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.

Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing.

Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.

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.

Comparable biological and molecular determinants in HIV type 1-infected long-term nonprogressors and recently infected individuals.

Human immunodeficiency virus type 1 (HIV-1) isolability, rate of replication, phenotype, plasma viremia, and specific intracellular transcripts were cross-sectionally analyzed in 61 HIV-1-seropositive individuals to evaluate the correlations between the virological and molecular correlates of protection and progression in different clinical subsets: recently infected subjects (RIs), long-term nonprogressors (LTNPs), late progressors (LPs), and typical progressors (TPs). Comparison of the major virological and molecular features of HIV-1 infection has defined distinct profiles for different subsets of patients. LTNPs or RIs, as well as LPs or TPs, exhibited similar titers of coculture p24 antigen; the differences between the former and the latter were statistically significant at all the time points tested (p = 0.0001; 0.0003 and 0.0001). Whereas LTNPs and RIs revealed comparable low levels of indexes of viral replication, LPs and TPs showed higher genome and mRNA copy numbers (p = 0.0004 and p = 0.0008, respectively). We demonstrated close biological and molecular similarities between RIs and LTNPs on the one hand, and LPs and TPs on the other. In LTNPs both viral biological properties and viral load are important determinants of the course of the disease.

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.

Viral load, viral phenotype modification, zidovudine susceptibility and reverse transcriptase mutations during the first 6 months of zidovudine monotherapy in HIV-1-infected people.

We studied 14 zidovudine-naive, HIV-1-infected patients attending an infectious diseases clinic in Milan during zidovudine therapy for 6 months. We monitored CD4 cell counts, immune complex-dissociated p24 antigen, viral phenotype and viral load in plasma. The virus infecting a subset of patients was examined for zidovudine susceptibility and zidovudine resistance-associated mutations. A significant correlation was established between the increase in the CD4 cell count and the decrease in viral load (Spearman's coefficients < -0.5). Patients who were p24 antigen positive had a higher viral load (P < 0.005 at baseline and after 6 months of therapy). Patients with non-syncytium-inducing (NSI) virus had higher CD4 cell counts over time than those with syncytium-inducing (SI) virus. We also examined the viral load in relation to viral phenotype. The median viral load in patients with NSI virus was higher than in SI controls at baseline, but not after 3 and 6 months of therapy. Sequential isolates of HIV-1 were obtained from nine patients and tested for resistance to zidovudine by monitoring the drug susceptibility and the reverse transcriptase-encoding sequence. Amino acid changes at codons 70 and 215 were present in some but not all isolates with zidovudine-resistant phenotype in vitro. It was possible to perform a correlation between zidovudine susceptibility and zidovudine-associated pol gene mutations only at the 6-month time point (Spearman's coefficient = 0.076). SI phenotype was associated with the development of a decreased zidovudine susceptibility. A correlation between zidovudine-associated pol gene mutations and SI phenotype was detected at the 6-month time point.

HIV type 1 phenotype correlates with the stage of infection in vertically infected children.

A cohort of 39 vertically infected children (class N, A, B, and C of the CDC HIV classification for pediatric infection) was studied by virus isolation and non-syncytium inducing (NSI)/syncytium inducing (SI) HIV-1 phenotype evaluation. The HIV-1 isolates were recovered from PBMCs and the MT-2 cell line was used to perform the syncytium assay. HIV-1 could be isolated in 34 of 39 (87%) infected children, regardless of the clinical and immunological stage of the disease. Class N and A subjects harbored exclusively NSI strains, whereas the SI phenotype was detected in two of eight class B and five of nine class C patients. All of the SI variants were observed in severely CD4-depleted children (class 3 patients). The capability of pediatric HIV-1 isolates to induce a cytopathic effect is associated with the clinical status and the degree of CD4 depletion. These data suggest that the biological properties of HIV-1 isolates in children do not differ from those observed in adults, and that viral phenotype strictly correlates with disease progression in vertically infected children.

HIV-1 reverse transcriptase codon 215 mutation and clinical outcome in children treated with zidovudine.

OBJECTIVE: In HIV-infected adults prolonged monotherapy with zidovudine may be associated with the appearance of HIV strains with decreased zidovudine sensitivity, owing to specific mutations in the reverse transcriptase (RT) gene, and this has been suggested to be a reason for reduced zidovudine efficacy. This study was undertaken to determine the appearance of mutation at codon 215 of the RT gene in proviral DNA from PBMCs in HIV-infected children. DESIGN: A prospective, open study. SETTING: A University Pediatric Department. PATIENTS AND METHODS: Nineteen HIV-infected symptomatic children were treated with zidovudine for a median of 24 months. Clinical and laboratory controls for HIV infection status were performed monthly. Mutant proviral sequences were evaluated at the start of therapy, every 3 months during the first 6 months of therapy, and every 6 months thereafter. Clinical outcome was defined as stable or deteriorating. RESULTS: No child had proviral sequences mutant at codon 215 before starting zidovudine. Ten of 13 children who had received zidovudine for more than 6 months developed mutant proviral sequences. All the children (10 of 10) with mutant proviral sequences had a deteriorating clinical condition, compared to none of those (0 of 9) without mutation at codon 215. CONCLUSION: The appearance of HIV-1 codon 215 mutation seems to be strongly associated with zidovudine therapy and with clinical progression of HIV disease in children.