Journal articles on the topic 'Neisseria Heparin Binding Antigen'
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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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Youssef, Abdel-Rahman. "Effect of Carcinoembryonic Antigen-related Cell Adhesion Molecule-binding Recombinant Polypeptide on the Killing of Neisseria meningitidis by Human Neutrophils." Journal of Umm Al-Qura University for Medical Sciences 7, no. 1 (June 1, 2021): 6–9. http://dx.doi.org/10.54940/ms10183116.
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Background: Neutrophils are an essential part of innate immunity and play a crucial role in controlling infection caused by Neisseria meningitidis. The Moraxella catarrhalis ubiquitous surface protein A1 (UspA1)-based recombinant polypeptide binds to the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) 1 receptor on host cells and blocks binding of the mucosal pathogens to human epithelial cells and T cells. Aim of the study: Since the CEACAM1 receptor is expressed on neutrophils, the aim of this study was to investigate the effect of CEACAM1-binding recombinant polypeptide on the ability of neutrophils to kill Neisseria meningitidis. Methods: The effect of CEACAM1-binding recombinant polypeptide on the phagocytic killing of Neisseria meningitidis by neutrophils was assessed by incubation of neutrophils with CEACAM1-binding recombinant polypeptide (UspA1 527–665) or with CEACAM1-non-binding polypeptide control (UspA1 659–863) for one hour before infection with Neisseria meningitidis. The surviving bacteria were released and counted. Results: 30 minutes after infection of neutrophils with Neisseria meningitidis, the survival of bacteria in presence of CEACAM1-binding recombinant polypeptide was 64% compared to 52% with control peptide and 43% without peptide. However, one-hour after infection, the surviving bacteria was 32% in presence of CEACAM1-binding recombinant polypeptide compared to 18% with control peptide and 22% without peptides. Conclusion: Although CEACAM1-binding polypeptide reduced the killing of Neisseria meningitidis by neutrophils, it did not entirely stop phagocytosis and killing of bacteria.
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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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Serruto, Davide, Tiziana Spadafina, Laura Ciucchi, Lisa A. Lewis, Sanjay Ram, Marta Tontini, Laura Santini, et al. "Neisseria meningitidisGNA2132, a heparin-binding protein that induces protective immunity in humans." Proceedings of the National Academy of Sciences 107, no. 8 (February 3, 2010): 3770–75. http://dx.doi.org/10.1073/pnas.0915162107.
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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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DUENSING, Thomas D., and Jos P. M. van PUTTEN. "Vitronectin binds to the gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge." Biochemical Journal 334, no. 1 (August 15, 1998): 133–39. http://dx.doi.org/10.1042/bj3340133.
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Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA–heparin–vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.
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Seib, Kate L., Maria Scarselli, Maurizio Comanducci, Daniela Toneatto, and Vega Masignani. "Neisseria meningitidisfactor H-binding protein fHbp: a key virulence factor and vaccine antigen." Expert Review of Vaccines 14, no. 6 (February 23, 2015): 841–59. http://dx.doi.org/10.1586/14760584.2015.1016915.
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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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Ram, Sanjay, Meabh Cullinane, Anna M. Blom, Sunita Gulati, Daniel P. McQuillen, Brian G. Monks, Catherine O'Connell, et al. "Binding of C4b-Binding Protein to Porin." Journal of Experimental Medicine 193, no. 3 (January 29, 2001): 281–96. http://dx.doi.org/10.1084/jem.193.3.281.
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We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface–bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp–Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp–Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal α-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp α-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.
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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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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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Chen, T., R. J. Belland, J. Wilson, and J. Swanson. "Adherence of pilus- Opa+ gonococci to epithelial cells in vitro involves heparan sulfate." Journal of Experimental Medicine 182, no. 2 (August 1, 1995): 511–17. http://dx.doi.org/10.1084/jem.182.2.511.
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Neisseria gonorrhoeae attaches to host epithelial cells via pili and opacity-associated (Opa) outer membrane proteins. Pilus- gonococci (Gc) of strain MS11 adhere to both human and nonhuman cells, but only when particular Opa proteins are expressed; OpaA+ variants adhere best, OpaC+ variants are next best, and the seven other Opa+ variants adhere poorly or not at all. The adherence of OpaA+ Gc to Chinese hamster ovary (CHO) cells is inhibited by heparin or heparan sulfate (HS), but not by chondroitin sulfate. OpaA+ Gc do not adhere to CHO cells devoid of HS proteoglycans; low concentrations of heparin restore OpaA+ Gc adherence to these HS-deficient CHO cells and high concentrations inhibit it. 3H-heparin binding to whole Gc parallels their adherence abilities (OpaA+ > OpaC+ > OpaH+ > Opas B, D, E, F, G, I = Opa- = 0). Opa proteins separated by SDS-PAGE also bind 3H-heparin. These data suggest that adherence of pilus-, Opa+ Gc involves HS-proteoglycan of eukaryotic cells.
