Poster presentations

Host-Pathogen Interactions

H1 Neisseria meningitidis serogroup B targets human dendritic cells to modify T helper cell responses in vitro

Alastair Copland1,2, Hannah E. Jones2, Mona Bajaj-Elliott2, Nigel J. Klein2, Garth L. J. Dixon2,3

1Division of Infection and Immunity, University College London, London, UK
2Infectious Diseases and Microbiology Unit, UCL Institute of Child Health, London, UK
3Department of Microbiology, Great Ormond Street Hospital for Children NHS Trust, London, UK

Keywords: dendritic cells, interleukin-17, T helper cells, signal transduction, immune evasion

Neisseria meningitidis serogroup B (Nm) is an important cause of meningitis and septicaemia. To date, no broadly-protective vaccine is yet available. Several recent studies have assessed the interaction between human dendritic cells (DCs) and Nm, as DCs are key mediators of adaptive immunity and vital for efficacious vaccine responses. We have previously reported that live and killed Nm differentially mature DCs, with live Nm inhibiting the full maturation of human DCs. Here we examine how Nm inhibits phenotypic DC maturation and also describe how this affects T helper (Th) cell responses.

DCs were infected with live and killed Nm and analysed for surface maturation markers CD40 and CD80. Killed Nm increased the surface expression of these markers and induced full phenotypic maturation, while live Nm prevented the increase in expression (p=<0.05). Further experiments showed that live Nm was able to block the activity of potent TLR agonists including the viral mimetic poly(I:C) and peptidoglycan from Staphylococcus aureus, with DCs unable to increase the expression of maturation markers. We next sought to investigate whether signalling pathways necessary for DC maturation were being affected by live Nm. Detection of IκB-⍺ by Immunoblotting was used as a measure of NF-κB activity. At 2 hours, killed Nm induced IκB-⍺ degradation, whereas there was no apparent degradation of IκB-⍺ in DCs infected by live bacteria. This raises the possibility that live NMB may interfere with NF-κB signalling pathway, which is requiredfor DC maturation.

As an effective Th cell repertoire is critical for pathogen clearance, we wanted to know whether the modification of DC signalling affected Th polarisation. An in vitro DC-CD4 co-culture model was used to assess the development of Th1, Th2 and Th17 cells by DCs pulsed with live or killed Nm. Flow cytometry experiments showed that live Nm induced a stronger Th2 response than killed Nm, and also produced a lower percentage of Th1 cells. Furthermore, live Nm was found to inhibit the development of Th17 cells when compared to killed Nm and positive controls.

Our data suggest that live Nm actively modifies DCs via a crucial signalling pathway and this modulates the resulting Th response. We speculate that this may be a novel form of meningococcal immune evasion by the DC-mediated inhibition of antibacterial T cells. Candidate immunological receptors and bacterial ligands esponsible for this effect are currently under investigation.

Anti-inflammatory and cyto-protective properties of commensal Neisseriae

Keith Page and Victoria Davenport

Faculty of Life Sciences, University of the West of England, Bristol, UK
Contact email: victoria.davenport@uwe.ac.uk

Keywords: Nasopharyngeal, inflammation, cytotoxicity, invasion, commensal

Objective of the study

N. lactamica is a human tropic commensal of the nasopharynx that may convey protection against invasive and/or colonising N. meningitidis in the population. We have previously demonstrated that N. lactamica attenuates inflammation induced by N. meningitidis in nasopharyngeal derived epithelial cells, by suppressing NFκB activity in a PPARγ-dependent manner. This study aimed to determine whether Neisseria commensals commonly found in the upper respiratory tract of healthy adults, N. cinerea and N. polysacchareae, share N. lactamica’s ability to attenuate host cell innate inflammation induced by N. meningitidis. We further aimed to investigate the effects of commensal Neisseriae on host cell invasion and host cell death by N. meningitidis and the Gram positive pathogen S. pneumoniae.

Methods used

The human nasopharygeal epithelial cell line Detroit 562 (D562) was grown to confluence and challenged with N. meningitidis (strain MC58) in the presence or absence of commensal Neisseriae (N. cinerea, N. polysaccharea, N. lactamica). Cells were challenged for 3h with bacteria, washed and cultured for a further 21h in the presence of antibiotics to generate supernatants for the evaluation of IL-6 cytokine secretion by sandwich ELISA. Standard gentamicin protection assays were performed to determine the effect of commensals on N. meningitidis invasion but using a lux-transformed N. meningitidis (Lux-MC58) and light output which was monitored by luminometer. Fluorescent molecular probes, Yopro1 and propidium iodide, were used to determine pathogen-induced host cell death and levels of protection afforded by commensal Neisseriae against Streptococcus pneumonia (Sp14) and Neisseria meningitidis.

Summary of results

We demonstrate for the first time that N. lactamica (p<0.001) and N. cinerea (p<0.01) but not N. polysacchareae were able to significantly attenuate host IL-6 cytokine secretion induced by N. meningitidis. Nasopharyngeal cell invasion by N. meningitidis was also inhibited by both N. lactamica and N. cinerea (p<0.0001) but not N. polysaccharea. Both necrotic and apoptotic host cell death was induced in nasopharyngeal cells challenged with N. meningitidis. Whilst both N. lactamica (p<0.001) and N. polysacchareae (p<0.0001) were able to attenuate necrotic cell death, only N. lactamica was able to suppress induction of apoptotic cell death induced by N. meningitidis (p<0.001). In contrast both N. lactamica (p<0.01) and N. cinerea (p<0.001) were able to suppress the induction of apoptosis by S. pneumoniae.

Conclusions

Commensal Neisseriae which colonise the nasopharynx of children and adults afford host cell protection against related Gram-negative and unrelated Gram-positive pathogenic bacteria that naturally colonise the same niche.

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Role of factor H binding protein (fHbp) in intracellular survival of Neisseria meningitidis after phagocytosis by human macrophages

Pike A1, Keats S1, Green L1, Serruto D2, Read RC1

1Dept of Infection & Immunity, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK
2Universita degli Studi di Siena, Siena Italy

Keywords: Meningococcus, Factor H binding protein, Innate immunity, Phagocytosis, Macrophage

Project Rationale & Hypothesis

Factor H binding protein (fHbp) is a surface antigen of N. meningitidis which inhibits complement deposition on the bacterial surface and may also be protective against endogenous defensins. fHbp is a major component of new generation meningococcal vaccines.

Objectives

To determine whether fHbp influences meningococcal phagocytosis by primary human monocyte-derived macrophages (MDMs), and if fHbp enhances intracellular survival of N. meningitidis after phagocytosis by MDMs.

Methodology

N. meningitidis was coincubated with 14 day-old MDMs at a range of multiplicities of infections, under opsonic and non-opsonic conditions. Bacterial association to the macrophage surface and internalisation were quantified by immunofluorescence. Intracellular survival of bacteria over 2 hours after phagocytosis was measured using a standard gentamicin protection assay. Uptake and survival of wild type organisms were compared with fHbp null mutants and a complemented strain.

Findings

fHbp significantly reduced internalisation of meningococci by MDMs opsonised by complement C6 deficient serum, but not under nonopsonic conditions. No difference was observed in intracellular survival of non-opsonised bacterial strains even when the MDMs were activated with Interferon-γ. We conclude that meningococcal fHbp inhibits opsonic uptake of N. meningitidis by macrophages, but has no effect on resistance to intracellular microbicidal mechanisms.

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Complement factor H genotype and protein levels associated with the clinical sepsis phenotype in children with meningococcal disease

Rodrigues CMC1, Wright VJ1, Inwald D1, Khor CC2, Davila S2, Hibberd ML2, Levin M1

1Department of Paediatric Infectious Diseases, 2nd Floor Wright-Fleming Institute, St Mary’s Campus, Imperial College London, UK
2Infectious diseases, Genome Institute of Singapore, Singapore
Contact email: c.rodrigues@imperial.ac.uk

Keywords: Complement factor H, meningococcal disease, sepsis, genotype, clinical markers

Introduction

Sepsis secondary to Neisseria meningitidis infection is a feared presentation for parents and professionals alike, due to the potentially rapidand devastating decline in these patients. The disease process and subsequent morbidity can be profound producing cardiovascular, coagulation, renal and limb impairment.

A recent genome wide association study (GWAS)[1] has identified variants in the complement factor H (CFH) gene and associated regions, which predispose to meningococcal disease (MD) susceptibility. CFH polymorphisms and MD associations have also been noted in 2 other UK based studies[2],[3].

Aim

We aimed to establish if serum CFH protein level and sepsis phenotype are associated with CFH genotype, thereby affecting risk of MD.

Method

A retrospective analysis using CFH data from the GWAS[1] of 475 paediatric patients with MD and 4073 population controls was undertaken. Genotypes, serum CFH levels (measured on an initial cohort of 60 patients) and several parameters of the sepsis phenotype, including clinical and biochemical markers, were investigated.

Results

Mean serum CFH levels were higher in the convalescent samples compared to the matched acute samples (p=0.0005). There was a trend for higher mean CFH levels in convalescent samples when compared to adult controls (p=0.0583).

When acute CFH levels were compared to the sepsis parameters, there was significant positive correlation with: ICU free days (p=0.0499), white cell count (p=0.0001), CRP (p=0.0087) and negative correlation with: Rotterdam score (p=0.0332), GMSPS (p=0.0070), PRISM (p=0.0135), INR (p=0.0235), APTT (p=0.0037). Mean acute serum CFH levels were lower in the group requiring inotropes (p=0.0327).

Only CRP level (p=0.0032) and platelet count (p=0.0095) were significantly different by genotype for the 2 most significant SNPs identified in GWAS[1] (rs1065489 and rs11582939).

Acute and convalescent CFH levels were not found to be statistically different in each of the genotypes for the SNPs rs1065489 or rs11582939.

Conclusion

CFH polymorphisms increase susceptibility to MD. Both CFH genotype and protein levels are associated with clinical sepsis parameters, but the mechanism needs to be elucidated. In the presence of infection, there is depletion of the serum CFH, which may increase complement activation. Excessive complement action, may in part be responsible for the multisystem failures seen in sepsis, as suggested by the correlation between poorer coagulation and cardiac function as well as outcome measures with lower CFH levels in the acute setting. Further study of CFH protein concentration and function in relation to genotype are required to define the mechanisms.

References:

1. Davila S, Wright VJ et al., Nat Genet. 2010 Sep;42(9):772-6.
2. Agbeko RS et al., Pediatr Crit Care Med. 2010 Sep;11(5):561-7
3. Haralambous E et al., Scand J Infect Dis. 2006;38(9):764-71

Gene Expression in relation to severity of Meningococcal Disease

V. Wright1, N. Yang2, L. Ling2, C. Hemingway1, N. Pathan1, H. Betts1, D. Inwald1, S. Nadel1, M.L Hibberd2, M. Levin1

1Paediatrics, Imperial College London, UK;
2Infectious Disease, Genome Institute of Singapore, Singapore

Keywords: RNA expression profiling

Background and aims

Meningococcal sepsis and meningitis remains one of the most devastating infections affecting young children and adolescents worldwide. Considerable progress has been made in understanding the pathophysiology and immunopathogenesis of the disorder, but mechanisms underlying shock and multi-organ failure still remain poorly understood. We aimed to understand the host response to meningococcal infection using whole-genome RNA expression profiling, both in acute disease and after recovery.

Methods

We used the Illumina Sentrix BeadChip array to examine patterns of gene expression in peripheral blood from 39 Caucasian paediatric patients with meningococcal meningitis or meningococcal sepsis. We compared gene expression in the acute phase to 21 Caucasian controls, as well as comparing expression levels between different clinical forms and different phases of the disease.

Results

We identified distinct subsets of genes differentially expressed by >2-fold between cases and controls, acute and convalescence, and meningococcal sepsis and meningococcal meningitis. Multiple biological pathways were found to be activated in the acute samples, including those mainly involved with the inflammatory response and in cell-to-cell signaling and interaction.

Conclusions

RNA expression analysis provides a way of studying the complex inflammatory and metabolic processes in critically ill children. Our analysisidentifies inflammatory genes and pathways involved in meningococcal sepsis and meningitis, and provides a comprehensive insight into the complexity of the host response to meningococcal infection.

Meningococcal surface protein mutations that disrupt human serum protein binding - a strategy for enhanced immunogen design

Lan Zhang1, Arthur Fridman2, Irene Pak2, Jing Lin1, Hui Xu1, Xin-Min Wang1, Zhiyun Wen1, Bob Lucas1, Eberhard Durr1, Chris Mensch1,Thomas Rosahl3, Ansu Bagchi2, Jan ter Meulen1, Craig Przysiecki1

1Vaccine Research, Merck, West Point, PA, USA
2Informatics IT, Merck Global Service, Rahway, NJ, USA
3ETS, Capability Del. In Vivo, Merck, Rahway, NJ, USA
Contact email: lan_zhang2@merck.com

Keywords: meningococcal B vaccine, pathogen-host interaction, transgenic mice

Many bacterial human pathogen surface proteins bind specifically to human sera components or cell surface proteins with high affinity. These interactions are important for bacterial virulence during infection or in the maintenance of the carriage state. By altering host ligand binding sites on these bacterial surface proteins, it may be possible to increase the titers and breadth of specific humoral immune responses by potentially increasing the number of accessible epitopes. Meningococcal factor H binding protein (fHbp) binds human factor H (fH), a serum intrinsic complement pathway inhibitor. fHbp has been identified as an important vaccine target and is included in several vaccines currently in clinical trials to prevent serogroup B meningococcal infection. We have designed fHbp analogs with potentially reduced binding to fH as vaccine candidates, by disruption of salt bridges and hydrogen bonds based on known protein structures. These analog proteins were expressed recombinantly in E. coli and purified. The overall folding of these mutants was analyzed by their CD spectra and their ability to bind anti-fHbp monoclonal antibodies. fH binding ability of these analogs was also evaluated using ELISA. Based on these in vitro screening experiments, selected fHbp analogs are injected into human fH transgenic mice (as well as wildtype mice) to evaluate the effect of the mutations on eliciting a functional immune response, antibodies with serum bactericidal activity. To identify additional human sera binding partners of bacterial surface proteins, a qualitative Ni-NTA resin pull-down assay was developed using recombinant HIS-tagged bacterial surface proteins with in-gel proteomics analysis. We have obtained proof-of-concept data using recombinant HIS-tagged fHbp to pull-down human fH from normal human serum.

Epidemiology and Surveillance


2011 - Invasive Bacterial Diseases caused by N.meningitidis, H.influenzae, S. pneumoniae and L.monocytogenes at University Hospital for Infectious Diseases “Dr Fran Mihaljević” Zagreb, Croatia

Suzana Bukovski

University Hospital for Infectious Diseases “Dr Fran Mihaljević” Zagreb, Croatia
Contact email: sloukovski@bfm.hr; suzana.bukovski@mefos.hr

Keywords: IBD, sepsis, meningitis, S.pneumoniae, N.meningitidis

Introduction

Since 2005 at University Hospital for Infectious Diseases “Dr Fran Mihaljević” invasive bacterial disease (IBD) caused by N.meningitidis, H.influenzae, S. pneumoniae and L.monocytogenes was diagnosed using simultaneously traditional cultivation method and molecular method, in house real time polymerase chain reaction (PCR). The cultivation, as a “gold standard”, gives us important information about the antibiotic susceptibility pattern of IBD causing microorganisms.

Materials and methods

Data about patients treated at our hospital from January till mid September 2011 with positive blood or/and cerebrospinal fluid (CSF) samples for N.meningitidis, H.influenzae, S. pneumoniae and L.monocytogenes were included. Two methods for the detection of etiological agent are used, cultivation with antibiotic susceptibility testing and in house real-time PCR detection based on specific primers.

Results

During the analysed period 74 patients with IBD treated at our hospital had positive blood or/and CSF. There were 23 children and 51 adults. S.pneumoniae was the predominant etiological agent and it caused 49/74 IBD mainly in adults (35/49) while N. meningitidis caused 18/74 IBD, in 9 adults and 9 children. L.monocytogenes caused IBD in 5 adults and H.influenzae was the cause of 2 adults IBD cases. It was recorded that sepsis was the dominant presentation of IBD in children (21/23) and it was caused by S.pneumoniae in 13/21 children while in 8/21 children N.meningitidis caused sepsis. Group B N.meningitidis predominated (7/8 isolates) and 1 isolate was N.meningitidis group C. One N.meningitidis isolate was recorded as penicillin intermediate (0.094 μg/mL) and 1 was recorded as penicillin resistant (0.5 μg/mL). All isolates were susceptible to ciprofloxacin, rifampicin and ceftriaxone. Sepsis was the predominant presentation of IBD in adults too (27/51). Among S.pneumoniae cases meningitis was the predominant clinical presentation in adults (18/20). L.monocytogenes caused meningitis in three adults and sepsis and meningitis in two adults. H.influenzae non b type caused sepsis in one adult and sepsis and meningitis in one adult.

Conclusion

Once again the main cause of IBD at our hospital was S.pneumoniae. It was the main cause of sepsis in adults and in children. H.influenzae cases were recorded in adults and all were non-vaccinable type. N.meningitidis causes mainly sepsis in children and N.meningitidis group B was the predominant isolate. Therefore our focus could be on a meningococcal vaccine against N.meningitidis group B.

