Improved quantification of HIV-1 infected CD4 + T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection

The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10-2 to 8×10-5, and by nearly two-fold (p<0.001) at the optimal MOI tested (10-2). The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs. © 2013 Elsevier B.V

Therapeutic vaccination following early antiretroviral therapy elicits highly functional T cell responses against conserved HIV-1 regions

'Kick and kill' cure strategies aim to induce HIV protein expression in latently infected cells (kick), and thus trigger their elimination by cytolytic T cells (kill). In the Research in Viral Eradication of HIV Reservoirs trial (NCT02336074), people diagnosed with primary HIV infection received immediate antiretroviral therapy (ART) and were randomised 24 weeks later to either a latency-reversing agent, vorinostat, together with ChAdV63.HIVconsv and MVA.HIVconsv vaccines, or ART alone. This intervention conferred no reduction in HIV-1 reservoir size over ART alone, despite boosting virus-specific CD4+ and CD8+ T cells. The effects of the intervention were examined at the cellular level in the two trial arms using unbiased computational analysis of polyfunctional scores. This showed that the frequency and polyfunctionality of virus-specific CD4+ and CD8+ T cell populations were significantly increased over 12 weeks post-vaccination, compared to the ART-only arm. HIV-specific IL-2-secreting CD8+ T cells also expanded significantly in the intervention arm and were correlated with antiviral activity against heterologous HIV in vitro. Therapeutic vaccination during ART commenced in primary infection can induce functional T cell responses that are phenotypically similar to those of HIV controllers. Analytical therapy interruption may help determine their ability to control HIV in vivo

Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses

Ajuts: R01/R56 NIH Grant AI-52779 (GDT), NIH F31 Fellowship (1F31AI106519-01)(TLP), Center for AIDS Research (P30 AI 64518) i Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, grant number UM1-AI100645-01 (AM)Abstract.Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control

Recovering faces from memory: the distracting influence of external facial features.

Recognition memory for unfamiliar faces is facilitated when contextual cues (e.g. head pose, background environment, hair and clothing) are consistent between study and test. By contrast, inconsistencies in external features, especially hair, promote errors in unfamiliar face-matching tasks. For the construction of facial composites, as carried out by witnesses and victims of crime, the role of external features (hair, ears and neck) is less clear, although research does suggest their involvement. Here, over three experiments, we investigate the impact of external features for recovering facial memories using a modern, recognition-based composite system, EvoFIT. Participant-constructors inspected an unfamiliar target face and, one day later, repeatedly selected items from arrays of whole faces, with ‘breeding’, to ‘evolve’ a composite with EvoFIT; further participants (evaluators) named the resulting composites. In Experiment 1, the important internal-features (eyes, brows, nose and mouth) were constructed more identifiably when the visual presence of external features was decreased by Gaussian blur during construction: higher blur yielded more identifiable internal-features. In Experiment 2, increasing the visible extent of external features (to match the target’s) in the presented face-arrays also improved internal-features quality, although less so than when external features were masked throughout construction. Experiment 3 demonstrated that masking external-features promoted substantially more identifiable images than using the previous method of blurring external-features. Overall, the research indicates that external features are a distractive rather than a beneficial cue for face construction; the results also provide a much better method to construct composites, one that should dramatically increase identification of offenders

Discovery of a radio transient in M81

We report the discovery of a radio transient in the spiral galaxy M81. The transient was detected in early 2015 as part of a two-year survey of M81 made up of 12 epochs using the Karl G. Jansky Very Large Array. While undetected on 2014 September 12, the source was first detected on 2015 January 2, from which point it remained visible at an approximately constant luminosity of LR, ν = 1.5 ± 0.1 × 1024 erg s−1 Hz−1 at the observing frequency of 6 GHz for at least 2 months. Assuming this is a synchrotron event with a rise-time between 2.6 and 112 d, the peak luminosity (at equipartition) corresponds to a minimum energy of 1044 ≾ Emin ≾ 1046 erg and jet power of Pmin ∼ 1039 erg s−1, which are higher than most known X-ray binaries. Given its longevity, lack of short-term radio variability, and the absence of any multiwavelength counterpart (X-ray luminosity Lx ≾ 1036 erg s−1), it does not behave like known Galactic or extragalactic X-ray binaries. The M81 transient radio properties more closely resemble the unidentified radio transient 43.78+59.3 discovered in M82, which has been suggested to be a radio nebula associated with an accreting source similar to SS 433. One possibility is that both the new M81 transient and the M82 transient may be the birth of a short-lived radio bubble associated with a discrete accretion event similar to those observed from the ULX Holmberg II X-1. However, it is not possible to rule out other identifications including long-term supernova shockwave interactions with the surrounding medium from a faint supernova or a background active galaxy

Rapid-response radio observations of short GRB 181123B with the Australia Telescope Compact Array

