Charlotte C Ip

Isolation, structural characterization, and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.

Origin of the Isoelectric Heterogeneity of Monoclonal Immunoglobulin h1B4

The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD 18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, each has been isolated by a combination of high-resolution anion-exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.

A Real-Time PCR Assay to Identify and Discriminate Among Wild-Type and Vaccine Strains of Varicella-Zoster Virus and Herpes Simplex Virus in Clinical Specimens, and Comparison With the Clinical Diagnoses

A real‐time PCR assay was developed to identify varicella‐zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild‐type VZV (VZV‐WT), Oka vaccine strain VZV (VZV‐Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β‐globin gene to assess specimen adequacy. Discrimination of all VZV‐WT strains, including Japanese isolates and the Oka parent strain, from VZV‐Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross‐reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. J. Med. Virol. 81:1310–1322, 2009. Published 2009 Wiley‐Liss, Inc.

Application of capillary ion electrophoresis and ion chromatography for the determination of O-acetate groups in bacterial polysaccharides

Many bacterial polysaccharides possess O-linked acetate groups as constituents of their repeating units which often can serve as immunological determinants. It is therefore important to develop analytical methods for process monitoring as well as product characterization when such O-acetylated polysaccharides are used as components of vaccines. This is the case in a polysaccharide conjugate vaccine under development for treatment of diseases caused by Streptococcus pneumoniae. An ion chromatographic (IC) method utilizing suppressed conductivity detection (SCD) was developed to quantitatively measure O-acetate groups in the capsular polysaccharides from S. pneumoniae types 18C and 9V following hydrolytic release of O-acetate from the polysaccharide backbones using 2 mM sodium hydroxide. IC was carried out using an OmniPac PAX-500 column and 0.98 mM NaOH in 2% methanol as the mobile phase. Capillary ion electrophoresis (CIE) with indirect photometric detection was evaluated as an alternative method. The CIE method utilized a 72 cm x 75 microns I.D. fused-silica capillary and an electrolyte composed of 5 mM potassium hydrogenphthalate, 0.5 mM tetradecyltrimethylammonium bromide, and 2 mM sodium tetraborate, pH 5.88. A comparison of CIE and IC-SCD in terms of reproducibility, accuracy, linearity, and sensitivity will be presented.