Characterization of Neutralizing Profiles in HIV-1 Infected Patients from whom the HJ16, HGN194 and HK20 mAbs were Obtained

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an ‘extended incubation phase’ PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development

The predominance of Human Immunodeficiency Virus type 1 (HIV-1) circulating recombinant form 02 (CRF02_AG) in West Central Africa may be related to its replicative fitness

BACKGROUND: CRF02_AG is the predominant HIV strain circulating in West and West Central Africa. The aim of this study was to test whether this predominance is associated with a higher in vitro replicative fitness relative to parental subtype A and G viruses. Primary HIV-1 isolates (10 CRF02_AG, 5 subtype A and 5 subtype G) were obtained from a well-described Cameroonian cohort. Growth competition experiments were carried out at equal multiplicity of infection in activated T cells and monocyte-derived dendritic cells (MO-DC) in parallel. RESULTS: Dual infection/competition experiments in activated T cells clearly indicated that CRF02_AG isolates had a significant replication advantage over the subtype A and subtype G viruses. The higher fitness of CRF02_AG was evident for isolates from patients with CD4+ T cell counts >200 cells/μL (non-AIDS) or CD4+ T cell counts <200 cells/μL (AIDS), and was independent of the co-receptor tropism. In MO-DC cultures, CRF02_AG isolates showed a slightly but not significantly higher replication advantage compared to subtype A or G isolates. CONCLUSION: We observed a higher ex vivo replicative fitness of CRF02_AG isolates compared to subtype A and G viruses from the same geographic region and showed that this was independent of the co-receptor tropism and irrespective of high or low CD4+ T cell count. This advantage in replicative fitness may contribute to the dominant spread of CRF02_AG over A and G subtypes in West and West Central Africa

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody (R) ALX-0061 supports its clinical development in rheumatoid arthritis

Introduction: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody (R) with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. Methods: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. Results: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. Conclusions: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA

Monocytes Contribute to Differential Immune Pressure on R5 versus X4 HIV through the Adipocytokine Visfatin/NAMPT

Background: The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations. Results: We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma. Conclusions: As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses. © 2012 Van den Bergh et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

<p>Abstract</p> <p>Background</p> <p>Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans <it>et al</it>., <it>JAIDS </it>2008, <b>47:</b>69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.</p> <p>Results</p> <p>Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the <it>tat </it>gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.</p> <p>Conclusions</p> <p>These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.</p

Advanced Virgo Plus: Future Perspectives

While completing the commissioning phase to prepare the Virgo interferometer for the next joint Observation Run (O4), the Virgo collaboration is also finalizing the design of the next upgrades to the detector to be employed in the following Observation Run (O5). The major upgrade will concern decreasing the thermal noise limit, which will imply using very large test masses and increased laser beam size. But this will not be the only upgrade to be implemented in the break between the O4 and O5 observation runs to increase the Virgo detector strain sensitivity. The paper will cover the challenges linked to this upgrade and implications on the detector's reach and observational potential, reflecting the talk given at 12th Cosmic Ray International Seminar - CRIS 2022 held in September 2022 in Napoli

The Advanced Virgo+ status

The gravitational wave detector Advanced Virgo+ is currently in the commissioning phase in view of the fourth Observing Run (O4). The major upgrades with respect to the Advanced Virgo configuration are the implementation of an additional recycling cavity, the Signal Recycling cavity (SRC), at the output of the interferometer to broaden the sensitivity band and the Frequency Dependent Squeezing (FDS) to reduce quantum noise at all frequencies. The main difference of the Advanced Virgo + detector with respect to the LIGO detectors is the presence of marginally stable recycling cavities, with respect to the stable recycling cavities present in the LIGO detectors, which increases the difficulties in controlling the interferometer in presence of defects (both thermal and cold defects). This work will focus on the interferometer commissioning, highlighting the control challenges to maintain the detector in the working point which maximizes the sensitivity and the duty cycle for scientific data taking

Virgo Detector Characterization and Data Quality during the O3 run

The Advanced Virgo detector has contributed with its data to the rapid growth of the number of detected gravitational-wave signals in the past few years, alongside the two LIGO instruments. First, during the last month of the Observation Run 2 (O2) in August 2017 (with, most notably, the compact binary mergers GW170814 and GW170817) and then during the full Observation Run 3 (O3): an 11 months data taking period, between April 2019 and March 2020, that led to the addition of about 80 events to the catalog of transient gravitational-wave sources maintained by LIGO, Virgo and KAGRA. These discoveries and the manifold exploitation of the detected waveforms require an accurate characterization of the quality of the data, such as continuous study and monitoring of the detector noise. These activities, collectively named {\em detector characterization} or {\em DetChar}, span the whole workflow of the Virgo data, from the instrument front-end to the final analysis. They are described in details in the following article, with a focus on the associated tools, the results achieved by the Virgo DetChar group during the O3 run and the main prospects for future data-taking periods with an improved detector.Comment: 86 pages, 33 figures. This paper has been divided into two articles which supercede it and have been posted to arXiv on October 2022. Please use these new preprints as references: arXiv:2210.15634 (tools and methods) and arXiv:2210.15633 (results from the O3 run