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Semchenko, Evgeny A., Tsitsi D. Mubaiwa, Christopher J. Day, and Kate L. Seib. "Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells." Journal of Infectious Diseases 221, no. 10 (November 29, 2019): 1612–22. http://dx.doi.org/10.1093/infdis/jiz628.
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Abstract The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.
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Lucidarme, Jay, Stefanie Gilchrist, Lynne S. Newbold, Stephen J. Gray, Edward B. Kaczmarski, Lynne Richardson, Julia S. Bennett, Martin C. J. Maiden, Jamie Findlow, and Ray Borrow. "Genetic Distribution of Noncapsular Meningococcal Group B Vaccine Antigens in Neisseria lactamica." Clinical and Vaccine Immunology 20, no. 9 (June 26, 2013): 1360–69. http://dx.doi.org/10.1128/cvi.00090-13.
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ABSTRACTThe poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp),Neisseriaadhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genusNeisseriathat also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal,Neisseria lactamica. All the isolates possessednhbabut were devoid offhbpandnadA. Thenhbaalleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.
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Vacca, Irene, Elena Del Tordello, Gianmarco Gasperini, Alfredo Pezzicoli, Martina Di Fede, Silvia Rossi Paccani, Sara Marchi, et al. "Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells." PLOS ONE 11, no. 10 (October 25, 2016): e0162878. http://dx.doi.org/10.1371/journal.pone.0162878.
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Semchenko, Evgeny A., Aimee Tan, Ray Borrow, and Kate L. Seib. "The Serogroup B Meningococcal Vaccine Bexsero Elicits Antibodies to Neisseria gonorrhoeae." Clinical Infectious Diseases 69, no. 7 (December 14, 2018): 1101–11. http://dx.doi.org/10.1093/cid/ciy1061.
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Abstract Background Neisseria gonorrhoeae and Neisseria meningitidis are closely-related bacteria that cause a significant global burden of disease. Control of gonorrhoea is becoming increasingly difficult, due to widespread antibiotic resistance. While vaccines are routinely used for N. meningitidis, no vaccine is available for N. gonorrhoeae. Recently, the outer membrane vesicle (OMV) meningococcal B vaccine, MeNZB, was reported to be associated with reduced rates of gonorrhoea following a mass vaccination campaign in New Zealand. To probe the basis for this protection, we assessed the cross-reactivity to N. gonorrhoeae of serum raised to the meningococcal vaccine Bexsero, which contains the MeNZB OMV component plus 3 recombinant antigens (Neisseria adhesin A, factor H binding protein [fHbp]-GNA2091, and Neisserial heparin binding antigen [NHBA]-GNA1030). Methods A bioinformatic analysis was performed to assess the similarity of MeNZB OMV and Bexsero antigens to gonococcal proteins. Rabbits were immunized with the OMV component or the 3 recombinant antigens of Bexsero, and Western blots and enzyme-linked immunosorbent assays were used to assess the generation of antibodies recognizing N. gonorrhoeae. Serum from humans immunized with Bexsero was investigated to assess the nature of the anti-gonococcal response. Results There is a high level of sequence identity between MeNZB OMV and Bexsero OMV antigens, and between the antigens and gonococcal proteins. NHBA is the only Bexsero recombinant antigen that is conserved and surfaced exposed in N. gonorrhoeae. Bexsero induces antibodies in humans that recognize gonococcal proteins. Conclusions The anti-gonococcal antibodies induced by MeNZB-like OMV proteins could explain the previously-seen decrease in gonorrhoea following MeNZB vaccination. The high level of human anti-gonococcal NHBA antibodies generated by Bexsero vaccination may provide additional cross-protection against gonorrhoea.
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Soler-Garcia, Aleix, Mariona Fernández de Sevilla, Raquel Abad, Cristina Esteva, Laia Alsina, Julio Vázquez, Carmen Muñoz-Almagro, and Antoni Noguera-Julian. "Meningococcal Serogroup B Disease in Vaccinated Children." Journal of the Pediatric Infectious Diseases Society 9, no. 4 (October 21, 2019): 454–59. http://dx.doi.org/10.1093/jpids/piz071.
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Abstract Background Neisseria meningitidis serogroup B (MenB) is the most frequent cause of invasive meningococcal disease (IMD) in Spain. The multicomponent vaccine against MenB (4CMenB) was approved in Spain in January 2014. Methods We present 4 cases of children who developed MenB-associated IMD despite previous vaccination with 4CMenB. Extensive immunologic diagnostic work-up was performed in order to rule out any immunodeficiency. Also, molecular characterization of the MenB strain was conducted to determine whether bacterial antigens matched vaccine antigens. Results Among the 4 patients (2 girls), 2 had previous risk factors for IMD (recurrent bacterial meningitis of unknown origin and treatment with eculizumab). All patients developed meningitis, but only 2 developed septic shock; they were all cured without sequelae. No other primary or secondary immunodeficiencies were detected. MenB sequence type 213 was identified in 3 cases. With the exception of neisserial heparin-binding antigen peptide 465 present in 1 isolate, the rest of the isolated strains harbored vaccine antigen variants that did not match antigen variants included in the vaccine. Conclusions We present 4 children who developed MenB-associated IMD despite previous vaccination with 4CMenB. In 2 cases, the antibodies induced by 4CMenB likely were not effective against the isolated strains. A high level of suspicion for IMD seems advisable regardless of the patient’s vaccination history.
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Ispasanie, Emma, Gerd Pluschke, Abraham Hodgson, Ali Sie, Calman MacLennan, and Oliver Koeberling. "Characterization of vaccine antigens of meningococcal serogroup W isolates from Ghana and Burkina Faso from 2003 to 2009." F1000Research 3 (November 3, 2014): 264. http://dx.doi.org/10.12688/f1000research.3881.1.
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Neisseria meningitidis is a major cause of bacterial meningitis and a considerable health problem in the 25 countries of the ‘African Meningitis Belt’ that extends from Senegal in West Africa to Ethiopia in the East. Approximately 80% of cases of meningococcal meningitis in Africa have been caused by strains belonging to capsular serogroup A. After the introduction of a serogroup A conjugate polysaccharide vaccine, MenAfriVac™, that began in December 2010, the incidence of meningitis due to serogroup A has markedly declined in this region. Currently, serogroup W of N. meningitidis accounts for the majority of cases. Vaccines based on sub-capsular antigens, such as Generalized Modules for Membrane Antigens (GMMA), are under investigation for use in Africa. To analyse the antigenic properties of a serogroup W wave of colonisation and disease, we investigated the molecular diversity of the protein vaccine antigens PorA, Neisserial Adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and factor H binding protein (fHbp) of 31 invasive and carriage serogroup W isolates collected as part of a longitudinal study from Ghana and Burkina Faso between 2003 and 2009. We found that the isolates all expressed fHbp variant 2 ID 22 or 23, differing from each other by only one amino acid, and a single PorA subtype of P1.5,2. Of the isolates, 49% had a functional nhbA gene and 100% had the nadA allele 3, which contained the insertion sequence IS1301 in five isolates. Of the W isolates tested, 41% had high fHbp expression when compared with a reference serogroup B strain, known to be a high expresser of fHbp variant 2. Our results indicate that in this collection of serogroup W isolates, there is limited antigenic diversification over time of vaccine candidate outer membrane proteins (OMP), thus making them promising candidates for inclusion in a protein-based vaccine against meningococcal meningitis for Africa.