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Determination of Pneumococcal Serotypes in Meningitis Cases in Niger, 2003 – 2011

Abdel-Kader Alio Sanda, Jean-François Jusot, Bassira Alkassoum, and Jean-Marc Collard

Centre de Recherche Médicale et Sanitaire (CERMES), Niamey, Niger
Contact email: jmcollard@cermes.org

Keywords: pneumococcal meningitis, microbiological surveillance, sequential multiplex PCR, pneumococcal conjugate vaccine.

Abstract background

Invasive pneumococcal disease and particularly pneumococcal meningitis (PM) causes an enormous burden in sub-Saharan Africa. Little is known about the circulating serotypes of Streptococcus pneumoniae responsible for meningitis cases in Niger. Since 2003, a total of 1016 cerebrospinal fluids (CSF) have been detected positive for PM based on a lytA PCR assay but only a limited number of strains (about 10% of the collected CSF) were isolated on culture and were serotypable. In order to characterise the circulating pneumococcal serotypes responsible for meningitis cases in Niger prior to the introduction of a potential pneumococcal conjugate vaccine, we formulated a new multiplex PCR algorithm for serotyping pneumococci directly on refrigerated/frozen CSF.

Abstract methods

Serotyping was performed using the sequential multiplex PCR method (SM-PCR) described by Pai et al (1) with a national algorithm in which 32 different serotypes were grouped into eight PCRs. All PCRs included four primer pairs for different serotypes. The national algorithm was adapted according to the distribution of 86 pneumococcal strains isolated between 2003 and 2008 and serotyped by the useof the Quellung reaction (Hamidou AA, unpublished data).

Abstract results

Between 2003 and June 2011, the serotype of S. pneumoniae present in 740 CSF was determined (83 were non-recoverable, 68 were cpsA negative and 127 were non-typeable with the SM-PCR). In total, 26 different serotypes were identified. Serotype 1 (n=388) was the most prevalent and accounted for 44.8% of infections (non-typeable included), followed by serotypes 12F/(12A/44/46) (6.5%), 6/(6A/6B/6C) (5.2%), 14 (5.1%), 5 (4.6%), 23F (4.0%), 45 (3.6%), 2 (3.0%) and 18/(18A/18B/18C/18F) (2.8%).

Abstract conclusions

This study represents the first comprehensive report outlining the national baseline serotype distribution of PM in Niger before the introduction of conjugated vaccine against pneumococci. All years during the study period, serotype 1 was the most common and accounted from 34 to 53% of CSF investigated. This is a similar finding to that of the study carried out in Burkina Faso (2) and to a lesser extent to Malawi (3). This report shows the importance of a sustained and efficient microbiological surveillance of PM complemented by an accurate analytical methodology for determining pneumococci serotypes.

References

1. Pai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol. 2006;44:124-31.
2. Njanpop-Lafourcade B, Sanou O, van der Linden M, Levina N, Karanfil M, Yaro S, Tamekloe TA, Mueller JE. Serotyping of pneumococcal meningitis cases in the meningitis belt by use of multiplex PCR with cerebrospinal fluid. J Clin. Microbiol. 2010; 48:612-4.
3. Cornick JE, Everett DB, Broughton C, Denis BB, Banda DL, Carrol ED, Parry CM. Invasive Streptococcus pneumoniae in children, Malawi, 2004-2006. Emerg Infect Dis 2011; 17:1107-9.

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Epidemiology of Serogroup B Invasive Meningococcal Disease in Ontario, Canada, 2000-2010

Vica Dang1,3, Frances Jamieson1,3, Sarah Wilson1,2, Prasad Rawte1, Natasha S Crowcroft1,2, Karen Johnson1, and Shelley L Deeks1,2

1Public Health Ontario, Toronto, Canada,
2Dalla Lana School of Public Health, University of Toronto, Toronto, Canada,
3Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Canada

Keywords: Invasive Meningococcal Disease, Neisseria meningitidis, serogroup B, epidemiology, Ontario (Canada)

Objectives

The introduction of a meningococcal quadrivalent (serogroups A, C, Y and W-135) vaccine into Ontario’s publicly-funded immunization program has left serogroup B as the only invasive meningococcal disease (IMD) serogroup that is not yet vaccine preventable. Vaccines targeting this serogroup are close to being approved, which prompted an assessment of the epidemiology of serogroup B disease in Ontario, Canada. This is important to assess the potential impact of vaccines, as well as to provide baseline data prior to vaccine availability.

Methods

Information on confirmed IMD cases was retrieved from Ontario’s integrated Public Health Information System and linked to IMD laboratory records from Public Health Ontario’s Laboratory via probabilistic linkage. The surveillance period was from January 1, 2000 to December 31, 2010. Capture re-capture method was used to estimate sensitivity of the linked IMD data. Rates were calculated with denominator data obtained from Statistics Canada.

Results

A total of 713 IMD cases were identified over the 11-year period in either data source, of which 77.3% (551/713) were successfully linked.The sensitivity of linked IMD data was estimated to be 98.9% (713/721). Over the surveillance period serogroup B was the most common IMD serogroup (259/713, 36.3%); incidence ranged from 0.11 to 0.27 per 100,000 per year, and fluctuated over time. There was a male predominance (55.1%) and cases ranged in age from 13 days to 101 years, with a median age of 17 years. Age distribution was positively skewed; 21.4% (55/257) of cases occurred in infants (< 1 year), of which 70.9% were < 6 months of age. The highest incidence occurred in infants (3.70 per 100,000; 95% CI, 2.72 to 4.67), followed by children 1-4 years (0.66 per 100,000; 95% CI, 0.46 to 0.87). There was geographic variation in rates, with the incidence in Toronto (0.11 per 100,000; 95% CI, 0.07 to 0.15) significantly lower than both Central West regions (0.22 per 100,000; 95% CI, 0.17 to 0.28) and Eastern (0.22 per 100,000; 95% CI, 0.16 to 0.29). The case-fatality ratio (CFR)was 10.7% overall; individuals ≥ 65 years had the highest CFR (21.1%), followed by infants (13.5%). The most common antigenic variant was B:14:P1.14 (10.4%; 22/242).

Conclusions

Between 2000 and 2010, serogroup B was the most common cause of IMD in Ontario and infants had the highest risk of disease. These findings can aid in decision-making regarding serogroup B vaccination programs in Ontario, including considerations of target age.

The Epidemiology and Surveillance of Meningococcal Disease in England and Wales

Gray SJ1, Campbell H2, Marsh WJ3, Carr AD1, Newbold LS1, Mallard RH1, Guiver M1, Borrow R1, Ramsay M2 and Kaczmarski EB1.

1Health Protection Agency (HPA) Meningococcal Reference Unit (MRU), NW Regional HPA Laboratory, Manchester Royal Infirmary, Manchester M13 9WL, UK
2 Immunisation Department, Centre for Infections, HPA, Colindale, London, UK
3Virology-Molecular Department, Manchester Medical Microbiology Partnership, Manchester Royal Infirmary, Manchester, UK
Email: steve.gray@hpa.org.uk

Keywords: meningococcal disease, epidemiology, surveillance, serogroups, age groups

Background and Aims:

The HPA performs surveillance of invasive meningococcal disease for England and Wales to ascertain case numbers and characterise strains.

Materials and Methods:

Clinicians notify suspected cases of meningococcal meningitis/septicaemia to the Office for National Statistics. Microbiology laboratories submit isolates for phenotypic characterisation and since, October 2007, by porA sequencing. MICs of penicillin, cefotaxime, rifampicin and ciprofloxacin are determined. Clinical samples are submitted for non-culture detection and serogroup confirmation by PCR.

Results and Conclusions:

The increase in serogroup C cases from 1995-9 resulted in the introduction (November 1999) of serogroup C conjugate (MenC) vaccine into the UK population. Laboratory confirmed cases rose from 1,448 in 1995 to peak at 2,804 in 1999 falling to 917 in 2010. During 2010, 489 cases (53%) of invasive meningococcal disease were confirmed by PCR alone (comprising 16,607 samples representing 11,418 patients investigated by PCR). Serogroup B accounted for 88% of all confirmed cases (2010), 7% were serogroup Y (more than doubling from 28 cases in 2004 to 68 in 2010) whereas only 2% (20 cases) were confirmed as serogroup C and 3% serogroup W135. Phenotypic and genotypic shifts have been observed, specifically the relative contribution of clonal complexes ST-41/44, ST-269, ST-32, ST-213 and ST-11to meningococcal epidemiology.

Surveillance has demonstrated a sustained reduction in serogroup C infections since 1999 to 2005 remaining at <20 cases per year, a direct consequence of the MenC programme and resultant herd immunity. A small but detectable increase in serogroup Y cases is now being closely monitored.

