AS03-adjuvanted H7N9 inactivated split virion vaccines induce cross-reactive and protective responses in ferrets.

Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model.

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice.

We investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 µg hemagglutinin (HA)/dose) or low-dose (LD) formulations (0.03 µg or 0.

The E119D neuraminidase mutation identified in a multidrug-resistant influenza A(H1N1)pdm09 isolate severely alters viral fitness in vitro and in animal models.

We recently isolated an influenza A(H1N1)pdm09 E119D/H275Y neuraminidase (NA) variant from an immunocompromised patient who received oseltamivir and zanamivir therapies. This variant demonstrated cross resistance to zanamivir, oseltamivir, peramivir and laninamivir. In this study, the viral fitness of the recombinant wild-type (WT), E119D and E119D/H275Y A(H1N1)pdm09 viruses was evaluated in vitro and in experimentally-infected C57BL/6 mice and guinea pigs.

Comparison of AS03 and Alum on immune responses elicited by A/H3N2 split influenza vaccine in young, mature and aged BALB/c mice.

Introduction: There is great interest in developing more effective influenza vaccines for the elderly. Oil-in-water adjuvants can boost humoral responses to seasonal vaccines in elderly subjects but relatively little is known about their mechanism of action.

Methods: We compared humoral and cellular immune profiles in young adult (2 months), mature (11-12 months) or aged (16-17 month) female BALB/c mice following two doses of Alum or AS03-adjuvanted A/H3N2 split-virus antigen (A/Uruguay/716/2007) at 0.

A chimeric haemagglutinin-based influenza split virion vaccine adjuvanted with AS03 induces protective stalk-reactive antibodies in mice.

Seasonal influenza virus vaccines are generally effective at preventing disease, but need to be well matched to circulating virus strains for maximum benefit. Influenza viruses constantly undergo antigenic changes because of their high mutation rate in the immunodominant haemagglutinin (HA) head domain, which necessitates annual re-formulation and re-vaccination for continuing protection. In case of pandemic influenza virus outbreaks, new vaccines need to be produced and quickly distributed.

Impact of a large deletion in the neuraminidase protein identified in a laninamivir-selected influenza A/Brisbane/10/2007 (H3N2) variant on viral fitness in vitro and in ferrets.

Viral fitness of a laninamivir-selected influenza A/Brisbane/10/2007-like (H3N2) isolate (LRVp9) containing a 237-amino acid neuraminidase deletion and a P194L hemagglutinin mutation was evaluated in vitro and in ferrets. LRVp9 and the wild-type (WT) virus showed comparable replication kinetics in MDCK-ST6GalI cells. Cultured virus was recovered between days 2 and 5 post-infection in nasal washes (NW) from the 4 WT-infected ferrets whereas no virus was recovered from the LRVp9-infected animals.

AS03-adjuvanted H7N1 detergent-split virion vaccine is highly immunogenic in unprimed mice and induces cross-reactive antibodies to emerged H7N9 and additional H7 subtypes.

Avian H7 is one of several influenza A virus subtypes that have the potential to cause pandemics. Herein we describe preclinical results following administration of an investigational H7N1 inactivated detergent-split virion vaccine adjuvanted with the AS03 Adjuvant System. The adjuvanted H7N1 vaccine was highly immunogenic compared to the non-adjuvanted H7N1 vaccine in unprimed mice with less than 100ng of hemagglutinin antigen per dose.

AS03-Adjuvanted, Very-Low-Dose Influenza Vaccines Induce Distinctive Immune Responses Compared to Unadjuvanted High-Dose Vaccines in BALB/c Mice.

During the 2009-2010 influenza pandemic, an adjuvanted, dose-sparing vaccine was recommended for most Canadians. We hypothesize that differences exist in the responses to AS03-adjuvanted, low antigen (Ag) dose versus unadjuvanted, full-dose vaccines. We investigated the relationship between Ag dose and the oil-in-water emulsion Adjuvant System AS03.

Adjuvanted inactivated influenza A(H3N2) vaccines induce stronger immunogenicity in mice and confer higher protection in ferrets than unadjuvanted inactivated vaccines.

Influenza viruses are major respiratory pathogens and the development of improved vaccines to prevent these infections is of high priority. Here, we evaluated split inactivated A(H3N2) vaccines (A/Uruguay/716/2007) combined or not with adjuvants (AS03, AS25 and Protollin) and administered by three different routes, intramuscular (i.m.

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus.

Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, zanamivir, and peramivir.

