-stimulated donor PBMCs as described elsewhere (11). Culture supernatants were harvested at the time when peak RT production was evident and were stored at 70 C for subsequent use in susceptibility assays. Aliquots of infected cocultured cells were also stored at 70 C for subsequent DNA extraction. This DNA was used for genotypic analysis and the recombinant virus assay (RVA) (see below). Phenotypic drug susceptibility assay. A protocol similar to the ACTG/DoD consensus assay (12) was used to determine the drug sensitivity of PBMC isolates (33). Titers of virus supernatants were determined by RT activity, and % tissue culture infectious doses was used to infect phytohemagglutinin-stimulated PBMCs, which were then cultured in the presence of appropriate concentrations of drug. Supernatants were assayed for RT activity on day 7, and the 50% inhibitory concentration (IC 50 ) was calculated. IC 50 s were scored as follows: 1 M AZT sensitive (S); 1 to 5 M partially resistant (PR); and 5 M highly AZT resistant (R). DNA sequence analysis of HIV-1 RT. The 5 region of the HIV-1 RT coding region was amplified by a nested PCR procedure using DNA extracted from the same PBMC cocultures used to obtain virus isolates for susceptibility testing. The outer primer pair was A-35/NE1-35, and the inner primer pair comb2/comb3 was used with previously reported PCR conditions (21). Most of the samples were sequenced once from a single set of PCR amplifications, using standard manual procedures as follows. The amplification products were purified by using the Geneclean (Bio 101) kit and then alkali denatured (0.2 mm NaOH plus 0.2 mm EDTA, incubation at 37 C for 30 min), neutralized (0.3 M sodium acetate [ph 4.8]), and precipitated in ethanol. Sequencing was performed by dideoxy-chain termination, using T7 DNA polymerase (Sequenase; U.S. Biochemical) and the primers comb2 and B (21). For automated sequencing, PCR amplification was performed as described above except that the 3 PCR primer was biotinylated. Single-stranded product was obtained by using a magnetic bead separation system (Dynabeads M-280 streptavidin; Dynal); 80 l of Dynabeads (5 g/ml) was used per PCR product. These preparations were washed twice (10 mm Tris-HCl [ph 8], 1 mm EDTA, 2 M NaCl), mixed with 80 l of PCR product, and incubated at 37 C for 15 min. The bound DNA was denatured by addition of 25 l of 0.1 M NaOH for 15 min at room temperature. The negative strand bound to the beads was used for solid-phase sequencing. Reactions were performed by using the PRISM Sequenase terminator single-stranded DNA sequencing kit (Applied Biosystems) and resolved on an ABI 373 DNA sequencer as instructed by the manufacturer. Codons were classified as mixtures if bands were detected for both wild-type and mutant nucleotides at a single position on sequencing gels or if a single peak from ABI sequencing comprised 33% of the total peak area (as determined by using ABI Sequence Navigator software). For AZT susceptibility assessment, samples were scored as follows: no AZT resistance mutations (or AZT resistance mutations together with an additional mutation likely to suppress phenotypic resistance) S; one to three AZT resistance mutations (but not Leu41 plus Tyr215) PR; and Leu41 plus Tyr215 or four of five AZT resistance mutations R. When mixtures of mutant and wild-type codons were detected, the phenotypic prediction was one category less resistant than the prediction made when a pure mutant population was detected. For example, Met/Leu41 plus Tyr215 would be scored as PR rather than R (as in the case of Leu41 plus Tyr215). Construction of recombinant HIV-1 strains and RVA. Full-length RT coding regions were obtained by nested PCR of DNA extracted (from the same PBMC cocultures used to obtain virus isolates for susceptibility testing) by using PCR primers and reaction conditions described previously (15). These clinical sample RT coding regions were subsequently transferred into the otherwise wild-type HXB2-D background by homologous recombination. The T-cell line C8166 was cotransfected by electroporation with a mixture of the RT-deleted proviral clone phiv RTBstEII and a PCR-derived RT fragment as described previously (15). Generally, viral replication was first evident in the cultures about 6 days posttransfection. Extracellular virus was harvested from culture supernatants after about 14 days and stored in aliquots at 70 C. AZT susceptibility (IC 50 ) was determined by a plaque reduction assay in the HeLaLacZ-1 cell line by infection of cell monolayers as described previously (13). The IC 50 s from RVA data were scored as follows: S 0.05 M, PR 0.05 to 0.5 M, and R 0.5 M. AZT susceptibility analysis of defined mixtures of recombinant viruses. The AZT susceptibility (IC 50 ) of mixtures of HIV HXB2 and the AZT-resistant isolate RTMN (13) at HXB2/RTMN ratios of 100:0, 25:75, 40:60, 50:50, 60:40, 75:25, and 0:100 were determined by a plaque reduction assay in the HeLa- LacZ-1 cell line by infection of cell monolayers as described previously (13). IC 50 curves derived for a clonal viral isolates can be generally fitted to a four-parameter logistic, N i a i 1 drug IC 50 i s i where N i is the number of plaques derived from virus i at a given drug concentration, a i is the number of plaques formed in the absence of drug, [drug] is the drug concentration, and s i is a slope factor for virus i (Graphit Software; Erithacus Software Limited, Middlesex, England). Given that the observable parameter in the