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Modifications to a Published ctrA PCR Assay for the Improved Non-Culture Confirmation of Meningococcal Disease in England and Wales

Guiver M1, Corless CE2, WJ Marsh3, Gray SJ1, Newbold LS1, Borrow R1 and Kaczmarski EB1

1Health Protection Agency (HPA) Meningococcal Reference Unit (MRU), NW Regional HPA Laboratory, Manchester Royal Infirmary, Manchester M13 9WL, UK.
2Department of Microbiology, 7th Floor Duncan Building, Royal Liverpool University Hospital, Liverpool. L7 8XP, UK.
3Virology-Molecular Department, Manchester Medical Microbiology Partnership, Manchester Royal Infirmary, Manchester, M13 9WL, UK.
Contact email: malcolm.guiver@hpa.org.uk

Keywords: meningococcal disease, PCR, ctrA, non-culture, modified primers

Background and Aims:

The HPA MRU has used a realtime Taqman™ ctrA PCR to detect capsulated Neisseria meningitidis in clinical samples (non-culture confirmation) since 1997. In 2003 it was observed that a small number of culture proven cases were matched with clinical samples negative with the screening ctrA assay (Corless et al., 2001). It is with interest we note recent publications from other groups reporting the same phenomenon (Cavrini et al., 2010 and Jaton et al., 2010).

Methods and Results:

Nucleotide sequencing was performed on a 142bp region spanning the ctrA real-time PCR assay primer binding regions on 7 cultured isolates that were ctrA assay negative. The isolates were B:NT:P1.9 (4) B:NT:P1.5,2 (2) and B:NT:P1.7 (1) but all MLST CC ST-269.

The nucleotide sequence data identified 4 nucleotide substitutions in the reverse primer sequence (A702G, T705G, A708C and A717G) and a single substitution in the probe binding region (G606A) based on the full length ctrA gene sequence (AF520903). A modification of the published assay (Corless et al., 2001) to incorporate an additional reverse primer (5’-TTGCCGCGGATTGGCCACCA-3’) has been made to enable detection of this variant strain.

Results and Conclusions:

Since March 2003 approximately 80,000 samples have been screened with the modified ctrA assay and there have been no obvious culture proven only cases where appropriately matched clinical samples have been ctrA negative.

This is an illustration of the potential for the emergence of variants that may present problems in PCR based assays and highlights the need for continued surveillance of isolates.

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Austria 2005-2010, Epidemiology of Invasive Pneumococcal Disease

S. Heuberger1, A. Kormann-Klement1, U. Orendi1, Y-L. Liu2 und D. Schmid2

1National Reference Centre for Meningococci, Pneumococci and H.influenzae, Austrian Agency for Health and Food Safety, Graz, Austria
2Competence Centre for Infectious Diseases Epidemiology, Vienna, Austria
Contact email: sigrid.heuberger@ages.at

Keywords: Pneumococci, Epidemiology, Incidence, Disease

Background

The National Reference Centre for Pneumococci (NRCM) was founded by the Ministry of Health in 2004. The NRCM collects data on cases of invasive pneumococcal disease (IPD) for annual analysis. The heptavalent pneumococcal vaccine (PCV/7) was introduced 2004 and is offered free of charge to children in high-risk groups. The 23-valent polysaccharide vaccine (PPV23) is recommended for persons aged ≥ 65 years and older.

Methods

The IPD case definition is consistent with 2008/426/EG. The isolates referred to the NRCM are routinely serotyped by serum-agglutination. The statistical analysis of seasonal and long term trends by the yearly incidence of invasive pneumococcal disease in Austria was done with regression analysis.

Results

From 2005-2010 the incidence of invasive pneumococcal disease (IPD) increased from 1.57 to 3.87 cases / 100,000 population (2005: 1.57, 2006: 2.03, 2007: 4.22, 2008: 2.99, 2009: 3.54, 2010: 3.87). The cases with the clinical manifestation meningitis decreased from 23.3% in 2005 to 15.1% in 2010. After low case fatality rates in the years 2005, 2006 and 2007 (2.4%, 3.9%, 3.6%), an abrupt rise was seen in 2008 (14.07%) and 2009 (17.9%). In 2010 the case fatality rate declined to 7.9%.The age specific incidence shows in all years the typical international pattern with the highest incidence rates in the under one year and 80+ age groups. The lowest incidence was observed in the age group 10-14 years. Serotype data was available for 1168 invasive isolates. A total of 47 different serotypes were identified. The five most frequent serotypes were 3 (15.6%), 14 (10.1%), 1 (8.7%), 7F (7.0%) and 4 (5.6%). All other serotypes were under 5%. The PCV/7 covered serotypes decreased from 46% (50/108) in 2005 to 16% in 2010 (48/291). The additional serotypes in PCV10, PCV13 and PPV23 showed only minimal fluctuations. The non-vaccine serotypes increased from 7% (8/108) in 2005 to 15% (48/291) in 2010.

Conclusions

After introduction of PCV/7 an abrupt reduction of the incidence of IPD in the age group 0-5 years in 2006-2007 was registered. This was followed by a subsequent return to almost prevaccine levels in 2009-2010. In the 0-5 age group a steady rise in non-PCV7 serotypes from 2005 to 2010 and also a subtle recurrence of the PCV7 serotypes 2007-2010 is seen. These results must be interpreted with caution because the completeness of the serotype data and the sensitivity of laboratory surveillance varied in the years 2005-2010.

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Approaches to modelling serogroup A meningococcal disease in Africa

Tom Irving1,2,3,*, Caroline Colijn4, Konstantin Blyuss5, Caroline Trotter2

1 Bristol Centre For Complexity Sciences, University of Bristol
2 School of Social and Community Medicine, University of Bristol
3 Department of Engineering Mathematics, University of Bristol
4 Department of Mathematics, Imperial College London
5 Department of Mathematics, University of Sussex
* Canynge Hall, 53 Whatley Road, Bristol, BS8 2PS. Email tom.irving@bristol.ac.uk

Keywords: Africa, meningococcal disease, modelling, meningitis belt, epidemics

The causes of the frequent but unpredictable large epidemics of meningococcal disease that affect the African meningitis belt are poorly understood, but are likely to be a combination of climatic effects and waning population immunity. With the ongoing introduction of the serogroup A vaccine in sub-Saharan Africa, it is important to be able to gain insights into the pattern of these epidemics and to investigate how they might change.

Mathematical modelling has been widely used to gain insights into infectious diseases and to help formulate vaccination policy. We outlineour past and future modelling approaches. Deterministic models suggest that population immunity is more important than climatic factors in causing epidemics. Properly parameterised stochastic models will be used to further investigate these findings.

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Epidemiology of Invasive Meningococcal Disease in Moscow, 2005-2010

T.A. Maxina1, I.S. Koroleva1 I.M. Zakroeva1, G.V. Beloshitsky1, I.N. Lytkina2, and A.P. Pyaeva2

1Federal Center for Surveillance on Bacterial Meningitis, Central Research Institute of Epidemiology, Moscow, Russia
2Directorate of Federal Service on Surveillance for Consumer Rights Protection and Human Well-Being for Moscow, Russia
Contact email: irina-korol@yandex.ru

Keywords: surveillance, morbidity, mortality, meningococci, serogroup

Invasive meningococcal disease (IMD) remains a significant problem for public health in Moscow. Twice during the last 15 years, (in 1996and 2003) a significant increase in IMD morbidity was observed in the city with respective incidence 3.8 and 3.6 per 100,000 population. Official records on IMD cases in Moscow were retrospectively analyzed for the period 2005-2010.

From 2005 to 2010, 1348 cases of IMD were identified (with an incidence of 2.1 per 100,000 people in 2005, 2.5 in 2006, 2.3 in 2007, 2.8 in 2008, 1.9 in 2009, 1.3 in 2010). Mortality from IMD was characterized by the absence of down and up trends. In the studied period 114 deaths due to IMD were registered (case-fatality ratio: 7.2% in 2005, 11.7% in 2006, 11.1% in 2007, 4.4% in 2008, 6.5% in 2009, 11.1% in 2010). A seasonal rise of IMD incidence was observed with the peak during winter-spring period. The proportion of laboratory-confirmed IMD cases in Moscow from 2005 to 2010 was in the range of 52 to 67%. One of the important parameters of epidemiologic surveillance for IMD is serogroup distribution. During the studied period mainly meningococci of serogroups A, B, and C were identified in laboratory cnfirmed IMD with predominance of serogroup A (except 2009), although meningococci of serogroups Y, Z, W-135 were also detected in some cases. Unlike other years, in 2009 IMD incidence caused by meningococci of serogroups A and C was nearly equal. IMD incidence among children ≤14 years of age in Moscow (7.5 per 100,000) was five times higher than in persons over 15 years of age (1.5 per 100,000). Average annual incidence of IMD in males exceeded those in females (1.3 and 0,9 per 100,000, respectively). Comparison of morbidity between children not attending day care centers (not organized), attending day care centers (organized), and schoolchildren showed that during the studied period IMD incidence in not organized children was 5.3-fold higher than in the organised children (0.63 and 0.12 per100,000 of the cohort, respectively) and 2.3-fold than in schoolchildren (0.27 per 100,000 of the cohort).

