Sofosbuvir and Velpatasvir for HCV Genotype 1, 2, 4, 5, and 6 Infection.

BACKGROUND: A simple treatment regimen that is effective in a broad range of patients who are chronically infected with the hepatitis C virus (HCV) remains an unmet medical need. METHODS: We conducted a phase 3, double-blind, placebo-controlled study involving untreated and previously treated patients with chronic HCV genotype 1, 2, 4, 5, or 6 infection, including those with compensated cirrhosis. Patients with HCV genotype 1, 2, 4, or 6 were randomly assigned in a 5:1 ratio to receive the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor velpatasvir in a once-daily, fixed-dose combination tablet or matching placebo for 12 weeks. Because of the low prevalence of genotype 5 in the study regions, patients with genotype 5 did not undergo randomization but were assigned to the sofosbuvir-velpatasvir group. The primary end point was a sustained virologic response at 12 weeks after the end of therapy. RESULTS: Of the 624 patients who received treatment with sofosbuvir-velpatasvir, 34% had HCV genotype 1a, 19% genotype 1b, 17% genotype 2, 19% genotype 4, 6% genotype 5, and 7% genotype 6. A total of 8% of patients were black, 19% had cirrhosis, and 32% had been previously treated for HCV. The rate of sustained virologic response among patients receiving sofosbuvir-velpatasvir was 99% (95% confidence interval, 98 to >99). Two patients receiving sofosbuvir-velpatasvir, both with HCV genotype 1, had a virologic relapse. None of the 116 patients receiving placebo had a sustained virologic response. Serious adverse events were reported in 15 patients (2%) in the sofosbuvir-velpatasvir group and none in the placebo group. CONCLUSIONS: Once-daily sofosbuvir-velpatasvir for 12 weeks provided high rates of sustained virologic response among both previously treated and untreated patients infected with HCV genotype 1, 2, 4, 5, or 6, including those with compensated cirrhosis. (Funded by Gilead Sciences; ClinicalTrials.gov number, NCT02201940.).

Sofosbuvir and Velpatasvir for HCV Genotype 2 and 3 Infection.

BACKGROUND: In phase 2 trials, treatment with the combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor velpatasvir resulted in high rates of sustained virologic response in patients chronically infected with hepatitis C virus (HCV) genotype 2 or 3. METHODS: We conducted two randomized, phase 3, open-label studies involving patients who had received previous treatment for HCV genotype 2 or 3 and those who had not received such treatment, including patients with compensated cirrhosis. In one trial, patients with HCV genotype 2 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir, in a once-daily, fixed-dose combination tablet (134 patients), or sofosbuvir plus weight-based ribavirin (132 patients) for 12 weeks. In a second trial, patients with HCV genotype 3 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir for 12 weeks (277 patients) or sofosbuvir-ribavirin for 24 weeks (275 patients). The primary end point for the two trials was a sustained virologic response at 12 weeks after the end of therapy. RESULTS: Among patients with HCV genotype 2, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 99% (95% confidence interval [CI], 96 to 100), which was superior to the rate of 94% (95% CI, 88 to 97) in the sofosbuvir-ribavirin group (P=0.02). Among patients with HCV genotype 3, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 95% (95% CI, 92 to 98), which was superior to the rate of 80% (95% CI, 75 to 85) in the sofosbuvir-ribavirin group (P<0.001). The most common adverse events in the two studies were fatigue, headache, nausea, and insomnia. CONCLUSIONS: Among patients with HCV genotype 2 or 3 with or without previous treatment, including those with compensated cirrhosis, 12 weeks of treatment with sofosbuvir-velpatasvir resulted in rates of sustained virologic response that were superior to those with standard treatment with sofosbuvir-ribavirin. (Funded by Gilead Sciences; ASTRAL-2 ClinicalTrials.gov number, NCT02220998; and ASTRAL-3, NCT02201953.).

An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.

Italian guidelines for the use of antiretroviral agents and the diagnostic-clinical management of HIV-1 infected persons. Update 2016.

The Italian Society of Infectious and Tropical Diseases (SIMIT) of the Technical Health Committee, Ministry of Health (Sections L and M) of Italy have supported recommendations for the Italian guidelines for the use of antiretroviral agents and the diagnostic-clinical management of HIV-1 infected persons. This publication summarizes the latest updates to the 2016 version of the Italian Guidelines for the management of HIV-1 infected patients and the use of antiretroviral drugs. In particular, new recommendations were released concerning the following topics: estimate of the HIV continuum of care in Italy, optimal timing and preferred drug combinations for starting antiretroviral therapy, treatment optimization, and pre-exposure prophilaxis (PrEP). For a complete review of clinical and therapeutic relevant topics we refer the reader to the extended version of the Guidelines.

Evolutionary analysis identifies an MX2 haplotype associated with natural resistance to HIV-1 infection.

The protein product of the myxovirus resistance 2 (MX2) gene restricts HIV-1 and simian retroviruses. We demonstrate that MX2 evolved adaptively in mammals with distinct sites representing selection targets in distinct branches; selection mainly involved residues in loop 4, previously shown to carry antiviral determinants. Modeling data indicated that positively selected sites form a continuous surface on loop 4, which folds into two antiparallel α-helices protruding from the stalk domain. A population genetics-phylogenetics approach indicated that the coding region of MX2 mainly evolved under negative selection in the human lineage. Nonetheless, population genetic analyses demonstrated that natural selection operated on MX2 during the recent history of human populations: distinct selective events drove the frequency increase of two haplotypes in the populations of Asian and European ancestry. The Asian haplotype carries a susceptibility allele for melanoma; the European haplotype is tagged by rs2074560, an intronic variant. Analyses performed on three independent European cohorts of HIV-1-exposed seronegative individuals with different geographic origin and distinct exposure route showed that the ancestral (G) allele of rs2074560 protects from HIV-1 infection with a recessive effect (combined P = 1.55 × 10(-4)). The same allele is associated with lower in vitro HIV-1 replication and increases MX2 expression levels in response to IFN-α. Data herein exploit evolutionary information to identify a novel host determinant of HIV-1 infection susceptibility.

Variants in the CYP7B1 gene region do not affect natural resistance to HIV-1 infection.

BACKGROUND: The genetic bases of natural resistance to HIV-1 infection remain largely unknown. Recently, two genome-wide association studies suggested a role for variants within or in the vicinity of the CYP7B1 gene in modulating HIV susceptibility. CYP7B1 is an appealing candidate for this due to its contribution to antiviral immune responses. We analyzed the frequency of two previously described CYP7B1 variants (rs6996198 and rs10808739) in three independent cohorts of HIV-1 infected subjects and HIV-1 exposed seronegative individuals (HESN). FINDINGS: rs6996198 and rs10808739 were genotyped in three case/control cohorts of sexually-exposed HESN and HIV-1-infected individuals from Italy, Peru and Colombia. Comparison of the allele and genotype frequencies of the two SNPs under different models showed that the only significant difference was seen for rs6996198 in the Peruvian sample (nominal p = 0.048, dominant model). For this variant, a random-effect meta-analysis yielded non-significant results (dominant model, p = 0.78) and revealed substantial heterogeneity among cohorts. No significant effect of the rs10808739 allelic status on HIV-1 infection susceptibility (additive model, p = 0.30) emerged from the meta-analysis. CONCLUSIONS: Although our study had limited power to detect association due to the small sample size, comparisons among the three cohorts revealed very similar allelic and genotypic frequencies in HESN and HIV-1 positive subjects. Overall, these data indicate that the two GWAS-defined variants in the CYP7B1 region do not strongly influence HIV-1 infection susceptibility.

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study.

