Affinity-Purified Human Immunoglobulin G That Binds a Lacto-N-Neotetraose-Dependent Lipooligosaccharide Structure Is Bactericidal for Serogroup BNeisseria meningitidis

Abstract

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

“…SDS-PAGE-We screened LOS made by the mutants by SDS-PAGE (30) of PK-treated whole cell lysates from overnight growth (39) with use of a Mini-Protean 3 cell (Bio-Rad) (14). For optimal resolution, LOS were electrophoresed through 27 cm of a 13.1% polyacrylamide gel with an SE600 series vertical slab gel unit (Hoefer, San Francisco, CA) (41).…”

“…The plasmids carrying the inactivated genes then were transformed into 7946. Transformants were selected on GC agar plates that contained 50 g/ml kanamycin, and the mutants were confirmed by PCR, SDS-PAGE of proteinase K (PK) lysates (30,39), and mass spectrometry of O-deacylated LOS (14,40).…”

“…Structures assigned to the serotypes of the strains (16 -18, 20) are in Table 2. All organisms were cultured as described previously (12)(13)(14).…”

“…We recently reported a means of affinity-purifying human IgG that binds different LOS structures and showed that these antibodies can kill group B endemic case isolates in the presence of human complement (14). One IgG pool bound LOS made by the Neisseria gonorrhoeae strain, 1291, which have the paraglobosyl lacto-N-neotetraose (nLc 4 ) 4 structure as their ␣ chain (15).…”

“…In order to define the specificity of LOS IgG and the breadth of protection that they might afford, we used LOS from pyocin C-selected mutants of strain 1291 that have successive saccharide deletions from the nLc 4 ␣ chain to affinity-purify LOS IgG (14,15). We then performed genetic analyses of endemic group B case strains and mass spectrometry of intact LOS molecules to provide structural information beyond immunotype.…”