Introduction of mass vaccination with meningococcal conjugated vaccines (MCV) in the USA and Europe led to a dramatic reduction in IMD incidence. There is a rationale for use of multivalent MCVs in Russia due to substantial proportion of children below 2 years of age in the total number of IMD cases as well as the dominance of A and C and presence of W-135 and Y serogroups in the structure of laboratory confirmed cases.

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The epidemiology of invasive meningococcal disease in Scotland 1999-2010

Eisin McDonald1, Barbara Denham2, Alison Smith-Palmer1, Giles Edwards2, Claire Cameron1

1Health Protection Scotland, Meridian Court, 5 Cadogan Street, Glasgow, G2 6QE
2The Scottish Haemophilus Legionella Meningococcus and Pneumococcus Reference Laboratory (SHLMPRL), Stobhill Hospital, 133 Balornock Road, Glasgow, G21 3UW

Contact email: eisin.shakir@nhs.net

Keywords: Epidemiology, Meningococcal disease, Serogroup B, Clinical presentation, Case fatality ratio

Meningococcal disease is a notifiable disease caused by infection with the bacterium Neisseria meningitidis. It is a significant cause of morbidity and mortality, particularly in children and young people. Meningococcal Invasive Disease Augmented Surveillance (MIDAS) was introduced in Scotland in 1999 to monitor the impact of the meningococcal C vaccine. This poster presents the epidemiology of invasive meningococcal disease in Scotland from 1999-2010 and discusses the potential implications for meningococcal B vaccine introduction.

There has been an average of 190 cases of meningococcal disease (3.4 cases per 100,000 population) reported each year in Scotland, including both clinical and laboratory confirmed cases. This has decreased steadily from 350 cases (6.8 cases per 100,000 population) in 1999 to 99 cases (1.9 cases per 100,000) in 2010. Approximately half of all cases reported are in children under five years of age (1083/2286; 47.4%) and the highest incidence is observed in children under one year of age with an average 82.8 cases per 100,000 population.

Laboratory confirmed cases of invasive meningococcal disease submitted to the Scottish Haemophilus, Legionella, Pneumococcal and Meningococcal Reference Laboratory (SHLMPRL) are routinely serogrouped. Serogroup B has been the most commonly detected serogroup in Scotland (903 cases), followed by C (208 cases), Y (42 cases) and W135 (34 cases).

The most commonly reported clinical presentation for cases was meningitis (799 cases; 35%), followed by septicaemia (641 cases; 28%) and both meningitis and septicaemia (387 cases; 17%). There were 136 deaths reported in the time period equating to an overall case fatalityratio (CFR) of 5.9%. However, CFR was found to vary by age and meningococcal serogroup.

In summary, meningococcal disease has declined in recent years in Scotland but remains a significant source of morbidity and mortality, especially among young children. As group B disease accounts for around three quarters of laboratory confirmed cases, any ability to prevent these infections could have a substantial impact on overall incidence of disease.

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Bacterial meningitis in babies 0-90 days of age: a UK and Republic of Ireland prospective study

I.O. Okike¹, K. Henderson², R. Blackburn², B.Muller-Peabody², A. Johnson², P.T. Heath¹

¹Child Health and Vaccine Institute, St George’s, University of London
²HCAI & AMR Department, Health Protection Services Colindale, HPA

Keywords: bacteria, meningitis, neonates, burden, management

Background and aims

Meningitis in the first 3 months of life is associated with significant mortality and morbidity. Previous UK studies were conducted in the 1980s and 1990s.

It is important to define the current burden of disease and how they are being managed and identify any opportunities for improvements in diagnosis and management in order to prioritise treatment and prevention strategies and establish an evidence based management guideline.

Methods

Cases are identified prospectively by active surveillance through the British Paediatric Surveillance Unit (BPSU), routine microbiological surveillance through the Health Protection Agency, and via parents of cases through meningitis and Group B streptococcus (GBS) support charities. The surveillance period is July 2010 – July 2011 (burden of disease study) and September 2011- March 2012 (healthcare delivery study-HCD).

Only cases notified outside the standard BPSU methodology in England are used for the healthcare delivery study. This study is based on a detailed medical notes review and parent-completed questionnaire. With parental consent, recruited babies will also be contacted at two years of age for a neurodevelopmental assessment.

Results

Over the first 10 months of the study 213 cases meeting the case definition of bacterial meningitis were reported; 189 (88.7 %) and 9(4.2%) from England and Wales respectively. 130 (61%) were male and the median age of disease was 14 days (range 0-88). 123 isolates were obtained from the cerebrospinal fluid; GBS (63, 51.2%) was the most common bacterium followed by Streptococcus pneumoniae (12, 9.8%), Neisseria meningitidis (9, 7.3%), Escherichia coli (9, 7.3%) and other Gram positives (12, 9.8%). 35 (16.4%) had CSF pleocytosis, a negative CSF culture but a positive blood culture, while 58 (27.2%) had clinical signs, CSF pleocytosis (≥20 or ≥10 cells/mm3 for babies≤28 days or 29-90 days respectively), but no organism identified.

Conclusions

There remains a significant burden of meningitis in the first 3 months of life which has not changed over the last 3 decades. New strategies for prevention are required.

The HCD study will provide data on the timing of events (symptoms/ signs) leading up to the diagnosis of bacterial meningitis in babies 0-90 days old in England. Both studies will contribute to new guidelines on the management of neonatal meningitis.

Funded by Meningitis Research Foundation.

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Continuing increase in 19A and 6C serotype carriage in children after the introduction of PCV-7 in the Netherlands

S.M.P.J. Prevaes1,2, N. Y. Rots3, J. Spijkerman1,2, E.J.M. van Gils1,2, R.H. Veenhoven2, A.J.Wijmenga-Monsuur3, D. Bogaert1, J.P. Bruin4, E.P. IJzerman4, G.P.J.M. van den Dobbelsteen3, E.A.M. Sanders1

1Department of Pediatric Immunology and Infectious Diseases, Wilhelmina Children Hospital, University Medical Center, Utrecht,
2Research Center Linnaeus Institute,
3National Institute of Public Health and the Environment, Bilthoven,
4Regional Laboratory of Public Health, Haarlem The Netherlands

Keywords: pneumococcal vaccine, carriage, The Netherlands, subtypes, 19A

Background

Pneumococcal conjugate vaccine (PCV-7) was implemented in the Dutch NIP in 2006. We studied the ongoing effect of PCV-7 introduction on nasopharyngeal pneumococcal carriage over time.

Methods

In follow-up of a randomized controlled trial (RCT) (NCT00189020), we performed two cross-sectional studies in 2009 and 2010 collecting nasopharyngeal swabs at 11 and 24 months of age from PCV-7 vaccinated children and from one of the parents of the latter group (n 330 per group). Swabs were cultured for S. pneumoniae and serotyped by Quellung.

Results

In 11-month-olds PCV-7 carriage rates were 38%, 8% and 3% in 2005, 2009 and 2010, respectively, whereas in 24-month-olds vaccine serotype carriage was 36%, 3% and 3% (p<0.001 for all comparisons vs. 2005). Carriage of non-PCV-7 serotypes increased in 11-month olds from 29% in 2005 to 39% and 50% in 2009 and 2010, respectively (p<0.01 for all comparisons vs. 2005) and from 30% to 45% and 61% in 24-month-old children (p<0.001: vs. 2005). A significant increase in carriage of 19A was found in 11 and 24-month-old children over time (11 months: 2%, 10% and 12%, 24 months: 3%, 6% and 14%, respectively). Moreover, the latter serotype also became the most dominant serotype. Besides, 6C carriage increased as well as other non-vaccine serotypes over time. In parents a similar shift of serotypes was seen.

Conclusions

Within 4.5 years after introduction of PCV-7, vaccine serotypes are virtually eliminated in children and their parents. However, non-PCV-7 serotypes, in particular 19A, have emerged. Pneumococcal disease surveillance remains important.

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Seroprevalence of serum bactericidal antibodies against group W135 and Y meningococci in England in 2009

Caroline Trotter1, Helen Findlow2, Helen Chadha2, Kelly Townsend2, Dani Thompson2, Lesley Mabey2, Stephanie Slater2, Stephen Clark2,Kate Nolan2, Ray Borrow2

1School of Social and Community Medicine, University of Bristol, Bristol, UK
2Vaccine Evaluation Unit, Health Protection Agency North West, Manchester, UK

Keywords: seroprevalence, serum bactericidal antibody activity, N meningitidis group Y, N meningitidis group W135, UK

Background and aims

Serological surveillance has been used in the United Kingdom to inform vaccine policy for several infections, including group C meningococci. Quadrivalent meningococcal conjugate vaccines, containing groups A, C, W135 and Y, are now available. The aim of this study is to establish a baseline for immunity for groups W135 and Y in England.