HeLaLacZ plaque reduction assay is the number of viral plaques formed, it is worthwhile considering that the total number of plaques derived from a mixture of two HIV genotypes at any given concentration of inhibitor will be the sum of the plaques derived from the wild-type (N w ) and from the mutant (N m ), where the number of individual plaques is described by the equation presented above. RESULTS Influence of combination therapy on the development of AZT resistance. We have previously assessed the rate of development of AZT resistance in this trial by using a PBMC susceptibility assay and by determining the status of RT codon 215 by using a selective PCR procedure (30). These studies showed that AZT-ddI or AZT-ddC combination therapy was unlikely to influence significantly the appearance of such mutants. We wished to confirm these data by using other techniques whereby it was possible to perform a more complete

3 5924 LARDER ET AL. J. VIROL. FIG. 1. Pre- and posttherapy AZT susceptibility determined by three different approaches. (A) Phenotypic determination of AZT IC 50 as determined by a PBMC assay. Solid bars represent isolates obtained from patients on AZT monotherapy (median, 0.24 [interquartile range {IQR}, 0.1 to 0.43] at week 0; median, 4.3 [IQR, 1.2 to 7.2] at week 48). Open bars represent isolates from the AZT-ddC combination (median, 0.11 [IQR, 0.04 to 0.34] at week 0; median, 3.7 [IQR, 1.7 to 6] at week 48). Hatched bars represent isolates from the AZT-ddI combination therapy (median, 0.19 [IQR, 0.1 to 0.5] at week 0; median, 2.2 [IQR, 1.0 to 3.6] at week 48). (B) Phenotypic determination of AZT IC 50 as determined by RVA. Solid bars represent isolates obtained from patients on AZT monotherapy (median, [IQR, 0.02 to 0.04] at week 0; median, 0.35 [IQR, 0.07 to 0.9] at week 48). Open bars represent isolates from the AZT-ddC combination (median, [IQR, 0.03 to 0.05] at week 0; median, 0.55 [IQR, 0.1 to 1.3] at week 48). Hatched bars represent isolates from the AZT-ddI combination therapy (median, 0.04 [IQR, 0.03 to 0.05] at week 0; median, 0.86 [IQR, 0.08 to 1.6] at week 48). (C) Predicted AZT susceptibility based on genotypic determination of mutations at codons 41, 67, 70, 215, and 219. Assignments of S (open bars), PR (hatched bars), and R (solid bars) were made by using predicted levels of AZT resistance as described in Materials and Methods. with the PBMC assay are shown in Fig. 1A to serve as a comparison with the other susceptibility determinations. The median IC 50 for AZT was between 0.11 and 0.24 M, at week 0, and had increased more than 10-fold, to between 2.2 and 4.3 M, by week 48. Clearly, significant AZT resistance had developed in each treatment group (P 0.05 by Wilcoxon ranks sum comparing week 0 with week 48 for all treatment arms individually or together), with no obvious difference between monotherapy and combination therapy (P 0.05, fusing Wilcoxon ranks sum data comparing groups with each other at week 0 or 48). It was possible to obtain IC 50 data from only around 50% of week 48 samples because the virus isolation rate was low at this time. We therefore attempted to construct recombinant viruses starting from DNA extracted from cocultured PBMCs in order to increase the number of isolates that could be assayed for AZT susceptibility. Using this technique, we obtained viruses from around 70% of week 48 DNA samples (the overall success rate with all samples was about 75%). The median IC 50 s for the treatment groups were 0.03 to 0.04 M at week 0 and 0.35 to 0.86 M by week 48 (Fig. 1B). Therefore, this more extensive phenotypic analysis showed a greater than 10-fold increase in AZT IC 50 over the course of therapy, reflecting our findings with the PBMC assay. To substantiate these data, we sequenced selected regions of the RT of all available week 48 DNA samples (n 100) with matched week 0 isolates where possible (n 95). As expected, the week 0 samples were predominantly wild type with respect to the residues known to be responsible for AZT resistance (Fig. 1C). Such viruses were thus predicted to be phenotypically S. Of interest, a number of AZT resistance mutations were seen at study entry, confirming our previous observations from codon 215 selective PCR (30), in addition to identifying other mutations and multiple mutants (including six samples with a codon 70 mutation, one with mutations of codons 41 and 215, one with mutations of codons 70 and 219, four with three mutations, and three with four mutations). By week 48, the amount of virus predicted to be AZT sensitive in each therapy group had declined considerably (to between 15 and 35%), and as expected, the number of isolates with AZT resistance mutations had increased (Fig. 1C), consistent with the increase in IC 50 seen with the phenotypic assays. However, there were no analysis, using either extensive DNA sequencing (not concentrating on only a single codon) or phenotypic susceptibility assessment of a large number of samples. Median IC 50 s of HIV-1 isolates obtained at weeks 0 and 48 FIG. 2. Comparison of genotypic predictions and phenotypic assignments after 48 weeks of antiretroviral therapy. Predicted AZT susceptibility by RT genotype compared with phenotypic AZT susceptibility as determined by both PBMC assay and RVA (when data were available) after week 48 of treatment. Assignments of S (open bars), PR (hatched bars), and R (solid bars) were made by using predicted levels of AZT resistance as described in Materials and Methods.

