To identify HIV-1 isolates resistant to multiple antiretroviral agents, we examined isolates from four heavily treated patients with HIV-1 infection who had started receiving antiretroviral therapy before potent three-drug combinations were available and had not achieved plasma HIV-1 RNA suppression despite treatment with several combination regimens. We reasoned that because these patients had ongoing HIV-1 replication, they were at risk for developing HIV-1 strains resistant to each of the drugs they had received.

Methods

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Patients

We obtained HIV-1 isolates from four consecutive patients who had received prolonged antiretroviral therapy and had not achieved plasma HIV-1 RNA suppression despite therapy with several drug combinations. Between August 1996 and February 1997, three viral samples each were obtained from patients 1,2, and 3. Patient 4 died before a follow-up sample was obtained for repeated sequencing and viral culture.

Virus Isolation

Peripheral blood mononuclear cells from the patients were co-cultured with phytohemagglutinin-stimulated peripheral blood mononuclear cells from HIV-seronegative blood donors. When the HIV-1 p24 antigen concentration in the culture exceeded 20 ng/mL, aliquots of supernatant were harvested for drug susceptibility testing. Wild-type control HIV-1 isolates (NL43 and H112) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.

Reverse Transcriptase and Protease Sequencing

We extracted HIV-1 RNA from plasma and from aliquots of cultured virus stock [9]. Nested polymerase chain reaction was then used to generate a 1.3-kb fragment encompassing HIV-1 protease and the first 250 residues of reverse transcriptase. Direct dideoxyterminator sequencing of the polymerase chain reaction product was done in both directions by using overlapping internal primers. Sequences were compared with the HIV-1 subtype B consensus sequence [10] and submitted to GenBank (AF047280-AF047321) [11].

Drug Susceptibility Testing

Susceptibility tests were done on one isolate each from patients 1 (August 1996), 2 (November 1996), and 3 (January 1997). Patient and wild-type control isolates were tested in triplicate with zidovudine (Glaxo Wellcome, Research Triangle Park, North Carolina), didanosine (Bristol-Myers Squibb, Wallingford, Connecticut), zalcitabine (Roche Laboratories, Nutley, New Jersey), stavudine (Bristol-Myers Squibb), saquinavir (Roche Laboratories), indinavir (Merck & Co., Whitehouse Station, New Jersey), and nelfinavir (Agouron Pharmaceuticals, La Jolla, California) and in duplicate with lamivudine (Glaxo Wellcome) and nevirapine (Boehringer-Ingleheim, Ridgefield, Connecticut).

A 50% tissue culture infectious dose of virus stock of 30 to 100 was used to infect 1 000 000 peripheral blood mononuclear cells from HIV-seronegative blood donors in the presence and absence of increasing drug concentrations [12]. After 4 days, HIV-1 p24 antigen production was measured in culture supernatant and the drug concentrations required to inhibit p24 antigen production by 90% (IC₉₀) compared with drug-free controls were calculated. Drug concentrations were 0.0005, 0.005, 0.05, 0.5, and 5 µmol/L for zidovudine; 0.6, 1.2, 2.5, 5, and 10 µmol/L for didanosine; 0.06, 0.12, 0.25, 0.5, and 1.0 µmol/L for stavudine and zalcitabine; and 0.016, 0.08, 0.4, 2, and 10 µmol/L for lamivudine, nevirapine, saquinavir, indinavir, and nelfinavir.

Biological Cloning

Serial fourfold dilutions of the initial virus stock of each isolate were co-cultured with 1 000 000 peripheral blood mononuclear cells from HIV-seronegative blood donors and monitored for HIV-1 p24 antigen production. Cultures in which fewer than one third of wells were positive were considered to represent infection with a single replication-competent virus.

Statistical Analysis

Statistical descriptions of groups of IC₉₀ were done using log-transformed IC₉₀. Nucleotide distances between all pairs of sequences were calculated to exclude the possibility of laboratory contamination or transmission between patients [13].

Results

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Antiretroviral Treatment History and Clinical Course

By August 1996, each patient had received drug treatment for 4 to 9 years (Figure 1). Each patient began receiving zidovudine and then added or substituted other drugs as they became available. Eventually, each patient received at least four of the five available nucleoside analogue reverse transcriptase inhibitors and two or three of the four available protease inhibitors. No patient received a non-nucleoside reverse transcriptase inhibitor or nelfinavir, the fourth approved protease inhibitor.