BACKGROUND: Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. FINDINGS: Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. CONCLUSIONS: Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy.

Five-year retrospective Italian multicenter study of visceral leishmaniasis treatment.

The treatment of visceral leishmaniasis (VL) is poorly standardized in Italy in spite of the existing evidence. All consecutive patients with VL admitted at 15 Italian centers as inpatients or outpatients between January 2004 and December 2008 were retrospectively considered; outcome data at 1 year after treatment were obtained for all but 1 patient. Demographic characteristics, underlying diseases, diagnostic procedures, treatment regimens and outcomes, as well as side effects were recorded. A confirmed diagnosis of VL was reported for 166 patients: 120 (72.3%) immunocompetent, 21 (12.6%) patients with immune deficiencies other than HIV infection, and 25 (15.1%) coinfected with HIV. Liposomal amphotericin B (L-AmB) was the drug almost universally used for treatment, administered to 153 (92.2%) patients. Thirty-seven different regimens, including L-AmB were used. The mean doses were 29.4 ± 7.9 mg/kg in immunocompetent patients, 32.9 ± 8.6 mg/kg in patients with non-HIV-related immunodeficiencies, and 40.8 ± 6.7 mg/kg in HIV-infected patients (P < 0.001). The mean numbers of infusion days were 7.8 ± 3.1 in immunocompetent patients, 9.6 ± 3.9 in non-HIV-immunodeficient patients, and 12.0 ± 3.4 in HIV-infected patients (P < 0.001). Mild and reversible adverse events were observed in 12.2% of cases. Responsive patients were 154 (93.3%). Successes were 98.4% among immunocompetent patients, 90.5% among non-HIV-immunodeficient patients, and 72.0% among HIV-infected patients. Among predictors of primary response to treatment, HIV infection and age held independent associations in the final multivariate models, whereas the doses and duration of L-AmB treatment were not significantly associated. Longer treatments and higher doses of L-AmB were not able to significantly modify treatment outcomes either in the immunocompetent or in the immunocompromised population.

A common polymorphism in TLR3 confers natural resistance to HIV-1 infection.

TLR3 recognizes dsRNA and activates antiviral immune responses through the production of inflammatory cytokines and type I IFNs. Genetic association studies have provided evidence concerning the role of a polymorphism in TLR3 (rs3775291, Leu412Phe) in viral infection susceptibility. We genotyped rs3775291 in a population of Spanish HIV-1-exposed seronegative (HESN) individuals who remain HIV seronegative despite repeated exposure through i.v. injection drug use (IDU-HESN individuals) as witnessed by their hepatitis C virus seropositivity. The frequency of individuals carrying at least one 412Phe allele was significantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87; 95% confidence interval, 1.06-3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Similar results were obtained: the frequency of individuals carrying at least one 412Phe allele was significantly higher compared with that of a matched control sample (odds ratio, 1.79; 95% confidence interval, 1.05-3.08; p = 0.029). In vitro infection assays showed that in PBMCs carrying the 412Phe allele, HIV-1(Ba-L) replication was significantly reduced (p = 0.025) compared with that of Leu/Leu homozygous samples and was associated with a higher expression of factors suggestive of a state of immune activation (IL-6, CCL3, CD69). Similarly, stimulation of PBMCs with a TLR3 agonist indicated that the presence of the 412Phe allele results in a significantly increased expression of CD69 and higher production of proinflammatory cytokines including IL-6 and CCL3. The data of this study indicate that a common TLR3 allele confers immunologically mediated protection from HIV-1 and suggest the potential use of TLR3 triggering in HIV-1 immunotherapy.

Von Willebrand factor hyperactivity affects the outcome of lower limb revascularization in subjects with type 2 diabetes mellitus complicated by diabetic foot vasculopathy: An observational pilot study.