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Plikaytis, Brian D., Maria Stella, Giuseppe Boccadifuoco, Lisa M. DeTora, Mauro Agnusdei, Laura Santini, Brunella Brunelli, et al. "Interlaboratory Standardization of the Sandwich Enzyme-Linked Immunosorbent Assay Designed for MATS, a Rapid, Reproducible Method for Estimating the Strain Coverage of Investigational Vaccines." Clinical and Vaccine Immunology 19, no. 10 (August 8, 2012): 1609–17. http://dx.doi.org/10.1128/cvi.00202-12.
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ABSTRACTThe meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or “relative potency” (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.
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Vu, David M., Tracy T. Wong, and Dan M. Granoff. "Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and Neisserial heparin binding antigen." Vaccine 29, no. 10 (February 24, 2011): 1968–73. http://dx.doi.org/10.1016/j.vaccine.2010.12.075.
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Maritan, Martina, Roberta Cozzi, Paola Lo Surdo, Daniele Veggi, Matthew James Bottomley, and Enrico Malito. "Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA." Acta Crystallographica Section F Structural Biology Communications 73, no. 6 (May 11, 2017): 305–14. http://dx.doi.org/10.1107/s2053230x17006021.
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Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein fromNeisseria meningitidisand is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.
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Domina, Maria, Veronica Lanza Cariccio, Salvatore Benfatto, Mario Venza, Isabella Venza, Danilo Donnarumma, Erika Bartolini, et al. "Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries." PLOS ONE 11, no. 8 (August 10, 2016): e0160702. http://dx.doi.org/10.1371/journal.pone.0160702.
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Newcombe, Jane, Tom A. Mendum, Chuan-peng Ren, and Johnjoe McFadden. "Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS." Microbiology 160, no. 2 (February 1, 2014): 429–38. http://dx.doi.org/10.1099/mic.0.071829-0.
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Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.
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Semchenko, Evgeny A., Christopher J. Day, and Kate L. Seib. "The Neisseria gonorrhoeae Vaccine Candidate NHBA Elicits Antibodies That Are Bactericidal, Opsonophagocytic and That Reduce Gonococcal Adherence to Epithelial Cells." Vaccines 8, no. 2 (May 13, 2020): 219. http://dx.doi.org/10.3390/vaccines8020219.
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Due to the continuing emergence of multidrug resistant strains of Neisseria gonorrhoeae there is an urgent need for the development of a gonococcal vaccine. We evaluated the gonococcal Neisseria heparin binding antigen (NHBA) as a potential vaccine candidate, in terms of its sequence conservation and expression in a range of N. gonorrhoeae strains, as well as its immunogenicity and the functional activity of antibodies raised to either the full length NHBA or a C-terminal fragment of NHBA (NHBA-c). The gene encoding NHBA is highly conserved and expressed in all N. gonorrhoeae strains investigated. Recombinant NHBA is immunogenic, and mice immunized with either NHBA or NHBA-c adjuvanted with either Freund’s or aluminium hydroxide (alum) generated a humoral immune response, with predominantly IgG1 antibodies. Antibodies generated by both NHBA and NHBA-c antigens promoted complement activation and mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity, with slightly higher titers seen for the NHBA-c antigen. Anti-NHBA was also able to block the functional activity of NHBA by reducing binding to heparin and adherence to cervical and urethral epithelial cells. These data suggest that the gonococcal NHBA is a promising vaccine antigen to include in a vaccine to control N. gonorrhoeae.
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Abad, R., A. Biolchi, M. Moschioni, M. M. Giuliani, M. Pizza, and J. A. Vázquez. "A Large Portion of Meningococcal Antigen Typing System-Negative Meningococcal Strains from Spain Is Killed by Sera from Adolescents and Infants Immunized with 4CMenB." Clinical and Vaccine Immunology 22, no. 4 (January 28, 2015): 357–60. http://dx.doi.org/10.1128/cvi.00669-14.
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ABSTRACTA new vaccine (the 4CMenB 4-component protein vaccine [Bexsero], which includes PorA, factor H-binding protein [fHbp], neisserial heparin-binding antigen [NHBA], andNeisseriaadhesin A [NadA]) against serogroup B meningococci has recently been approved for use in people older than age 2 months in Europe, Australia, and Canada. Preapproval clinical efficacy studies are not feasible for invasive meningococcal disease because its incidence is low/very low, and the serum bactericidal antibody (SBA) titer (or the human SBA [hSBA] titer when human complement is used in the assay) has been used as a surrogate marker of protection. However, the hSBA assay cannot be used on a large scale, and therefore, a meningococcal antigen typing system (MATS) was developed. MATS combines conventional PorA genotyping with an enzyme-linked immunosorbent assay (ELISA) that quantifies both the expression and the cross-reactivity of antigenic variants. The assay has been used to evaluate the potential of the 4CMenB meningococcal group B vaccine to cover group B strains in several countries. Some recent data suggest that MATS is a conservative predictor of strain coverage. We used pooled sera from adolescents and infants to test by the hSBA assay 10 meningococcal group B strains isolated in Spain that were negative for the 3 antigens (n= 9) or that had very low levels of the 3 antigens (n= 1) by MATS. We found that all strains were killed by sera from adolescents and that 5 of the 10 strains were also killed, although at a low titer, by sera from infants. Our data confirm that MATS underestimates vaccine coverage.
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Rossi, Raffaella, Peter T. Beernink, Serena Giuntini, and Dan M. Granoff. "Susceptibility of Meningococcal Strains Responsible for Two Serogroup B Outbreaks on U.S. University Campuses to Serum Bactericidal Activity Elicited by the MenB-4C Vaccine." Clinical and Vaccine Immunology 22, no. 12 (September 30, 2015): 1227–34. http://dx.doi.org/10.1128/cvi.00474-15.
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ABSTRACTIn 2013 and 2014, two U.S. universities had meningococcal serogroup B outbreaks (a total of 14 cases) caused by strains from two different clonal complexes. To control the outbreaks, students were immunized with a serogroup B meningococcal vaccine (Novartis) that was not yet licensed in the United States. The vaccine (referred to as MenB-4C) contains four components capable of eliciting bactericidal activity. Both outbreak strains had high expression levels of two of the vaccine antigens (subfamily B factor H binding protein [FHbp] and neisserial heparin binding antigen [NHba]); the university B outbreak strain also had moderate expression of a third antigen, NadA. We investigated the bactericidal activity of sera from mice immunized with FHbp, NHba, or NadA and sera from MenB-4C-immunized infant macaques and an adult human. The postimmunization bactericidal activity of the macaque or human serum against isolates from university B with FHbp identification (ID) 1 that exactly matched the vaccine FHbp sequence variant was 8- to 21-fold higher than that against isolates from university A with FHbp ID 276 (96% identity to the vaccine antigen). Based on the bactericidal activity of mouse antisera to FHbp, NadA, or NHba and macaque or human postimmunization serum that had been depleted of anti-FHbp antibody, the bactericidal activity against both outbreak strains largely or entirely resulted from antibodies to FHbp. Thus, despite the high level of strain expression of FHbp from a subfamily that matched the vaccine antigen, there can be large differences in anti-FHbp bactericidal activity induced by MenB-4C vaccination. Further, strains with moderate to high NadA and/or NHba expression can be resistant to anti-NadA or anti-NHba bactericidal activity elicited by MenB-4C vaccination.
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Garcia, Yara Ruiz, Woo-Yun Sohn, Mariagrazia Pizza, and Rafik Bekkat-Berkani. "02. Beyond B Antigen Coverage: The Potential of the 4CMenB Vaccine for Cross-protection Against Pathogenic Neisseria Infections." Open Forum Infectious Diseases 8, Supplement_1 (November 1, 2021): S125. http://dx.doi.org/10.1093/ofid/ofab466.205.