4 VOL. 70, 1996 HIV-1 SUSCEPTIBILITY DURING COMBINATION THERAPY 5925 FIG. 3. Median AZT IC 50 compared with predicted IC 50 for isolates from patients after 48 weeks of antiretroviral therapy. Median AZT IC 50 s as determined by PBMC assay and RVA are indicated for isolates with predicted AZT IC 50 s as determined in Fig. 1C when data from both assays were available. Open bars represent the PBMC assay; hatched bars represent the RVA. Median IC 50 s (with interquartile ranges) were 0.85 (0.3 to 1.1), 4.4 (1.3 to 6.1), and 3.7 (2.2 to 5.5) for genotypes designated S, PR, and R, respectively, in the PBMC assay and were 0.04 (0.02 to 0.07), 0.28 (0.13 to 1.3), and 1.3 (0.75 to 1.7) for genotypes designated S, PR, and R in the RVA. differences between the treatment groups with respect to the number of isolates predicted to have PR or R phenotypes from these genotypic data. Comparison of AZT resistance data between different assays. Since AZT resistance data were collected from this trial by using three distinct methods of analysis, we considered it worthwhile to make direct comparisons between the different assays. First, we scored IC 50 s from both phenotypic assays as S, PR, or R (see Materials and Methods) so that they could be compared with the sequence data. Comparisons of phenotypic data with genotypic data are shown in Fig. 2 for all week 48 samples that could be analyzed by all three procedures (45 in total). Data from both phenotypic assays were in relatively good agreement with the phenotypic predictions from sequence data. However, in both cases, a considerable proportion of phenotypically sensitive viruses were scored as PR according to the genotype (56% with the PBMC assay and 42% with the RVA). These were mainly isolates with various combinations of mixed bases at the AZT resistance codons (that is, the presence of more than one base could be detected at a single position in the RT) and thus were less amenable to precise phenotypic predictions. Isolates found to be PR by PBMC assay or RVA were generally scored as PR or R from the DNA sequence (although a small proportion of each were genotypically S). In this analysis, the RVA data were in somewhat better apparent agreement with the genotypic prediction than the data for PBMC assay, in which the isolates were AZT resistant (Fig. 2). Median IC 50 s for isolates obtained with both assays are shown in Fig. 3 according to genotypic assignment. This analysis showed that the RVA median IC 50 s for each of the three genotypic assignments (i.e., S, PR, and R) fell within the groups. This was also the case for the PBMC IC 50 data exception those for the PR group, in which case the median IC 50 was 4.4 M (Fig. 3). IC 50 s differed significantly (P 0.05) by the Wilcoxon ranks sum test for all groups of S, PR, and R tested pairwise, except for the PR and R groups in the PBMC assay. We also compared absolute IC 50 s derived from the same week 48 samples between both phenotypic assays. It was apparent from this analysis that there was only a modest correlation between the individual values of the AZT IC 50 determined by using these two phenotypic assays (Fig. 4), even though the assignments of AZT sensitivity independently agreed reasonably well with the genotypic assessments. Comparative genotypic and AZT susceptibility data from sequential isolates. To confirm the relationships between genotypic and phenotypic AZT susceptibility data described above, we next compared sequential isolates from eight individuals (four who received AZT alone, one who received AZT plus ddi, and three who received AZT plus ddc). These isolates were selected because they encompassed a spectrum of different genotypes. In a case in which none of the isolates had any AZT resistance mutations (patient 510 [Table 1]), IC 50 s were between 0.01 and 0.04 M for the RVA and between 0.18 and 0.88 M for the PBMC assay (and thus within a range considered to be S by both phenotypic assays). In general, week 0 isolates assayed by the PBMC method or RVA were AZT sensitive, which correlated with the DNA sequence data, since no AZT resistance mutations were observed (Table 1). The range of IC 50 s was lower for the RVA (0.02 to 0.05 M) than for the PBMC assay (0.06 to 0.79 M). As expected, there was a clear relationship between the accumulation of AZT resistance mutations and an increase in phenotypic resistance. For example, isolates from patient 323 were initially genotypically and phenotypically S and by the RVA became sequentially less sensitive upon the emergence of Leu41 plus Tyr215 (IC M) and then more resistant when Asn67 plus Arg70 also appeared (IC 50 1 M). In this case, we were able to obtain PBMC IC 50 data from only weeks 0, 12, and 36. However, there was a clear increase in IC 50 with the week 36 isolate, which showed evidence of mutations at codons 41, 67, 70, and 215. A number of anomalies were apparent from these analyses, reinforcing the notion that direct comparison of RVA and PBMC IC 50 values were hard to make. The most obvious from Table 1 are the phenotypic data from patient 506. By RVA analysis, all isolates remained substantially sensitive to AZT (IC 50 s ranged from 0.03 to 0.06 M). However, PBMC IC 50 s for the same isolates differed considerably and appeared to evolve from S (IC M) to R (IC M). These data were not consistent with the DNA sequence analysis of the isolates, since most only had the codon 70 mutation and at week 48, additional mixtures of wild type and mutant at codons 41 and 215 were seen. It is worth noting that it was not always easy to predict phenotypic AZT susceptibility from the genotypic data which showed the existence of a number of genotypic mixtures. For example, the week 36 isolate of patient 104, which had mixtures at codons 41, 67, and 215, was correctly predicted as PR since both FIG. 4. Correlation between individual determinations of AZT resistance by PBMC assay and RVA. AZT IC 50 as determined by PBMC assay and RVA for all time points for which data from both assays were available. The solid line represents a linear regression to the data, r 0.58 (P 0.001).