Aim of this study is to evaluate any differences in VWF antigen, VWF activity and ADAMTS-13 activity before and after successful and non-successful Percutaneous Transluminal Angioplasty (PTA) in subjects with type 2 diabetes (T2DM) complicated by Chronic limb-threatening ischemia (CLTI) in diabetic foot vasculopathy. METHODS: In this prospective observational pilot study, we enrolled 35 T2DM subjects who underwent lower limb PTA. Transcutaneous oximetry was performed in all patients before and 6 weeks after PTA. The change in oxygen partial pressure (TcpO2) before and after PTA was expressed as TcpO2-delta (ΔTcpO2). VWF antigen, VWF activity and ADAMTS-13 activity were measured before and 6 weeks after PTA; changes were expressed as delta and ratio from baseline. RESULTS: Subjects with ∆TcpO2 < 15 mmHg presented higher ΔVWF activity (p = 0.050) and lower ADAMTS-13 activity ratio (p = 0.080). Subjects with ∆TcpO2 < 30 mmHg showed lower ADAMTS-13 activity Δ and ratio (p = 0.028). CONCLUSIONS: VWF antigen levels and VWF activity may potentially affect PTA outcome. Higher levels of VWF could derive from VWF release as consequence of PTA-induced mechanical endothelial damage and/or oxidative stress-induced modifications of VWF structure with impairment of VWF-ADAMTS13 interactions.

Insulin pump treatment vs. multiple daily insulin injections in patients with poorly controlled Type 2 diabetes mellitus: a comparison of cardiovascular effects.

AIMS: Both hyperglycaemia and large glycaemic variability are associated with worse outcomes in patients with Type 2 diabetes mellitus (T2DM), possibly causing sympatho-vagal imbalance and endothelial dysfunction. Continuous subcutaneous insulin injection (CSII) improves glycemic control compared to multiple daily insulin injections (MDI). We aimed to assess whether CSII may improve cardiac autonomic and vascular dilation function compared to MDI. METHODS: We enrolled T2DM patients without cardiovascular disease with poor glycaemic control, despite optimized MDI therapy. Patients were randomized to continue MDI (with multiple daily peripheral glucose measurements) or CSII; insulin dose was adjusted to achieve optimal target ranges of blood glucose levels. Patients were studied at baseline and after 6 months by: 1) flow-mediated dilation (FMD) and nitrate-mediated dilation (NMD) of the brachial artery; 2) heart rate variability (HRV) by 24-hour ECG Holter monitoring (HM). 7-day continuous glucose monitoring (CGM) was performed in 9 and 8 patients of Group 1 and 2, respectively. RESULTS: Overall, 21 patients were enrolled, 12 randomized to CSII (Group 1) and 9 to MDI (Group 2). The daily dose of insulin and Hb1AC did not differ significantly between the 2 groups, both at baseline and at follow-up. Glucose variability showed some significant improvement at follow-up in the whole population, but no differences were observed between the 2 groups. Both FMD and NMD, as well as HRV parameters, showed no significant differences between the 2 groups at 6-month follow-up. CONCLUSIONS: In this randomized small study we show that, in T2DM patients, CSII achieves a similar medium-term glycemic control compared to MDI, without any adverse effect on the cardiovascular system.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Overactivation of plasmacytoid dendritic cells inhibits antiviral T-cell responses: a model for HIV immunopathogenesis.

A delicate balance between immunostimulatory and immunosuppressive signals mediated by dendritic cells (DCs) and other antigen-presenting cells (APCs) regulates the strength and efficacy of antiviral T-cell responses. HIV is a potent activator of plasmacytoid DCs (pDCs), and chronic pDC activation by HIV promotes the pathogenesis of AIDS. Cholesterol is pivotal in maintaining HIV envelope integrity and allowing HIV-cell interaction. By depleting envelope-associated cholesterol to different degrees, we generated virions with reduced ability to activate pDCs. We found that APC activation was dissociated from the induction of type I IFN-α/ß and indoleamine-2,3-dioxygenase (IDO)-mediated immunosuppression in vitro. Extensive cholesterol withdrawal, resulting in partial protein and RNA loss from the virions, rendered HIV a more powerful recall immunogen for stimulating memory CD8 T-cell responses in HIV-exposed, uninfected individuals. These enhanced responses were dependent on the inability of cholesterol-depleted HIV to induce IFN-α/ß.

