Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C

IntroductionOuter membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined.MethodsTen paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays.Results and discussionPost-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, β-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design

Regulatory Role of the MisR/S Two-Component System in Hemoglobin Utilization in Neisseria meningitidis▿ †

Outer membrane iron receptors are some of the major surface entities that are critical for meningococcal pathogenesis. The gene encoding the meningococcal hemoglobin receptor, HmbR, is both independently transcribed and transcriptionally linked to the upstream gene hemO, which encodes a heme oxygenase. The MisR/S two-component system was previously determined to regulate hmbR transcription, and its hemO and hmbR regulatory mechanisms were characterized further here. The expression of hemO and hmbR was downregulated in misR/S mutants under both iron-replete and iron-restricted conditions, and the downregulation could be reversed by complementation. No significant changes in expression of other iron receptors were detected, suggesting that the MisR/S system specifically regulates hmbR. When hemoglobin was the sole iron source, growth defects were detected in the mutants. Primer extension analysis identified a promoter upstream of the hemO-associated Correia element (CE) and another promoter at the proximal end of CE, and processed transcripts previously identified for other cotranscribed CEs were also detected, suggesting that there may be posttranscriptional regulation. MisR directly interacts with sequences upstream of the CE and upstream of the hmbR Fur binding site and thus independently regulates hemO and hmbR. Analysis of transcriptional reporters of hemO and hmbR further demonstrated the positive role of the MisR/S system and showed that the transcription of hmbR initiated from hemO was significantly reduced. A comparison of the effects of the misS mutation under iron-replete and iron-depleted conditions suggested that activation by the MisR/S system and iron-mediated repression by Fur act independently. Thus, the expression of hemO and hmbR is coordinately controlled by multiple independent regulatory mechanisms, including the MisR/S two-component system

Image_1_Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C.tif

IntroductionOuter membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined.MethodsTen paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays.Results and discussionPost-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, β-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design.</p

Table_1_Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C.docx

IntroductionOuter membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined.MethodsTen paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays.Results and discussionPost-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, β-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design.</p