[Show abstract][Hide abstract] ABSTRACT: A prophylactic vaccine to prevent congenital transmission of human Cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants would be highly desirable. Neutralizing antibodies against HCMV confer significant protection, and gB is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been a failure to induce high titer persistent neutralizing antibody responses that could prevent infection of epithelial cells. We used enveloped virus-like-particles (eVLPs), in which particles were produced in cells after expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV) G protein (gB-G eVLPs). gB-G expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity for epithelial cell neutralizing activity than that induced with soluble recombinant gB protein. Analysis of gB antibody binding titers and T helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. Cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a pre-fusion gB conformation like that of infected cells and viral particles. Two among five gB-G eVLP-induced monoclonal antibodies examined in detail had neutralizing activity, one of which possessed particularly potent epithelial cell neutralizing activity. These data differentiate gB-G eVLPs from past gB antigens and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection.
[Show abstract][Hide abstract] ABSTRACT: The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-
2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.
BioMed Research International 03/2011; 2011(6):174615. DOI:10.1155/2011/174615 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon alpha-2 (IFN alpha-2) products have been widely used as antivirals for the treatment of serious diseases such as hepatitis B and C. However, reports of adverse reactions following treatment have prompted investigations into the cause of these undesirable events. In this study size-exclusion HPLC (SE-HPLC) methods coupled with intrinsic fluorescence detection were developed for evaluating the stability and degradation profiles of IFN alpha-2 drug substances and drug products. The method allowed baseline resolution of the active ingredient from the excipients present in the finished products that included large amounts of albumin. Limits of detection (S/N>or=3) for IFN alpha-2a and IFN alpha-2b were 32 ng/mL and 28 ng/mL, respectively and good repeatability of chromatographic profiles (%RSD<2.1) was obtained. High molecular weight (HMW) aggregates with apparent molecular weight of approximately 650 kDa as well as dimers, denatured and reduced variants were successfully identified and separated from native IFN alpha-2 proteins. This chromatographic method, which quantitatively measures physical and chemical changes taking place in solution formulations, was found to be capable of monitoring IFN alpha-2a and IFN alpha-2b stability. Potency assay results revealed up to 87% decrease in biological activity of the physically and chemically altered variants compared to the original IFNs.
Journal of Chromatography A 10/2009; 1217(19):3297-306. DOI:10.1016/j.chroma.2009.10.081 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of pure nonaqueous solvents in capillary electrophoresis (CE) can alter the separation selectivity and enhance the solubility of hydrophobic compounds and enables the use of higher voltages. However, control of the electro-osmotic flow (EOF) is essential. In this work, we report the use of self-assembled coatings for EOF modification and elimination of analyte adsorptions onto silica capillaries in pure formamide. Bilayer capillary coatings derived from the double chain cationic surfactant dimethyldioctadecylammoniurn bromide (2C(18)DAB) reverses the EOF in buffers such as acetate, formate, and phosphate. Reversed EOF of > 1.1 x 10(-4) cm(2)/Vs enables the separation of the pharmaceutical drugs propranolol, metoprolol, chloroquine, and chloropheniramine in less than 5 min with efficiencies of 0.2-0.5 million plates/m (66 000 to 165 000 plates). Chemical and physical factors affecting the coating stability and their influence on separation speed and efficiency of the cationic drugs in formamide are also investigated.
Canadian Journal of Chemistry 06/2007; 85(7-8):540-546. DOI:10.1139/v07-077 · 1.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This report describes the creation of semipermanent capillary coatings that are compatible with organic-water solvent systems in CE. The coatings are created by simply rinsing the fused-silica capillary with long double-chain cationic surfactants, such as dimethyl-ditetradecyl ammonium bromide (2C(14)DAB), dihexadecyldimethyl ammonium bromide (2C(16)DAB), and dimethyldioctadecyl ammonium bromide (2C(18)DAB). These surfactants generate semipermanent bilayer coatings on the capillary surface, which display a high degree of stability in buffers containing up to 60% v/v of organic solvents, such as methanol and ACN. The coating stability increases with increasing hydrophobicity of the surfactant, i.e., with increasing chain length. For instance, the EOF changes by only 1.2% in a 2C(18)DAB-coated capillary after 130 capillary volumes of rinsing with 60% v/v methanol containing buffer. The bilayer coatings allow separations to be performed without the need to regenerate the coating between runs or to maintain the EOF modifier in the run buffer. Rapid separations (<2 min) of anions and basic drugs with migration time reproducibility of less than 0.5% RSD and efficiencies of 0.4-0.6 million plates/m are obtained. In addition, selectivity changes for small anions and cationic drugs are also observed when the organic solvent content is adjusted.
[Show abstract][Hide abstract] ABSTRACT: In capillary electrophoresis (CE) analysis of small inorganic anions, the ability to control the electroosmotic flow (EOF) and the ability to alter the electrophoretic mobility of the ions are essential to improve resolution and separation speed. In this work, a CE method for separation of small inorganic anions using indirect detection in mixed methanol/water buffers is presented. The suitability of different UV absorbing probes commonly used for indirect detection including chromate, iodide, phthalate, benzoate, trimellitate, and pyromellitate, in mixed methanol/water buffers is examined. The effect of the electrolyte buffer system, including the pH, buffer concentration and the organic solvent on the electrophoretic mobility of the probes and analytes are also investigated. The EOF was reversed using cationic surfactant, cetyltrimethylammonium bromide (CTAB) so ions were separated under co-EOF mode. The organic solvent alters the electrophoretic mobility of the probes and the analytes differently and hence choice of the appropriate probe is essential to achieve high degree of detection sensitivity. Separations of six anions in less than 2.5 min were accomplished in buffers containing up to 30% MeOH. Adjustment of the methanol content helps to improve the selectivity and resolution of inorganic anions. Limit of detection, reproducibility and application of the method for quantification of anions in water samples will also be discussed.
Journal of Chromatography A 09/2005; 1085(1):155-63. DOI:10.1016/j.chroma.2004.12.004 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Manipulation of the electroosmotic flow (EOF) is essential for achieving optimized separations of small anions by capillary electrophoresis (CE). In this work, efficient suppression or reversal of EOF is achieved upon addition of small amounts of the cationic surfactants, cetyltrimethylammonium bromide (CTAB) or didodecyldimethylammonium bromide (DDAB) to the electrophoretic buffer. Highly stable and reversed EOF are achieved using the surfactants in the presence of up to 50% MeOH. In aqueous and low methanol containing solutions (up to 30%, v/v) surface aggregation of the surfactants at the capillary wall occurs at a concentration below the critical micelle concentration (CMC). The impact of MeOH on reversed EOF is predominantly a function of the diminished zeta potential of the silica, and to a lesser extent on the CMC in the bulk solution of the surfactant. Fast baseline separation and selectivity changes for small inorganic anions are observed when mixed aqueous-organic buffers are employed. Changes in EOF, micellar properties of the surfactant and selectivity for inorganic anions upon addition of various percent of methanol are also discussed.
Journal of Chromatography A 03/2004; 1027(1-2):185-91. DOI:10.1016/j.chroma.2003.10.081 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Dept. of Chemistry, University of Alberta. Thesis (Ph. D.)--University of Alberta, 2006. Includes bibliographical references.