Loss of lamivudine resistance in a zidovudine and lamivudine dual-resistant HIV-1 isolate after discontinuation of in vitro lamivudine drug pressure.

We examined the in vitro phenotypic and genotypic profiles of an extensively passaged human immunodeficiency virus type 1 clinical isolate which has been selected for lamivudine resistance, with an M184V mutation in a zidovudine-resistant genetic background, and then cultured with zidovudine alone. Our passaging strategy led to a decrease in lamivudine IC50 values, which were comparable to those prior to lamivudine exposure, and the genotypic restoration of the wild-type sequence at codon 184 of reverse transcriptase.

Loss of antiviral effect owing to zidovudine and lamivudine double resistance in HIV-1-infected patients in an ongoing open-label trial.

In order to compare the resistance pattern to zidovudine plus lamivudine in zidovudine-experienced patients, we studied three HIV-1-infected patients enrolled in NUCB3004, an open-label trial. Over a 24-week follow-up, the patients were studied for drug sensitivity, reverse transcriptase genotype, viral load (HIV-1 RNA level) and viral phenotype (syncytium inducing (SI) or non-syncytium inducing). Virus isolates derived from peripheral blood mononuclear cells (PBMCs) were tested for changes in drug susceptibility. Proviral DNA in the patients' PBMCs and RNA from plasma and culture supernatant were subjected to amplification and sequencing. All three HIV-1 strains showed a decreased susceptibility to either zidovudine or lamivudine after 24 weeks of therapy. The pattern of DNA genotypic resistance to lamivudine in patient A showed a mutation at codon 184 of the reverse transcriptase-encoding gene (methionine to valine). No HIV-1 strains with lamivudine-related mutations in proviral DNA were found among the isolates obtained from patients B and C. In these two patients, the mutation at codon 184 of the reverse transcriptase-encoding gene appeared in RNA, both in plasma and in culture supernatant. Viral phenotyping revealed the maintenance of the SI phenotype at week 24. Two out of the three patients experienced a reduction in HIV-1 RNA levels after 24 weeks of therapy, and in two out of three there was a rebound in viral load at week 28 together with the onset of the codon 184 mutation in RNA. The degree of phenotypic resistance to both zidovudine and lamivudine correlated with the amino acid changes in RNA and the rapid increase in viral load.

Virological response in multidrug-experienced HIV-1-infected subjects failing highly active combination regimens after shifting from lamivudine to didanosine.

The aim of this study (the RESCUE trial) was to verify the effect of a shift from a lamivudine-containing to a didanosine-containing regimen on viral replication in HIV-1-infected subjects who had experienced prior treatment failure. Sixteen patients (didanosine-experienced in 14/16 cases) were consecutively enrolled: eight patients shifted from lamivudine to didanosine without other changes in their drug regimen. The other eight shifted from lamivudine to didanosine and changed one or more of their other drugs according to their physician's judgement. At the time of the regimen shift, all the subjects exhibited a high-level phenotypic resistance to both zidovudine and lamivudine with changes at codons 70-219 in 100% of cases, at codon 215 in 13 of 16 patients, and the M184V substitution in 13/16 patients. Phenotypic susceptibility to didanosine was maintained in the majority of cases (14/16) despite the high prevalence of changes at codon 184. A statistically significant decrease in viral load (P<0.005) without a significant increase in CD4 lymphocytes (P=0.514) was observed after 3 and 6 months from the introduction of the didanosine-containing regimen. These findings suggest the possibility of achieving a viral load response to didanosine-containing regimens in patients with reverse transcriptase (RT) M184V mutations who were previously treated with this drug and its possible use in salvage combinations.

In vitro inhibition of HIV-1 by Met-SDF-1beta alone or in combination with antiretroviral drugs.

Compounds that can block the CXCR4 chemokine receptor are a promising new class of antiretroviral agents. In these experiments we studied the effect of a modified form of the native stromal cell-derived factor-1 (SDF-1), Met-SDF-1beta. The in vitro susceptibility of two different CXCR4-tropic HIV-1 strains was determined. Antiviral effect was assessed by the reduction of p24 antigen production in PHA-stimulated peripheral blood mononuclear cells with exposure to the modified SDF-1 molecule. The 50% inhibitory concentrations (IC50) were derived from six separate experiments. The IC50 against the two HIV-1 isolates was in 1.0-2.8 microg/ml range for Met-SDF-1beta. Met-SDF-1beta showed synergy to additivity with either zidovudine or nelfinavir at IC75 IC90 and IC95. Additivity was seen when Met-SDF-1beta was combined with efavirenz. No cellular toxicity was observed at the highest concentrations when these agents were used either singly or in combination. This compound is a promising new candidate in a receptor-based approach to HIV-1 infection in conjunction with currently available combination antiretroviral drug therapies.

Lack of in vitro anti-gp160 antibody production is a correlate of nonprogression among HIV type 1-infected individuals.

The aim of our study was to investigate the possible correlation of in vitro antibody production (IVAP) directed to the gp160 protein of HIV-1 with CD4+ slopes, plasma viremia, and disease progression in long-term nonprogressors (LTNPs). Nineteen subjects with a long-term nonprogressive HIV-1 infection were studied and followed for 2 years. During the follow-up, in vitro anti-gp160 producers showed negative CD4+ slopes in the majority of cases (9 of 12), whereas 5 of 7 nonproducers showed positive CD4+ slopes. Plasma viremia values, which were not significantly different in the two groups at baseline, became significantly higher in anti-gp160 producers when compared with nonproducers during the follow-up (p = 0.012). Finally, a trend toward progression was observed in the group of producers but not in nonproducers. These findings suggest that the in vitro production of anti-gp160 antibodies by peripheral B cells is not a correlate of protection, and may represent an early predictor of progression in LTNPs.

In vitro evolution of the human immunodeficiency virus type 1 gag-protease region and maintenance of reverse transcriptase resistance following prolonged drug exposure.

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions.

Combination of CCR5 and CXCR4 inhibitors in therapy of human immunodeficiency virus type 1 infection: in vitro studies of mixed virus infections.

We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stroma-derived factor 1beta (SDF-1beta), Met-SDF-1beta, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1beta had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.

Susceptibility to PNU-140690 (Tipranavir) of human immunodeficiency virus type 1 isolates derived from patients with multidrug resistance to other protease inhibitors.

In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs). The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690. We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC(50)) and IC(90), PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M. Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC(50)s, >0.1 microM). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T. Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC(90) of 0.18 +/- 0.02 microM in multiple experiments. The average mean +/- standard error of mean IC(90) for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 +/- 0.055 microM (range, 0.31 to 0.86 microM). Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies.