Novel Bivalent Viral-Vectored Vaccines Induce Potent Humoral and Cellular Immune Responses Conferring Protection against Stringent Influenza A Virus Challenge

Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional vaccines do not typically induce broad-spectrum immunity. In addition to seasonal infections, emerging pandemic influenza viruses present a continued threat to global public health. Pandemic influenza viruses have consistently higher attack rates and are typically associated with greater mortality compared with seasonal strains. Ongoing strategies to improve vaccine efficacy typically focus on providing broad-spectrum immunity; although B and T cells can mediate heterosubtypic responses, typical vaccine development will augment either humoral or cellular immunity. However, multipronged approaches that target several Ags may limit the generation of viral escape mutants. There are few vaccine platforms that can deliver multiple Ags and generate robust cellular and humoral immunity. In this article, we describe a novel vaccination strategy, tested preclinically in mice, for the delivery of novel bivalent viral-vectored vaccines. We show this strategy elicits potent T cell responses toward highly conserved internal Ags while simultaneously inducing high levels of Abs toward hemagglutinin. Importantly, these humoral responses generate long-lived plasma cells and generate Abs capable of neutralizing variant hemagglutinin-expressing pseudotyped lentiviruses. Significantly, these novel viral-vectored vaccines induce strong immune responses capable of conferring protection in a stringent influenza A virus challenge. Thus, this vaccination regimen induces lasting efficacy toward influenza. Importantly, the simultaneous delivery of dual Ags may alleviate the selective pressure that is thought to potentiate antigenic diversity in avian influenza viruses

Preliminary Assessment of the Efficacy of a T-Cell–Based Influenza Vaccine, MVA-NP+M1, in Humans

A single vaccination with MVA-NP+M1 boosts T-cell responses to conserved influenza antigens in humans. Protection against influenza disease and virus shedding was demonstrated in an influenza virus challenge study

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Novel characterization of a protein implicated in pigmentary glaucoma and glioblastomas

NMB is a putative melanosomal integral membrane protein that is expressed in a diverse range of cell types including retinal pigment epithelium, osteoblasts, and macrophages. The GPNMB gene was identified fourteen years ago, but characterization of the NMB gene product remains lacking. Despite being linked to multiple pathologies including pigmentary glaucoma and glial tumors, the function, subcellular location, and sorting of NMB have yet to be fully described. Here we show basic biochemical and cytological characterization of NMB in an endogenous retinal pigment epithelial system. To elucidate the trafficking and subcellular localization of the protein, we transfected pigment and non-pigment cells with NMB constructs and analyzed transfectants by indirect immunofluorescence microscopy. Our data demonstrate that in ARPE-19 pigment cells NMB is segregated away from lysosomes at steady state. Expression of NMB in non-pigmented COS-7 cells reveals partial overlap with LAMP-1 positive structures indicating co-localization with lysosomes. The NMB cytoplasmic domain contains within it a dileucine-based motif that is known to function as endocytic signal in other melanosomal proteins. Localization and continuous antibody uptake experiments with a site-directed mutant show that loss of the dileucine-based signal leads to an endocytic defect resulting in the accumulation of NMB on the cell surface. From these data we propose that NMB is sorted via the cell surface to its intracellular site of residence

Novel characterization of a protein implicated in pigmentary glaucoma and glioblastomas

NMB is a putative melanosomal integral membrane protein that is expressed in a diverse range of cell types including retinal pigment epithelium, osteoblasts, and macrophages. The GPNMB gene was identified fourteen years ago, but characterization of the NMB gene product remains lacking. Despite being linked to multiple pathologies including pigmentary glaucoma and glial tumors, the function, subcellular location, and sorting of NMB have yet to be fully described. Here we show basic biochemical and cytological characterization of NMB in an endogenous retinal pigment epithelial system. To elucidate the trafficking and subcellular localization of the protein, we transfected pigment and non-pigment cells with NMB constructs and analyzed transfectants by indirect immunofluorescence microscopy. Our data demonstrate that in ARPE-19 pigment cells NMB is segregated away from lysosomes at steady state. Expression of NMB in non-pigmented COS-7 cells reveals partial overlap with LAMP-1 positive structures indicating co-localization with lysosomes. The NMB cytoplasmic domain contains within it a dileucine-based motif that is known to function as endocytic signal in other melanosomal proteins. Localization and continuous antibody uptake experiments with a site-directed mutant show that loss of the dileucine-based signal leads to an endocytic defect resulting in the accumulation of NMB on the cell surface. From these data we propose that NMB is sorted via the cell surface to its intracellular site of residence

Improved adjuvanting of seasonal influenza vaccines: Pre-clinical studies of MVA-NP+M1 co-administration with inactivated influenza vaccine.

Licensed seasonal influenza vaccines induce antibody responses against influenza hemagglutinin that are limited in their ability to protect against different strains of influenza. Cytotoxic T lymphocytes (CTLs) recognizing the conserved internal nucleoprotein (NP) and matrix protein (M1) are capable of mediating a cross-subtype immune response against influenza. Modified vaccinia virus Ankara encoding NP and M1 (MVA-NP+M1) is designed to boost pre-existing T-cell responses in adults in order to elicit a cross-protective immune response. We examined the co-administration of hemagglutinin (HA) protein formulations and candidate MVA-NP+M1 influenza vaccines in murine, avian, and swine models. Antibody responses post-immunization were measured by ELISA and pseudotype neutralization assays. Here we demonstrate that MVA-NP+M1 can act as an adjuvant enhancing antibody (Ab) responses to HA while simultaneously inducing potent T-cell responses to conserved internal antigens. We show that this regimen leads to the induction of cytophilic Ab isotypes that are capable of inhibiting hemagglutination and in the context of H5 exhibit cross-clade neutralization. The simultaneous induction of T cells and antibody responses has the potential to improve seasonal vaccine performance and could be employed in pandemic situations

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection