Loss of gut barrier integrity triggers activation of islet-reactive T cells and autoimmune diabetes.

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in "at-risk" individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.

Fecal Clostridiales distribution and short-chain fatty acids reflect bowel habits in irritable bowel syndrome.

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.

Testicular microbiome in azoospermic men-first evidence of the impact of an altered microenvironment.

STUDY QUESTION: Given the relevant role of the extracellular microenvironment in regulating tissue homeostasis, is testicular bacterial microbiome (BM) associated with germ cell aplasia in idiopathic non-obstructive azoospermia (iNOA)? SUMMARY ANSWER: A steady increase of dysbiosis was observed among testis with normal spermatogenesis vs. iNOA with positive sperm retrieval and iNOA with complete germ cell aplasia. WHAT IS KNOWN ALREADY: Tissue-associated BM has been reported to be a biologically important extracellular microenvironment component for numerous body habitats, but not yet for the human testis. STUDY DESIGN, SIZE, DURATION: Cross-sectional study, investigating tissue-associated BM in the testis of (i) five men with iNOA and negative sperm retrieval at microdissection testicular sperm extraction (microTESE); (ii) five men with iNOA and positive sperm retrieval at microTESE; and (iii) five normozoospermic men upon orchiectomy. Every testicular specimen was histologically classified and analyzed in terms of bacterial community. PARTICIPANTS/MATERIALS, SETTING, METHODS: Massive ultra-deep pyrosequencing was applied to investigate testis microbiome. Metagenome was analyzed using Quantitative Insights Into Microbial Ecology (QIIME). Tissue-associated bacterial load was quantified by digital droplet PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Normozoospermic men showed small amounts of bacteria in the testis, with Actinobacteria, Bacteroidetes, Firmicutes Proteobacteria as the dominating phyla; iNOA individuals had increased amounts of bacterial DNA (P = 0.02), associated with decreased taxa richness due to the lack of Bacteroidetes and Proteobacteria (P = 2 × 10-5). Specimens with negative sperm retrieval at microTESE depicted complete germ cell aplasia and a further decrease in terms of Firmicutes and Clostridia (P < 0.05), a complete lack of Peptoniphilus asaccharolyticus, but increased amount of Actinobacteria. LIMITATIONS, REASONS FOR CAUTION: The limited number of specimens analyzed in this preliminary study deserves external validation. The paraneoplastic microenvironment could have an impact on the residential bacterial flora. WIDER IMPLICATION OF THE FINDINGS: Human testicular microenvironment is not microbiologically sterile, containing low amounts of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. A dysbiotic bacterial community was associated with iNOA and complete germ cell aplasia. Novel findings on testicular BM could support future translational therapies of male-factor infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by URI-Urological Research Institute free funds. Authors declared no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Natural Product Kuwanon-L Inhibits HIV-1 Replication through Multiple Target Binding.

In recent years many advances have been made in the fight against HIV-1 infection. However, the lack of a vaccine, together with the increasing resistance to the highly active anti-retroviral therapy (HAART), make HIV-1 infection still a serious global emergency. Thus, new compounds with original modes of action are continuously required, and natural products have ever been a very interesting class of pharmacologically active molecules. Some of them have been used since ancient times against viral infections. Here we present a work in which we suggest that kuwanon-L, a natural product active as an HIV-1 integrase (IN) inhibitor, might exert its overall antiviral activity through binding to multiple viral targets. Specific enzymatic tests, together with a time-of-addition (TOA) experiment, support our hypothesis of binding both to IN and to reverse transcriptase (RT). Overall, this compound can be considered an attractive lead for the development of new classes of antiviral agents able to overcome the problem of resistance, due to its ability to exert its action by binding simultaneously to multiple viral targets.

One drug for two targets: Biological evaluation of antiretroviral agents endowed with antiproliferative activity.

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certainly the main cause. We envisaged the possibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors mainly due to HIV infection like Burkitt's Lymphoma. From cellular assays and gene expression analysis we identified two promising compounds. These derivatives have the dual action required inhibiting HIV replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribution, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile.

Inhibition of HIV-1 Reverse Transcriptase Dimerization by Small Molecules.

