Eulalia Grau

Measurement of Intracellular Didanosine and Tenofovir Phosphorylated Metabolites and Possible Interaction of the Two Drugs in Human Immunodeficiency Virus-Infected Patients

ABSTRACT Recent work has demonstrated the existence of a systemic interaction between didanosine (ddI) and tenofovir disoproxyl fumarate (TDF) that leads to a significant increase in plasma ddI levels when coadministered with TDF (40 to 50% increase). These two drugs are, respectively, nucleoside and nucleotide analogues of adenosine and efficiently inhibit the human immunodeficiency virus (HIV) reverse transcriptase when transformed to their triphosphate moieties in the intracellular (IC) medium (ddA-TP and TFV-DP, respectively). Since ddI and TDF partly share the same IC metabolic pathway leading to the active triphosphates, we investigated a putative IC interaction. We used high-performance liquid chromatography-tandem mass spectrometry techniques to determine ddA-TP and TFV-DP IC levels in HIV-infected patients cotreated with both drugs, in comparison with patients treated with just one of the two drugs. These measurements revealed no significant differences in IC levels of the corresponding triphosphates when ddI (250 mg, once a day [QD]) was coadministered with TDF (300 mg, QD) compared to ddI 400 mg (QD) administered without TDF, thus supporting the dose adaptation proposed for this combination. However, we observed that both ddA-TP and TFV-DP have very long IC half-lives, resulting in unusual IC pharmacokinetic profiles with no significant changes in triphosphate concentrations between two dosings. In the case of TFV-DP, this t1/2 of elimination was roughly estimated to be 180 h (7.5 days). This characteristic is certainly interesting in terms of efficacy but could have some drawbacks in terms of virus resistance for patients discontinuing these drugs.

Coinfection and Superinfection in Patients with Long-Term, Nonprogressive HIV-1 Disease

Human immunodeficiency virus 1 (HIV-1) dual infections are considered important because they have been related to AIDS progression. We identified dual infections in 2 patients with long-term, nonprogressive HIV-1 disease; the first patient was diagnosed as being already coinfected, on the basis of the first sample analyzed, but a previous superinfection could not be excluded; the second patient was diagnosed as having a superinfection, on the basis of the 9-year difference between the viral dating of the 2 strains. Dual infections occur in patients with long-term, nonprogressive disease, with no immediate clinical manifestations. Such occurrences could indicate a general phenomenon in natural HIV-1 infections.

ZNRD1 (Zinc Ribbon Domain-Containing 1) Is a Host Cellular Factor That Influences HIV-1 Replication and Disease Progression

BACKGROUND Human immunodeficiency virus (HIV) takes advantage of multiple host proteins to support its own replication. The gene ZNRD1 (zinc ribbon domain-containing 1) has been identified as encoding a potential host factor that influenced disease progression in HIV-positive individuals in a genomewide association study and also significantly affected HIV replication in a large-scale in vitro short interfering RNA (siRNA) screen. Genes and polymorphisms identified by large-scale analysis need to be followed up by means of functional assays and resequencing efforts to more precisely map causal genes. METHODS Genotyping and ZNRD1 gene resequencing for 208 HIV-positive subjects (119 who experienced long-term nonprogression [LTNP] and 89 who experienced normal disease progression) was done by either TaqMan genotyping assays or direct sequencing. Genetic association analysis was performed with the SNPassoc package and Haploview software. siRNA and short hairpin RNA (shRNA) specifically targeting ZNRD1 were used to transiently or stably down-regulate ZNRD1 expression in both lymphoid and nonlymphoid cells. Cells were infected with X4 and R5 HIV strains, and efficiency of infection was assessed by reporter gene assay or p24 assay. RESULTS Genetic association analysis found a strong statistically significant correlation with the LTNP phenotype (single-nucleotide polymorphism rs1048412; P = .0004), independently of HLA-A10 influence. siRNA-based functional analysis showed that ZNRD1 down-regulation by siRNA or shRNA impaired HIV-1 replication at the transcription level in both lymphoid and nonlymphoid cells. CONCLUSION Genetic association analysis unequivocally identified ZNRD1 as an independent marker of LTNP to AIDS. Moreover, in vitro experiments pointed to viral transcription as the inhibited step. Thus, our data strongly suggest that ZNRD1 is a host cellular factor that influences HIV-1 replication and disease progression in HIV-positive individuals.

Differential prevalence of the HLA-C -- 35 CC genotype among viremic long term non-progressor and elite controller HIV+ individuals.