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Abstract Background Two human pathogenic Neisseria species exist: N. meningitidis (Nm) and N. gonorrhoeae (Ng). Although causing disparate clinical syndromes, invasive meningococcal disease (IMD) and gonorrhea, they are genetically similar and share key protein antigens. The 4CMenB vaccine, licensed against meningococcal B disease, comprises 4 antigenic components (factor H binding protein (fHbp), variant 1.1, subfamily B; Neisseria heparin binding antigen (NHBA) peptide 2; Neisserial adhesin A (NadA) variant 3; and Porin A (PorA) P1.4), and potentially protects against non-B invasive meningococcal and gonococcal strains. In this review, we summarize the similarities between these antigens and those in Nm serogroups A, C, W, X and Y and Ng. Methods Published data in humans were analyzed to conduct a narrative literature review of the potential extent of meningococcal vaccine-induced protection against non-B meningococcal strains and Ng. Techniques applied to indirectly measure this effect are based on genotype-phenotype modelling, strain coverage, bactericidal killing and direct impact on disease reduction. Results Data were identified from countries in America, Europe, Africa and Oceania. The genes encoding for fHbp and NHBA are also present in strains belonging to the five non-B serogroups, while NadA is present in several strains of serogroups C, W and Y, and PorA P1.4 mainly in serogroup W. At the genome level, Ng and Nm share up to 90% homology. Most of the outer membrane vesicle antigens, like PilQ, Omp85 (BamA), NspA, MtrE, MetQ, LbpA, PorB, FetA, OpcA and NHBA, are highly conserved in Ng. In addition, a synergistic effect might enhance immunogenicity against non-B serogroups as shown against serogroup B. Conclusion 4CMenB components are present and conserved in several Ng and Nm strains. Recent results demonstrate that 4CMenB reduces MenW disease incidence in infants and might generate cross-protection against other non-B serogroups. In addition, 4CMenB has been proven to be effective in reducing gonococcal infections in adolescents. Research on future genomic and proteomic characterizations of IMD and gonorrhea strains will provide information on the molecular basis of the underlying broad strain coverage, while informing decisions regarding prevention and immunization programmes. Disclosures Yara Ruiz Garcia, MSc, PhD, GSK group of companies (Employee) Woo-Yun Sohn, MD, GSK group of companies (Employee, Shareholder) Mariagrazia Pizza, Biological Sciences, PhD, GSK group of companies (Employee, Shareholder) Rafik Bekkat-Berkani, M.D, GSK group of companies (Employee, Shareholder)
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Bos, Martine P., David Kao, Daniel M. Hogan, Christopher C. R. Grant, and Robert J. Belland. "Carcinoembryonic Antigen Family Receptor Recognition by Gonococcal Opa Proteins Requires Distinct Combinations of Hypervariable Opa Protein Domains." Infection and Immunity 70, no. 4 (April 2002): 1715–23. http://dx.doi.org/10.1128/iai.70.4.1715-1723.2002.
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ABSTRACT Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.
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Di Fede, Martina, Massimiliano Biagini, Elena Cartocci, Carlo Parillo, Alessandra Greco, Manuele Martinelli, Sara Marchi, Alfredo Pezzicoli, Isabel Delany, and Silvia Rossi Paccani. "Neisseria Heparin Binding Antigen is targeted by the human alternative pathway C3-convertase." PLOS ONE 13, no. 3 (March 26, 2018): e0194662. http://dx.doi.org/10.1371/journal.pone.0194662.
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Chen, Tie, Fritz Grunert, Andrew Medina-Marino, and Emil C. Gotschlich. "Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins." Journal of Experimental Medicine 185, no. 9 (May 5, 1997): 1557–64. http://dx.doi.org/10.1084/jem.185.9.1557.
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Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLaCEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.
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Sarantis, Helen, and Scott D. Gray-Owen. "Defining the Roles of Human Carcinoembryonic Antigen-Related Cellular Adhesion Molecules during Neutrophil Responses to Neisseria gonorrhoeae." Infection and Immunity 80, no. 1 (November 7, 2011): 345–58. http://dx.doi.org/10.1128/iai.05702-11.
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ABSTRACTSymptomatic infection of humans withNeisseria gonorrhoeaeis characterized by a neutrophil-rich cervical or urethral exudate, suggesting that neutrophils are important both for the clearance of these bacteria and for the pathogenesis of gonorrhea.Neisseriainteracts with neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by human neutrophils, and yet their coexpression has precluded understanding of the relative contribution of each CEACAM to functional responses of neutrophils during neisserial infection. In this work, we directly address the role of each CEACAM during infection by introducing individual human CEACAMs into a differentiated murine MPRO cell line-derived neutrophil model. Murine neutrophils cannot bind the human-restrictedNeisseria; however, we show that introducing any of the Opa-binding CEACAMs of human neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry ofNeisseriainto murine neutrophils. While CEACAM1- and CEACAM6-expressing neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly proceeds without appreciable neutrophil activation. In stark contrast, neisserial engulfment via CEACAM3 recapitulates the oxidative burst and intracellular granule release seen during human neutrophil infection. Finally, by coexpressing multiple CEACAMs in our model, we show that the expression of CEACAM1 and CEACAM6 potentiate, rather than hinder, CEACAM3-dependent responses of neutrophils, exposing a cooperative role for this family of proteins during neisserial infection of neutrophils. These observations illustrate a divergence in function of CEACAMs in neutrophils and implicate the human-restricted CEACAM3 in the neutrophil innate response to neisserial infection.
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Sakuma, T., S. Higashiyama, S. Hosoe, S. Hayashi, and N. Taniguchi. "CD9 Antigen Interacts with Heparin-Binding EGF-Like Growth Factor through Its Heparin-Binding Domain." Journal of Biochemistry 122, no. 2 (August 1, 1997): 474–80. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a021776.
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NAITO, Mariko, Tomohiko FUKUDA, Kiyotoshi SEKIGUCHI, and Takeshi YAMADA. "The domains of human fibronectin mediating the binding of α antigen, the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection." Biochemical Journal 347, no. 3 (April 25, 2000): 725–31. http://dx.doi.org/10.1042/bj3470725.
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We have recently shown that α antigen (α-Ag), the immunodominant antigen of mycobacteria, has a novel fibronectin (FN)-binding motif that is unique among mycobacteria [Naito, Ohara, Matsumoto and Yamada (1998) J. Biol. Chem. 273, 2905-2909]. In this study, we examined the domains of human FN that interacted with α-Ag. Fragments of FN generated by either proteolysis or recombinant DNA techniques were compared for their ability to bind to α-Ag. Fragments containing either the C-terminal heparin-binding domain or the central cell-binding domain consistently bound to α-Ag. The fragment of the C-terminal heparin-binding domain, upon mutation that resulted in the loss of its heparin-binding activity, could not bind with α-Ag. These findings suggested that the mutated site, i.e. the main heparin-binding site of FN, was also the principal site for binding to α-Ag. The α-Ag-binding domains of FN could bind whole mycobacterial bacilli, suggesting that these two domains are important contributors to mycobacterial infection.
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Lujan, Eduardo, Rolando Pajon, and Dan M. Granoff. "Impaired Immunogenicity of Meningococcal Neisserial Surface Protein A in Human Complement Factor H Transgenic Mice." Infection and Immunity 84, no. 2 (November 23, 2015): 452–58. http://dx.doi.org/10.1128/iai.01267-15.
