The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components

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

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

Risk of Gonococcal Infection During Vaginal Exposure is Associated With High Vaginal pH and Active Menstruation.

BackgroundAn understanding of the biological reasons why 25% to 35% of women resist infection during vaginal intercourse with a man infected with Neisseria gonorrhoeae could lead to novel control measures. We sought modifiable biological bases for infection resistance by comparing women in the same core-mixing group who did or did not become infected after sexual exposure.MethodsWe enrolled 61 female contacts of index men with gonorrhea seen at Baltimore City Health Department clinics from January 2008 through May 2012. Exposure and sexual practices and histories, co-infections, physical signs on exam, patient symptom report, and menstrual history were collected.ResultsThirty-eight (62.3%) of the exposed women developed cervical infections. Multiple logistic regression found that a vaginal pH of 4.5 or higher at presentation to clinic was significantly associated with gonococcal infection (adjusted odds ratio, 5.5; P = 0.037) in women who presented within one menstrual cycle, 35 days. In this group of women, there was a significant association between acquiring an N. gonorrhoeae cervical infection and sexual exposure during menstruation (adjusted odds ratio 12.5; P = 0.05).ConclusionsModification of vaginal pH could be explored as novel strategy for reducing the risk of N. gonorrhoeae infections in women

Risk of Gonococcal Infection During Vaginal Exposure is Associated With High Vaginal pH and Active Menstruation.

BackgroundAn understanding of the biological reasons why 25% to 35% of women resist infection during vaginal intercourse with a man infected with Neisseria gonorrhoeae could lead to novel control measures. We sought modifiable biological bases for infection resistance by comparing women in the same core-mixing group who did or did not become infected after sexual exposure.MethodsWe enrolled 61 female contacts of index men with gonorrhea seen at Baltimore City Health Department clinics from January 2008 through May 2012. Exposure and sexual practices and histories, co-infections, physical signs on exam, patient symptom report, and menstrual history were collected.ResultsThirty-eight (62.3%) of the exposed women developed cervical infections. Multiple logistic regression found that a vaginal pH of 4.5 or higher at presentation to clinic was significantly associated with gonococcal infection (adjusted odds ratio, 5.5; P = 0.037) in women who presented within one menstrual cycle, 35 days. In this group of women, there was a significant association between acquiring an N. gonorrhoeae cervical infection and sexual exposure during menstruation (adjusted odds ratio 12.5; P = 0.05).ConclusionsModification of vaginal pH could be explored as novel strategy for reducing the risk of N. gonorrhoeae infections in women

Biochemical Analysis of Lpt3, a Protein Responsible for Phosphoethanolamine Addition to Lipooligosaccharide of Pathogenic Neisseria

The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62ΔLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62ΔLgtA, producing strain F62ΔLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62ΔLgtA indicated that this strain contained two PEA modifications on its LOS. F62ΔLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62ΔLgtAlpt3::Tn5