Isabel Olivares

In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants.

Nevirapine-resistant variants were generated by serial passages in MT-2 cells in the presence of increasing drug concentrations. In passage 5, mutations V106A, Y181C and G190A were detected in the global population, associated with a 100-fold susceptibility decrease. Sequence analysis of biological clones obtained from passage 5 and subsequent passages showed that single mutants, detected in first passages, were progressively replaced in passage 15 by double mutants, correlating with a 500-fold increase in phenotypic resistance. Fitness determination of single mutants confirmed that, in the presence of nevirapine, every variant was more fit than wild-type with a fitness order Y181C>V106A>G190A>wild-type. Unexpectedly, in the absence of the drug, the Y181C resistant mutant was more fit than wild-type, with a fitness gradient Y181C>wild-type >G106A>or=V190A. Using a molecular clone in which the Y181C mutation was introduced by in vitro mutagenesis, the greater fitness of the Y181C mutant was confirmed in new competition cultures. These data exemplify the role of resistance mutations on virus phenotype but also on virus evolution leading, occasionally, to resistant variants fitter than the wild-type in the absence of the drug.

A combination of defective DNA and protective host factors are found in a set of HIV-1 ancestral LTNPs.

We studied viral evolution in three HIV-1 ancestral patients from a group of LTNPs; although some minor sequences showing viral evolution were detected in all patients, the extremely low viral evolution of their viruses was shown by the phylogenetic analysis of the env sequences. Complete nucleotide sequencing of viral DNA showed the major presence of deletions. In two patients, deletions of 1088 and 228 nucleotides mapped to 5 LTR-gag region; in the other, a 247 nucleotide deletion was positioned in pol gene up to the vif ORF. These deleted genomes became dominant during follow up. Patients viruses displayed 13 common mutations in conserved residues, from the 5 LTR to the nef gene. These mutations provided evidence of a common origin. Regarding host characteristics, one patient had HLA B2705/B5801; another B1402/B5701; whereas a third showed B3901/B4402 and was Delta32-CCR5 heterozygous. These HIV controllers presented a combination of deleted viral genomes and host protective factors.

Molecular epidemiology of HIV-1 in Madrid

Thirteen HIV-1 isolates from patients of different risk groups in Madrid (Spain) have been analyzed at the genetic level. Two distinct lineages of subtype B have been detected among the HIV-1 circulating in this area: one was related to SF-2/RF strains, whereas the other consists of a more heterogeneous group related to reference strain III-B. Variants of each lineage appeared to circulate preferentially within a risk group: III-B among intravenous drug users, and RF/SF-2 among male homosexuals.

Human immunodeficiency virus type 1 chronic infection is associated with different gene expression in MT-4, H9 and U937 cell lines.

To investigate cellular factors involved in HIV-1 chronic infection, three cell lines chronically infected with the same HIV-1 viral isolate (s61) were studied by cDNA microarray analysis. Two T cell lines, H61 and M61, showed the characteristics of a persistent infection whereas U61 cell line displayed a latent infection pattern. Analysis of genes with altered expression in the three cell lines revealed evidence of apoptosis control by up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes. In addition, cell cycle control was affected in the two persistent T cell lines particularly through the down-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Moreover, each cell line showed specific characteristics, like in M61 cells, genes related with cellular activation and with cell migration and motility. In U61 cells, genes associated with immune response were activated. Genes with altered expression in our experiments, and not previously related with HIV such as ANXA 1 or CFLAR were detected and validated. This work revealed that different cell mechanism such as control of apoptosis and cell cycle are important for in vitro HIV-1 chronic infections, and discovered new genes previously not related with HIV-1 replication.

Primary genetic characterization of HIV-1 isolates from WHO-sponsored vaccine evaluation sites by the RNase-A mismatch method.

We have analyzed by the RNase-A mismatch method 35 isolates from four WHO-sponsored vaccine evaluation sites as a secondary laboratory of the WHO Network for HIV Isolation and Characterization. The application of an estimator for the establishment of genetic distances based on the RNase-A digestion patterns in combination with the phylogenetic analysis has allowed us to construct a tree with five well defined groups of viruses. Because the clustering with known reference strains, samples from Brazil could be grouped as subtype B and the majority of those from Thailand were subtype E. Some of the samples from Uganda were classified as subtype D. Isolates from Rwanda and some from Uganda were identified as subtype A viruses. These results coincide with data obtained by heteroduplex mobility assay and nucleotide sequencing in env regions. The RNase-A mismatch method combined with phylogenetic analysis permitted the primary genetic classification of 33 of 35 samples from the WHO Network.

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2()-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2()-difluoro-2()-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.

Identification of a cluster of HIV-1 controllers infected with low replicating viruses.

Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection

ABSTRACT HIV-1 elite controllers (EC) maintain undetectable viral loads (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes, including a proportion that loses HIV-1 control over time. In this work, we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, with persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1 infection with a longitudinal retrospective study design. Gag-specific T-cell responses were assessed by in vitro intracellular polycytokine production quantified by flow cytometry. Viral diversity determinations and sequence dating were performed in proviral DNA by PCR amplification at limiting dilution of env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity, and high proinflammatory cytokine levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r = −0.8; P = 0.02). RANTES was a potential biomarker of transient control. This study identified virological and immunological factors, including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in better clinical management of these individuals and in the development of future curative approaches. IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so-called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work, we found differences in virological and immune factors, including soluble inflammatory biomarkers, between subjects with persistent control of viral replication and EC that will lose virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose natural control of viral replication and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV infection.

Evidence of ongoing replication in a human immunodeficiency virus type 1 persistently infected cell line.

Human immunodeficiency virus type 1 (HIV-1) persistently infected cell lines are characterized by the continuous viral production without cytopathic effect. However, it is not completely clear if this production is contributed only by viral transcription or also by new cycles of viral replication. We studied an HIV-1 persistently infected cell line, designated H61-D, providing evidence of new replication cycles as sustained by: (i) a decrease in viral production, measured by p24 protein, after treatment of the culture with 3-azydo-3-deoxythymydine; (ii) detection of new integration events in the course of cell culture, and (iii) finding of two-long-terminal repeat circles in the cells. H61-D cells were not infected by cell-free virus, but infection was possible by co-culture with another productive-infected cell line. In conclusion, ongoing viral replication is taking place in H61-D persistent cells and new infections are mediated by a cell-to-cell spread mechanism.

Sequence analysis of HIV-1vif gene in Spanish isolates

We have determined the nucleotide sequence of the HIV-1vif gene in viruses obtained from symptomatic patients of distinct risk groups in Madrid. The genetic diversity among the isolates was estimated in 4.6% (±1.4 standard deviation), a similar value to that obtained for thegag gene 3.9% (±0.8 standard deviation) andenv 4.1% (±1 standard deviation) (Rojas et al., Virus Res31, 331–342, 1994). Amino acid sequence analysis revealed the presence of hypermutable residues at positions 101 and 167, close to antigenically relevant sequential epitopes (comprising amino acids 87–94 and 172–178). Phylogenetic analysis supports the existence of two virus lineages circulating preferentially within different risk groups.