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Neisserial surface protein A (NspA) is a highly conserved outer membrane protein previously investigated as a meningococcal vaccine candidate. Despite eliciting serum bactericidal activity in mice, a recombinant NspA vaccine failed to elicit serum bactericidal antibodies in a phase 1 clinical trial in humans. The discordant results may be explained by the recent discovery that NspA is a human-specific ligand of the complement inhibitor factor H (FH). Therefore, in humans but not mice, NspA would be expected to form a complex with FH, which could impair human anti-NspA protective antibody responses. To investigate this question, we immunized human FH transgenic BALB/c mice with three doses of recombinant NspA expressed inEscherichia colimicrovesicles, with each dose being separated by 3 weeks. Three of 12 (25%) transgenic mice and 13 of 14 wild-type mice responded with bactericidal titers of ≥1:10 in postimmunization sera (P= 0.0008, Fisher's exact test). In contrast, human FH transgenic and wild-type mice immunized with a control meningococcal native outer membrane vesicle vaccine had similar serum bactericidal antibody responses directed at PorA, which is not known to bind human FH, and a mutant factor H binding protein (FHbp) antigen with a >50-fold lower level of FH binding than wild-type FHbp antigen binding.Thus, human FH can impair anti-NspA serum bactericidal antibody responses, which may explain the poor immunogenicity of the NspA vaccine previously tested in humans. A mutant NspA vaccine engineered to have decreased binding to human FH may increase protective antibody responses in humans.
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Muenzner, Petra, Christoph Dehio, Taku Fujiwara, Mark Achtman, Thomas F. Meyer, and Scott D. Gray-Owen. "Carcinoembryonic Antigen Family Receptor Specificity of Neisseria meningitidis Opa Variants Influences Adherence to and Invasion of Proinflammatory Cytokine-Activated Endothelial Cells." Infection and Immunity 68, no. 6 (June 1, 2000): 3601–7. http://dx.doi.org/10.1128/iai.68.6.3601-3607.2000.
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ABSTRACT The carcinoembryonic antigen (CEA) family member CEACAM1 (previously called biliary glycoprotein or CD66a) was previously shown to function as a receptor that can mediate the binding of Opa protein-expressing Neisseria meningitidis to both neutrophils and epithelial cells. Since neutrophils and polarized epithelia have both been shown to coexpress multiple CEACAM receptors, we have now extended this work to characterize the binding specificity of meningococcal Opa proteins with other CEA family members. To do so, we used recombinant Escherichia coli expressing nine different Opa variants from three meningococcal strains and stably transfected cell lines expressing single members of the CEACAM family. These infection studies demonstrated that seven of the nine Opa variants bound to at least one CEACAM receptor and that binding to each of these receptors is sufficient to trigger the Opa-dependent bacterial uptake by these cell lines. The other two Opa variants do not appear to bind to either CEACAM receptors or heparan sulfate proteoglycan receptors, which are bound by some gonococcal Opa variants, thus implying a novel class of Opa proteins. We have also extended previous studies by demonstrating induction of CEACAM1 expression after stimulation of human umbilical vein endothelial cells with the proinflammatory cytokine tumor necrosis factor alpha, which is present in high concentrations during meningococcal disease. This induced expression of CEACAM1 leads to an increased Opa-dependent bacterial binding and invasion into the primary endothelia, implying that these interactions may play an important role in the pathogenesis of invasive meningococcal disease.
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Sekiguchi, Asuka, Miwako Narita, Toshio Yano, Naoko Sato, Anri Saito, Norihiro Watanabe, Ayumi Yokoyama, et al. "Enhancing Effects of Heparin/Low Molecular Weight Heparin/Heparan Sulfate on Antigen Presentation and Antigen-Specific Cytotoxic T Lymphocyte (CTL) Induction by Monocyte-Derived Dendritic Cells." Blood 104, no. 11 (November 16, 2004): 3816. http://dx.doi.org/10.1182/blood.v104.11.3816.3816.
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Abstract Heparin is bound with heparin-binding sites on certain cells, which induces proliferation and differentiation signals. In addition, heparin is bound with heparin-binding domains of various cytokines, which enhances the interaction between cytokines and target cells. Monocytes have been demonstrated to posses heparin-binding sites on cell surfaces. In the present study, we investigated the effects of heparin (including low molecular weight heparin) and heparan sulfate on antigen presentation and antigen-specific CTL induction of monocyte-derived DCs. Peripheral blood CD14+ cells were cultured to generate immature and mature DCs with various concentrations of heparin, low molecular weight heparin or heparan sulfate. Cultured cells were analyzed for DC-associated surface phenotypes by flow cytometry and evaluated for allogeneic antigen presenting ability by mixed leukocyte culture. In order to evaluate the effects of heparin on monocyte-derived DCs to generate antigen-specific CTL, DCs were generated from HLA-A2402 donors by serum-free culture with heparin, transfected with in vitro transcribed WT-1 mRNA on day 6 and cultured with the addition TNF-α/IL-1α/IL-6/IFN-γ/PGE2 for further 1 day. WT-1 mRNA-transfected DCs were used for priming autologous lymphocytes in co-culture at the stimulator:responder ratio of 1:10. Lymphocytes were primed with the same DCs 2-3 times in the interval of 5-7 days. CD8+ T cells were separated and used as effector cells in 51Cr-release assay. WT-1 expressing and HLA-A24+ cell line MegO1 was used as target cells. In order to evaluate the association of MHC molecules in the cytotoxicity, 51Cr-lebelled target cells were treated with anti-MHC class I or class II monoclonal antibody before cytotoxicity assay. In order to evaluate the antigen specificity of the generated CTL, unlabelled target cells were added to the cytotoxicity assay. By the addition of heparin, the expression of CD1a and CD80 on both immature and mature DCs was markedly enhanced and the allogeneic antigen presenting ability was elevated in both immature and mature DCs. By the addition of low molecular weight heparin, the expression of CD1a was enhanced and antigen presenting ability was elevated also. By the addition of heparan sulfate, similar results of elevated antigen presentation were obtained. By the priming of lymphocytes with WT-1 mRNA transfected DCs generated from monocytes by the serum-free culture with heparin, cytotoxic capability against WT-1 expressing target cells was demonstrated in the primed lymphocytes. The cytotoxic capability of the lymphocytes was blocked by the treatment of the target cells with anti-MHC class I monoclonal antibody and the addition of unlabelled target cells in the cytotoxicity reaction. The present study demonstrated that heparin/low molecular weight heparin/heparan sulfate could enhance the antigen presentation and antigen-specific CTL induction of monocyte-derived DCs. These findings suggest the usefulness of heparin for generating efficient DCs for DC-based immunotherapy and the involvement of heparan sulfate in immunological defense mechanism.
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Khandelwal, Sanjay, Johnson M. Alexandra, C. Garren Hester, Michael M. Frank, and Gowthami M. Arepally. "Mechanism of Complement Activation By PF4/ Heparin Complexes." Blood 128, no. 22 (December 2, 2016): 137. http://dx.doi.org/10.1182/blood.v128.22.137.137.