Methods

Serum samples collected in 2009 from individuals across all ages (N=1191) were obtained from the Health Protection Agency Seroepidemiology Unit, which collects residual sera from participating laboratories across the country. Serum bactericidal activity (SBA) against serogroup Y (strain M03 241125) and W135 (strain M01 240070) was determined using a standardized complement-mediated SBA assay, with complement derived from baby rabbits. The age-specific geometric mean titres (GMTs) and percentage of individuals with SBA ≥8 (the putative protective level) were calculated, together with 95% confidence intervals.

Results and conclusions

Antibody prevalence varied according to age and serogroup. In general, titres were low in younger children with 7% and 13% of children under 5 years achieving titres ≥8 against group W135 and Y respectively. GMTs peaked in 20-24year olds for group W135 (GMT= 7.1 95% CI 4.7, 10.9) and in 30-44 year olds for group Y (GMT=8.6, 95% CI 5.9, 12.7). Unlike seroprevalence against group B meningococci, there was not an obvious peak in titres in teenagers.

Natural immunity against group W135 and Y meningococci in England is low.

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Clinical Diagnosis, Treatment and Sequelae

Oculomotor nerve palsy secondary to childhood bacterial meningitis: a systematic literature review of outcome and management effectiveness

Archer, J1 and Brown, R2

1Foundation Year Two, Paediatrics, Peterborough City Hospital, UK
2Consultant Paediatrician, Peterborough City Hospital, UK

Keywords: Bacterial meningitis, oculomotor nerve palsy, neurological sequelae, paediatric, amblyopia

Background

Oculomotor nerve palsy is a recognised sequela of meningitis reported in 3% (123/4102) of cases. We were struck by the lack of literature available to guide best practice management and inform parents regarding prognosis. Reviews of oculomotor palsies and of sequelae post bacterial meningitis use categories such as ‘post-infectious’ aetiologies or ‘neurological sequlae’. Potentially valuable information is lost.

Objectives

To evaluate:
  1. In patients ≤ 16 years what is the course and outcome of oculomotor nerve palsy secondary to bacterial meningitis?
  2. To what extent can different management strategies influence the above?
Method

A review was conducted of English language reports which included information of oculomotor palsies secondary to bacterial meningitis for subjects ≤16 years at diagnosis. Studies of encephalitis and aseptic meningitis were excluded. Papers were identified from an electronic database (OvidMEDLINE(R) 1950 to June 2011) and reference lists. The inclusion of descriptive studies was necessary. Due to heterogeneity of studies, calculating a summary estimate of effect was not possible.

Results and Conclusions

8 reports (9 cases) were reviewed. Data from our unpublished case report was included.

In all but two cases (6/8) the oculomotor palsy completely resolved. Time to resolution was a median 46 days (range 5 days to 1 year). This suggests it is largely a short-lived resolving sequela.

The time to onset of the oculomotor palsy post symptoms’ onset was a median 4 days (range 2 to 11 days). Five cases proceeded to CT head of which four were initially normal. Palsies were all unilateral. 5/7 were partial.

Pupillary involvement was inconsistent. Non-reactive mydriasis may be a sign of impending tentorial herniation, suggesting the need for a low threshold for ICP management. Some cases did not show other signs of raised ICP, supporting suggestions that nerve encasement by subarachnoid space exudate and vasculitis are also causes to target treatment.

Cranial nerve palsies and pupillary involvement are significantly associated with death and sequelae at follow-up suggesting that oculomotor palsy should prompt management escalation.

Amblyopia is common in oculomotor palsy. Cases respond to part-time patching. This highlights the need to remember the risk of amblyopia in the susceptible age-group.

Post bacterial meningitis, while results are inconsistent a meta-analysis suggests adjuvant dexamethasone may reduce long-term neurologicaldeficits and a double-blind placebo-controlled trial suggests oral glycerol may reduce neurological sequelae.

The small sample size limits conclusions, suggesting the need for further study including contacting authors for unpublished data.

Angular deformity of the ankle with distal fibula sparing following meningococcal septicaemia

F Monsell, A McBride, J Barnes, R Kirubanandan

Bristol Royal Hospital for Sick Children, Bristol, UK

Keywords: Meningococcal Septicaemia, deformity, ankle, physis, varus deformity

Progressive angular deformity of an extremity due to differential physeal arrest is the most common late orthopaedic sequelae following meningococcal septicaemia in childhood. 10 patients (14 ankles) with distal physeal arrest as a sequelae of meningococcal septicaemia have been reviewed. Radiographic analysis of their ankles has demonstrated a distinct pattern of deformity. In 13 out of 14 cases we have found that the distal fibula physis is completely unaffected and that continued distal fibula growth contributed to a varus deformity. We recommend that planning of surgical management should take in to account of this consistent finding during correction of these deformities.

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Distal tibial growth arrest following meningococcal septicaemia; management and outcome in a series of seven ankles

F Monsell, J Barnes, A McBride, R Kirubanandan

Bristol Royal Hospital for Sick Children, Bristol, UK

Keywords: Meningococcal Septicaemia, deformity, external fixator, growth arrest, Limb alignment

Survivors of infantile meningococcal septicaemia often develop progressive skeletal deformity as a consequence of physeal damage at multiple sites, particularly in the lower limb. Distal tibial physeal arrest typically occurs with sparing of the distal fibular physis leading to a rapidly progressive varus deformity. Isolated case reports include this deformity, but to our knowledge there is no previous literature that specifically reports the development of this deformity and potential treatment options.

We report our experience of 6 patients (7 ankles) with this deformity, managed with corrective osteotomy using a programmable circular fixator.

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Development of a tmRNA based NASBA Assay for the detection of Neisseria meningitidis

Eoin Clancy1,2,3, Helena Coughlan1,2,3, Terry Smith2,3 and Thomas Barry1,2,3

1Microbiology, School of Natural science, National University of Ireland, Galway, Ireland
2Molecular Diagnostics Research Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
3Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

Keywords: Bacterial meningitis; Molecular diagnostics; tmRNA; NASBA; μTAS

Failure to promptly diagnose and treat bacterial meningitis can result in significant morbidity and mortality. Bacterial meningitis is most commonly caused by one of three bacteria; Haemophilus influenzae type b (Hib), Neisseria meningitidis, and Streptococcus pneumoniae.

A culture-independent means of rapid, low-cost multiplexed nucleic acid sequence-specific detection of these three bacteria would have a major impact, ultimately saving many lives and potentially reducing the use of broad spectrum antimicrobial agents to treat this disease. We have recently demonstrated the potential of the ssrA gene and its corresponding RNA transcript, tmRNA, as a molecular based target for the specific detection of a number of bacterial pathogens[1-3].

We are currently developing a suite of tmRNA isothermal in vitro amplification assays capable of detecting and differentiating the three main causative bacteria associated with bacterial meningitis. Once validated, these assays will be integrated onto a micro- Total Analysis System (μTAS) that will separate, capture, and detect the bacterial meningitis-specific targets from whole blood samples in less than one hour. In this poster, we demonstrate the development and use of a tmRNA based isothermal NASBA in vitro diagnostics assay for the sensitive and specific detection of N. meningitidis.

1. O'Grady, J., et al., tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA. Fems Microbiology Letters, 2009. 301(2): p. 218-223.
2. McGuinness, S., et al., Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat. Meat Science, 2009. 83(3): p. 555-562.
3. Wernecke, M., et al., Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs. BMC Infectious Diseases, 2009. 9: p. 148-156.

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Meeting the health, social and educational needs of children who have survived meningitis and septicaemia: the parents perspective

Laura Clark1,2, Linda Glennie2, Suzanne Audrey1, Caroline Trotter1

1School of Social and Community Medicine, University of Bristol
2Meningitis Research Foundation, Bristol
Contact email: laura.J.Clark@hotmail.co.uk

Keywords: children, aftercare, needs, meningitis, parent

There is little published research on the needs and provision of aftercare for children surviving meningitis and septicaemia. To investigate this further we used a mixed methods study design, employing a survey and in-depth follow-up interviews of a sample of survey participants. Participants were recruited from Meningitis Research Foundation’s member database and social media sites. Eligible participants were parents of children who survived meningitis or septicaemia between January 2000 and May 2010, living in the UK or Ireland. In stage one, participants completed a multiple choice questionnaire, either online or by post. In stage two, twenty participants were invited to take part in a follow-up interview, based on their answer to the survey question, ‘overall, to what extent does / did the aftercare and support received meet your child's needs?’ Interviews were designed to explore the factors affecting parents’ opinions of whether their child’s needs for aftercare had been met. Here we report on findings from follow-up interviews with 18 parents. We identified three main themes from interviews; accessing services, communication, and relevance or appropriateness of services. Our findings lend support to the NICE guideline recommending a follow-up appointment with a paediatrician 4-6 weeks after discharge and go further in identifying a need for parental support to cope with an often uncertain long term prognosis for their child. Difficulty navigating services emerged as an important issue and an understanding of the link between meningitis and cognitive and psychosocial problems amongst educationalists remains inadequate, so improvements in these areas would contribute to meeting the needs of parents and children.