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Coppens, Isabelle, Sylvie Alonso, Rudy Antoine, Françoise Jacob-Dubuisson, Geneviève Renauld-Mongénie, Eric Jacobs, and Camille Locht. "Production of Neisseria meningitidisTransferrin-Binding Protein B by RecombinantBordetella pertussis." Infection and Immunity 69, no. 9 (September 1, 2001): 5440–46. http://dx.doi.org/10.1128/iai.69.9.5440-5446.2001.
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ABSTRACT Neisseria meningitidis serogroup B infections are among the major causes of fulminant septicemia and meningitis, especially severe in young children, and no broad vaccine is available yet. Because of poor immunogenicity of the serogroup B capsule, many efforts are now devoted to the identification of protective protein antigens. Among those are PorA and, more recently, transferrin-binding protein B (TbpB). In this study, TbpB of N. meningitidiswas genetically fused to the N-terminal domain of the Bordetella pertussis filamentous hemagglutinin (FHA), and thefha-tbpB hybrid gene was expressed in B. pertussis either as a plasmid-borne gene or as a single copy inserted into the chromosome. The hybrid protein was efficiently secreted by the recombinant strains, despite its large size, and was recognized by both anti-FHA and anti-TbpB antibodies. A single intranasal administration of recombinant virulent or pertussis-toxin-deficient, attenuated B. pertussis to mice resulted in the production of antigen-specific systemic immunoglobulin G (IgG), as well as local IgG and IgA. The anti-TbpB serum antibodies were of the IgG1, IgG2a, and IgG2b isotypes and were found to express complement-mediated bactericidal activity againstN. meningitidis. These observations indicate that recombinant B. pertussis may be a promising vector for the development of a mucosal vaccine against serogroup B meningococci.
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Malito, Enrico, Paola Lo Surdo, Daniele Veggi, Laura Santini, Heather Stefek, Brunella Brunelli, Enrico Luzzi, Matthew J. Bottomley, Peter T. Beernink, and Maria Scarselli. "Neisseria meningitidis factor H-binding protein bound to monoclonal antibody JAR5: implications for antibody synergy." Biochemical Journal 473, no. 24 (December 9, 2016): 4699–713. http://dx.doi.org/10.1042/bcj20160806.
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Factor H-binding protein (fHbp) is an important antigen of Neisseria meningitidis that is capable of eliciting a robust protective immune response in humans. Previous studies on the interactions of fHbp with antibodies revealed that some anti-fHbp monoclonal antibodies that are unable to trigger complement-mediated bacterial killing in vitro are highly co-operative and become bactericidal if used in combination. Several factors have been shown to influence such co-operativity, including IgG subclass and antigen density. To investigate the structural basis of the anti-fHbp antibody synergy, we determined the crystal structure of the complex between fHbp and the Fab (fragment antigen-binding) fragment of JAR5, a specific anti-fHbp murine monoclonal antibody known to be highly co-operative with other monoclonal antibodies. We show that JAR5 is highly synergic with monoclonal antibody (mAb) 12C1, whose structure in complex with fHbp has been previously solved. Structural analyses of the epitopes recognized by JAR5 and 12C1, and computational modeling of full-length IgG mAbs of JAR5 and 12C1 bound to the same fHbp molecule, provide insights into the spatial orientation of Fc (fragment crystallizable) regions and into the possible implications for the susceptibility of meningococci to complement-mediated killing.
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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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Duensing, Thomas D., Jenny S. Wing, and Jos P. M. van Putten. "Sulfated Polysaccharide-Directed Recruitment of Mammalian Host Proteins: a Novel Strategy in Microbial Pathogenesis." Infection and Immunity 67, no. 9 (September 1, 1999): 4463–68. http://dx.doi.org/10.1128/iai.67.9.4463-4468.1999.
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ABSTRACT Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1α) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.
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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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Nazábal, C., T. Carmenate, S. Cruz, S. González, R. Silva, A. Musacchio, M. Delgado, and G. Chinea. "Mapping of monoclonal antibodies specific to P64k: A common antigen of several isolates of Neisseria meningitidis." Canadian Journal of Microbiology 47, no. 2 (February 1, 2001): 158–64. http://dx.doi.org/10.1139/w00-133.
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P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.Key words: Neisseria meningitidis, P64k, monoclonal antibodies, epitope mapping.
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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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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