The development and evolution of insulin pumps: from early beginnings to future prospects.

Diabetes technology has proliferated extensively over the past few decades with vast ameliorations in glucose monitoring and in insulin delivery systems. From a treatment based on daily insulin injections, we have moved to increasingly advanced technologies. Despite such advancements which have allowed better glycemic control, decreased diabetes-related complications, and improved the quality of life among diabetic patients, it has left many individuals unsatisfied with the current rate of commercial artificial pancreas development, stemming the need for further research into novel technologies. Accordingly, the Juvenile Diabetes Research Foundation has marked three generations for the development of an artificial pancreas comprising historical landmarks and future prospects which aim to produce an advanced technological system that attempts to mimic the endogenous pancreas, eliminating the need for user input. This review presents a synopsis of the development and evolution of insulin pumps, starting with the earliest technologies available such as continuous subcutaneous insulin infusion and continuous glucose monitoring as separate components, to currently available integrated advanced closed-loop hybrid systems and possible future technologies. The aim of the review is to provide insight of the advantages and limitations of past and currently available insulin pumps with the hope of driving research into novel technologies that attempt to mimic endogenous pancreatic function as closely as possible.

Genetic diversity at endoplasmic reticulum aminopeptidases is maintained by balancing selection and is associated with natural resistance to HIV-1 infection.

Human ERAP1 and ERAP2 encode two endoplasmic reticulum aminopeptidases. These enzymes trim peptides to optimal size for loading onto major histocompatibility complex class I molecules and shape the antigenic repertoire presented to CD8(+) T cells. Therefore, ERAP1 and ERAP2 may be considered potential selection targets and modulators of infection susceptibility. We resequenced two genic regions in ERAP1 and ERAP2 in three HapMap populations. In both cases, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, and reduced population genetic differentiation. The genealogy of ERAP1 and ERAP2 haplotypes was split into two major branches with deep coalescence times. These features suggest that long-standing balancing selection has acted on these genes. Analysis of the Lys528Arg (rs30187 in ERAP1) and Asn392Lys (rs2549782 in ERAP2) variants in an Italian population of HIV-1-exposed seronegative (ESN) individuals and a larger number of Italian controls indicated that rs2549782 significantly deviates from Hardy-Weinberg equilibrium (HWE) in ESN but not in controls. Technical errors were excluded and a goodness-of-fit test indicated that a recessive model with only genetic effects adequately explains HWE deviation. The genotype distribution of rs2549782 is significantly different in the two cohorts (P = 0.004), mainly as the result of an over-representation of Lys/Lys genotypes in the ESN sample (P-value for a recessive model: 0.00097). Our data suggest that genetic diversity in ERAP1 and ERAP2 has been maintained by balancing selection and that variants in ERAP2 confer resistance to HIV-1 infection possibly via the presentation of a distinctive peptide repertoire to CD8(+) T cells.

High throughput fabrication of plasmonic nanostructures in nanofluidic pores for biosensing applications.