5 5926 LARDER ET AL. J. VIROL. TABLE 1. AZT susceptibility by phenotypic and genotypic assessment of sequential isolates Patient Wk Amino acid at RT codon a : phenotypic assays scored the isolate as partially resistant. However, the week 24 isolate from patient 319, which had mixtures at codons 41 and 215, was phenotypically S by both assays. It is evident from Table 1 that fairly complex genotypic mixtures can occur at codon 215. Assignment of amino acids in these cases is difficult, especially as mutation at codon 215 involves changes at two nucleotides; however, a number of these isolates could be scored as having a mixture of Thr/Ser and Tyr (e.g., patient 104, weeks 36 and 48). It has previously been suggested that Ser might be a possible intermediate residue during the change from wild-type Thr to mutant Tyr or Phe, although it was shown that Ser215 did not confer AZT resistance in an otherwise wild-type background (18). Thus, it is unlikely that Ser215 occurs to any great extent, but these data probably reflect genotypic mixtures of Thr and Tyr. Analysis of susceptibility of viruses with mixed genotypes. To examine the effects of genotypic mixtures on the phenotypic drug resistance, the AZT IC 50 s of defined genotypic mixtures of wild-type (HXB2) HIV-1 and the AZT-resistant mutant RTMN were determined at several different ratios. The equation in Materials and Methods has the practical implication IC 50 ( M) RVA PBMC assay Met Asp Lys Thr Leu Asp Lys Tyr Leu Asp/Asn Lys Tyr Met/Leu Asp/Asn Lys Thr/Ser/Tyr Met/Leu Asp Lys Thr/Ser/Tyr Met Asp Lys Thr Met Asp Lys Thr Met Asp Lys/Arg Ser/Tyr Met Asp Lys Tyr Met Asp Lys Tyr Met Asp Lys Thr Met Asp Lys Thr Met/Leu Asp Lys Thr/Ser Leu Asp Lys Tyr Leu Asp Lys Tyr/Ser Met Asp Lys Thr Met Asp Lys Thr Leu Asp Lys Tyr Met/Leu Asp/Asn Lys/Arg Tyr Leu Asn Arg Tyr Met Asp Lys Thr Met Asp Lys/Arg Thr Met Asp Arg Thr/Ser Met Asp Arg Tyr Met/Leu Asp Arg Tyr Met Asp Lys Thr Met Asp Lys/Arg Thr Met Asp Arg Thr Met Asp Arg Thr Met/Leu Asp Arg Thr/Ser Met Asp Lys Thr Met Asp Lys Thr Met Asp Lys/Arg Tyr Met/Leu Asn Lys Tyr Leu Asn Lys Tyr Met Asp Lys Thr Met Asp Lys Thr Met Asp Lys Thr Met Asp Lys Thr Met Asp Lys Thr a In all cases, codon 219 encoded Lys. that IC 50 s when mixed genotypes are present are not additive, and the relationship between IC 50 and the proportion of resistant virus is distinctly sublinear (data not shown). For example, the IC 50 of mixtures of HXB2 and RTMN at ratios of 40:60, 50:50, and 60:40 were 0.03, 0.04, and 0.07 M, respectively, much lower than would be expected from simply averaging the proportional contributions IC 50 s for HXB2 and RTMN (0.02 and 0.32 M in assays run concurrently with these mixtures). A similar nonlinearity between IC 50 and the proportion of resistant virus has been observed earlier for mixtures of two and three viral genotypes (Table 1) (13). Analysis of resistance to ddi and ddc. We next determined the ddi and ddc susceptibilities of the isolates from the three treatment groups by DNA sequence analysis and the PBMC assay. We examined all available week 48 isolate DNA sequences paired with week 0 samples, specifically to determine the status of those codons in RT where mutation confers resistance to ddi and/or ddc (namely, codons 65, 69, 74, 75, and 184). Almost all of the week 0 isolates were wild type at these codons (there was one mixed base and one mutant at position 184 in 95 samples), suggesting that these isolates were pheno-

6 VOL. 70, 1996 HIV-1 SUSCEPTIBILITY DURING COMBINATION THERAPY 5927 isolates had none of the recognized AZT or ddn resistance mutations. In view of the description of a possible distinct set of nucleoside analog resistance mutations, including mutations at codons 75, 77, and 151 (31, 32), we specifically examined these isolates for the presence of these changes. No clear patterns of substitutions were noted from this analysis. More importantly, no amino acid variation from the HXB2-D sequence was seen at RT codon 75, 77, or 151, indicating that these isolates had not developed these mutations following exposure to combinations of AZT plus ddi or ddc. FIG. 5. Prevalence of Cys181c mutation conferring resistance to NNRTIs. The proportion of isolates from patients with mutations or mixtures of mutant and wild type at codon 181 are indicated for each of the three treatment arms at study entry and after 48 weeks of therapy. Open bars represent wild type at codon 181; solid bars represent mutant at codon 181. typically susceptible to both ddi and ddc. Similarly, no mutations were seen in the 48 week isolates from the AZT monotherapy group, whereas 2 of 39 were mutant in the AZT-ddI group (one each at codons 74 and 184), and 1 of 29 was at mutant codon 74 and 2 of 29 were mutant at codon 