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Abstract The mechanisms underlying the PF4/heparin immune response are poorly understood. In recent studies, we showed that PF4/heparin complexes, but not PF4 alone or heparin alone, activate complement (C') in a heparin-dependent manner and lead to selective binding of C'-coated antigen (PF4/heparin complexes) to B cells via CD21. In additional studies, we showed that heparinized patients have circulating B cells with C'-coated PF4/heparin complexes (Khandelwal, Blood 2016). To investigate the mechanism by which PF4/heparin complexes activate complement, we performed studies in whole blood using previously described methods assessing binding of C'-fixed PF4/heparin complexes to B cells in whole blood. We incubated blood with inhibitors or conditions known to selectively block specific complement activation pathways, followed by incubation with PF4 and heparin for 1 hour (hr) at 370 C. Binding of PF4/heparin complexes and C' fragments to B-cells was determined by flow cytometry using the murine monoclonal antibody to PF4/heparin complexes (KKO) and antibodies to specific C' activation products (Khandelwal, Blood 2016). To distinguish activation by classical and lectin pathways from alternative pathway of activation, we incubated blood with C1 esterase inhibitor, a protein that inhibits C' activation by the classical and lectin pathways. As shown in Figure 1, whole blood incubated with PF4/heparin is associated with C' activation as measured by C3c binding to B cells (Figure 1, column 3), but not if blood is incubated with buffer or PF4 alone (Figure 1, columns 1 & 2). With the addition of C1 esterase inhibitor (10 or 20 U/ml) prior to incubation with PF4/heparin complexes, we noted a dose-dependent reduction in C' activation (>85% reduction with 10 and 20 U/ml) suggesting that C' activation by PF4/heparin complexes occurs via the classical or lectin pathways. To further confirm that the alternate pathway is not involved in PF4/heparin mediated C' activation, we used sensitivity of the alternative pathway to Mg2+ using differential chelation with EDTA and EGTA. EDTA, a chelator of both Ca2+ and Mg2+, inhibits complement activation by all three pathways, whereas EGTA, which preferentially sequesters Ca2+ over Mg2+, permits alternative pathway activation. As shown in Figure 2, incubation of PF4/heparin complexes without chelators allowed for maximal antigen binding to B cells (~100%), while incubation with EDTA (Figure 2, column 3) abolished antigen binding, as did EGTA with and without additional Mg2+ supplementation. We next investigated the role of the classical pathway, a pathway triggered by immune complex mediated-binding and activation of C1. By flow cytometry, we were unable to show C1q deposition although we were able to demonstrate C3c/C4c deposition on antigen-coated B cells from healthy donors (data not shown). We also showed that C' activation by PF4/heparin complexes was preserved in human serum low in immunoglobulins (IgG and IgM) as well as plasma from mMT mice lacking circulating immunoglobulins (data not shown). To investigate the role of the lectin pathway, we performed competitive inhibition assays by incubating whole blood with PF4/heparin complexes in the presence of mannan. As shown in Figure 3, we show that mannan (500 mg/mL) inhibited binding of antigen to B cells. In other studies, we show that an anti-MBL antibody partially reduced binding of KKO/C' to B cells. Together, these preliminary studies demonstrate a lack of involvement of the classical and alternative pathways in C' activation and indicate a contribution by the lectin pathway in C' activation by PF4/heparin complexes. Further studies are underway to elucidate the role of the lectin pathway in complement activation by PF4/heparin complexes. Insights from these studies will lead to novel interventions that can block the initial steps of immune activation by heparin. Disclosures Arepally: Biokit: Patents & Royalties.
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Bos, Martine P., Motomu Kuroki, Anna Krop-Watorek, Daniel Hogan, and Robert J. Belland. "CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains." Proceedings of the National Academy of Sciences 95, no. 16 (August 4, 1998): 9584–89. http://dx.doi.org/10.1073/pnas.95.16.9584.
Full textAbstract:
Neisseria gonorrhoeaestrain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed inEscherichia colireflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants, suggesting that the differential recognition of CD66 receptors by Opa variants is dictated by the presence of specific binding regions on the N-domain of the receptor.
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Jalkanen, S., and M. Jalkanen. "Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin." Journal of Cell Biology 116, no. 3 (February 1, 1992): 817–25. http://dx.doi.org/10.1083/jcb.116.3.817.
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The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.
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Menozzi, F. D., J. H. Rouse, M. Alavi, M. Laude-Sharp, J. Muller, R. Bischoff, M. J. Brennan, and C. Locht. "Identification of a heparin-binding hemagglutinin present in mycobacteria." Journal of Experimental Medicine 184, no. 3 (September 1, 1996): 993–1001. http://dx.doi.org/10.1084/jem.184.3.993.
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Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.
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Almus, FE, LV Rao, UR Pendurthi, L. Quattrochi, and SI Rapaport. "Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin." Blood 77, no. 6 (March 15, 1991): 1256–62. http://dx.doi.org/10.1182/blood.v77.6.1256.1256.
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Abstract We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF- 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.
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Almus, FE, LV Rao, UR Pendurthi, L. Quattrochi, and SI Rapaport. "Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin." Blood 77, no. 6 (March 15, 1991): 1256–62. http://dx.doi.org/10.1182/blood.v77.6.1256.bloodjournal7761256.
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We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF- 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.
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Kavan, Daniel, Markéta Vančurová, Dana Ulbrichová, Ivana Hladíková, Miloslav Pospíšil, and Karel Bezouška. "Identification of Heparin-Binding Sites in the Fibronectin Type III Domains of the Leukocyte Common Antigen (CD45)." Collection of Czechoslovak Chemical Communications 69, no. 3 (2004): 645–58. http://dx.doi.org/10.1135/cccc20040645.
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Leukocyte common antigens (CD45) are large receptors that are abundantly expressed at the surface of all leukocytes. These receptors are type I membrane glycoproteins possessing two large C-terminal intracellular domains with protein tyrosine phosphatase activity. While the role of these enzyme domains in leukocyte signaling is well documented, the role of the N-terminal extracellular portion of CD45, composed of sequences formed by alternatively spliced exons, the cysteine rich domain, and three type III fibronectin repeats, remains unclear. The presence of fibronectin domains would predict the occurrence of heparin-binding sites, which may account for the documented affinity of CD45 for acid polysaccharides. We addressed this hypothesis using soluble recombinant proteins corresponding to the individual fibronectin domains (FN1 to FN3), and to the entire extracellular portion of CD45 (sCD45). Binding of these proteins to heparin was examined by frontal affinity chromatography. We found that while the sCD45 bound to heparin with Kd of 3.2 × 10-8 mol/l, the binding of FN2 and FN3 was somewhat weaker (Kd was 1.4 and 7.4 × 10-7 mol/l, respectively). The FN1 domain did not interact with heparin. Our results bring definitive evidence for the existence of binding sites for acid polysaccharides in the extracellular domain of CD45. These binding sites may be important for surface interactions of CD45 and for leukocyte signaling.
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Brissette, Catherine A., Tomasz Bykowski, Anne E. Cooley, Amy Bowman, and Brian Stevenson. "Borrelia burgdorferi RevA Antigen Binds Host Fibronectin." Infection and Immunity 77, no. 7 (April 27, 2009): 2802–12. http://dx.doi.org/10.1128/iai.00227-09.
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ABSTRACT Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.
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Beernink, Peter T., Arunas Leipus, and Dan M. Granoff. "Rapid Genetic Grouping of Factor H-Binding Protein (Genome-Derived Neisserial Antigen 1870), a Promising Group B Meningococcal Vaccine Candidate." Clinical and Vaccine Immunology 13, no. 7 (July 2006): 758–63. http://dx.doi.org/10.1128/cvi.00097-06.
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ABSTRACT The most important antigen component of a promising multicomponent group B meningococcal recombinant protein vaccine is based on genome-derived neisserial antigen 1870, which recently was renamed factor H-binding protein (FHBP) to reflect one of its critical functions as a complement regulatory protein. Neisseria meningitidis strains can be subdivided into three FHBP variant groups based on divergence of FHBP amino acid sequences. Within each variant group, amino acid sequences are >90% conserved. To develop an FHBP-based group B vaccine, it is important to know the distribution of FHBP variant 1, 2, and 3 strains in different geographic regions, since antibodies against FHBP are bactericidal against strains within the homologous group but show minimal activity against strains from other groups. We have devised a high-throughput, quantitative PCR-based method that allows rapid and precise assignment of FHBP genes into each of the three major variant lineages. Among 48 group B isolates from patients hospitalized in California in 2003 to 2004, 83%, 13%, and 4%, respectively, had variant 1, 2, and 3 genes. Thus, a vaccine based on the variant 1 protein has the potential to prevent the majority of cases of group B disease. The quantitative PCR-based method will be useful for determining and monitoring the prevalence of meningococcal isolates with genes encoding different FHBP variant proteins. The technique also is suitable for monitoring variation of genes encoding other protein antigens targeted for vaccination.
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Tripodi, A., A. Krachmalnicoff, and P. M. Mannucci. "Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding." Thrombosis and Haemostasis 56, no. 03 (1986): 349–52. http://dx.doi.org/10.1055/s-0038-1661681.
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SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.
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Holen, Halvor L., Lillian Zernichow, Kristine E. Fjelland, Ida M. Evenroed, Kristian Prydz, Heidi Tveit, and Hans-Christian Aasheim. "Ephrin-B3 binds to a sulfated cell-surface receptor." Biochemical Journal 433, no. 1 (December 15, 2010): 215–23. http://dx.doi.org/10.1042/bj20100865.
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The ephrins are a family of proteins known to bind the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinase family. In the present paper, we provide data showing that ephrin-B3 binds a sulfated cell-surface protein on HEK-293T (human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40) and HeLa cells, a binding that is nearly completely blocked by treatment of these cell lines with chlorate or heparinase, or by addition of the heavily sulfated glycosaminoglycan heparin. This indicates that heparan sulfate on these cells is essential for cell-surface binding of ephrin-B3. Heparin did not affect ephrin-B3 binding to EphB receptors expressed on transfected HEK-293T cells, indicating further that ephrin-B3 binds an alternative receptor which is a heparan sulfate proteoglycan. Site-directed mutagenesis analysis revealed that Arg178 and Lys179 are important for heparin binding of ephrin-B3 and also for ephrin-B3 binding to cells. These amino acids, when introduced in the non-heparin-binding ephrin-B1, conferred the heparin-binding property. Functional studies reveal that ephrin-B3 binding to cells induces cellular signalling and influences cell rounding and cell spreading. In conclusion, our data provide evidence for an unknown ephrin-B3-binding cell-surface proteoglycan involved in cellular signalling.
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Sachais, Bruce S., Rustem I. Litvinov, Serge V. Yarovoi, Lubica Rauova, Jillian L. Hinds, Ann H. Rux, Gowthami M. Arepally, et al. "Dynamic antibody-binding properties in the pathogenesis of HIT." Blood 120, no. 5 (August 2, 2012): 1137–42. http://dx.doi.org/10.1182/blood-2012-01-407262.
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Abstract Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4K50E. Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4K50E dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4K50E. This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.
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Krauel, Krystin, Claudia Weber, Sven Brandt, Ulrich Zähringer, Uwe Mamat, Andreas Greinacher, and Sven Hammerschmidt. "Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes." Blood 120, no. 16 (October 18, 2012): 3345–52. http://dx.doi.org/10.1182/blood-2012-06-434985.
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AbstractThe positively charged chemokine platelet factor 4 (PF4) forms immunogenic complexes with heparin and other polyanions. Resulting antibodies can induce the adverse drug effect heparin-induced thrombocytopenia. PF4 also binds to bacteria, thereby exposing the same neoantigen(s) as with heparin. In this study, we identified the negatively charged lipopolysaccharide (LPS) as the PF4 binding structure on Gram-negative bacteria. We demonstrate by flow cytometry that mutant bacteria with progressively truncated LPS structures show increasingly enhanced PF4 binding activity. PF4 bound strongest to mutants lacking the O-antigen and core structure of LPS, but still exposing lipid A on their surfaces. Strikingly, PF4 bound more efficiently to bisphosphorylated lipid A than to monophosphorylated lipid A, suggesting that phosphate residues of lipid A mediate PF4 binding. Interactions of PF4 with Gram-negative bacteria, where only the lipid A part of LPS is exposed, induce epitopes on PF4 resembling those on PF4/heparin complexes as shown by binding of human anti-PF4/heparin antibodies. As both the lipid A on the surface of Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host defense mechanism.
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Fabris, Fabrizio, Immacolata Cordiano, Federica Salvan, Leopoldo Saggin, Giuseppe Cella, Guido Luzzatto, and Antonio Girolami. "Heparin-Induced Thrombocytopenia: Prevalence in a Large Cohort of Patients and Confirmed Role of PF4/Heparin Complex as the Main Antigen for Antibodies." Clinical and Applied Thrombosis/Hemostasis 3, no. 3 (July 1997): 203–9. http://dx.doi.org/10.1177/107602969700300309.
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We performed a retrospective study on the prevalence of heparin-induced thrombocytopenia (HIT) in 233 patients receiving hog mucosa heparin therapy. Of these, 82 patients received s.c. calcium heparin, 130 patient received unfractionated (UF) i.v. heparin, and 21 patients received low molecular weight heparins (LMWH). An additional four patients, referred to our consultation and diagnosed by us as having clinically active type II HIT (HIT-II) were also studied. The mean platelet count of the 233 patients receiving heparin showed a significant decrease after 2 days of heparin treatment and a following significant increase 6 days later (basal: 257 ± 147 x 109 platelets/L; day 2: 239 ± 122, p < 0.0002; day 6: 286 ± 119, p < 0.004). Of the 212 patients receiving UF heparin, 13 (6%) fulfilled the criteria for HIT-II: seven of these had received i.v. heparin (mean daily dose 26,600 ± 4,082 IU ± SD) and six had received s.c. heparin (mean daily dose 21,428:t 6,900 IU). Their mean basal platelet count was 226 ± 100 SD × 109 platelets/L and the nadir during heparin treatment was 78 ± 39 x 10 9 platelets/L. Thrombotic complications occurred in four (30.7%) of the 13 patients with HIT-II. Since the immunological mechanism has been demonstrated for HIT-II and since platelet factor 4 (PF4) was identified as the co-factor for the binding of heparin-related antibodies, we set up our own enzyme-linked immunosorbent assay (ELISA) for testing antibodies against PF4/heparin complex bound through electrostatic bridges to the solid phase. The highest binding capacity of HIT-related IgG to the multimolecular complex was obtained at 20 μg/ml for PF4 and 3 μg/ml for heparin, corresponding to 250 ng of PF4 and 42 ng of heparin in each microtiter well. Such binding was inhibited in a dose-dependent manner by increasing amounts of heparin, protamine hydrochloride, and a monoclonal antibody anti-human PF4 clone 1OB2. We observed that HIT-related antibodies bound also to PF4/LMWH complexes but the optimal PF4/glycosaminoglycan ratio appeared more critical for LMWH (enoxaparin, fraxiparin, and pamaparin) than for UF heparin. Sera from eight patients with HIT-II were tested by PF4/heparin ELISA; six of these had IgG against the complex PF4/heparin and three also had IgM. The persistence of HIT-related antibodies was investigated in three patients: in one such antibodies were still detectable 3 years after the acute episode, while in the other two, they disappeared after 6 months and 1 year, respectively. Key Words: Heparin-related anti body—Platelet factor 4 (PF4)—Heparin—Low molecular weight heparin—Thrombocytopenia—Thrombosis.
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Newman, Peter, Rebecca Swanson, and Beng Chong. "Heparin-induced Thrombocytopenia: IgG Binding to PF4-Heparin Complexes in the Fluid Phase and Cross-reactivity with Low Molecular Weight Heparin and Heparinoid." Thrombosis and Haemostasis 80, no. 08 (1998): 292–97. http://dx.doi.org/10.1055/s-0037-1615190.
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SummaryEarly diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.
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Pu, Feifei, Jing Feng, Fei Niu, and Ping Xia. "Diagnostic value of recombinant heparin-binding hemagglutinin adhesin protein in spinal tuberculosis." Open Medicine 15, no. 1 (February 28, 2020): 114–18. http://dx.doi.org/10.1515/med-2020-0017.
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AbstractBackground and aimTo explore the diagnostic value of recombinant heparin-binding hemagglutinin adhesin (HBHA) protein antigen in spinal tuberculosis.Materials and methodsForty patients with spinal tuberculosis were included in the experimental group and 40 healthy people were included in the control group. Serum IgG antibody expression level was detected with recombinant HBHA protein as the antigen, using enzyme-linked immunosorbent assay (ELISA) detection.ResultsPatients with spinal tuberculosis and healthy volunteers were included in this study. A total of 40 eligible patients with spinal tuberculosis were included (24 males and 16 females, aged 18-72 years, with an average age of 41.24 ± 15.74 years). Forty healthy people were included (21 males and 19 females, aged 18-70 years, with an average age of 41.33 ± 12.36 years). On comparing the groups, no significant difference was found in the general data (P >0.05). IgG antibody level in the experimental group was higher than that in the control group, and the difference was significant (P < 0.00001).ConclusionsDetection of serum HBHA protein antibody is of great value in the auxiliary diagnosis of spinal tuberculosis, and high HBHA expression can be used as an indicator for diagnosis of spinal tuberculosis.
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Krauel, Krystin, Christian Pötschke, Claudia Weber, Wolfram Kessler, Birgitt Fürll, Till Ittermann, Stefan Maier, Sven Hammerschmidt, Barbara M. Bröker, and Andreas Greinacher. "Platelet factor 4 binds to bacteria, inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia." Blood 117, no. 4 (January 27, 2011): 1370–78. http://dx.doi.org/10.1182/blood-2010-08-301424.
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AbstractA clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n = 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism.
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Borghi, Sara, Ana Antunes, Andreas F. Haag, Marco Spinsanti, Tarcisio Brignoli, Enea Ndoni, Vincenzo Scarlato, and Isabel Delany. "Multilayer Regulation of Neisseria meningitidis NHBA at Physiologically Relevant Temperatures." Microorganisms 10, no. 4 (April 18, 2022): 834. http://dx.doi.org/10.3390/microorganisms10040834.
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Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30–32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.
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Lee, Hannah S. W., Ian C. Boulton, Karen Reddin, Henry Wong, Denise Halliwell, Ofer Mandelboim, Andrew R. Gorringe, and Scott D. Gray-Owen. "Neisserial Outer Membrane Vesicles Bind the Coinhibitory Receptor Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 and Suppress CD4+ T Lymphocyte Function." Infection and Immunity 75, no. 9 (July 9, 2007): 4449–55. http://dx.doi.org/10.1128/iai.00222-07.
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ABSTRACT Pathogenic Neisseria bacteria naturally liberate outer membrane “blebs,” which are presumed to contribute to pathology, and the detergent-extracted outer membrane vesicles (OMVs) from Neisseria meningitidis are currently employed as meningococcal vaccines in humans. While the composition of these vesicles reflects the bacteria from which they are derived, the functions of many of their constituent proteins remain unexplored. The neisserial colony opacity-associated Opa proteins function as adhesins, the majority of which mediate bacterial attachment to human carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). Herein, we demonstrate that the Opa proteins within OMV preparations retain the capacity to bind the immunoreceptor tyrosine-based inhibitory motif-containing coinhibitory receptor CEACAM1. When CD4+ T lymphocytes were exposed to OMVs from Opa-expressing bacteria, their activation and proliferation in response to a variety of stimuli were effectively halted. This potent immunosuppressive effect suggests that localized infection will generate a “zone of inhibition” resulting from the diffusion of membrane blebs into the surrounding tissues. Moreover, it demonstrates that OMV-based vaccines must be developed from strains that lack CEACAM1-binding Opa variants.
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Vilstrup, Joachim, Amanda Simonsen, Thea Birkefeldt, Dorthe Strandbygård, Jeppe Lyngsø, Jan Skov Pedersen, and Søren Thirup. "Crystal and solution structures of fragments of the human leucocyte common antigen-related protein." Acta Crystallographica Section D Structural Biology 76, no. 5 (April 15, 2020): 406–17. http://dx.doi.org/10.1107/s2059798320003885.
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Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1–4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1–4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1–3) and the four C-terminal FNIII domains (LARFN5–8) both bound heparin, while LARFN1–4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1–3 and LARFN5–8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.
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Radtke, Klaus-P., Judith S. Greengard, José A. Fernández, Bruno O. Villoutreix, and John H. Griffin. "A Two-Allele Polymorphism in Protein C Inhibitor with Varying Frequencies in Different Ethnic Populations." Thrombosis and Haemostasis 75, no. 01 (1996): 062–69. http://dx.doi.org/10.1055/s-0038-1650222.
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SummarycDNAs for protein C inhibitor (PCI), prepared from human liver RNA, contained two forms of PCI, designated PCI*A and PCPB 1 . While PCI*A is identical to the published PCI sequence, PCPB differs in 4 of 1221 bp and two amino acids, A36V and K86E. Frequencies for the PCI*B allele, determined from genomic DNA, differed among ethnic groups. Frequency distribution and historical migration of modem man suggest that PCI*A arose from the PCI*B allele. Antigen levels in plasma homozygous for PCI*A or PCI*B equalled that of pooled normal plasma. K86E in PCI*B causes a charge alteration in helix D which is likely involved in heparin binding in antithrombin III but not likely involved in glycosaminoglycan binding in PCI. Kinetic studies showed that plasmas homozygous for PCI*A and PCPB are similar in their APC inhibiting properties and in their heparin sensitivity, consistent with the idea that helix D in PCI is not involved in heparin binding
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Li, Zhong Q., Weiyi Liu, Kwang S. Park, Brue S. Sachais, Gowthani M. Arepally, Douglas B. Cines, and Mortimer Poncz. "Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody." Blood 99, no. 4 (February 15, 2002): 1230–36. http://dx.doi.org/10.1182/blood.v99.4.1230.
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Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.
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Cadelis, François, Pierre Julien, Paul J. Lupien, Jean-Paul Valet, Yves Deshaies, and M. R. Ven Murthy. "A monoclonal antibody against human lipoprotein lipase inhibiting heparin binding without affecting catalytic activity." Biochemistry and Cell Biology 74, no. 3 (May 1, 1996): 383–89. http://dx.doi.org/10.1139/o96-041.
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A fragment of the human lipoprotein lipase (LPL) cDNA (405 bp, 5′ terminal end) was cloned in an expression vector to produce a ~17 kDa fusion peptide and was used as antigen to produce a high titre anti-LPL monoclonal antibody (10C3 MAb). This antibody reacts with both native and denatured forms of LPL from different tissue and animal sources. Competition studies with heparin indicate that 10C3 MAb is specific for an epitope at a heparin binding site. The antibody does not inhibit LPL enzyme activity, indicating that the antigenic epitope is not situated within or in the proximity of the LPL catalytic region. With these characteristics, 10C3 MAb should prove to be a useful immunochemical tool in clinical as well as in fundamental investigations on the metabolism of triglyceride-rich lipoproteins and in studies on the functional anatomy of LPL.Key words: lipoprotein lipase, monoclonal antibody, LPL cDNA, heparin, immunoassay, ELISA.
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Greinacher, Andreas. "Treatment of Heparin-Induced Thrombocytopenia." Thrombosis and Haemostasis 82, no. 08 (1999): 457–67. http://dx.doi.org/10.1055/s-0037-1615866.