We introduce the Australia Telescope Compact Array (ATCA) rapid-response mode by presenting the first successful trigger on the short-duration gamma-ray burst (GRB) 181123B. Early-time radio observations of short GRBs may provide vital insights into the radio afterglow properties of Advanced LIGO- and Virgo-detected gravitational wave events, which will in turn inform follow-up strategies to search for counterparts within their large positional uncertainties. The ATCA was on target within 12.6 hr post-burst, when the source had risen above the horizon. While no radio afterglow was detected during the 8.3 hr observation, we obtained force-fitted flux densities of $7 \pm 12$ and $15 \pm 11~\mu$Jy at 5.5 and 9 GHz, respectively. Afterglow modelling of GRB 181123B showed that the addition of the ATCA force-fitted radio flux densities to the Swift X-ray Telescope detections provided more stringent constraints on the fraction of thermal energy in the electrons (log$\epsilon_e = -0.75^{+0.39}_{-0.40}$ rather than log$\epsilon_e = -1.13^{+0.82}_{-1.2}$ derived without the inclusion of the ATCA values), which is consistent with the range of typical $\epsilon_e$ derived from GRB afterglow modelling. This allowed us to predict that the forward shock may have peaked in the radio band $\sim10$ days post-burst, producing detectable radio emission $\gtrsim3-4$ days post-burst. Overall, we demonstrate the potential for extremely rapid radio follow-up of transients and the importance of triggered radio observations for constraining GRB blast wave properties, regardless of whether there is a detection, via the inclusion of force-fitted radio flux densities in afterglow modelling efforts

Understanding the multiframe caricature advantage for recognizing facial composites.

Eyewitnesses often construct a ‘composite’ face of a person they saw commit a crime, a picture that police use to identify suspects. We described a technique (Frowd et al., 2007, Visual Cognition, 15, 1-31) based on facial caricature to facilitate recognition of these images: correct naming substantially improves when composites are seen with progressive positive caricature, where distinctive information is enhanced, and then with progressive negative caricature, the opposite. Over the course of four experiments, the underpinnings of this mechanism are explored. Positive-caricature levels were found to be largely responsible for improving naming of composites, with some benefit from negative-caricature levels. Also, different frame-presentation orders (forward, reverse, random, repeated) facilitated equivalent naming benefit relative to static composites. Overall, the data indicate that composites are usually constructed as negative caricatures

Adjuvant bevacizumab in patients with melanoma at high risk of recurrence (AVAST-M): preplanned interim results from a multicentre, open-label, randomised controlled phase 3 study

Background Bevacizumab, a monoclonal antibody that targets VEGF, has shown restricted activity in patients with advanced melanoma. We aimed to assess the role of bevacizumab as adjuvant treatment for patients with resected melanoma at high risk of recurrence. We report results from the preplanned interim analysis. Methods We did a multicentre, open-label, randomised controlled phase 3 trial at 48 centres in the UK between July 18, 2007, and March 29, 2012. Patients aged 16 years or older with American Joint Committee on Cancer stage (AJCC) stage IIB, IIC, and III cutaneous melanoma were randomly allocated (1:1), via a central, computer-based minimisation procedure, to receive intravenous bevacizumab 7·5 mg/kg, every 3 weeks for 1 year, or to observation. Randomisation was stratifi ed by Breslow thickness of the primary tumour, N stage according to AJCC staging criteria, ulceration of the primary tumour, and patient sex. The primary endpoint was overall survival; secondary endpoints included disease-free interval, distant-metastases interval and quality of life. Analysis was by intention-to-treat. This trial is registered as an International Standardised Randomised Controlled Trial, number ISRCTN81261306. Findings 1343 patients were randomised to either the bevacizumab group (n=671) or the observation group (n=672). Median follow-up was 25 months (IQR 16ñ37) in the bevacizumab group and 25 months (17ñ37) in the observation group. At the time of interim analysis, 286 (21%) of 1343 enrolled patients had died: 140 (21%) of 671 patients in the bevacizumab group, and 146 (22%) of 672 patients in the observation group. 134 (96%) of patients in the bevacizumab group died because of melanoma versus 139 (95%) in the observation group. We noted no signifi cant di┎ erence in overall survival between treatment groups (hazard ratio [HR] 0·97, 95% CI 0·78ñ1·22; p=0·76); this fi nding persisted after adjustment for stratifi cation variables (HR 1·03; 95% CI 0·81ñ1·29; p=0·83). Median duration of treatment with bevacizumab was 51 weeks (IQR 21ñ52) and dose intensity was 86% (41ñ96), showing good tolerability. 180 grade 3 or 4 adverse events were recorded in 101 (15%) of 671 patients in the bevacizumab group, and 36 (5%) of 672 patients in the observation group. Bevacizumab resulted in a higher incidence of grade 3 hypertension than did observation (41 [6%] vs one [<1%]). There was an improvement in disease-free interval for patients in the bevacizumab group compared with those in the observation group (HR 0·83, 95% CI 0·70ñ0·98, p=0·03), but no signifi cant di┎ erence between groups for distant-metastasis-free interval (HR 0·88, 95% CI 0·73ñ1·06, p=0·18). No signifi cant di┎ erences were noted between treatment groups in the standardised area under the curve for any of the quality-of-life scales over 36 months. Three adverse drug reactions were regarded as both serious and unexpected: one patient had optic neuritis after the fi rst bevacizumab infusion, a second patient had persistent erectile dysfunction, and a third patient died of a haemopericardium after receiving two bevacizumab infusions and was later identifi ed to have had signifi cant predisposing cardiovascular risk factors. Interpretation Bevacizumab has promising tolerability. Longer follow-up is needed to identify an e┎ect on the primary endpoint of overall survival at 5 years. Funding Cancer Research U

Identification of Effective Subdominant Anti-HIV-1 CD8+ T Cells Within Entire Post-infection and Post-vaccination Immune Responses

Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called “beneficial” regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control