Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells

We have investigated the immunostimulatory, anti-inflammatory, and antioxidant activities of various Echinacea raw materials and commercially available products on murine macrophages and human peripheral blood mononuclear cells (PBMCs). To emulate oral dosing, a simulated digestion protocol was employed as a means of sample preparation. Echinacea-induced macrophage activation was used as a measure of immunostimulatory activity determined via quantitative assays for macrophage-derived factors including tumor necrosis factor alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, and nitric oxide. Echinacea herb and root powders were found to stimulate murine macrophage cytokine secretion as well as to significantly enhance the viability and/or proliferation of human PBMCs in vitro. In contrast, Echinacea extracts chemically standardized to phenolic acid or echinacoside content and fresh pressed juice preparations were found to be inactive as immunostimulatory agents but did display, to varying degrees, anti-inflammatory and antioxidant properties.     

Adjuvant solution for pandemic influenza vaccine production

Extensive preparation is underway to mitigate the next pandemic influenza outbreak. New vaccine technologies intended to supplant egg-based production methods are being developed, with recombinant hemagglutinin (rHA) as the most advanced program for preventing seasonal and avian H5N1 Influenza. Increased efforts are being focused on adjuvants that can broaden vaccine immunogenicity against emerging viruses and maximize vaccine supply on a worldwide scale. Here, we test protection against avian flu by using H5N1-derived rHA and GLA-SE, a two-part adjuvant system containing glucopyranosyl lipid adjuvant (GLA), a formulated synthetic Toll-like receptor 4 agonist, and a stable emulsion (SE) of oil in water, which is similar to the best-in-class adjuvants being developed for pandemic flu. Notably, a single submicrogram dose of rH5 adjuvanted with GLA-SE protects mice and ferrets against a high titer challenge with H5N1 virus. GLA-SE, relative to emulsion alone, accelerated induction of the primary immune response and broadened its durability against heterosubtypic H5N1 virus challenge. Mechanistically, GLA-SE augments protection via induction of a Th1-mediated antibody response. Innate signaling pathways that amplify priming of Th1 CD4 T cells will likely improve vaccine performance against future outbreaks of lethal pandemic flu.H5N1 is a highly pathogenic avian influenza virus that can cause severe disease and death in humans, and world health authorities agree that the potential for pandemic H5N1 infection is high. Vaccination remains the most effective mechanism for preventing influenza, but there are complex challenges in implementing a pandemic preparedness plan, including: an inability to rapidly deploy the vast numbers of safe and effective doses needed on a worldwide scale; the fact that the immunogenicity of current nonadjuvanted H5N1 vaccines are relatively weak and require large antigen doses; and the potency of stockpiled prepandemic vaccines may be severely limited given the anticipated antigenic drift/shift associated with the emergence of a novel strain of pandemic H5N1.The US government has outlined provisions for new technologies that maximize immunogenicity and manufacturing capacity of vaccines for influenza, including the use of recombinant protein-based vaccines and adjuvants, which augment immunity and dose-sparing capacity. The most advanced egg-free flu vaccine candidate is a recombinant multimeric H5 hemagglutinin protein (rH5) produced by using a baculovirus expression vector system in SF+ insect cells (1, 2). Previous clinical studies suggested that two 90-μg doses of rH5 induced modest responses equivalent to conventional subvirion-based H5N1 vaccines (3, 4). This finding has prompted efforts to test rH5 with an adjuvant. Currently, the leading H5N1 vaccine adjuvants are oil-in-water (o/w) emulsions, which augment neutralizing antibody titers, increase the breadth of cross-reactive antibodies, and possess significant dose-sparing activity (5, 6). Importantly, these adjuvants are particularly effective in priming naïve individuals in the absence of preexisting memory.Vaccine adjuvants regulate adaptive immunity by stimulating dendritic cell maturation and antigen presentation (7, 8). A leading adjuvant target on DC is the family of innate Toll-like receptors, particularly the LPS receptor, Toll-like receptor 4 (TLR4). Glucopyranosyl lipid adjuvant (GLA) is a formulated form of the synthetic TLR4 agonist PHAD (Avanti Polar Lipids), which is analogous to the detoxified LPS derivative monophosphoryl lipid A (MPL), a component of the human papillomavirus vaccine Cervarix (9). Experimental vaccines containing GLA demonstrate enhanced immunogenicity in a variety of disease models (8), and in the context of influenza, GLA formulated in a stable emulsion (GLA-SE) improved Fluzone-dependent antibody titers in mice and nonhuman primates, relative to an emulsion alone (10–13). Given the critical importance of immunological priming for pandemic vaccine preparedness, we set out to test whether adjuvanting a recombinant H5 antigen with GLA-SE would broaden protective immunity against H5N1.     

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8 months at 4 °C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14 weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.     

Time course comparison of cell-cycle protein expression following partial hepatectomy and WY14,643-induced hepatic cell proliferation in F344 rats

During recent years, there has been an extensive research focus in the area of cell-cycle control in eukaryotes and the relationship that exists between cell proliferation and cancer. The eukaryotic cell-cycle is governed by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDK) and their partner cyclin proteins. This study was performed to identify differences in cell-cycle control protein expression following physical and chemical stimuli of hepatic cell growth. Protein levels of cell cycle mediators, cyclin dependent kinases (CDK 1,2,4,5), cyclin proteins (A,B,D1-D3 and E), proliferating cell nuclear antigen (PCNA), tumor suppressor proteins (p53 and Rb), and CDK inhibitory proteins (p16Ink4, p21Waf1 and p27Kip1) were examined in F344 rats following 70% partial hepatectomy or a single dose of WY14,643 over 96- and 48-h time courses, respectively. CDK1 (p34cdc2) and PCNA protein concentrations, quantified by ELISA, were significantly increased beginning at the 24-h time point and maximal at 48 h (6.9- and 3.7-fold for partial hepatectomy and 4.2- and 3.3-fold for WY14,643, respectively). Differential effects were observed with the G1 cell-cycle mediators CDK4, CDK5, and cyclin D3, p21Waf1 and p27Kip1 CDK inhibitory protein concentrations rose in accordance with the induction of DNA synthesis and histone H1 kinase activity. In addition, there were dramatic differences in p53 protein expression patterns following partial hepatectomy versus WY14,643 dosing. Because non-genotoxic hepatocarcinogens are known to induce cellular proliferation, data generated from this study may aid in elucidating the specific hepatocarcinogenic signal transduction pathways stimulated by non-genotoxic carcinogens.      

Differential gene expression technologies for identifying surrogate markers of drug efficacy and toxicity

Advances in the rapidly evolving discipline of pharmacogenomics have forced the biotechnology and pharmaceutical industries to integrate differential gene expression profiling into their drug discovery and development strategies. Here we highlight the use of differential gene expression technologies for the elucidation of both drug efficacy and toxicity as well as novel candidate genes for pharmacogenetic analyses to assess individual variability to drug response. This will include an overview of the different technologies created to facilitate pharmacogenomic analyses and to highlight advantages and disadvantages of these emerging methodologies. Two high-throughput differential gene expression technologies, microarrays and GeneCalling^®, will be presented in detail.     

Characterization of a recombinant influenza vaccine candidate using complementary LC-MS methods

Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MS(E) (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MS(E) was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.     

Assessment of CA1 injury after global ischemia using supervised 2D analyses of nuclear pyknosis

Selective neuronal vulnerability is a common theme in both acute and chronic diseases affecting the nervous system. This phenomenon is particularly conspicuous after global cerebral ischemia wherein CA1 pyramidal neurons undergo delayed death while surrounding hippocampal regions are relatively spared. While injury in this model can be easily demonstrated using either histological or immunological stains, current methods used to assess the cellular injury present in these biological images lack the precision required to adequately compare treatment effects. To address this shortcoming, we devised a supervised work-flow that can be used to quantify ischemia-induced nuclear condensation using microscopic images. And while we demonstrate the utility of this technique using models of ischemic brain injury, the approach can be readily applied to other paradigms in which programmed cell death is a major component.