The truncated LOS of a mutant would not be an optimal immunogen for induction of the IgG preparations that were affinity purified for this study. 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-that causes much of the meningococcal disease in industrialized nations (36). Polysialyl structures identical to the group B capsular polysaccharide are found on human tissue, including neural cells (14, 21), which has diminished enthusiasm for a group B conjugate vaccine. The lack of a group B polysaccharide vaccine led to the development of vaccines composed of outer membrane proteins (OMP) in the form of outer membrane vesicles. Efficacy trials (3, 4, 7, 31, 32, 42, 48) have shown that these vaccines induce a short-lived immunoglobulin G (IgG) response in older children and adults but that they are poorly immunogenic in younger children, who are at greatest risk of disease. The protective antibody response is usually subtype specific, which limits the use of these vaccines because of the large antigenic variability of OMP among meningococci (36). Outer membrane lipooligosaccharides (LOS) are alternative vaccine candidates. Anti-LOS antibodies have been detected in normal human serum (NHS) and sera from patients convalescing from disease (8, 12, 18, 28, 33). Although the LOS in the outer membrane vesicle vaccines was depleted, about 8% LOS relative to protein remained and induced IgG antibodies in humans (35). This has led to an interest in the development of LOS vaccines (6, 24). Meningococcal LOS are short, surface-exposed glycolipids (17, 20, 24). They all contain lipid A that is integrated into the bacterial outer membrane, a proximal core segment that is conserved irrespective of the capsular serogroup or LOS serotype, and three variable, short, distal oligosaccharide chains, designated the , , and chains. The core region consists of TA-02 a highly conserved structure that is composed of two heptoses (Hep1 and Hep2) and two 3-deoxy-d-and strains used does not have designated L types, but strain 1291 expresses LNnT paraglobosyl LOS that is identical to the L7 LOS made by meningococci. In addition, an isogenic LOS mutant of strain 7994 (L1,8) was prepared. Meningococcal strain NMB-SS3 (a gift from M. Apicella, University of Iowa) is an LOS mutant that lacks galactose because of TA-02 inactivation of mutant of this strain. Microtiter wells were coated with strain 7994 and 7994-and then were reacted with 10 g/ml LOS-specific IgG and developed. Positive control wells coated with organisms were reacted with MAb B33 (provided by G. Brooks, University of California at San Francisco), which binds to all outer membrane Opa proteins (19). The assay was repeated using purified OMC. Flow cytometry analyses. (i) 2,588.56; this values are ?(H). cA sialylated molecular ion (291.26) of this species is also present. dSee reference 22. Strain 35E is the L2 prototype strain; the structures of LOS made by a different L2 strain have been described previously (15). The MALDI-TOF molecular ions of 35E were consistent with the previously described structures. The 2 2,954.71 is the 2,954.71 molecule (3,245.95) also were present. Strain 8532V is an LOS variant of the L8 strain 8532 that does not bind the L8-specific MAb 2-1-L8 (53a); its LOS yielded a single ?(H) molecular ion at 2,465.51. The strains 7946, TA-02 7994, and 7993 as determined by flow cytometry. The open curves indicate the binding of the secondary antibody alone, and the solid curves indicate the binding of IgG. does not have designated L types, but strain 1291 expresses LNnT paraglobosyl LOS that is identical to that of the L7 LOS made by meningococci. Whole-cell ELISA. Whole-cell ELISA was used to confirm the binding of the 1291 LOS IgG observed by flow cytometry to additional group B case isolates expressing LNnT LOS. Strains 7948 (L3,7), 7949 (L3,7,8), 8003 (L3,7), and 7971 (L2), as well as strain 7946 (L3, 7), bound the 1291 LOS IgG (L7 comparative) very well (Fig. ?(Fig.4).4). The other three IgG preparations (35E [L2] 6940 [L1], and 8532V [L8 variant]) also bound to these LNnT-bearing strains, but the amount of binding was much less than that RAB25 of 1291 IgG, consistent with the results of flow cytometry. Open in a separate windows FIG. 4. Binding of four LOS-specific IgG preparations (8532V, 35E, 1291, and 6940) to group B strains as determined by whole-cell ELISA. Microtiter wells were coated with the bacteria and reacted with 10 g/ml of the IgG. Strains 7946, 7948, and 8003 are L3,7 strains. Strain 7949 is an L3,7,8 strain, and strain 7971 is.