Reduced airborne transmission of oseltamivir-resistant pandemic A/H1N1 virus in ferrets.

Background: The H275Y neuraminidase mutation conferring oseltamivir resistance has been reported in several pandemic A/H1N1 (pH1N1) isolates. We sought to evaluate transmission of this mutant virus through the direct contact and the airborne (aerosol and droplet) routes in the ferret model.

Methods: Groups of four ferrets were infected with either wild-type (WT) or oseltamivir-resistant pH1N1 (H275Y) strains.

Oseltamivir-resistant pandemic A/H1N1 virus is as virulent as its wild-type counterpart in mice and ferrets.

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK alpha2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets.

The stem cell marker CD133 (prominin-1) is phosphorylated on cytoplasmic tyrosine-828 and tyrosine-852 by Src and Fyn tyrosine kinases.

CD133 (prominin-1) is a transmembrane glycoprotein expressed at the surface of normal and cancer stem cells, progenitor cells, rod photoreceptor cells, and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to fundamental properties of stem cells such as self-renewal and differentiation remains unknown. CD133 contains a short C-terminal cytoplasmic domain with five tyrosine residues, including a consensus tyrosine phosphorylation site that has not yet been investigated.

Impaired tyrosine phosphorylation of membrane type 1-matrix metalloproteinase reduces tumor cell proliferation in three-dimensional matrices and abrogates tumor growth in mice.

Pericellular proteolysis of the extracellular matrix by membrane type 1-matrix metalloproteinase (MT1-MMP) confers tumor cells with the ability to proliferate within three-dimensional (3D) matrices and sustains tumor growth in mice. In this study, we show that in addition to its matrix-degrading activity, phosphorylation of MT1-MMP on its unique tyrosine residue located within its cytoplasmic sequence (Tyr573) may also participate to these processes. Fibrosarcoma cells expressing a proteolytically active but non-phosphorylable mutant of MT1-MMP showed a markedly reduced proliferation rate when embedded within 3D type I collagen matrices, this antiproliferative effect being correlated with arrest in the G(0)/G(1) phase of the cell cycle.

Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling.

Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo.

Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins.

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro.

Proteomic analysis of human plasma proteins by two-dimensional gel electrophoresis and by antibody arrays following depletion of high-abundance proteins.

Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma.

VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases.

Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation.

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src.

Direct-acting fibrinolytic enzymes in shark cartilage extract: potential therapeutic role in vascular disorders.

Fibrinogen and fibrin are molecules with overlapping roles in blood clotting, fibrinolysis, wound healing, inflammation, matrix and cellular interactions and neoplasia. There is currently much interest in the possible use of fibrinolytic agents in human therapeutics. In this study, we report the presence of fibrinolytic activities in shark cartilage extract (SCE).

The antiangiogenic agent Neovastat (AE-941) induces endothelial cell apoptosis.

Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent, has been shown to inhibit key components of the angiogenic process, including matrix metalloproteinases and vascular endothelial growth factor-mediated signaling events. In this study, we report the presence of a proapoptotic activity within this compound. Neovastat treatment of bovine aortic endothelial cells caused cell death with characteristics of apoptosis, including chromatin condensation and DNA fragmentation.

The antiangiogenic agent neovastat (AE-941) inhibits vascular endothelial growth factor-mediated biological effects.

Purpose: Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, which exerts direct effects on vascular endothelial cells, including endothelial cell proliferation and survival, tubulogenesis, and vascular permeability. In this study, we examined whether Neovastat, a naturally occurring multifunctional antiangiogenic drug, could inhibit the endothelial cell response to VEGF stimulation.

Results: We demonstrated that Neovastat was able to block the VEGF-dependent microvessel sprouting from Matrigel-embedded rat aortic rings, and it also blocked the VEGF-induced endothelial cell tubulogenesis in vitro.

Differential regulation of matrix metalloproteinase activities in abdominal aortic aneurysms.

Background: The increased synthesis of matrix metalloproteinases (MMPs) by aortic smooth muscle cells (SMCs) is thought to be involved in the etiopathogenesis of abdominal aortic aneurysms (AAAs), but the functional regulation and the activation states of these MMPs remain unclear. In this study, we assessed the expression levels and the functional regulation of several MMPs in the pathogenesis of AAAs.

Methods: Human healthy aorta and AAA specimens were homogenized, and the proteolytic activities of MMP-2 and MMP-9 and of the macrophage metalloelastase (MMP-12) were assessed with zymography.