Phages and HIV-1: From Display to Interplay

The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures

A hepatitis A, B, C and HIV prevalence and risk factor study in ever injecting and non-injecting drug users in Luxembourg associated with HAV and HBV immunisations

<p>Abstract</p> <p>Background</p> <p>In Luxembourg, viral hepatitis and HIV infection data in problem drug users (PDUs) are primarily based on self-reporting. Our study aimed to determine the prevalence of HAV, HBV, HCV and HIV infections in ever injecting (IDUs) and non-injecting drug users (nIDUs) including inherent risk factors analysis for IDUs. Secondary objectives were immunisation against HAV and HBV, referral to care and treatment facilities as well as reduction in risk behaviour.</p> <p>Methods</p> <p>A nationwide, cross-sectional multi-site survey, involving 5 in-, 8 out-treatment and 2 prison centres, included both an assisted questionnaire (n = 368) and serological detection of HIV and Hepatitis A, B, C (n = 334). A response rate of 31% resulted in the participation of 310 IDUs and 58 nIDUs.</p> <p>Risk factors such as drug use, sexual behaviour, imprisonment, protection and health knowledge (HAV, HBV status and immunisations, HCV, HIV), piercing/tattoo and use of social and medical services were studied by means of chi2 and logistic models.</p> <p>Results</p> <p>Seroprevalence results for IDUs were 81.3% (218/268, 95%CI=[76.6; 86.0]) for HCV, 29.1% (74/254, 95%CI=[25.5;34.7 ]) for HBV (acute/chronic infection or past cured infection), 2.5% (5/202, 95%CI=[0.3; 4.6]) for HIV-1 and 57.1% (108/189, 95%CI=[50.0; 64.1]) for HAV (cured infections or past vaccinations). Seroprevalence results for nIDUs were 19.1% (9/47, 95%CI=[7.9;30.3]) for HCV, 8.9% (4/45, 95%CI=[0.6;17.2]) for HBV (acute/chronic infection or past cured infection), 4.8% (2/42, 95%CI=[-1.7;11.3]) for HIV-1 and 65.9% (27/41, 95%CI=[51.4;80.4]) for HAV. Prisoners showed the highest rates for all infections. Age, imprisonment and setting of recruitment were statistically associated with HCV seropositivity. Age, speedball career and nationality were significantly associated with HBV seropositivity. Only 56% of the participants in outpatient centres collected their serology results and 43 doses of vaccine against HAV and/or HBV were administered.</p> <p>Conclusions</p> <p>Despite the existing national risk-reduction strategies implemented since 1993, high prevalence of HCV and HBV infections in injecting drug users is observed. Our study showed that implementing risk-prevention strategies, including immunisation remains difficult with PDUs. Improvement should be looked for by the provision of field healthcare structures providing tests with immediate results, advice, immunisation or treatment if appropriate.</p

HIV-1 tropism determination using a phenotypic Env recombinant viral assay highlights overestimation of CXCR4-usage by genotypic prediction algorithms for CRRF01_AE and CRF02_AG

Background: Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno([coreceptor]) and webPSSM. Methods: Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno([coreceptor]) (5-20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile (R) Enhanced-Sensitivity-Tropism-Assay. Results: The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno([coreceptor]) was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno([coreceptor]) was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno([coreceptor]) and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A. Conclusions: Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors

Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir

<p>Abstract</p> <p>Background</p> <p>Human Immunodeficiency Virus type 2 is naturally resistant to some antiretroviral drugs, restricting therapeutic options for patients infected with HIV-2. Regimens including integrase inhibitors (INI) seem to be effective, but little data on HIV-2 integrase (IN) polymorphisms and resistance pathways are available.</p> <p>Materials and methods</p> <p>The <it>integrase </it>coding sequence from 45 HIV-2-infected, INI-naïve, patients was sequenced and aligned against the ROD (group A) or EHO (group B) reference strains and polymorphic or conserved positions were analyzed.</p> <p>To select for raltegravir (RAL)-resistant variants <it>in vitro</it>, the ROD strain was cultured under increasing sub-optimal RAL concentrations for successive rounds. The phenotype of the selected variants was assessed using an MTT assay.</p> <p>Results</p> <p>We describe <it>integrase </it>gene polymorphisms in HIV-2 clinical isolates from 45 patients. Sixty-seven percent of the integrase residues were conserved. The HHCC Zinc coordination motif, the catalytic triad DDE motif, and AA involved in IN-DNA binding and correct positioning were highly conserved and unchanged with respect to HIV-1 whereas the connecting residues of the N-terminal domain, the dimer interface and C-terminal LEDGF binding domain were highly conserved but differed from HIV-1. The N155 H INI resistance-associated mutation (RAM) was detected in the virus population from one ARV-treated, INI-naïve patient, and the 72I and 201I polymorphisms were detected in samples from 36 and 38 patients respectively. No other known INI RAM was detected.</p> <p>Under RAL selective pressure <it>in vitro</it>, a ROD variant carrying the Q91R+I175M mutations was selected. The Q91R and I175M mutations emerged simultaneously and conferred phenotypic resistance (13-fold increase in IC₅₀). The Q91R+I175M combination was absent from all clinical isolates. Three-dimensional modeling indicated that residue 91 lies on the enzyme surface, at the entry of a pocket containing the DDE catalytic triad and that adding a positive charge (Gln to Arg) might compromise IN-RAL affinity.</p> <p>Conclusions</p> <p>HIV-2 polymorphisms from 45 INI-naïve patients are described. Conserved regions as well as frequencies of HIV-2 IN polymorphisms were comparable to HIV-1. Two new mutations (Q91R and I175M) that conferred high resistance to RAL were selected <it>in vitro</it>, which might affect therapeutic outcome.</p

In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres

Hypertension burden in Luxembourg: Individual risk factors and geographic variations, 2013 to 2015 European Health Examination Survey

Hypertension is a modifiable risk factor for cardiovascular disease, but it remains the main cause of death in Luxembourg. We aimed to estimate the current prevalence of hypertension, associated risk factors, and its geographic variation in Luxembourg.Cross-sectional, population-based data on 1497 randomly selected Luxembourg residents aged 25 to 64 years were collected as part of the European Health Examination Survey from 2013 to 2015. Hypertension was defined as systolic/diastolic blood pressure ≥140/90 mm Hg, self-report of a physician diagnosis or on antihypertensive medication. Standard and Bayesian regressions were used to examine associations between hypertension and covariates, and also geographic distribution of hypertension across the country.Nearly 31% of Luxembourg residents were hypertensive, and over 70% of those were either unaware of their condition or not adequately controlled. The likelihood of hypertension was lower in men more physically active (odds ratio [95% credible region] 0.6 [0.4, 0.9]) and consuming alcohol daily (0.3 [0.1, 0.8]), and higher in men with a poor health perception (1.6 [1.0, 2.7]) and in women experiencing depressive symptoms (1.8 [1.3, 2.7]). There were geographic variations in hypertension prevalence across cantons and municipalities. The highest odds ratio was observed in the most industrialized region (South-West) (1.2 [0.9, 1.6]) with a positive effect at 90% credible region.In Luxembourg, the vast majority of people with hypertension are either unaware of their condition or not adequately controlled, which constitutes a major, neglected public health challenge. There are geographic variations in hypertension prevalence in Luxembourg, hence the role of individual and regional risk factors along with public health initiatives to reduce disease burden should be considered

Predominance of the heterozygous CCR5 delta‐24 deletion in African individuals resistant to HIV infection might be related to a defect in CCR5 addressing at the cell surface

Introduction The chemokine receptor CCR5 is the main co-receptor for R5-tropic HIV-1 variants. We have previously described a novel 24-base pair deletion in the coding region of CCR5 among individuals from Rwanda. Here, we investigated the prevalence of hCCR5 Delta 24 in different cohorts and its impact on CCR5 expression and HIV-1 infection in vitro. Methods We screened hCCR5 Delta 24 in a total of 3232 individuals which were either HIV-1 uninfected, high-risk HIV-1 seronegative and seropositive partners from serodiscordant couples, Long-Term Survivors, or HIV-1 infected volunteers from Africa (Rwanda, Kenya, Guinea-Conakry) and Luxembourg, using a real-time PCR assay. The role of the 24-base pair deletion on CCR5 expression and HIV infection was assessed in cell lines and PBMC using mRNA quantification, confocal analysis, flow and imaging cytometry. Results and Discussion Among the 1661 patients from Rwanda, 12 individuals were heterozygous for hCCR5 Delta 24 but none were homozygous. Although heterozygosity for this allele may not confer complete resistance to HIV-1 infection, the prevalence of the mutation was 2.41% (95%CI: 0.43; 8.37) in 83 Long-Term Survivors (LTS) and 0.99% (95%CI: 0.45; 2.14) in 613 HIV-1 exposed seronegative members as compared with 0.35% (95% Cl: 0.06; 1.25) in 579 HIV-1 seropositive members. The prevalence of hCCR5 Delta 24 was 0.55% (95%CI: 0.15; 1.69) in 547 infants from Kenya but the mutation was not detected in 224 infants from Guinea-Conakry nor in 800 Caucasian individuals from Luxembourg. Expression of hCCR5 Delta 24 in cell lines and PBMC showed that the hCCR5 Delta 24 protein is stably expressed but is not transported to the plasma membrane due to a conformational change. Instead, the mutant receptor was retained intracellularly, colocalized with an endoplasmic reticulum marker and did not mediate HIV-1 infection. Co-transfection of hCCR5 Delta 24 and wtCCR5 did not indicate a transdominant negative effect of CCR5 Delta 24 on wtCCR5. Conclusions Our findings indicate that hCCR5 Delta 24 is not expressed at the cell surface. This could explain the higher prevalence of the heterozygous hCCR5 Delta 24 in LTS and HIV-1 exposed seronegative members from serodiscordant couples. Our data suggest an East-African localization of this deletion, which needs to be confirmed in larger cohorts from African and non-African countries

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries

Background: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. Methods: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. Results: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in &lt; 25%. No sequence had resistance to all currently available drugs. Conclusion: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.</p

The HIV/AIDS epidemic in sub-Saharan Africa: thinking ahead on programmatic tasks and related operational research

Until now, we have all been desperately trying to run behind the HIV/AIDS epidemic and catch up with it, but despite all our efforts, the epidemic remains well ahead of us. In 2010, the antiretroviral treatment (ART) gap was about 60%, AIDS-related deaths were almost two million a year, and on top of these figures, for every one person started on ART, there were two new HIV infections. What is needed to change this situation is to think ahead of the epidemic in terms of the programmatic tasks we will be faced with and try to act boldly in trying to implement those tasks. From a programmatic perspective, we: a) highlight what needs to fundamentally change in our thinking and overall approach to the epidemic; and b) outline a number of key task areas for implementation and related operational research