View larger version (23K): [in this window] [in a new window]   Figure 1. Serial plasma HIV-1 RNA levels and CD4+ lymphocyte counts of four patients with multidrug-resistant HIV-1 isolates. The time course for each patient begins at the start of plasma HIV-1 RNA level monitoring. The drug regimens received by each patient are listed along the x-axis. Arrows indicate plasma HIV-1 isolates that were sequenced; dashed lines show the limit of detection of the plasma HIV-1 RNA assays used during the study (500 log10 copies/mL). Drugs were given at standard dosages with several exceptions. Patients 2 and 4 received periods of reduced-dose zidovudine (300 mg/d); patient 3 received saquinavir, 1200 mg/d, for 6 months; and patients 1, 3, and 4 received a combination of ritonavir (600 to 1200 mg/d) and saquinavir (600 to 1800 mg/d) for several months. Before HIV-1 RNA monitoring began, patient 1 received zidovudine from 1987 to 1990, zidovudine-didanosine and zidovudine-zalcitabine from 1990 to 1993, and zidovudine-lamivudine from 1994 to 1995. Patient 2 received zidovudine from 1989 to 1992, zidovudine-didanosine and zidovudine-zalcitabine from 1992 to 1993, and zalcitabine-saquinavir in 1994. Patient 3 received zidovudine from 1992 to 1993 and didanosine from 1993 to 1994. Patient 4 received zidovudine in 1990, zidovudine-zalcitabine in 1991, didanosine and zidovudine-didanosine in 1993, and stavudine in 1994. 3TC = lamivudine; AZT = zidovudine; d4T = stavudine; ddC = zalcitabine; ddi = didanosine; IND = indinavir; RIT = ritonavir; SQV = saquinavir.

Patient 1 had localized cutaneous Kaposi sarcoma diagnosed in 1992. Patient 4 had several opportunistic infections between 1994 and 1996 and died in December 1996. Patients 2 and 3 had no HIV-related complications. Isolates of HIV-1 from patient 3 were syncytium-inducing; isolates from patients 1 and 2 were not syncytium-inducing. Despite treatment with several drug combinations, only two patients had reductions in plasma HIV-1 RNA levels. These levels were not reduced below the limit of detection in any patient.

Drug Susceptibility

Isolates from three patients were available for susceptibility testing. Each isolate had high-level resistance to zidovudine (90-fold to 470-fold) and lamivudine (>400-fold) and low-level resistance to didanosine (3-fold), zalcitabine (3-fold to 4-fold), and stavudine (3-fold to 5-fold) (Table 1 and Table 2). Each isolate was also resistant to the protease inhibitors saquinavir (90-fold to 200-fold), indinavir (50-fold to 100-fold), and nelfinavir (30-fold to 50-fold) (Table 1 and Table 2). Ritonavir was not tested because indinavir-resistant isolates are usually also resistant to ritonavir [5, 6, 14]. Each isolate was susceptible to the non-nucleoside reverse transcriptase inhibitor nevirapine.

Protease and Reverse Transcriptase Sequences

The four patients had isolates sharing seven protease mutations associated with drug resistance (L10I, G48V, I54V/T, L63P/H/Q, A71V/L, V77I, and V82A) [5, 6, 14]. Three patients had isolates sharing eight reverse transcriptase mutations, including zidovudine-resistance mutations (M41L, D67N, L210W, and T215Y) and the lamivudine-resistance mutation (M184V) [4, 14]. Other shared mutations were K43E/N, E44D/A, and V118I [4, 14].

In addition to the shared mutations, the protease-inhibitor resistance mutations M46I and L90M and the reverse transcriptase mutation L74I were present in the isolate from patient 4. The isolate from patient 2 had the zalcitabine-resistance mutation T69D [14]. Mutations V60I, K102Q, Q207E, H208Y, and K219N were each present in isolates from two patients.

Co-Linearity and Stability of Reverse Transcriptase and Protease Mutations

To determine whether the reverse transcriptase and protease mutations occurred in the same HIV-1 genomes, we sequenced the reverse transcriptase and protease genes of two biological clones from patients 1, 2, and 3 and found that all six clones had the reverse transcriptase and protease mutations shown in the Table 1 and (Table 2).

Patients 1, 2, and 3 each had three different isolates sequenced between August 1996 and January 1997. During this period, there was little intrapatient sequence divergence in either the reverse transcriptase or the protease genes (0.8% and 0.6%, respectively). With one exception (the August 1996 isolate from patient 3 lacked M184V), each isolate had the mutations shown in the Table 1 and (Table 2).

Interpatient Sequence Divergence

Isolates from the four patients had a mean nucleotide sequence divergence of 6.4% for the protease gene and 5.6% for the reverse transcriptase gene, suggesting that the shared mutations did not result from laboratory contamination or transmission of a single HIV-1 strain.

Previous HIV-1 Isolates

A June 1990 isolate from patient 1 had the zidovudine-resistance mutations M41L and T215Y and was highly resistant to zidovudine. However, it was susceptible to the other reverse transcriptase inhibitors and to the protease inhibitors. A March 1995 isolate from patient 1, obtained after 6 months of saquinavir therapy, had the protease mutations G48V, L63P, A71V, and T74A and had an approximately 10-fold decreased susceptibility to both saquinavir and nelfinavir but no reduced susceptibility to indinavir.

