Abstract
Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-alpha-d-Glcp-(1-->3)-[l-alpha-d-Hepp-(1-->7)]-l-alpha-d-Hepp 4P-(1-->3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (KD values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (KD values) determined by SPR were in the range of 3.6 x 10-5 to 3.2 x 10-8 m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest KD values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5-10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding.
Highlights
Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate
Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-␣-D-Glcp-(133)-[L␣-D-Hepp-(137)]-L-␣-D-Hepp 4P-(133)-R2, which is common to LPS from all E. coli, Salmonella, and Shigella
The concept of treatment of septic shock in humans by active or passive vaccination with cross-protective antibodies was developed after a report by Braude and Douglas, who showed that an antiserum in rabbits was cross-protective against the local Shwartzman reaction induced by heterologous bacteria (8)
Summary
Extraction of LPS, and Isolation of Oligosaccharides— E. coli F470 (R1 core-type) (15), E. coli F576 (R2 core-type) (15), E. coli F653 (R3 core type) (16), E. coli F2513 (R4 core type) (16), Salmonella enterica sv. 250 g of NaBH4 was added, and the samples were incubated for 1 h at 4 °C in the dark followed by dialysis against water once and three times against PBS, pH 7.2. MAb WN1 222-5 was dialyzed against PBS, pH 7.2, and the concentration was determined by UV measurement (1 mg mlϪ1 ϭ A280 of 1.35). Purified and desalted deacylated LPS oligosaccharide were dissolved at a concentration of 0.35 mM in dialysis buffer and loaded into the syringe of the microcalorimeter. Both solutions were thoroughly degassed prior to loading. The antibody concentration in the cell was corrected after the curve fitting as described in the ITC Data Analysis in the Origin Version 2.9 manual provided by the manufacturer. One-dimensional 1H, 13C, and 31P and two-dimensional homonuclear 1H,1H (DQF-COSY, NOESY, TOCSY), heteronuclear 1H,13C, and 1H,31P NMR correlation spectra (HMQC) were recorded using Bruker standard pulse programs and analyzed with Bruker Xwinnmr software
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
