Background: Neisseria meningitidis is a gram-negative bacterium with the potential to cause invasive disease. Invasive meningococcal disease (IMD) can be fatal if delay to antibiotic therapy. There are six serogroups, which are capable of causing invasive disease in humans; A, B, C, W, X and Y. Since 2015, serogroup W and serogroup Y account for the majority of IMD cases reported in Sweden. Aim: To investigate the clinical presentations of IMD caused by Neisseria meningitidis serogroup W and Y. Method: Two databases, PubMed and Cochrane, were used to find articles that described the clinical picture of IMD. Articles with description of clinical features of the studied serogroups and with eight cases or more in every study were included. In addition, only original articles were included. Results: A total of 633 articles were found and 11 fulfilled all the inclusion criteria. Five out of seven articles found meningococcemia as the predominating presentation of serogroup W IMD. Two out of the four articles that studied serogroup Y IMD found meningitis at a higher number. Conclusion: The results of this systematic review suggest meningococcemia as a relatively common presentation of serogroup W IMD while meningitis and pneumonia might occur more frequently in serogroup Y IMD. However, these results should be interpreted carefully because the included articles were mostly retrospective studies and future prospective studies are needed to better identify clinical presentations of serogroup W and Y IMD.

As vacinas antimeningocócicas têm se demonstrado efetivas contra os sorogrupos A e C, no entanto ainda não existe vacina contra o sorogrupo B devido à similaridade entre a estrutura capsular do polissacáride B e o ácido polisiálico que faz parte do tecido cerebral humano, podendo levar à autoimunidade. O objetivo deste estudo foi investigar as propriedades adjuvantes dos toxóides Stx1 e Stx2 (STEC) de Escherichia coli, administrados em preparações antigênicas com vesículas de membrana externa nativa (NOMV) de Neisseria meningitidis B, comparando duas vias de imunização prime-boost ou intramuscular, em camundongos BALB/c com idade entre 6-8 semanas. A determinação dos níveis de anticorpos empregando a técnica de ELISA, mostrou elevadas concentrações de anticorpos IgG em soros de animais imunizados pela via intramuscular com Stx1+NOMV, mas não com NOMV, o que sugere que por esta via (intramuscular apenas) Stx1 possa ter atuado como adjuvante. No ensaio de Immunoblotting, soros de animais imunizados com Stx1+NOMV reconheceram maior número de antígenos de NOMV quando comparado ao grupo que recebeu Stx2+NOMV. O sistema prime-boost mostrou-se efetivo quando comparamos os níveis de anticorpos presentes no soro após a dose intramuscular (reforço), entretanto, não melhor do que quando utilizamos duas doses apenas pela via intramuscular. Este estudo poderá contribuir no desenvolvimento de tecnologias associadas a novas preparações antigênicas utilizando antígenos de membrana externa de N. meningitidis B , empregando toxóides como adjuvantes. / The meningococcal vaccines have been shown to be effective against serogroups A and C, however there is still no vaccine against serogroup B. The capsular polysaccharide from serogroup B meningococci polysialic acid moiety mimetic of many human glycoproteins including the neural cell adhesion molecules and may lead to autoimmunity. This study aimed to investigate the adjuvant properties of toxoids Stx1 and Stx2 (STEC) from Escherichia coli and native outer membrane vesicles (NOMV) of Neisseria meningitidis B, comparing two ways of immunization prime-boost or only intramuscular in BALB/c mice. The results showed high concentrations of IgG antibodies in sera of animals immunized intramuscularly with Stx1+NOMV, suggesting that in this way may have Stx1 acted as an adjuvant. In the Immunoblotting assay, sera from animals immunized with Stx1+NOMV recognized more antigens of NOMV when compared to the group that received Stx2+NOMV. The prime-boost was effective however, no better than only two doses intramuscularly. This study may contribute to the development of new technologies and strategies against N. meningitidis B employing toxoids as adjuvants.

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host’s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.