Susceptibility to HIV infection and disease progression are complex traits modulated by environmental and genetic factors, affecting innate and adaptive immune responses, among other cellular processes. A single nucleotide polymorphism (SNP) 35 kb upstream of the HLA-C gene locus (-35C/T) was previously shown to correlate with increased HLA-C expression and improved control of HIV-1. Here, we genotyped the -35C/T SNP in 639 subjects (180 uninfected patients, 304 HIV progressors and 155 LTNP) and confirmed the association of the -35C/T variant with the LTNP phenotype. The genotype frequencies in the general population subjects did not differ significantly from those seen in HIV progressors (p-value=0.472). However, a significant higher frequency of the protective CC genotype was identified when LTNP were compared either with HIV progressors alone (p-value<0.0001) or progressors and uninfected subjects together (p-value<0.0001). When considering aviremic LTNP alone (elite controllers; viral load below 50 copies/ml), the -35 CC genotype was not overrepresented compared to HIV progressors. Conversely, a significant association was found with the viremic LTNP groups (viral loads below 10,000 copies/ml). These results suggest that other factors alone or in combination with the -35 CC genotype may play an important role in differentiating the elite controller status from LTNP. Combination of different genetic variants may have additive or epistatic effects determining the HIV course of infection.

Rate and predictors of progression in elite and viremic HIV-1 controllers.

Background:The proportion of HIV controllers developing virologic, immunological or clinical progression and the baseline predictors of these outcomes have not been assessed in large cohorts. Methods:A multicenter cohort of HIV controllers was followed from baseline (the first of the three HIV-1 RNA levels < 50 in elite controller or from 50 to 2000 copies/ml in viremic controllers) up to August 2011, to the development of a progression event (loss of viral load control, CD4+ decline, AIDS or death) or to the censoring date (lost to follow-up or initiation of antiretroviral therapy). Predictive models of progression at baseline and a risk score for the combined HIV-1 progression end point were calculated. Results:Four hundred and seventy-five HIV-1 controllers of whom 204 (42.9%) were elite controller with 2972 person-years of follow-up were identified. One hundred and forty-one (29.7%) patients lost viral load control. CD4+ cell count declined in 229 (48.2%) patients. Thirteen patients developed an AIDS event and four died. Two hundred and eighty-seven (60.4%) developed a combined HIV-1 progression. Baseline predictors for the progression end points and for elite and viremic controller patients were very similar: risk for HIV-1 acquisition, baseline calendar year, CD4+ nadir, viral load before baseline and hepatitis C virus coinfection. The probability of a combined HIV-1 progression at 5 years was 70% for elite controllers with the highest score compared with 13% for those with the lowest. Conclusion:HIV-1 disease progression in elite and viremic controllers is frequent. We propose a baseline clinical score to easily classify these patients according to risk of progression. This score could be instrumental for taking clinical decisions and performing pathogenic studies.

Low-replicating viruses and strong anti-viral immune response associated with prolonged disease control in a superinfected HIV-1 LTNP elite controller.

Objective To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. Methodology and Principal Findings We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. Conclusions The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.

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.

Prevalence of HIV-1 dual infection in long-term nonprogressor-elite controllers

Introduction:Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor–elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC. Methods:For DI detection, nucleotide sequences in env gene from viruses from 20 LTNP-EC were analyzed by maximum likelihood. Epidemiological and clinical parameters and host factors of patients were also studied. Results:DI was detected in 4 (20%) of the 20 LTNP-EC, of which 3 maintained the elite controller status. CD4+ T-cell counts were not different between single and DI patients although higher CD8+ T-cell counts were observed in DI patients, and, consequently, the CD4+/CD8+ ratios were lower in LTNP-EC DI patients. Conclusions:Prevalence of HIV-1 DIs in LTNP-EC is similar to other groups of HIV-1 patients; in addition, DI was not associated with loss of disease control in the patients. These DI LTNP-EC patients showed, in comparison with single infected patients, higher numbers of CD8+ T cells and lower CD4+/CD8+ ratios.

Characterization of broadly neutralizing antibody responses to HIV-1 in a cohort of long term non-progressors

Background Only a small fraction of HIV-1-infected patients develop broadly neutralizing antibodies (bNAbs), a process generally associated to chronic antigen stimulation. It has been described that rare aviremic HIV-1-infected patients can generate bNAbs but this issue remains controversial. To address this matter we have assessed bNAb responses in a large cohort of long-term non-progressors (LTNPs) with low or undetectable viremia. Methods Samples from the LTNP cohort of the Spanish AIDS Research Network (87 elite and 42 viremic controllers) and a control population of 176 viremic typical-progressors (TPs) were screened for bNAbs using Env-recombinant viruses. bNAb specificities were studied by ELISA using mutated gp120, neutralization assays with mutated viruses, and peptide competition. Epitope specificities were also elucidated from the serum pattern of neutralization against a panel of diverse HIV-1 isolates. Results Broadly neutralizing sera were found among 9.3% LTNPs, both elite (7%) and viremic controllers (14%). Within the broadly neutralizing sera, CD4 binding site antibodies were detected by ELISA in 4/12 LTNPs (33%), and 16/33 of TPs (48%). Anti-MPER antibodies were detected in 6/12 LTNPs (50%) and 14/33 TPs (42%) whereas glycan-dependent HIV-1 bNAbs were more frequent in LTNPs (11/12, 92%) as compared to TPs (12/33, 36%). A good concordance between standard serum mapping and neutralization-based mapping was observed. Conclusion LTNPs, both viremic and elite controllers, showed broad humoral immune responses against HIV-1, including activity against many major epitopes involved in bNAbs-mediated protection.