184 in the AZT-ddC group. PBMC-derived virus isolates with a sufficiently high titer were also tested for ddi and ddc susceptibility by using the PBMC assay described in Materials and Methods. No preexisting dideoxynucleoside (ddn) resistance was seen in week 0 samples by this method. In addition, very few of the week 48 isolates tested were considered phenotypically resistant to ddi (2 of 17, or 11%, in the AZT-ddI group) or ddc (1 of 12, or 8%, in the AZT-ddC group) (data not shown). No other mutations conferring ddn resistance at codon 65 or 69 were observed at any time in the study. Thus, by both assessment techniques, it appeared that very little ddn resistance was present by 48 weeks of the study. Frequency of RT Cys181 mutation associated with NNRTI resistance. During DNA sequence analysis of isolates from this trial, we observed that codon 181 was mutated in a number of cases from the wild-type Tyr to Cys. This was of potential significance because Cys181 has been recognized previously as an NNRTI resistance mutation (24). The frequency of codon 181 mutations (mutant Cys or mixtures of Tyr and Cys) observed in week 0 and 48 isolates is shown in Fig. 5. The majority of changes seen at codon 181 comprised mixtures of an A and G nucleotide at the second position of the triplet, which led to the predicted mix of Tyr and Cys residues. It is of interest of the samples which could be evaluated, the majority of isolates showing evidence of the 181 mutation at week 0 were wild type at week 48. Isolates from only two individuals had an altered codon 181 both at week 0 and week 48. In addition, the total number of isolates with evidence of mutation at codon 181 (mutant or mixtures) was approximately 10% of the total both samples analyzed at weeks 0 and 48 (10 and 12, respectively). The possibility of detecting the codon 181 mutation as a result of PCR contamination appeared remote, since we observed this variant in a number of complex, unrelated RT genetic backgrounds as deduced from DNA sequence analysis. Amino acid variation in RT of week 48 isolates with no AZT resistance mutations. DNA sequence analysis of the RT coding region of 100 week 48 isolates revealed that 13 of these DISCUSSION The primary objective of the trial described here was to determine if combination therapy with two nucleoside analogs (ddi and ddc) was able to delay the development of AZT resistance when compared directly with AZT monotherapy. Clearly, from data reported here and previously (27, 30), no such delay in AZT resistance was seen. However, since both of the drug combinations studied showed positive benefits with regard to increases in CD4 cell numbers and decreases in viral load and clinical benefit (9, 30, 35), it was considered worthwhile to obtain comprehensive drug resistance data from this cohort. In doing so, we gained some insight as to the virological basis of the observed benefits, in addition to compiling a large drug susceptibility data set from one genotypic assay and two phenotypic assays. The most obvious reason for the difference between the combination and monotherapy groups was that very little resistance to either ddi or ddc was seen, as deduced phenotypically and by DNA sequence analysis. These data are consistent with previous small studies of AZT plus ddi or ddc (27). Direct comparison of the rate of development of resistance to ddi or ddc monotherapy could not be made in the present study since these arms were not included in the trial. However, comparison with a previous ddi monotherapy study, in which about 50% of samples showed ddi resistance by 48 weeks, suggests the possibility that combining AZT with ddi slows ddi resistance (31). It is difficult to make similar comparisons with ddc monotherapy studies because few resistance data are available and when this was studied, the incidence of ddc resistance reported was very low (26). Nevertheless, it is clear that most isolates from individuals in our trial receiving either AZT plus ddi or AZT plus ddc after 48 weeks of combination therapy remained susceptible to ddi and ddc. Thus, it would be expected that these drugs should still have an inhibitory effect at this time. All three methods used here to assess AZT susceptibility gave very similar results regarding the degree of AZT resistance in each treatment group at specific times. Thus, the majority of isolates were AZT sensitive before treatment started, and there was a similarly high level of resistance in all groups after 48 weeks of therapy. These results are comparable to those obtained in earlier studies of AZT monotherapy. It was obviously interesting to try to correlate the results of these assays, especially with regard to the relationship between the phenotypic and genotypic assessments. In general, data from both phenotypic assays independently correlated well with the DNA sequence data, giving reassurance that genotyping could be used as a fairly accurate predictor of AZT susceptibility. We encountered some difficulties, however, when there were obvious mixed populations by DNA sequencing. In such cases, we found a number of examples, both with the PBMC assay and by RVA, in which phenotypic predictions from the genotype did not precisely match the actual phenotype. Thus, care is required when one is interpreting RT se-