Meningitis is an infection of the meninges, a layer of tissue surrounding the brain. In cases of pneumococcal meningitis (where the bacterium Streptococcus pneumoniae is the causat- ive agent) this causes severe inflammation, requiring intensive care and rapid antibiotic treatment. The contribution of variation in host and pathogen genetics to pneumococcal meningitis is unknown. In this thesis I develop and apply statistical genetics techniques to identify genomic variation associated with the various stages of pneumococcal meningitis, including colonisation, invasion and severity. I start by describing the development of a method to perform genome-wide association studies (GWAS) in bacteria, which can find variation in bacterial genomes associated with bacterial traits such as antibiotic resistance and virulence. I then applied this method to longitudinal samples from asymptomatic carriage, and found lineages and specific variants associated with altered duration of carriage. To assess meningitis versus carriage samples I applied similar analysis techniques, and found that the bacterial genome is crucial in determining invasive potential. As well as bacterial serotype, which I found to be the main effect, I discovered many independent sequence variants associated with disease. Separately, I analysed within host-diversity during the invasive phase of disease and found it to be of less relevance to disease progression. Finally, I analysed host genotype data from four independent studies using GWAS and heritability estimates to determine the contribution of human sequence variation to pneumococcal meningitis. Host sequence accounted for some variation in susceptibility to and severity of meningitis. The work concludes with a combined analysis of pairs of bacterial and human sequences from meningitis cases, and finds variation correlated between the two.

Abstract The aims of this work were to study the carriage of respiratory bacteria and to identify risk factors affecting pharyngeal colonisation by these pathogens among young Finnish men during military service, and also to investigate the role of mannose-binding lectin (MBL) concentrations and MBL2 gene polymorphisms in the carriage of respiratory bacteria. A total of 892 military recruits entering the Kainuu Brigade, including 224 men with asthma, were followed up prospectively to the end of their military service. Carriage of Streptococcus pneumoniae, Neisseria meningitidis and beta-haemolytic streptococci appeared to be higher during and at the end of military service than at the beginning. Smoking was found to be a significant risk factor for colonisation by these bacteria. S.pneumoniae was more common in the asthmatic than military conscripts in the non-asthmatic ones at the beginning of military service. A low MBL level increased the risk of carrying N. meningitidis and beta-haemolytic streptococci during military service among non-smokers but not among smokers. Low MBL levels producing MBL2 haplotypes seemed to be associated with the carriage of N. meningitidis and S. pneumoniae. Characterisation of all the oropharyngeal N.meningitidis isolates obtained (n=215) by phenotypic and genotypic methods showed that most of them belonged to the carriage-associated ST-60 clonal complex. Clonal complexes ST-41/44, ST-32, and ST-23, which have previously been associated with disease, also accounted for a third of the carriage strains. Furthermore, a significant association was indicated between an acute upper respiratory infection and oropharyngeal carriage of the virulent meningococcal ST-23 clone. In conclusion, the results reported here show a significant increase in bacterial carriage during military service and provide new information on the association between MBL and carriage of respiratory bacteria. These findings also highlight the importance of smoking cessation, especially among military conscripts, who have been found to be a risk group for invasive bacterial diseases, and they also point to the importance of meningococcal vaccination for military recruits and the need for an efficacious vaccine against serogroup B meningococci. / Tiivistelmä Hengitystieinfektiot ovat yleisiä varusmiespalvelun aikana. Myös oireeton bakteerien nielukantajuus on lisääntynyt. Useimmiten infektiot ovat lieviä virusinfektioita, mutta bakteerien nielukantajuus voi johtaa myös vaikeisiin bakteeritulehduksiin. Tämän väitöskirjatyön tarkoituksena oli tutkia bakteerien nielukantajuutta varusmiespalveluksen alkaessa ja päättyessä sekä mahdollisten