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IntroductionUnfractionated heparin (UFH) and low molecular weight heparin (LMWH) are the most widely used anticoagulants when parenteral anticoagulation with a short half-life is required. Both can be administered subcutaneously and intravenously, and both have been shown to be effective in a variety of clinical settings.1 UFH has several limitations. One is its poor bioavailability after subcutaneous injection and the marked variability in its anticoagulant response in patients with an acute thromboembolic complication.2,3 Another major issue associated with UFH is the induction of heparin-induced thrombocytopenia (HIT). Both limitations are closely linked,4 as the underlying cause is the high density of negative charges on the heparin molecule and its molecular weight. Both are responsible for the binding of heparin to plasma proteins other than antithrombin (AT), such as platelet factor 4 (PF4) and to several cell types. This leads to heparin-platelet interaction, the formation of HIT antigen (i.e., PF4/heparin complexes), and inhibition of the anticoagulant effect of heparin (aPTT-nonresponder).
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Westmark, Pamela, and John P. Sheehan. "Antithrombin- and Heparin-Binding Exosites Regulate Extravascular Binding and Clearance of Factor IXa." Blood 134, Supplement_1 (November 13, 2019): 2392. http://dx.doi.org/10.1182/blood-2019-131580.
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Introduction: Administration of increasing doses of FIX to hemophilia B (HB) mice reveals that the extravascular compartment is capable of binding several-fold more FIX than circulates in the plasma (Cooley et al., Blood 2019). FIX binds rapidly and reversibly to vascular endothelium and the extravascular matrix, in part mediated by interaction of the Gla domain with collagen IV (Col IV). Anticoagulant heparan sulphate and Col IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into Col IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT) binding exosites on the protease domain to the regulation of FIXa activity. We now compare the contribution of these protease exosites to in vivo recovery and clearance of the FIX(a) protease and zymogen in hemophilia A (HA) or HB mice. Methods: Recombinant human FIX variants expressed in HEK293 cells with substitutions in the AT- (R150A) and heparin-binding (K126A/K132A) exosites (chymotrypsinogen #) were purified to homogeneity and activated to FIXa with FXIa. FIX(a) pharmacokinetics (PK) in HA or HB mice were determined with injection of a weight-based dose into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX(a) content determined with a human FIX specific ELISA. FIX(a) concentrations were plotted versus time and fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1and k2are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters. Results: PK for clearance of predicted 30% plasma levels of human FIXa (5 min to 3 hr) or FIX (5 min to 25 hr) were examined in HA (Fig 1) or HB mice (Fig 2), respectively. HA mice were employed for FIXa to avoid in vivoclot formation triggered by active protease. Notably, FIXa plasma clearance was relatively prolonged with ~50% of recovered antigen levels present at 25-30 min post-injection. Similar to FIX, the pattern of FIXa plasma clearance suggested a 2-compartment model with initial and terminal phases of elimination. FIXa WT demonstrated approximately ~23% recovery of predicted levels at 5 min in HA mice, similar to the zymogen (21%) in HB mice (Table I). FIXa R150A (reduced AT affinity) demonstrated markedly enhance recovery (43%) in HA mice relative to WT, while FIX R150A (24%) in HB mice which was similar to WT. FIXa K126A/132A (reduced heparin affinity) demonstrated markedly enhanced recovery (55%) in HA mice, slightly enhanced relative to the similarly increased zymogen recovery (48%) in HB mice. FIXa WT-GGACK (active site inhibited) demonstrated similar recovery to the unmodified FIXa WT with a significantly faster rate of clearance (2.2 fold) in the initial phase (k1) and a significantly slower rate of clearance (3.6-fold) in the terminal phase (k2). Overall, the initial rate (k1) was not significantly different among the active FIXa variants in HA mice or among zymogen variants in HB mice. However, comparison of individual proteases variants to their respective zymogens demonstrated that the initial rate (k1) of plasma clearance was 2-3 fold faster for the proteases, while the terminal rate (k2) was 5-9 fold faster (FIXa R150 had the smallest relative increase). Conclusions: The 2-phase elimination pattern for FIXa suggests that significant amounts of protease may exist in a non-circulating extravascular pool, similar to the zymogen. Further, clearance of FIXa antigen from mouse plasma is prolonged, consistent with the half-life of FIXa activity in human plasma (~41 min). FIXa variants with reduced affinity for AT (R150A) or heparin (K126A/K132A) showed enhanced recovery compared to FIXa WT, suggesting that these protease exosites make independent contributions to the extravascular binding of FIXa. The heparin exosite makes a similar contribution to zymogen binding to extravascular sites, but the AT exosite does not, likely due to being unavailable in the zymogen. Active site inhibited FIXa (GGACK) demonstrates an accelerated initial elimination phase relative to FIXa WT, suggesting re-distribution of the protein is conformation-dependent. The subsequent terminal elimination phase was slowed, consistent with an inability to be inhibited and cleared by AT Disclosures Sheehan: Pfizer: Research Funding; Bioverativ: Consultancy; Genetech: Consultancy.
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Greinacher, Andreas, Susanne Alban, Veronika Dummel, Gerhard Franz, and Christian Mueller-Eckhardt. "Characterization of the Structural Requirements for a Carbohydrate Based Anticoagulant with a Reduced Risk of Inducing the Immunological Type of Heparin-associated Thrombocytopenia." Thrombosis and Haemostasis 74, no. 03 (1995): 886–92. http://dx.doi.org/10.1055/s-0038-1649842.
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SummaryHAT is the most frequent drug induced immune-thrombocytopenia. We recently identified multimolecular PF4/heparin complexes as the major antigen. In order to evaluate the structural requirements for formation of the antigenic complex, we chemically synthesized 13 glucan sulfates and used 5 heparin fractions (2.4-4.8 kD) and a synthesized pentasaccharide, representing the antithrombin III binding sequence of heparin, to further characterize the HAT antigen. In the presence of glucan sulfates and heparin, HAT antibodies caused platelet activation typically at low but not at high concentrations, as measured by 14C-5HT release. The concentration range giving the activation pattern depended on the degree of sulfation (DS) and molecular weight (MW) of the glucan sulfates but not on the type of glycosidic linkage of a polysaccharide. With linear glucan sulfates with a chain length of 35 monosaccharides, the critical DS to form the HAT antigen ranged between 0.60 and 1.20. Glycosidic branched glucan sulfates were able to form the HAT antigen at a lower DS and a lower MW than linear glucan sulfates. Platelet activation by HAT-antibodies in the presence of linear curdlan sulfate fractions was dependent on their MW. At a low concentration (0.01 µM) medium-size fractions (60 kD) caused platelet activation but neither small (12 kD) nor large fractions (>150 kD) did. At higher concentrations (2 µM) the opposite reaction pattern was observed. In the case of heparin, the optimal chain length for forming the HAT antigen is a hexadecasaccharide (4.8 kD). Antigen generation decreased with larger and smaller fractions. For 50% platelet activation by HAT antibodies increasing concentrations of heparin were necessary using heparins with decreasing MW: 0.02 ± 0.015 µM (4.8 kD), 0.09 ± 0.016 pM (3.0 kD), 0.8 ± 0.21 µM (2.4 kD). In the presence of the pentasaccharide, HAT antibodies did not cause platelet activation atany concentration tested, nor bound to PF4-pentasaccha- ride complexes in a PF4 based ELISA system. We conclude that generation of the HAT antigen is dependent on the ratio of MW and DS of a glucan sulfate. We conclude from this study that a carbohydrate based anticoagulant with a reduced risk of forming the HAT antigen should be linear with a DS<0.6 or a MW <2.4 kD. These data might be important for the design of new drugs for parenteral anticoagulation.