7 5928 LARDER ET AL. J. VIROL. quence data when mixed populations are apparent at the AZT resistance codons. Despite the good independent correlation between the genotypic assay and each of the phenotypic resistance assays, there was a less obvious relationship between absolute PBMC assay and RVA AZT IC 50 s. This seemed curious since both assays showed 10-fold increases in AZT IC 50 over the course of therapy (though the range of values was quite different between the assays). From these data, however, it is clear that direct comparisons of absolute IC 50 s for individual samples from both assays are very difficult to make. Therefore, it would seem that comparisons of the results of susceptibility analyses from different clinical trials should be made only when the same susceptibility assay has been used. The greater sensitivity of the RVA (15), which relies on PCR to amplify the RT coding region, may render this approach the more useful when virus isolation is difficult because of low overall viral load. For example, virus isolation from week 48 samples was about 50% by the PBMC assay and about 70% for the RVA. In addition, we found that there was considerably less variability in the RVA, which relies on a plaque assay in a cell line rather than infection of primary PBMC cultures. For example, it appears that the RVA may distinguish between genotypic groups designated PR and those designated R, while the PBMC assay may not. The fact that these viruses produced by recombination have a homogeneous genetic background (outside the RT) also probably contributes to lower variation in the assay. Obviously, with the RVA, it is also possible to use plasma-derived viral RNA as the starting material. We have recently started to compare recombinant viruses derived both from PBMC DNA and viral RNA for drug susceptibility. Such analyses of RNAderived recombinant viruses will allow direct measurement of viral phenotype from the same plasma sample used to obtain viral load (HIV RNA copy number). Although our main objective of the DNA sequence analysis of these isolates was to assess AZT and ddn resistance, we also had the opportunity to examine other RT codons. For example, we noticed a surprisingly high incidence of mutations at codon 181 characteristic of resistance to NNRTIs. These were present in both week 0 and week 48 samples in similar proportions but mainly in different patient isolates. The question as to how these mutations appeared is not clear. We carefully examined the possibility of these observations being spurious as a result of contamination of PCR assays. However, this seemed unlikely since the codon 181 mutation was detected in a wide variety of different genetic backgrounds, in terms of both AZT resistance mutations and background fingerprint changes. Furthermore, it is highly unlikely that any of the individuals had access to NNRTIs prior to enrollment into the trial because these inhibitors were not widely available at that time. It is possible that some individuals obtained illicit NNR TIs during the trial, but this was also not very likely, especially as we did not observe any of the other mutations characteristic of NNRTI resistance (data not shown). Thus, the most plausible explanation is that this mutation (i.e., Tyr181Cys) occurs naturally in HIV-1 populations at a relatively high frequency. This possibility is supported by previous reports of the detection of Cys181 in HIV-1 isolates from untreated individuals (5, 23). This of course implies that virus containing Cys181 has little growth disadvantage compared to virus with Tyr181. It might also explain why this mutation appears very rapidly after the initiation of therapy with NNRTIs such as Nevirapine and is rapidly selected by in vitro passage (26, 28). We were also interested in exploring the possibility that the unique mutations recently reported to be associated with multidrug resistance (31, 32) had occurred in some of the isolates that we sequenced. Isolates obtained at week 48 that had no conventional AZT or ddn resistance mutations were specifically examined for changes at codons 62, 75, 77, 116, and 151. Although a degree of heterogeneity was noted at various codons, none of the