hengitystieinfektioiden aikana ja näin saada uutta tietoa bakteerien nielukantajuuteen vaikuttavista tekijöistä. Lisäksi tavoitteena oli selvittää mannoosia sitovan lektiinin (MBL) sekä MBL2-geenin polymorfismien yhteyttä bakteerien nielukantajuuteen. Työn tarkoituksena oli myös feno- ja genotyypittää varusmiehiltä palveluksen aikana eristetyt meningokokkikannat ja verrata niitä vastaavana ajankohtana invasiivista tautia sairastaneista henkilöistä eristettyihin meningokokkikantoihin. Tutkimuksessa seurattiin prospektiivisesti 892 varusmiestä, jotka suorittivat asepalveluksen Kainuun Prikaatissa vuosina 2004–2006. Tutkimukseen osallistuneista varusmiehistä 224:llä oli astma. Tutkimuksessa havaittiin, että oireeton bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana. Lisäksi havaittiin, että tupakointi oli merkittävä itsenäinen riskitekijä pneumokokin, meningokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle varusmiespalveluksen aikana. Astmaatikkojen pneumokokin nielukantajuus varusmiespalveluksen alussa oli yleisempi kuin terveiden varusmiesten. Tutkimuksessa osoitettiin myös pienen seerumin MBL-pitoisuuden sekä MBL2-geenin polymorfismin eksoni 1:n alueella ja geenin säätelyalueella olevan riskitekijöitä meningokokin, pneumokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle tupakoimattomilla varusmiehillä. Meningokokin nielukannoista jopa kolmasosa kuului genotyyppiryhmään, jonka on aiemmissa tutkimuksissa havaittu liittyvän invasiiviseen tautiin. Tutkimuksessa osoitettiin myös tietyn hyperinvasiivisen meningokokin genotyypin (ST-23) liittyvän hengitystieinfektioepisodeihin. Tässä väitöskirjatyössä osoitettiin, että bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana ja että oireettomilla varusmiehillä tavataan myös hyperinvasiivisia meningokokkikantoja. Tutkimus antoi myös uutta tietoa hyperinvasiivisten meningokokin genotyyppien liittymisestä hengitystieinfektioihin sekä MBL:n vaikutuksesta bakteerien nielukantajuuteen. Tutkimushavainnot tukevat tupakoimattomuuden edistämisen tärkeyttä myös varusmiespalveluksen aikana.

Neisseria meningitidis (Nm) est un germe commensal du rhinopharynx ayant pour seul hôte l’homme. Malgré un portage asymptomatique largement répandu, et pour des raisons encore inconnues, elle peut échapper au système immunitaire de l’hôte et devenir pathogène provoquant ainsi méningite et septicémie pouvant être mortelles principalement chez les enfants. Au cours du processus infectieux, Nm alterne entre des phases de colonisation et de dissémination, et se retrouve alors confrontée à différents environnements. L’adaptation rapide à ces variations, par modulation de l’expression des gènes de virulence, représente un facteur important dans sa pathogénie. Les facteurs qui contribuent à la virulence de Nm sont essentiellement des structures présentes à la surface de la bactérie parmi lesquelles les pili et la capsule. Les gènes codant ces facteurs sont sous le contrôle de la protéine CrgA, régulateur transcriptionnel de la famille LysR qui intervient lors de l’adhésion de Nm aux cellules humaines. La protéine CrgA régule négativement sa propre expression ainsi de celle des gènes impliqués dans la synthèse de la capsule (sia) et des pili (pilE et pilC1). Par ailleurs, le PTS est un système de transduction du signal qui intervient, par phosphorylation ou via des interactions protéine/protéine, dans le transport des sucres et dans la régulation du métabolisme du carbone. Chez Nm, ce système est incomplet (constitué des protéines EI, HPr, et deux EIIA), il n’est donc pas fonctionnel pour le transport des sucres mais aurait pu conserver ses fonctions régulatrices. Nous avons montré que les protéines du PTS de Nm étaient actives in vitro et in vivo et que la cascade de phosphorylation du PTS était fonctionnelle. Nous avons également montré que l’inactivation du gène ptsH, codant la protéine HPr, entrainait une diminution significative de la synthèse de la capsule, une augmentation de l’adhésion du mutant aux cellules épithéliales humaines et une augmentation de l’expression de crgA. De ce fait, l’absence de l’HPr semble empêcher la répression de crgA et par conséquent celle des gènes sia. Par ailleurs, des expériences de co-immunoprécipitation, nous ont permis de mettre en évidence que l’HPr interagissait directement avec la protéine CrgA in vitro et in vivo. Ces résultats suggèrent que la protéine HPr interviendrait dans la régulation de l’expression des gènes de virulence de Nm via la régulation de l’expression de crgA. Ainsi, un lien entre