Minority Quasispecies of Drug-Resistant HIV-1 That Lead to Early Therapy Failure in Treatment-Naive and -Adherent Patients

Background.Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure. Methods.We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure. Results.Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07% 2.0%. A range of 1 4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times. Conclusions.Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrie

SHAPE-directed Discovery of Potent shRNA Inhibitors of HIV-1

The RNA interference (RNAi) pathway can be exploited using short hairpin RNAs (shRNAs) to durably inactivate pathogenic genes. Prediction of optimal target sites is notoriously inaccurate and current approaches applied to HIV-1 show weak correlations with virus inhibition. In contrast, using a high-content model for disrupting pre-existing intramolecular structure in the HIV-1 RNA, as achievable using high-resolution SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing information, we discovered strong correlations between inhibition of HIV-1 production in a quantitative cell-based assay and very simple thermodynamic features in the target RNA. Strongest inhibition occurs at RNA target sites that both have an accessible “seed region” and, unexpectedly, are structurally accessible in a newly identified downstream flanking sequence. We then used these simple rules to create a new set of shRNAs and achieved inhibition of HIV-1 production of 90% or greater for up to 82% of designed shRNAs. These shRNAs inhibit HIV-1 replication in therapy-relevant T cells and show no or low cytotoxicity. The remarkable success of this straightforward SHAPE-based approach emphasizes that RNAi is governed, in significant part, by very simple, predictable rules reflecting the underlying RNA structure and illustrates principles likely to prove broadly useful in understanding transcriptome-scale biological recognition and therapeutics involving RNA

A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1 replication

<p>Abstract</p> <p>Background</p> <p>Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.</p> <p>Results</p> <p>When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)₂R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.</p> <p>Conclusions</p> <p>This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.</p

Anaphylaxis in Elderly Patients-Data From the European Anaphylaxis Registry

Background: Elicitors and symptoms of anaphylaxis are age dependent. However, little is known about typical features of anaphylaxis in patients aged 65 years or more. Methods: The data from the Network for Online Registration of Anaphylaxis (NORA) considering patients aged ≥65 (elderly) in comparison to data from adults (18–64 years) regarding elicitors, symptoms, comorbidities, and treatment measures were analyzed. Results: We identified 1,123 elderly anaphylactic patients. Insect venoms were the most frequent elicitor in this group (p < 0.001), followed by drugs like analgesics and antibiotics. Food allergens elicited less frequently anaphylaxis (p < 0.001). Skin symptoms occurred less frequently in elderly patients (77%, p < 0.001). The clinical symptoms were more severe in the elderly (51% experiencing grade III/IV reactions), in particular when skin symptoms (p < 0.001) were absent. Most strikingly, a loss of consciousness (33%, p < 0.001) and preexisting cardiovascular comorbidity (59%, p < 0.001) were more prevalent in the elderly. Finally, adrenaline was used in 30% of the elderly (vs. 26% in the comparator group, p < 0.001) and hospitalization was more often required (60 vs. 50%, p < 0.001). Discussion and Conclusion: Anaphylaxis in the elderly is often caused by insect venoms and drugs. These patients suffer more often from cardiovascular symptoms, receive more frequently adrenaline and require more often hospitalization. The data indicate that anaphylaxis in the elderly tends to be more frequently life threatening and patients require intensified medical intervention. The data support the need to recognize anaphylaxis in this patient group, which is prone to be at a higher risk for a fatal outcome

On the role of four small hairpins in the HIV-1 RNA genome

An RNA secondary structure model for the complete HIV-1 genome has recently been published based on SHAPE technology. Several well-known RNA motifs such as TAR and RRE were confirmed and numerous new structured motifs were described that may play important roles in virus replication. The 9 kb viral RNA genome is densely packed with many RNA hairpin motifs and the collective fold may play an important role in HIV-1 biology. We initially focused on 16 RNA hairpin motifs scattered along the viral genome. We considered conservation of these structures, despite sequence variation among virus isolates, as a first indication for a significant function. Four relatively small hairpins exhibited considerable structural conservation and were selected for experimental validation in virus replication assays. Mutations were introduced into the HIV-1 RNA genome to destabilize individual RNA structures without affecting the protein-coding properties (silent codon changes). No major virus replication defects were scored, suggesting that these four hairpin structures do not play essential roles in HIV-1 replicatio

Design of modified U1i molecules against HIV-1 RNA

Several gene therapeutic approaches have been proposed to add to current antiretroviral therapy against HIV-1. U1 interference (U1i) is a promising new gene therapy tool that targets mRNAs with modified U1 snRNAs. For efficient inhibition, the 3'-terminal exon of pre-mRNAs must be recognized by the modified U1 snRNA. Subsequent interaction between the U1-associated 70K protein and poly(A) polymerase leads to inhibition of polyadenylation and consequently degradation of the pre-mRNA. We designed 14 new U1i inhibitors against HIV-1 mRNA regions that are 100% complementary to at least 70% of HIV-1 sequences listed in the HIV database. All U1i inhibitors were tested transiently in HIV-1 production assays as well as luciferase reporter experiments and three candidates were examined further in stably lentivirus-transduced T cell lines. We identified U1i-J that targets the region encoding the NF-kappa B binding sites as the most effective inhibitor that substantially reduced viral protein expression. The potency of J is determined in part by the presence of a duplicated target within the HIV-1 mRNA. The stably transduced SupT1 T cells were challenged with HIV-1 but no antiviral effect was detected. U1i inhibitors can be potent suppressors of HIV-1 production in transient assays but further optimization of this antiviral approach is needed. (C) 2012 Elsevier B.V. All rights reserve

SHAPE-directed Discovery of Potent shRNA Inhibitors of HIV-1

The RNA interference (RNAi) pathway can be exploited using short hairpin RNAs (shRNAs) to durably inactivate pathogenic genes. Prediction of optimal target sites is notoriously inaccurate and current approaches applied to HIV-1 show weak correlations with virus inhibition. In contrast, using a high-content model for disrupting pre-existing intramolecular structure in the HIV-1 RNA, as achievable using high-resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing information, we discovered strong correlations between inhibition of HIV-1 production in a quantitative cell-based assay and very simple thermodynamic features in the target RNA. Strongest inhibition occurs at RNA target sites that both have an accessible "seed region" and, unexpectedly, are structurally accessible in a newly identified downstream flanking sequence. We then used these simple rules to create a new set of shRNAs and achieved inhibition of HIV-1 production of 90% or greater for up to 82% of designed shRNAs. These shRNAs inhibit HIV-1 replication in therapy-relevant T cells and show no or low cytotoxicity. The remarkable success of this straightforward SHAPE-based approach emphasizes that RNAi is governed, in significant part, by very simple, predictable rules reflecting the underlying RNA structure and illustrates principles likely to prove broadly useful in understanding transcriptome-scale biological recognition and therapeutics involving RN