reported multidrug resistance mutations were seen in these samples. We assumed that these isolates had therefore remained wild type with respect to drug resistance. This might have been because only 50% of the conventional ddi dose (i.e., 100 mg twice a day) was used in this study or we did not sample at a sufficiently late time point. Alternatively, the set of mutations reported by others (10, 31, 32) might occur at a low frequency. In summary, our extensive drug susceptibility analysis of HIV isolates from individuals participating in a combination therapy trial have allowed us to obtain a large amount of information regarding drug resistance. The most likely reason for the observed benefit of the two-drug combinations compared with AZT alone was that the majority of isolates from individuals in the combination arms remained susceptible to ddi and ddc throughout the study period. A broad agreement between the predicted phenotype from the genotype and the actual observed phenotype was apparent when the different drug susceptibility assays were compared. However, direct comparisons between the two phenotypic assays could not be reliably made for individual samples, which suggests that the same assay procedure should be used in order to make valid comparisons of virus susceptibility data between different trials. As future clinical trials will involve double and triple drug combinations, the patterns of drug resistance will become increasingly more complex. Thus, it will probably be necessary to use both phenotypic and genotypic assays to determine a comprehensive picture of HIV-1 drug resistance. ACKNOWLEDGMENT We thank all of the investigators and participants involved in Protocol 34, REFERENCES 1. Balzarini, J., A. Karlsson, M. L. Perez-Perez, M. J. Camarasa, W. G. Tarpley, and E. de Clerq Treatment of human immunodeficiency virus type-1 (HIV-1) infected cells with combinations of HIV-1 specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy. Virology 192: Boucher, C. A., E. O Sullivan, J. W. Mulder, C. Ramautarsing, P. Kellam, G. Darby, J. M. Lange, J. Goudsmit, and B. A. Larder Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects. J. Infect. Dis. 165: Byrnes, V. W., V. V. Sardana, W. A. Schleif, J. H. Condra, J. A. Waterbury, J. A. Wolfgang, W. J. Long, C. L. Schneider, A. J. Schlabach, B. S. Wolanski, D. J. Graham, L. Gotlib, A. Rhodes, D. L. Titus, E. Roth, O. M. Blahy, J. C. Quintero, S. Staszewski, and E. A. Emini Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors. Antimicrob. Agents Chemother. 37: D Aquila, R. T., V. A. Johnson, S. L. Welles, A. J. Japour, D. R. Kuritzkes, V. DeGruttola, P. S. Reichelderfer, R. W. Coombs, C. S. Crumpacker, J. O. Kahn, et al Zidovudine resistance and HIV-1 disease progression during antiretroviral therapy. AIDS Clinical Trials Group Protocol 116B/117 Team and the Virology Committee Resistance Working Group. Ann. Intern. Med. 122: De Jong, M. D., R. Schuurman, J. M. A. Lange, and C. A. B. Boucher Replication of a pre-existing resistant HIV-1 subpopulation in vivo after introduction of a strong selective pressure. Antiviral Ther. 1: Fitzgibbon, J. E., R. M. Howell, C. A. Haberzettl, S. J. Sperber, D. J. Gocke, and D. T. Dubin Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2,3 -dideoxycytidine. Antimicrob. Agents Chemother. 36: Gu, Z., Q. Gao, H. Fang, H. Salomon, M. A. Partiak, E. Goldberg, J. Cameron, and M. A. Wainberg Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encodes human immunodeficiency virus resistance to 2,3 -dideoxycytidine and 2,3 -deoxythiacytidine. Antimicrob. Agents Chemother. 38:

8 VOL. 70, 1996 HIV-1 SUSCEPTIBILITY DURING COMBINATION THERAPY Gu, Z., Q. Gao, X.-J. Li, and M. A. Wainberg Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross resistance to 2,3 -dideoxycytidine and 2,3 dideoxy-3 -thiacytidine. J. Virol. 66: Hammer, S., D. Katzenstein, M. Hughes, H. Grundacker, M. Hirsch, T. Merigan, for the ACTG 175 Study Team Nucleoside monotherapy (MT) vs combination therapy (CT) in HIV infected adults: a randomised double blind placebo controlled trial in persons with CD4 counts between 200 and 500 cells/mm3. Presented at the 5th European Conference on Clinical Aspects and Treatment of HIV Infection, Copenhagen, Denmark. 10. Iverson, A. K. N., R. W. Shafer, K. Wehrly, M. A. Winters, J. I. Mullins, B. Chesebro, and T. C. Merigan Multidrug resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy. J. Virol. 70: Jackson, J. B., R. W. Coombs, K. Sannerud, F. S. Rhame, and H. H. Balfour, Jr Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J. Clin. Microbiol. 