Because HIV-1 reverse transcriptase is an enzyme whose catalytic activity depends on its heterodimeric structure, this system could be a target for inhibitors that perturb the interactions between the protein subunits, p51 and p66. We previously demonstrated that the small molecule MAS0 reduced the association of the two RT subunits and simultaneously inhibited both the polymerase and ribonuclease H activities. In this study, some analogues of MAS0 were rationally selected by docking studies and evaluated in vitro for their ability to disrupt dimeric assembly. Two inhibitors were identified with improved activity compared to MAS0. This study lays the basis for the rational design of more potent inhibitors of RT dimerization.

Kuwanon-L as a New Allosteric HIV-1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation.

HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents.

Performance of commonly used genotypic assays and comparison with phenotypic assays of HIV-1 coreceptor tropism in acutely HIV-1-infected patients.

OBJECTIVES: Although founder viruses in primary HIV-1 infections (PHIs) typically use the CCR5 coreceptor (R5-tropic), 3%-19% of subjects also harbour CXCR4-using viruses (X4-tropic), making tropism determination before CCR5 antagonist usage mandatory. Genotypic methods can be used to accurately determine HIV-1 tropism in chronically infected patients. METHODS: We compared the results of genotypic methods [geno2pheno, PSSMx4r5 including a novel nucleotide-input version (ntPSSM) and distant segments (ds)Kernel] to predict coreceptor usage in a cohort of 67 PHIs. Specimens with discrepant results were phenotypically tested after cloning the V3 gene region into proviral backbones. Recombinant viruses were used to infect U87 indicator cell lines bearing CD4 and either CCR5 or CXCR4. RESULTS: Geno2pheno10%, PSSMx4r5 and (ds)Kernel gave identical predictions in 85% of cases. Geno2pheno10% predicted the presence of CXCR4 viruses in 18% of patients. Two patients were predicted to carry X4-tropic viruses by all algorithms and X4-tropic viruses were detected in at least one of the recombinant AD8 or NL4-3 backbone-based assays. Ten samples resulted in discordant predictions with at least one algorithm. Full concordance between tropism prediction by using population sequencing and phenotypic assays was observed only with ntPSSM. Geno2pheno prediction and the phenotypic assay gave the same results in a minority of 'discordant' patients. CONCLUSIONS: Compared with both PSSMx4r5 versions, (ds)Kernel and our phenotypic assay, geno2pheno10% overestimated the frequency of X4-tropic viruses (18% versus 3%). ntPSSM was able to detect one additional X4 virus compared with (ds)Kernel that was confirmed with the phenotypic assay.

Comparison of the artus HIV-1 QS-RGQ and VERSANT HIV-1 RNA 1.0 assays for quantitative detection of human immunodeficiency virus type 1 in plasma samples.

Several integrated diagnostic platforms to quantify human immunodeficiency virus type-1 viremia have been developed in recent years. We evaluated the performances of the Artus HIV-1 QS-RGQ assay, using the complete QIAsymphony RGQ workflow. 192 clinical plasma specimens and external control panel samples were analyzed, using the Artus assay and the routine Siemens VERSANT HIV-1 RNA 1.0 assay. Three samples were excluded due to amplification inhibition. Among the remaining 189 specimens, 130 samples were detected as positive (above the limit of detection by both assays; median log10 difference: 0.01) and 18 samples were detected as negative. Eight samples (4.2%), all slightly above the limit of detection of the Versant assay, were negative with the Artus assay. The remaining 33 samples (beside 3 negative by Artus assay) were positive by both assays, but below the limit of detection at least in one of them. Results from the external panel samples showed a mean Log10 variation of -0.18 and -0.45 for the Versant and the Artus assays, respectively. As both assays showed highly correlated results, the QIAsymphony RGQ system, using the Artus HIV-1 QS-RGQ assay, could be considered a potential platform for HIV-1 RNA quantification in plasma.

2-Aminothiazolones as anti-HIV agents that act as gp120-CD4 inhibitors.

We report here the synthesis of 2-aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors.

In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir.

OBJECTIVES: The cross-resistance profiles of elvitegravir and dolutegravir on raltegravir-resistant variants is still controversial or not available in macrophages and lack extensive evaluations on wide panels of clonal variants. Thus, a complete evaluation in parallel with all currently available integrase inhibitors (INIs) was performed. METHODS: The integrase coding region was RT-PCR-amplified from patient-derived plasma samples and cloned into an HIV-1 molecular clone lacking the integrase region. Twenty recombinant viruses bearing mutations to all primary pathways of resistance to raltegravir were phenotypically evaluated with each integrase inhibitor in freshly purified CD4+ T cells or monocyte-derived macrophages. RESULTS: Y143R single mutants conferred a higher level of raltegravir resistance in macrophages [fold change (FC) 47.7-60.24] compared with CD4+ T cells (FC 9.55-11.56). All other combinations had similar effects on viral susceptibility to raltegravir in both cell types. Elvitegravir displayed a similar behaviour both in lymphocytes and macrophages with all the tested patterns. When compared with raltegravir, none to modest increases in resistance were observed for the Y143R/C pathways. Dolutegravir maintained its activity and cross-resistance profile in macrophages. Only Q148H/R variants had a reduced level of susceptibility (FC 5.48-18.64). No variations were observed for the Y143R/C (+/-T97A) or N155H variants. CONCLUSIONS: All INIs showed comparable antiretroviral activity in both cell types even if single mutations were associated with a different level of susceptibility in vitro to raltegravir and elvitegravir in macrophages. In particular, dolutegravir was capable of inhibiting with similar potency infection of raltegravir-resistant variants with Y143 or N155 pathways in both HIV-1 major cell reservoirs.

Infection and coinfection of human rhinovirus C in stem cell transplant recipients.

In 54 adult stem cell transplant recipients, the presence and persistence of human rhinoviruses (including the novel lineage C) were evaluated by molecular detection and phylogenetic analysis, independently from respiratory symptoms. In the same group of patients, the presence of other coinfecting respiratory pathogens, including the novel enterovirus 109, was also evaluated.

TLR7 agonist RO7020531 versus placebo in healthy volunteers and patients with chronic hepatitis B virus infection: a randomised, observer-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Toll-like receptor 7 (TLR7) agonists augment immune activity and have potential for the treatment of chronic hepatitis B virus (HBV) infection. We aimed to assess the safety and tolerability of RO7020531 (also called RG7854), a prodrug of the TLR7 agonist RO7011785, in healthy volunteers and patients with chronic HBV infection. METHODS: This randomised, observer-blind, placebo-controlled, phase 1 study was done in two parts. Part 1 was done at one site in New Zealand and part 2 was done at 12 sites in Bulgaria, Hong Kong, Italy, New Zealand, the Netherlands, Taiwan, Thailand, and the UK. In part 1, healthy volunteers were randomly assigned (4:1) within one of eight dose cohorts (3 mg, 10 mg, 20 mg, 40 mg, 60 mg, 100 mg, 140 mg, or 170 mg) to receive a single RO7020531 dose or placebo or randomly assigned (4:1) within one of three dose cohorts (100 mg, 140 mg, or 170 mg) to receive either RO7020531 or placebo every other day for 13 days. In part 2, nucleoside or nucleotide analogue-suppressed patients with chronic HBV infection were randomly assigned (4:1) within cohorts 1-3 (150 mg, 150 mg, or 170 mg) to receive either RO7020531 or placebo and treatment-naive patients with chronic HBV infection were randomly assigned (3:1) in cohort 4 to receive either 150 mg of RO7020531 or placebo. Patients were treated every other day for 6 weeks. Study medication was administered orally to participants after they had fasted. Study participants and investigational staff were masked to treatment allocation. The primary outcome was the safety and tolerability of RO7020531, as measured by the incidence and severity of adverse events and the incidence of laboratory, vital sign, and electrocardiogram abnormalities, and was analysed in all participants who received at least one dose of the study medication. This trial is registered with ClinicalTrials.gov, NCT02956850, and the study is complete. FINDINGS: Between Dec 12, 2016, and March 21, 2021, 340 healthy volunteers were screened in part 1, of whom 80 were randomly assigned in the single ascending dose study (eight assigned RO7020531 in each cohort and 16 assigned placebo) and 30 were randomly assigned in the multiple ascending dose study (eight assigned RO7020531 in each cohort and six assigned placebo), and 110 patients were screened in part 2, of whom 30 were randomly assigned in cohorts 1-3 (16 assigned RO7020531 150 mg, eight assigned RO7020531 170 mg, and six assigned placebo) and 20 were randomly assigned in cohort 4 (15 assigned RO7020531 and five assigned placebo). All randomly assigned participants received at least one dose of a study drug and were included in the safety analysis. All tested doses of RO7020531 were safe and had acceptable tolerability in healthy volunteers and patients. The most frequent treatment-related adverse events among the total study population were headache (15 [9%] of 160 participants), influenza-like illness (seven [4%] of 160 participants), and pyrexia (ten [6%] of 160 participants). Most adverse events were mild and transient. There were no severe or serious adverse events in healthy volunteers. In the patient cohorts, there was one severe adverse event (influenza-like illness with 170 mg of RO7020531) and one serious adverse event (moderate influenza-like illness with a 3-day hospitalisation in a treatment-naive patient receiving RO7020531). There were no treatment-related deaths. INTERPRETATION: Due to acceptable safety and tolerability, RO7020531 should continue to be developed for the treatment of patients with chronic HBV infection. FUNDING: F Hoffmann-La Roche.

Residual viraemia does not influence 1 year virological rebound in HIV-infected patients with HIV RNA persistently below 50 copies/mL.

OBJECTIVES: It is currently debated whether patients with residual viraemia are at higher risk of virological failure than those attaining <1 HIV RNA copy/mL. We therefore investigated the effect of residual viraemia on virological rebound. METHODS: We used a prospective, non-interventional, single-centre, study. This analysis was based on HIV-infected patients with two consecutive HIV RNA viral loads (VLs) of <50 copies/mL as tested by Versant bDNA, followed by two HIV RNA VLs of <50 copies/mL as tested using the Versant kinetic PCR molecular system (kPCR; limit of quantification = 1 copy/mL). Virological rebound was defined as two consecutive HIV RNA values of >50 copies/mL after baseline, and the time to virological rebound was calculated using the Kaplan-Meier method. RESULTS: There were 739 eligible patients; 446 (60.4%) had HIV RNA <1 copy/mL (group A) and 293 (39.6%) had residual viraemia (1-49 HIV RNA copies/mL; group B). After a follow-up (median 48.9 weeks), virological rebound occurred in four patients in group A (0.9%) and six patients in group B (2%); the time to virological rebound was similar in the two groups (log-rank test P = 0.231). CD4+ cell recovery (slope) was significantly less in the patients with residual viraemia; +14.3 (-7.7, 43.9) cells/mm(3) per year versus +21.2 (-2.5, 53.2) cells/mm(3) per year; P = 0.036. CONCLUSIONS: Residual viraemia assessed by kPCR was not associated with virological rebound during 1 year of follow-up. However, the patients attaining <1 HIV RNA copy/mL showed a small but statistically significant improvement in CD4+ cell recovery.

Viral tropism by geno2pheno as a tool for predicting CD4 decrease in HIV-1-infected naive patients with high CD4 counts.

OBJECTIVES: To investigate the value of tropism (determined by genotypic testing) to predict CD4 depletion in HIV-infected antiretroviral-naive patients with high CD4 counts. METHODS: Viral tropism was determined by geno2pheno (false positive rate = 10%) in 223 HIV-infected subjects naive to antiretrovirals with CD4 count ≥350 cells/µL and HIV-RNA >500 copies/mL enrolled in the ICONA Foundation Study for whom a stored plasma sample (baseline) was retrospectively tested. We monitored CD4 cell count and identified predictors of decline before antiretroviral therapy initiation, applying a mixed linear model with covariates (age, gender, tropism, HIV risk factor, calendar year of HIV infection, months from HIV diagnosis to baseline, hepatitis C virus status, CD4 and HIV-RNA at sample collection and duration of follow-up). RESULTS: Two hundred and twenty-three subjects met the eligibility criteria; 137 (61%) were male and the median age was 35 (31-40) years. Median follow-up was 16.4 (3.2-37.2) months. Median CD4 decrease during follow-up was -157 (-278 to -13) cells/µL. At baseline, 192 (86%) subjects were defined as harbouring R5 virus and 31 (14%) non-R5. Median CD4 count was 571 (458-729) cells/µL and median HIV-RNA was 4.08 (3.57-4.55) log(10) copies/mL. At multivariable analysis, a greater mean CD4 decrease was associated with non-R5 viral tropism (-159.9 ± 12.22, P = 0.0002) at baseline. Other significant covariates were female gender, older age, intravenous drug use, longer duration of follow-up, and higher CD4 cell count and higher HIV-RNA at sample collection. CONCLUSIONS: In patients with CD4 counts ≥350 cells/µL, non-R5 viral tropism by geno2pheno is predictive of CD4 decrease independent of their viral set point and CD4 counts.