One of the primary advantages of nanoscale sensors is that they often can provide conceptually new ways of performing sensing that are not feasible with their large-scale analogs. For example, the small size of nanoscale sensor elements, such as plasmonic metal nanoparticles, allows them to be combined with nanofluidic systems. Among the potential applications of such a combination is the efficient delivery of analyte to the sensor surface. With this in mind, in this work we look to address the challenge of creating and positioning nanoplasmonic sensor elements within nanofluidic pores. A scheme is presented that allows for the production of arrays of pores in a thin (220 nm) silicon nitride membrane with one plasmonic nanoparticle sensor element in each pore. The high throughput fabrication protocol is parallel and enables multiple sensor chips to be produced simultaneously, yet with accurate tuning of the dimension and shape of the nanoparticles. The presented system is shown to possess polarization-sensitive plasmonic resonances that can be tuned significantly in the visible wavelength range by just varying one process parameter. The thickness of the membrane could be optimized to minimize the influence of the optical membrane interference on the plasmonic readout. The sensitivity of the plasmon resonances to changes in refractive index, which forms the basis for using the system for biosensing, was found to be competitive with other nanoplasmonic sensors.

TLR activation pathways in HIV-1-exposed seronegative individuals.

TLRs trigger innate immunity that recognizes conserved motifs of invading pathogens, resulting in cellular activation and release of inflammatory factors. The influence of TLR activation on resistance to HIV-1 infection has not been investigated in HIV-1 exposed seronegative (ESN) individuals. PBMCs isolated from heterosexually ESN individuals were stimulated with agonists specific for TLR3 (poly I:C), TLR4 (LPS), TLR7 (imiquimod), and TLR7/8 (ssRNA40). We evaluated expression of factors involved in TLR signaling cascades, production of downstream effector immune mediators, and TLR-expression in CD4+ and CD14+ cells. Results were compared with those obtained in healthy controls (HCs). ESN individuals showed: 1) comparable percentages of CD14+/TLR4+ and CD4+/TLR8+ CD14+/TLR8+ cells; 2) higher responsiveness to poly I:C, LPS, imiquimod, and ssRNA40 stimulation, associated with significantly increased production of IL-1beta, IL-6, TNF-alpha, and CCL3; 3) augmented expression of mRNA specific for other targets (CCL2, CSF3, CSF2, IL-1alpha, IL-8, IL-10, IL-12, cyclooxygenase 2) demonstrated by broader TLRs pathway expression analyses; and 4) increased MyD88/MyD88s(short) ratio, mainly following TLR7/8 stimulation. We also compared TLR-agonist-stimulated cytokine/chemokine production in CD14+ PBMCs and observed decreased IFN-beta production in ESN individuals compared with HCs upon TLR7/8-agonist stimulation. These data suggest that TLR stimulation in ESN individuals results in a more robust release of immunologic factors that can influence the induction of stronger adaptive antiviral immune responses and might represent a virus-exposure-induced innate immune protective phenotype against HIV-1.

Performance of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group.

OBJECTIVE: The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory. METHODS: Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory. Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). RESULTS: Quantification of HIV-1 DNA was available for 35/45 (77.8%) samples, and gave a median value of 598 (IQR:252- 1,203) copies/10 PBMCs. A total of 56/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54/56 (96.4%). Results of tropism prediction by local centers were: 33/54 (61.1%) R5 and 21/54 (38.9%) X4/DM. CONCLUSION: There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment.

Under representation of the inhibitory KIR3DL1 molecule and the KIR3DL1+/BW4+ complex in HIV exposed seronegative individuals.

The activation of natural killer (NK) cells is modulated by surface molecules. We analyzed NK cells in human immunodeficiency virus (HIV)-exposed seronegative (HESN) individuals by means of molecular typing of HLA B, Cw, and killer cell immunoglobulin-like receptor (KIR) molecules. In HESN individuals, compared with HIV patients, the frequency of the inhibitory KIR3DL1 allele and of the KIR3DL1(+)/Bw4(+) inhibitory complex was reduced, whereas that of the activatory KIR3DS1(+) ligand and the activatory Bw4(+)/3DL1(-)/3DS1(+) complex was increased, resulting in a statistically significant diversion from Hardy-Weinberg equilibrium (KIR3DS1 homozygote) in HESN individuals. The reciprocal equilibrium between inhibitory and activatory NK receptors and their ligands favors NK activation in HESN individuals.