métabolisme du carbone et virulence a été mis en évidence chez Nm. / Neisseria meningitidis (Nm) is a commensal bacterium of the nasopharynx, which only colonizes humans. Despite a large number of asymptomatic carriers, and for reasons so far unknown, Nm occasionally becomes virulent, escaping the host’s immune system and causing septicaemia and meningitis, the latter being potentially lethal, mostly in children.During the infectious process, Nm alternates between phases of colonization and dissemination, each time facing different environments. This rapid adaptation to the changing environment occurs via the modulation of the expression of virulence genes and represents an important factor of pathogenicity. The structures involved in virulence in Nm are mainly present at the surface of the bacterium, including the pili and the capsule. The genes coding for these structures are controlled by the CrgA protein, a transcriptional regulator of the LysR family, which is induced during the adhesion of Nm to human cells. CrgA negatively regulates its own expression as well as the expression of those genes implicated in the synthesis of the capsule (sia) and pili (pilE and pilC1).Moreover, the PTS is a signal transduction system, which is involved, via phosphorylation or protein/protein interactions, in the transport of sugars and the regulation of the carbon metabolism. In Nm, the PTS is incomplete (only composed of the proteins EI, HPr and two EIIA), thus not functioning in the transport of sugars but it may have conserved regulatory functions.In this work, we demonstrate that the PTS proteins in Nm are active in vitro and in vivo and that the phosphorylation cascade of the PTS is functional. We further show that the inactivation of the ptsH gene, coding for the HPr protein, significantly reduces the synthesis of the capsule, enhances the adhesion of the mutants to human epithelial cells and increases the expression of crgA. Thus, the absence of HPr seems to inhibit the repression of crgA and as a consequence also the repression of the sia genes. Furthermore, from co-immunoprecipitation experiments we provide evidence that HPr directly interacts with the CrgA protein in vitro and in vivo. These results suggest that the HPr protein in Nm regulates the expression of the virulence genes via the regulation of crgA expression. Thus, we provide evidence of a link between carbon metabolism and virulence in Nm.

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No. of bitstreams: 2 Dissertação - Lísia Gomes Martins de Moura Tomich - 2016.pdf: 2901743 bytes, checksum: 22cd41bfc4499cfd4754d856635357af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-15 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / INTRODUCTION: Routine infant immunization with meningococcal C conjugate vaccine (MenC-V) started in Brazil in November 2010, administered at three, five and 12 months of age with no catch-up for older age-groups. However, by March 2010, a vaccination campaign with MenC-V was performed in Salvador in individuals under five years-old, and from 10 to 24 yearsold. In São Paulo state, the outbreaks occurred in teenagers and young adults prompting one-time vaccination campaign from 2010 to 2014 targeting these age-groups. OBJECTIVE: To assess the direct and indirect impact (herd effect) of vaccination on invasive meningococcal disease (MD) for capsular group C (MenC) four years after the introduction of MenC-V in three scenarios: i) Brazil as a whole (routine vaccination in childhood only); ii) Brazil except for Salvador (vaccination campaign with teenagers during the year of MenC-V introduction); and iii) São Paulo state (vaccination campaign for adolescents and young adults during 2010-2014 to control outbreaks). METHODS: We performed an ecological quasi-experimental design from 2008 to 2014 using data from the National Reference Laboratory for Meningitis, and data from the National Information System for Notifiable Diseases. A deterministic linkage was performed between the two databases to improve the accuracy of the detection of MD, especially in capsular groups. An interrupted time-series analysis was conducted using the Holt-Winters technique to control for pre-existing trends and seasonal variations. The MenC vaccination impact was evaluated as the percentage of reduction in the incidence rates of MenC in the post-vaccination period (2012 to 2014), using the pre-vaccination period (2008 to 2010) to estimate what would be expected on the post-vaccination period, whether the vaccination had not been introduced. For Salvador, we analyzed the effect of the vaccination on the number of MenC cases. RESULTS: A total of 18,136 invasive MD cases were analyzed. For Brazil as a whole, the vaccination reduced 67.4% (lower 95%CI 42.5%) the rates for MenC for infants under 12 months, 92.3% (lower 95%CI 77.7%) for the age-group 12-23 months, and 65.7% (lower 95%CI 28%) for children aged 2-4 years. Indirect impact (20-24.7%) was observed in the age-group 5-19 years. When excluding Salvador from the analysis of Brazil, the indirect impact was observed only for children in the age-group 5-9 years. In the scenario of São Paulo state, similarly to Brazil, significant impact was observed in the target age-groups, in addition to indirect impact in the age group 5-9 years. In Salvador, in addition to the effect on the vaccinated population a sharp and sustainable decline of MenC cases was observed in all age-groups not target for vaccination. Overall, 1,170 cases of MenC were averted in Brazil after the introduced of Men-C vaccination. CONCLUSION: The strategy of catch-up for adolescents and young adults, especially during the year of MenC-V introduction may lead to rapid and sustainable herd effect. / A vacina meningocócica conjugada contra o grupo capsular C (MenC-V) foi introduzida no calendário de imunização infantil brasileiro em novembro de 2010, sendo administrada aos três, cinco e 12 meses de idade sem catch-up para os demais grupos etários. Entretanto, em março de 2010, uma campanha de vacinação com MenC-V foi realizada em Salvador para indivíduos menores de cinco anos de idade e de 10 a 24 anos. No estado de São Paulo os surtos ocorreram em adolescentes e adultos jovens, determinando campanhas de vacinações de bloqueio nessa faixa etária nos anos de 2010 a 2014. OBJETIVO: Avaliar o impacto direto e indireto (rebanho) da vacinação nas taxas de incidência de doença meningocócica (DM) invasiva pelo grupo capsular C (MenC) após quatro anos da introdução da MenC-V em três cenários: i) Brasil como um todo (imunização de rotina somente de crianças); ii) Brasil exceto Salvador (campanha de vacinação em adolescentes no ano de introdução da MenCV); e iii) estado de São Paulo (vacina de rotina na infância e vacinações de bloqueio em adolescentes e adultos jovens para controlar surtos). MÉTODOS: Foi realizado um estudo ecológico quasi-experimental para avaliar o impacto da vacinação em série histórica de 2008 a 2014 usando os bancos de dados do Laboratório Nacional de Referência para Meningites Bacterianas, Instituto Adolfo Lutz (IAL) e o Sistema de Informação de Agravos de Notificação (Sinan). Um processo de vinculação (linkage) determinístico entre as duas bases foi realizado para melhorar a acurácia da detecção de casos de DM, especialmente de grupo capsulares. Uma análise de série temporal interrompida foi conduzida utilizando a técnica de Holt-Winters para controlar por tendência pré-existente e variações sazonais. O desfecho foi taxa de MenC. O impacto da vacinação foi avaliado pelo percentual de redução da incidência de MenC no período pós-vacinal (2012 a 2014), utilizando o período pré-vacinal (2008 a 2010) para estimar o que seria esperado no período pós-vacinal, caso a vacinação não tivesse sido introduzida. Para Salvador foi analisado o efeito da MenC-V no número de casos de MenC. RESULTADOS: Um total de 18.136 casos de DM invasiva foram analisados. Para o Brasil como um todo, a vacinação reduziu significativamente a DM por MenC na faixa etária alvo, com redução de 67,4% (limite inferior do IC95% 42,5%) em menores de 12 meses, 92,3% (limite inferior do IC95% 77,7%) para faixa etária de 12-23 meses e 65,7% (limite inferior do IC95% 28%) em crianças de 2-4 anos, e efeito rebanho foi observado na faixa etária de 5 a 19 anos com 20-24,7%. Quando se exclui Salvador na análise do Brasil, impacto indireto significativo foi observado somente em crianças de 5-9 anos. No cenário São Paulo, semelhante ao Brasil, observou-se impacto estatisticamente significante nas faixas etárias alvo do PNI, além do efeito rebanho na faixa etária de 5-9 anos de idade. Para Salvador, o impacto da vacinação apresentou um declínio acentuado e sustentável em todas as faixas etárias fora do alvo da vacinação. Ao todo, 1.170 casos de MenC foram evitados no período estudado. CONCLUSÃO: A estratégia de vacinação de catch-up em adolescentes e adultos jovens, especialmente no ano de introdução da MenC-V, promoveu um rápido e sustentável rebanho.

<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.