The role of infections and coinfections with newly identified and emerging respiratory viruses in children.

Acute respiratory infections are a major cause of morbidity in children both in developed and developing countries. A wide range of respiratory viruses, including respiratory syncytial virus (RSV), influenza A and B viruses, parainfluenza viruses (PIVs), adenovirus, rhinovirus (HRV), have repeatedly been detected in acute lower respiratory tract infections (LRTI) in children in the past decades. However, in the last ten years thanks to progress in molecular technologies, newly discovered viruses have been identified including human Metapneumovirus (hMPV), coronaviruses NL63 (HcoV-NL63) and HKU1 (HcoV-HKU1), human Bocavirus (HBoV), new enterovirus (HEV), parechovirus (HpeV) and rhinovirus (HRV) strains, polyomaviruses WU (WUPyV) and KI (KIPyV) and the pandemic H1N1v influenza A virus. These discoveries have heavily modified previous knowledge on respiratory infections mainly highlighting that pediatric population is exposed to a variety of viruses with similar seasonal patterns. In this context establishing a causal link between a newly identified virus and the disease as well as an association between mixed infections and an increase in disease severity can be challenging. This review will present an overview of newly recognized as well as the main emerging respiratory viruses and seek to focus on the their contribution to infection and co-infection in LRTIs in childhood.

Epidemiological, molecular and clinical features of enterovirus 109 infection in children and in adult stem cell transplant recipients.

BACKGROUND: A novel human enterovirus (HEV) type within the species HEV-C, named EV109, was discovered from cases of respiratory illness in Nicaragua in September 2010. The aim of this study, was to retrospectively examine the presence and the role of EV109 in respiratory samples from two patients populations; infants below the age of 2 years, hospitalized for acute respiratory diseases (ARDs) and adult hematopoietic stem cell transplantation recipients. RESULTS: A total of 1149 nasopharingeal aspirates were collected and tested for the presence of EV109 by reverse transcription-PCR (RT-PCR). In positive samples, the presence of the most common respiratory viruses was also assayed and clinical symptoms were evaluated. Samples from 2 of the 974 infants tested positive for EV109 RNA (0.2%) and belonged to patients with lower ARDs; co-infection with other viral pathogens under study was observed in both cases. In transplant recipients, one out of the 175 samples analyzed, from a patients with upper respiratory simptoms tested positive for HEV 109 in the absence of co-infecting viruses. Sequence analysis of amplified EV109 genomic regions, showed only a few nucleotide differences when compared with the Nicaraguan strains. CONCLUSIONS: Overall these results indicate that HEV109 variants have circulated and differentiated in different lineages worldwide. Although more cases and larger studies are needed, HEV109 infection may be associated to ARDs both in infants and in hematopoietic stem cell transplantation recipients. If these preliminary observations will be confirmed, improved molecular methods with a wider panel of potential pathogens will be useful for monitoring these categories of patients.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Cross-resistance profile of the novel integrase inhibitor Dolutegravir (S/GSK1349572) using clonal viral variants selected in patients failing raltegravir.

Novel integrase inhibitors are in advanced clinical development, and cross-resistance data are needed to consider the possibility to plan a sequential usage within this class of antiretroviral drugs. Ex vivo phenotypic assays were conducted on 11 wild-type and 27 fully replicating recombinant viruses obtained from 11 patients failing previous raltegravir-containing regimens. Dolutegravir maintained its activity in vitro on viruses with mutations in position 143 and 155. However, viruses with mutation Q148R associated with secondary mutations and the combination Q148H+G140S were instead associated with a reduced level of susceptibility to dolutegravir in vitro.