Document Detail

Biochemical characterizations of Escherichia coli-expressed protective antigen Ag473 of Neisseria meningitides group B.
MedLine Citation:
PMID:  20937316     Owner:  NLM     Status:  In-Process    
Polysaccharide-based vaccines against Neisseria meningitidis (Nm) serogroups A, C, Y and W135 have been available since 1970, but similar vaccine candidates developed for Nm group B (NmB) have not been successful due to both poor immunogenicity and their potential immunological cross-reactivity with human neurological tissue. In previous reports, a protective antigen and vaccine candidate, Ag473, was identified using proteomics and NmB-specific bactericidal monoclonal antibody. To initiate human phase one clinical trials, antigen production and characterization, pre-clinical toxicology and animal studies are required. In the present study, we report the biochemical characterization of Escherichia coli-expressed recombinant Ag473 (rAg473). Using MALDI-TOF mass analysis, chromatographically purified rAg473 was found to have two major isoforms that have molecular masses of 11,306 and 11,544amu, respectively. The isoforms were separated using RP-HPLC and pooled into two fractions. Based on the chromatogram, the ratio of lipoproteins in fractions #1 and #2 was found to be 1-2. GC-MS analysis of lipoproteins was performed, and the acylated fatty acids were identified. The results indicated that the first lipoproteins in fraction #1 contained the lipids palmitic acid (C16:0), cyclopropaneoctanoic acid (C17:1) and, predominately, stearic acid (C18:0). A different lipid composition of cyclopropaneoctanoic acid (C17:1), oleic acid (C18:1) and, predominately, palmitic acid (C16:0) was found in the second lipoprotein fraction. Both lipoprotein isoforms were tested and found to have Toll-like receptor (TLR) agonist activity in stimulating cytokine secretion from THP-1 cells. Circular dichroism (CD) analysis showed the secondary structure of rAg473 to be dominated by α-helices (48%), and the overall protein structure was stable up to 60°C and could refold after having been exposed to a temperature cycle from 20 to 90°C. In addition, the solubility of rAg473 (5mg/mL) was not affected after several freeze-thaw cycles. These biophysical and immunological properties make rAg473 a good vaccine candidate against NmB.
Jerry Wang-Chou Sung; Shih-Yang Hsieh; Chang-Ling Lin; Chih-Hsiang Leng; Shih-Jen Liu; Ai-Hsiang Chou; Li-Wei Lai; Li-Hsiu Lin; Yan Kwok; Chiou-Ying Yang; Pele Chong
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-10-23
Journal Detail:
Title:  Vaccine     Volume:  28     ISSN:  1873-2518     ISO Abbreviation:  Vaccine     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-12-03     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8406899     Medline TA:  Vaccine     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  8175-82     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier Ltd. All rights reserved.
Vaccine Research and Development Center, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan.
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