Discussion

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This report shows that HIV-1 has the potential to develop resistance to most available antiretroviral drugs. The patients described had HIV-1 isolates with high-level resistance to zidovudine, lamivudine, indinavir, saquinavir, and nelfinavir and lower-level resistance to didanosine, zalcitabine, and stavudine. No patient had received a non-nucleoside reverse transcriptase inhibitor, and all isolates were susceptible to drugs of this class. Additional preliminary studies have shown that these isolates are at least partly cross-resistant to the experimental nucleoside analogue reverse transcriptase inhibitor 1592U89 (Glaxo Wellcome) and the protease inhibitor 141W (Glaxo Wellcome) but are susceptible to the experimental non-nucleoside reverse transcriptase inhibitor efivarenz (Dupont Merck, Wilmington, Delaware) and the acyclic nucleoside phosphonates adefovir and PMPA [9-(2-phosphonomethoxypropyl)adenine] (Gilead Sciences, Foster City, California) [15].

Because our patients were selected as the first four consecutive patients found to have received many antiretroviral drugs and to have poor responses to their most recent antiretroviral regimens, the overall prevalence of multidrug-resistant HIV-1 is not known. Additional studies are needed to assess the prevalence, mechanisms, and clinical implications of multidrug resistance in larger and more diverse populations. However, drug resistance has rarely been reported in previously untreated patients who initially received potent drug combinations and were found to have marked reductions in HIV-1 replication [1, 2, 16].

Because of the complex treatment histories of the patients in this report and the many mutations in their HIV-1 isolates, it is difficult to correlate individual mutations precisely with resistance to specific drugs. Reverse transcriptase mutations M41L, D67N, L210W, and T215Y confer zidovudine resistance, and M184V confers lamivudine resistance [4]. Although M184V partially suppresses T215Y-mediated zidovudine resistance, high-level resistance to both drugs has been reported [4, 17]. Whether the other shared mutations (K43E/N, E44D/A, and V118I) contributed to dual zidovudine-lamivudine resistance in our patients is not known.

Although the level of resistance to didanosine, zalcitabine, and stavudine ranged from 3-fold to 5-fold, the possible clinical importance of this cannot be discounted because the maximum possible in vitro resistance to these three compounds is only about 10-fold [14]. The lamivudine-resistance mutation M184V confers low-level resistance to didanosine and zalcitabine [14] and may partly explain resistance to these compounds. In addition, isolates from patient 2 had the zalcitabine-resistance mutation T69D, and the isolate from patient 4 had a mutation at residue 74, a site involved in didanosine resistance.

Each isolate had at least three protease mutations in the substrate cleft or the flap that clamps onto substrates (G48V, V54I/T, and V82A). Mutation G48V confers saquinavir resistance, and mutations at residues 54 and 82 confer resistance to indinavir and ritonavir. Although these mutations have not been reported in patients receiving nelfinavir monotherapy, recent reports have documented extensive cross-resistance among the available protease inhibitors [5, 6, 18-20].

Recent data from our laboratory suggest that our findings are not anecdotal peculiarities. Between June and December 1997, the Stanford University Hospital Clinical Virology Laboratory sequenced HIV-1 isolates from approximately 150 patients at the request of their physicians. Of 69 patients who had each received at least four nucleoside analogue reverse transcriptase inhibitors, 18 (26%) shared at least six of the eight reverse transcriptase mutations described in this report. Of 45 patients who had each received at least three protease inhibitors, 13 (29%) shared at least five of the seven protease mutations described here, including G48V and V82A. The clinical status of these patients and other patterns of mutations in their isolates are currently being studied.

By sequencing six different isolates (three plasma isolates, one cultured isolate, and two biological clones) from three patients, we have shown the stability of multidrug resistance over time in vivo. Multidrug-resistant isolates provide insight needed for the development of antiretroviral drugs that will complement those already available. Such new drugs could prolong the lives of patients already infected with multidrug-resistant isolates and could help prevent the development of drug resistance if combined with other non-cross-resistant drugs during initial therapy.

Appendix: Terms Related to the Genetic and Functional Correlates of Antiretroviral Drug Resistance

Human immunodeficiency virus type 1 protease and reverse transcriptase amino acid residue numbers represent the amino acid positions in the gene sequence. The amino acid that precedes the residue number is the amino acid most commonly reported among North American and European HIV-1 isolates and is also referred to as the consensus or wild-type amino acid [10]. Abbreviations for all 20 amino acids are used in the paper: A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; V = valine; W = tryptophan; Y = tyrosine. For example, M41L refers to the presence of leucine at position 41 rather than the consensus amino acid methionine.

Genetic polymorphisms are variations of the consensus sequence that have often been seen in HIV-1 isolates from untreated persons [10, 11].

The 50% tissue culture infectious dose is the unit of virus infectivity commonly used to standardize the virus inoculum for drug susceptibility tests.

The IC₉₀ (the 90% inhibitory concentration) is the drug concentration required to inhibit virus replication by 90%.