26: Japour, A. J., D. L. Mayers, V. A. Johnson, D. R. Kuritzkes, L. A. Beckett, J. M. Arduino, J. Lane, R. J. Black, P. S. Reichelderfer, R. T. D Aquila, C. S. Crumpacker, The RV-43 Study Group, and The AIDS Clinical Trials Group Virology Committee Resistance Working Group Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 37: Kellam, P., C. A. B. Boucher, and B. A. Larder Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high level resistance to zidovudine. Proc. Natl. Acad. Sci. USA 89: Kellam, P., C. A. B. Boucher, J. M. G. H. Tijnagel, and B. A. Larder Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance. J. Gen. Virol. 75: Kellam, P., and B. A. Larder Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 38: Kellam, P., and B. A. Larder Retroviral recombination can lead to linkage of reverse transcriptase mutations that confer increased zidovudine resistance. J. Virol. 69: Larder, B. A., G. Darby, and D. D. Richman HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science 243: Larder, B. A., P. Kellam, and S. D. Kemp Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. AIDS 5: Larder, B. A., P. Kellam, and S. D. Kemp Convergent combination therapy can select viable multidrug resistant HIV-1 in vitro. Nature (London) 365: Larder, B. A., and S. D. Kemp Multiple mutations in HIV-1 reverse transcriptase confer high level resistance to zidovudine. Science 246: Larder, B. A., A. Kohli, P. Kellam, S. D. Kemp, M. Kronick, and R. D. Henfrey Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing. Nature (London) 365: Mellors, J., B. A. Larder, and R. Schinazi Mutations associated with HIV-1 reverse transcriptase and protease associated with drug resistance. Int. Antiviral News 3: Najera, I., D. D. Richman, I. Olivares, J. M. Rojas, M. A. Perucho, R. Najera, and C. Lopez-Galindez Natural occurrence of drug resistant mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates. AIDS Res. Hum. Retroviruses 10: Richman, D. D Resistance of clinical isolates of human immunodeficiency virus to antiretroviral agents. Antimicrob. Agents Chemother. 37: Richman, D. D Resistance, drug failure, and disease progression. AIDS. Res. Hum. Retroviruses. 10: Richman, D. D., D. Havlir, J. Corbeil, D. Looney, C. Ignatio, S. A. Spector, J. Sullivan, S. Cheeseman, K. Barringer, D. Pauletti, C.-K. Shih, M. Myers, and J. Griffin Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68: Richman, D. D., T. C. Meng, S. A. Spector, M. A. Fischl, L. Resnick, and S. Lai Resistance to AZT and ddc during long term combination therapy in patients with advanced infection with human immunodeficiency virus. J. Acquired Immune Defic. Syndr. 7: Richman, D. D., C. K. Shih, I. Lowy, et al Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc. Natl. Acad. Sci. USA 88: Rooke, R., M. Tremblay, H. Soudeynes, L. De Stephano, X.-J. Yao, M. Fanning, J. S. G. Montaner, M. O Shaughnessy, K. Gelman, C. Tsoukas, J. Gill, J. Reudy, and M. A. Wainberg Isolation of drug resistant variants of HIV-1 from patients on long term zidovudine therapy. AIDS 3: Schooley, R. T., C. Ramirez-Ronda, J. M. A. Lange, D. A. Cooper, J. Lavelle, L. Lefkowitz, M. Moore, B. A. Larder, M. St. Clair, P. Reiss, R. McKinnis, K. Pennington, P. R. Harrigan, I. Kinghorn, H. Steel, J. F. Rooney, and the Wellcome Resistance Study Collaborative Group Virologic and immunologic benefits of initial combination therapy with zidovudine and zalcitabine or didanosine compared to zidovudine monotherapy. J. Infect. Dis. 173: Shafer, R. W., M. J. Kozal, M. A. Winters, A. K. Iversen, D. A. Katzenstein, M. V. Ragni, W. A. Meyer 3rd, P. Gupta, S. Rasheed, R. Coombs, et al Combination therapy with zidovudine and didanosine selects for drug resistant human immunodeficiency virus type 1 strains with unique patterns of pol gene mutations. J. Infect. Dis. 169: Shirasaka, T., M. F. Kavlick, T. Ueno, W. Y. Gao, E. Kojima, M. L. Alcaide, S. Chokekijchai, B. M. Roy, E. Arnold, R. Yarchoan, et al Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc. Natl. Acad. Sci. USA 92: St. Clair, M. H., J. L. Martin, G. Tudor-Williams, M. C. Bach, C. L. Vavro, D. M. King, P. Kellam, S. D. Kemp, and B. A. Larder Resistance to ddi and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science 253: Volberding, P The need for additional options in the treatment of human immunodeficiency virus infection. J. Infect. Dis. 171(Suppl. 2):S150 S Yenni, P., on behalf of the DELTA International Co-ordinating Committee Preliminary results of the European/Australian DELTA trial: based on data up to 31 May Presented at the 5th European Conference on Clinical Aspects and Treatment of HIV Infection, Copenhagen, Denmark. 36. Zhang, D., A. M. Caliendo, J. J. Eron, K. M. DeVore, J. C. Kaplan, M. S. Hirsch, and R. T. D Aquila Resistance to 2,3 -dideoxycytidine conferred by a mutation in codon 65 of the human immunodeficiency virus type 1 reverse transcriptase. Antimicrob. Agents. Chemother. 38: