Summal~To learn how lipooligosaccharide (LOS) phase variations affect pathogenesis, we studied two male volunteers who were challenged intraurethrally with Neisseria gonorrhoeae that make a single LOS of 3,600 daltons and sequentially followed LOS expression by gonococci as urethritis developed. LOS variation occurred in vivo. Signs and symptoms of gonorrhea began with the appearance of variants making 4,700-dalton LOS that are immunochemically similar to glycosphingolipids of human hematopoietic cells (Man&ell, R. E., J. M. Griffiss, and B. A. Macher. 1989. J. Exp. Med. 168:107) and that have acceptors for sialic acid. A variant that appeared at the onset of leukorrhoea was shed by 34/36 men with naturally acquired gonorrhea at the time they sought medical attention; the other two shed the variant associated with dysuria. None shed the challenge variant. These data show that in vivo phase shifts to higher molecular mass LOS that mimic human cell membrane glycolipids are associated with the development of gonococcal leukorrhea. Outer membrane glycolipids of Gram-negative bacteria that cause disease along the respiratory or genital mucosae are relatively small (<7,000-dalton) lipooligosaccharides (LOS) 1 whose multiantennary oligosaccharide structures mimic those of human cell membrane glycosphingolipids (GSL) (1-4). During a study of the effect of piliation on infectivity, human volunteers developed gonorrhea after intraurethral challenge with a piliated Ndsseria gonorrhoeae strfm (5) that made a single LOS of 3,600 daltons. We made serial analyses of the LOS made by the organisms infecting two of the volunteers to learn whether LOS phase variations occurred during infection. We then confirmed the results by studying men with naturally acquired gonorrhea.

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A hybridoma produced by the polyethylene glycol fusion of the NS-1 variant of the P3x63Ag8 BALB/c plasmacytoma to splenocytes harvested from a BALB/ c mouse immunized with whole gonococci was found to be producing antibody to a common region on gonococcal lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay inhibition systems were established by utilizing this antibody, designated 3F11, and 100% inhibition occurred with both LPS and the LPSderived polysaccharides from each of the six prototype strains. Meningococcal LPS and LPS-derived polysaccharides partially inhibited the enzyme-linked immunosorbent assay, whereas similar preparations isolated from Escherichia coli 0:111, the J-5 mutant of this strain, and Salmonella minnesota Re595 failed MATERIALS AND METHODS Strains. Prototype strains representative of the six gonococcal LPS serotypes were obtained from our own collection. These were strain 1342 (serotype Gc,), strain 1291 (serotype GC2), strain 4505 (serotype GC3), strain 8551 (serotype Gc4), strain PID2 (serotype Gcs), and strain 3893 (serotype Gc6). Strain 4505r was also 751 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from

One of the lipooligosaccharide (LOS) structures of Neisseria gonorrhoeae contains a terminal Gal(␤1-4)GlcNAc residue which is a good candidate to serve as a ligand for human asialoglycoprotein receptors (ASGP-R). These receptors have been shown to be present on macrophages, sperm cells, and hepatocytes. The human tissue culture cell line used most often to study this receptor, HepG2, was used in our investigations only as a model. We also chose N. gonorrhoeae 1291 for these studies because, unlike many other gonococcal strains, this strain expresses one main species of LOS. The LOS structure expressed by this strain has also been fully characterized. Using well-established assays for the utilization of the ASGP-R, we found that incubation of HepG2 cells with gonococci expressing the terminal Gal(␤1-4)GlcNAc asialo-LOS carbohydrate structure competitively inhibited the ASGP-R from binding to one of its well-known ligands, asialo-␣-acid-1glycoprotein. The inhibition was specific to the ASGP-R, since binding of two other ligands to their specific receptors in the same model cell system was not affected. Immunoblot analysis for the ASGP-R suggested that gonococci seemed to stimulate the HepG2 cells to increase the expression of the major (46-kDa) receptor species. This observation was confirmed both by functional analysis, which showed that the concentration of total receptor molecules, as well as surface receptors, was about 60% higher after incubation with gonococci than in control cells and by Northern (RNA) blot analysis using a cDNA probe of the major human H1 subunit. Poly(A) RNA purified from control and HepG2 cells exposed to gonococci indicated the presence of increased amounts of mRNA coding for the ASGP-R after incubation with gonococci. This result supports the idea that the molecular mechanism controlling the receptor level after gonococcal exposure is under transcriptional regulation.

We developed a human inhibition monoclonal enzyme-linked immunosorbent assay (HIMELISA) to investigate the human immune response to the lipooligosaccharides (LOS) of Neisseria meningitidis. Monoclonal antibodies (MAb) were used to define seven epitopes on four LOS molecules of a meningococcal strain (126E) previously shown to express immunogenic LOS epitopes. The assay could distinguish epitope-specific antibody within whole sera. Neither the specificity nor the amount of the antibody measured by HIMELISA in sera of vaccinates changed during the immune response to meningococcal capsular polysaccharides, a chemically unrelated antigen. By using the HIMELISA, it was determined that sera from adults convalescing from meningococcal disease strongly inhibited MAb binding to two of the seven defined epitopes. The 3.6-kilodalton LOS of strain 126E expressed both of these epitopes. In addition, one of the inhibited epitopes was also expressed on the 4.0-kilodalton LOS of strain 126E. The convalescent-phase sera inhibited MAb binding to these two epitopes when they were expressed on LOS of diverse meningococcal strains. An acute-phase serum blocked MAb to the two epitopes to a lesser degree than did a convalescent-phase serum from the same patient. Immunoblotting the sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated LOS with convalescent-phase sera confirmed the specificity of the human anti-LOS antibody identified by HIMELISA.