Sequencing-based detection of low-frequency human immunodeficiency virus type 1 drug-resistant mutants by an RNA/DNA heteroduplex generator-tracking assay

Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.     

Replication-competent simian immunodeficiency virus (SIV) Gag escape mutations archived in latent reservoirs during antiretroviral treatment of SIV-infected macaques

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.     

Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.     

Antiretroviral drug resistance mutations sustain or enhance CTL recognition of common HIV-1 Pol epitopes

Antiretroviral drug resistance and escape from CTL are major obstacles to effective control of HIV replication. To investigate the possibility of combining drug and immune-based selective pressures against HIV, we studied the effects of antiretroviral drug resistance mutations on CTL recognition of five HIV-1 Pol epitopes presented by common HLA molecules. We found that these common drug resistance mutations sustain or even enhance the antigenicity and immunogenicity of HIV-1 Pol CTL epitopes. Variable patterns of cross-reactive and selective recognition of wild-type and corresponding variant epitopes demonstrate a relatively diverse population of CD8(+) T cells reactive against these epitopes. Variant peptides with multiple drug resistance mutations still sustained CTL recognition, and some HIV-infected individuals demonstrated strong CD8(+) T cell responses against multiple CTL epitopes incorporating drug resistance mutations. Selective reactivity against variant peptides with drug resistance mutations reflected ongoing or previous exposure to the indicated drug, but was not dependent upon the predominance of the mutated sequence in endogenous virus. The frequency and diversity of CTL reactivity against the variant peptides incorporating drug resistance mutations and the ability of these peptides to activate and expand CTL precursors in vitro indicate a significant functional interface between the immune system and antiretroviral therapy. Thus, drug-resistant variants of HIV are susceptible to immune selective pressure that could be applied to combat transmission or emergence of antiretroviral drug-resistant HIV strains and to enhance the immune response against HIV.     

High level HIV-1 DNA concentrations in brain tissues differentiate patients with post-HAART AIDS dementia complex or cardiovascular disease from those with AIDS

Highly active antiretroviral treatment (HAART) has had a significant impact on survival of individuals with acquired immunodeficiency syndrome (AIDS); however, with the longer life-span of patients with AIDS, there is increasing prevalence of AIDS dementia complex (ADC) and other non-AIDS-defining illness, and cardiovascular diseases (CVD) are also common. The influence of these varied disease processes on HIV-1 DNA concentration in brain tissues has not been thoroughly assessed in the post-HAART era. The purpose of the current study is to clarify the impacts of ADC and other complications of HIV disease on the viral load in the brains in AIDS patients with post-HARRT. We examined autopsy specimens from the brains of thirteen patients who died from complications of AIDS with quantitative polymerase chain reaction (QPCR). All but one patient had received HAART prior to death since 1995. Two patients died with severe CVD, multiple cerebrovascular atherosclerosis (CVA) throughout the brain and five patients died with ADC. Six patients had no ADC/CVA. A QPCR was used to measure the presence of HIV-1 DNA in six brain tissues (meninges, frontal grey matter, frontal white matter, temporal subcortex, cerebellum and basal ganglia). In the post-HARRT era, for non-ADC/CVA patients, HIV-1 DNA concentration in brain tissues was statistically higher than that in patients with ADC. In a new finding, two patients who suffered from severe CVD, especially CVA, also had high concentrations of HIV-1 in brain compartments not showing ADC related changes. To our knowledge, this is the first report of a relationship between the CVA and HIV-1 viral burden in brain. The current observations suggest that HAART-resistant HIV reservoirs may survive within ADC lesions of the brain as well as the macrophage rich atherosclerosis, which needs to be confirmed by more AIDS cases with CVA. Supported by the National Institutes of Health (Grant Nos. NIH ZMH1 BRB-S and UOI CA66259-09 TDC), National Science Foundation (Grant No. NSF DMI-0349669), abd Science & Technology Development Program of Shandong Province (Grant No. 2007GG30002003).     

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.     

In vitro human immunodeficiency virus eradication by autologous CD8(+) T cells expanded with inactivated-virus-pulsed dendritic cells

Despite significant immune recovery with potent highly active antiretroviral therapy (HAART), eradication of human immunodeficiency virus (HIV) from the bodies of infected individuals represents a challenge. We hypothesized that an inadequate or inappropriate signal in virus-specific antigen presentation might contribute to the persistent failure to mount efficient anti-HIV immunity in most HIV-infected individuals. Here, we conducted an in vitro study with untreated (n = 10) and HAART-treated (n = 20) HIV type 1 (HIV-1) patients which showed that pulsing of monocyte-derived dendritic cells (DC) with aldrithiol-2-inactivated autologous virus resulted in the expansion of virus-specific CD8(+) T cells which were capable of killing HIV-1-infected cells and eradicating the virus from cultured patient peripheral blood mononuclear cells independently of the disease stages and HAART response statuses of the patients. This in vitro anti-HIV effect was further enhanced by the HIV protease inhibitor indinavir (at a nonantiviral concentration), which has been shown previously to be able to up-regulate directly patient T-cell proliferation following immune stimulation. However, following a 2-day treatment with culture supernatant derived from immune-activated T cells (which mimics an in vivo environment of HIV-disseminated and immune-activated lymphoid tissues), DC lost their capacity to present de novo inactivated-virus-derived antigens. These findings provide important information for understanding the establishment of chronic HIV infection and indicate a perspective for clinical use of DC-based therapeutic vaccines against HIV.     

Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.