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Recognition and action to tackle acute bacterial meningitis in urban Blantyre, Malawi

N Desmond1,2, D Nyirenda1, E Molyneux3, M Mallewa1,2,3, D Lalloo2, R Heyderman1

1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi
2Liverpool School of Tropical Medicine, UK
3Queen Elizabeth Central Hospital, Blantyre, Malawi
Contact email: ndesmond@liverpool.ac.uk; nic.desmond@gmail.com

Keywords: Acute bacterial meningitis, qualitative, recognition, action, Malawi

Background

More than one million cases of acute bacterial meningitis occur annually in sub-Saharan Africa including Malawi. Late presentation has been identified as a major contributor to high case fatality rates since prompt treatment is vital to effective management. We present findings from a Meningitis Research Foundation (MRF) funded cross-sectional study to explore treatment seeking pathways and reasons for late presentation in adults and young children at Queen Elizabeth Central Hospital (QECH) in urban Blantyre, Malawi.

Methods

We conducted 16 in-depth case history interviews (IDI) with carers and patients of adults and children purposively sampled by outcome. We held four focus group discussions (FGDs) with communities in QECH catchment areas. Following interim analysis we conducted 20 semistructured interviews with randomly selected Health Care Workers (HCW) stratified proportionally by cadre.

Results

Recognition

Meningitis was commonly misdiagnosed at household level, often as malaria leading to prolonged periods of self-treatment or common indigenous illness requiring traditional medicine. Misdiagnoses at primary care level meant discharge with inappropriate treatment and deterioration of health status prior to re-presentation and subsequent delayed referral.

Barriers to prompt action

Treatment seeking decisions were embedded in social norms. Childhood illness was recognised earlier and men were slowest to acknowledge treatment need. Treatment seeking was delayed by economic constraints, perceptions of accessibility, long waiting times, erratic medical supplies and mistreatment by health providers. Treatment with chloramphenicol and referral followed later stage, emergency diagnosis. Policy priorities to reduce maternal mortality have had severe negative repercussions on other emergency referrals.

Conclusion

The prevention of meningitis is dependent on reactive, secondary prevention through prompt treatment seeking. There is need to develop and evaluate public health campaigns to address meningitis in Malawi, drawing on these findings and successful approaches implemented elsewhere.

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Bacterial Meningitis In Adults And Dexamethasone: A 10 Year Review From Aarhus, Denmark

Schou K, Damsgaard J, Soegaard O, Oestergaard L, Leutscher P, Laursen AL, Deutch S

Department of Infectious Diseases Q, Aarhus University Hospital, Skejby, 8200 Aarhus N

Keywords: Bacterial meningitis, dexamethasone, outcome, treatment, adults

Introduction including objectives

Patients suffering from bacterial meningitis have a substantial risk of sequelae and death. Since 2002, treatment with adjunctive intravenous dexathasone has proven beneficial for the outcome of bacterial meningitis in adults.

The aim of the study was to assess adjunctive intravenous dexamethasone in adult community-acquired bacterial meningitis (BM) in daily practice.

Furthermore, to compare the outcome of these patients (dexamethasone vs no dexamethasone) regarding the causative agents, clinical manifestations, neurological status, and the course of the disease.

Materials and methods

We reviewed the reports of all adults with BM admitted to Aarhus University Hospital from January 1999 to December 2008. Sociodemographic and clinical data were extracted from the patients’ medical files for further in-depth analysis. In particular, we assessed the risk of complications, mortality and use of antibiotics and dexamethasone.

Results

A total of 159 were diagnosed with BM. Twenty seven percent (n=43) received dexamethasone. Of those patients without dexamethasone, only 13.9% (n=16) had received antibiotics before admission. In comparison, the mortality was non-significantly increased for those treated with dexamethasone (16.3% (n=7)) compared with patients without dexamethasone treatment (14.6% (n=17)). In addition, a nonsignificant increase was found in the risk of complications, intensive care treatment and sequelae. Detailed data analysis are pending.

Conclusion

We found no substantial benefit of adjunctive dexamethasone treatment in adults with BM. Empirical use of dexamethasone in this setting appears controversial. In addition, a limited amount of adults had received dexamethasone.

Funded by Aarhus University Research Fund.

How sick is this child? Predicting serious infections using four vital signs

Susannah Fleming1, Matthew Thompson1,2, Lionel Tarassenko3

1Department of Primary Health Care, University of Oxford, Oxford, UK
2Department of Family Medicine, Oregon Health and Sciences University, Portland, USA
3Institute of Biomedical Engineering, University of Oxford, Oxford, UK
Contact email: susannah.fleming@phc.ox.ac.uk

Keywords: Vital signs, prediction, infection, data fusion

Introduction

Vital signs are known to be predictive of serious illness in children, but measuring them accurately and interpreting their values poses considerable challenges in primary and emergency care. Combining vital signs in a simple score may be a more useful and practical way of identifying children with serious illness.

Method

An existing dataset containing heart rate, temperature, respiratory rate and oxygen saturation measurements from 568 children admitted to a paediatric assessment unit with a suspected infection. The outcome measure used was the diagnosis of a serious infection, which was likely to be life-threatening if untreated, or which had a high chance of life-threatening complications or sequelae.

Classification was carried out using the heart rate, temperature, respiratory rate and oxygen saturation elements of an existing paediatric scoring system (PAWS), designed for use in secondary care. In addition, a variety of data fusion models, including regression and distribution modelling, were developed using the same four vital signs. Jack-knifing was used to obtain accurate estimates of model quality.

Results

The existing scoring system had a potential range of 0-12 points, and showed best separation of the data when a score ≥2 was used to identify serious infection. This gave a sensitivity of 73.1%, and specificity of 49.5%. The area under the ROC curve was 0.64, which was below the 10th percentile for area under the ROC curve in the four best-performing data fusion models.

The best-performing model produced a median area under the ROC curve of 0.69 (10th-90th percentile 0.66-0.72), with an optimal sensitivity of 64.6% (60.6-68.2) and specificity of 65.7% (61.6-69.7). This method fitted a four-dimensional Gaussian distribution to the vital signs data from each group, allowing the probability of membership into each class to be calculated for each child.

Conclusion

Data fusion of four vital signs (heart rate, temperature, respiratory rate, and oxygen saturation) provides moderate accuracy for predicting serious illness in children, and outperforms existing scoring systems. Although this type of score cannot easily be calculated by hand, it could be incorporated into commonly-available handheld phone applications, or into an integrated device incorporating a thermometer and pulse oximeter. Such a device would require a means of entering the patients age, as the technique uses evidence-based curves to allow for the normal variation of heart rate during childhood, and would also require entry of manually-measured respiratory rate.

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Purpura Fulminans in Childhood Meningococcal Septicaemia

Robert Fuller, Sian Falder

University of Liverpool, Liverpool, UK L69 3BX
Dept of Plastic Surgery, Alder Hey Children’s NHS Foundation Trust, Eaton Road, Liverpool L12 2AP

Keywords: Septicaemia, Purpura Fulminans, APTT, Amputations, Audit

Introduction

Acute infectious purpura fulminans (PF) occurs in severe acute sepsis and is most commonly associated with gram negative bacteria, particularly Neisseria meningitidis. It is characterised by progressive and patchy cutaneous haemorrhage leading to tissue death as a result of vascular necrosis and disseminated intravascular coagulation. PF has been associated with higher mortality, cosmetic deformities and multiple amputations. t-PA has been shown to reduce incidence of amputations but has high rate of intra-cerebral haemorrhage. There is controversy over the use of fasciotomies.

Aims

(1) To audit all cases of meningococcal septicaemia and PF in Alder Hey since 2007
(2) To ascertain whether there are any factors or early signs that may assist in early diagnosis of purpura fulminans or predict its development
(3) To compare our surgical management and outcomes of purpura fulminans to those of published literature.

Methods

This is a retrospective study of all patients admitted with suspected meningococcal septicaemia from 1st January 2007 to 31st May 2011. Electronic patient care records and laboratory data were retrieved to identify those patients who developed PF. Blood test results from days 1-7, medical and surgical interventions and outcome were reviewed.
 
125 patients were treated as suspected meningococcal sepsis. The mean age was 3 years 4 months (range was 1 month 24 days to 17 years; median 2 years 1 month). The number of admissions per year with meningococcal septicaemia decreased from 32 cases in 2007 to 23 cases in 2010, with 9 cases in the first 5 months of 2011. Mortality for meningococcal septicaemia was 4/125 (3.2%). Sixteen patients of the 125 developed PF (12.8%). Of these 16, 7 had surgical amputations (43.8%). None of these patients died. No patients underwent fasciotomy. The causative organism was N. meningitidis group B in 10 patients, and was unknown in the remaining 6. The study found that patients admitted who went on to develop PF had a significantly higher mean APTT on day one (95.6) compared to patients who did not develop PF (38.6).

Conclusions

(1) The number of cases of meningococcal septicaemia admitted to Alder Hey is decreasing
(2) Both the mortality and amputation rates at Alder Hey Hospital compare favourably with published rates
(3) Day one APTT may be useful in identifying patients who are at risk of developing PF and who may respond to an early intervention or be a suitable group for future prospective study into PF.

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Risk Evaluation and Plan to Prevent Meningococcal Infections in Eculizumab-treated Individuals

Lucia Lee1, Therese Cvetkovich2, Robert Kane2

1Center for Biologics Evaluation and Research,
2Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland, USA

Keywords: eculizumab, safety, meningococcal, vaccine, post-marketing

Background

Eculizumab is a monoclonal antibody that inhibits complement component C5. Eculizumab-treated individuals are at increased risk of developing invasive meningococcal disease due to drug-induced terminal complement deficiency. In 2007, eculizumab was approved by EMA and FDA for treatment of paroxysmal nocturnal hemoglobinura (PNH), and is being reviewed for treatment of atypical hemolytic uremic syndrome (aHUS).

Objective

To review the current post-marketing safety requirements for meningococcal disease prevention in eculizumab-treated individuals, and describe characteristics of identified disease cases.

Method

Published articles and product approval summaries were reviewed for meningococcal disease cases in clinical trials. Post-marketing reports of cases through the year 2010 were identified from the FDA Adverse Event Reporting System.

Results

As part of the risk management strategy for preventing invasive meningococcal infections, all patients treated with eculizumab must be vaccinated with an age appropriate meningococcal vaccine beginning at least 2 weeks before initiating eculizumab, and patients are to be revaccinated according to country-specific recommendations for individuals with late complement component deficiencies. Eculizumabtreated patients are provided a Patient Safety Card that describes possible signs and symptoms of invasive meningococcal infection for which the patient should seek immediate medical attention.

Of nineteen identified pre-licensure trial/post-marketing cases of meningococcal disease, the median age was 24 years (range 15–54). All but one individual had received a meningococcal vaccine as follows: tetravalent conjugate (n=4) or polysaccharide (PS, n=6), bivalent/monovalent PS or conjugate (combined n=8), and in n=3 the type of vaccine was not identified. The median time from first eculizumab dose to event onset was 5 months. Of 13 cases with known serogroup, seven were due to serogroup B. In 37 aHUS patients, no meningococcal infections occurred during the 26 weeks of eculizumab treatment. No cases of recurrent invasive meningococcal infection among eculizumab treated patients have been identified to date.

Conclusions

If approved, increased use of eculizumab for the treatment of aHUS would include greater numbers of pediatric patients. Invasive meningococcal infections can be anticipated among eculizumab-treated patients due to drug-induced complement deficiency, vaccine failure, and the lack of safe and effective licensed meningococcal serogroup B vaccines. The availability of multivalent infant meningococcal PS conjugate vaccines and serogroup B vaccines would optimize current strategies for meningococcal disease prevention.

In suspected cases of meningitis, do we need a CT scan before doing a lumbar puncture?

Wail Seleem1, Khalid Ibrahim1, Ahmed Sotouhi1, Hoda Badran1, Farooq Abdul-Aziz2, Nahla Sharf1

1Hamad Medical Corporation, Doha, Qatar
2Aspetar Medical Center, Doha, Qatar

Keywords: meningitis, CSF, lumbar puncture, CT head

In young children with suspected meningitis, lumbar puncture (LP) forms an important component of the diagnostic and therapeutic process. However, pre-request for CT scan can cause undesirable delay in effective treatment.

Aim

To find out if doing CT scan before lumbar puncture affects the clinical decisions in cases of suspected meningitis including the initiation of treatment.

Methodology

We retrospectively reviewed charts of children who were admitted with a diagnosis of meningitis between the age of 18 months and 14 years over a two year period in our tertiary hospital. A neuroradiologist subsequently reviewed all scans.

Results

A total of 116 cases were included, 79 (68.1%) were boys and 37 (31.9%) girls. 55.2% were in the age group 5 – 9 years, 25% were under 5. Mean age was 6.3±2.7 year. The majority (68.1%) were from the Middle East and 110 (94.8%) cases had LP done. CSF studies showed that 104 (92.8) cases were aseptic meningitis, 10 were bacterial and 2 were due to mycoplasma infection.

Only 6 scans showed abnormalities. 2 scans showed diffuse brain oedema; one suggested possible uncal herniation and one showed dilatation of the ventricular system. One scan showed mild brain oedema and one mild ventricular dilatation and in both cases LP was done.

No specific symptom or sign could predict the scan result with fever and vomiting occurring in all 6 cases.

On average LP was done 8.9 hours after presentation (CI 7.7 – 10.1).

CT scans were done 7.7 hours after presentation (CI 5.5 – 9.9).

Antibiotics were started 6.3 hours after presentation (CI 5.3 – 7.3).

No serious complication related to LP was reported in any of the cases.

Conclusion

CT scan is widely used in cases of children with suspected meningitis to decide on the safety of performing LP. The yield of the scans in these cases is very low. Performing the scans has resulted in significant delays in performing the LP and most importantly in initiating antibiotic therapy, a practice which can have serious consequences to the outcome of these cases.

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Public Health Management

Meningitis in the Facebook generation: Public health out of hours management of a complex meningitis case in a teenager

Charlotte Ashton and Barry Walsh (joint presenting authors)

South West London Health Protection Unit, Health Protection Agency, London, England

Keywords: Public Health; out of hours; contact tracing; anxiety; teenager

The Health Protection Agency provides 24/7 public health management of cases of infectious diseases including meningitis. Two of the salient actions following a probable or confirmed case of bacterial meningitis is to identify close ‘household’ contacts and arrange chemoprophylaxis for these individuals. The second is to manage concerns of contacts of the case who do not need antibiotics but are worried about becoming unwell.

The aim of this piece of work is to share the response and lessons learnt by SW London HPU (SWLHPU) from the management of a meningococcal meningitis case with multiple close contacts over a weekend.

SWLHPU received a call from a microbiologist on a Saturday lunchtime during the summer of 2011, notifying a case of probable bacterial meningitis in a teenager who had been admitted to ITU. As well as a number of close family contacts requiring prophylaxis we were informed that the case had recently returned from a holiday sharing an apartment with an extended group friends – all of whom required prophylaxis. There was a high level of anxiety particularly as several of the close contacts had meningitis like symptoms.

Sadly the teenager’s condition deteriorated and they died. This escalated anxiety within the friend network. There was considerable concern from friends who had had minimal contact with the case in the previous week and were presenting at a range of out of hours GP practices and A&E units across South London and Surrey and who wanted prophylaxis.

The case resulted in an extremely large workload over the weekend and a number of lessons can be shared with others around managing public health actions out-of-hours including: effective approaches to providing prophylaxis in a systematic manner to multiple close contacts through liaising closely with out of hours GPs; limiting anxiety and misinformation within the ‘Facebook generation’ who have access to a range of instantaneous routes of communication and through which rumours can spread rapidly; ensuring a consistent approach to provision of prophylaxis based on evidence based guidelines and effective communication of risk.

Following the case a number of local resources are being developed to support management of cases in the future including increasing awareness of management of close contacts amongst clinicians; working with the MRF to develop supporting material for the worried well and primary care; local processes for transferring information to hospitals out of hours and local factsheets to support management of risk communication.

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Investigation of the uptake of chemoprophylaxis by contacts of cases of meningococcal disease in the North East of England

Anne Halewood, Health Protection Nurse1, Tom Inns, Epidemiology and Surveillance Scientist2, Dr Naweed Sattar, Specialist Registrar in Rehabilitation Medicine3, Dr Tricia Cresswell, Consultant in Health Protection1

1North East Health Protection Unit, HPA, Newcastle upon Tyne, England
2North East Health Regional Epidemiology Unit, HPA, Newcastle upon Tyne, England
3Walkergate Park Hospital, Newcastle upon Tyne, England
Contact email: anne.halewood@hpa.org.uk

Keywords: chemoprophylaxis; meningococcal disease; compliance; audit

Objectives
  • To investigate the uptake of antibiotic chemoprophylaxis by contacts of cases of meningococcal disease, including timeliness of receipt of prescription, ease of access to prescription and medication, compliance with advice.
  • To make recommendations to improve uptake.
Methods

An audit was undertaken retrospectively involving the cohort of contacts of cases of meningococcal disease which occurred from 1 January to 31 March 2011. Contacts of cases who had died were excluded. A questionnaire was designed based on current North East Health Protection Unit (HPU) and national guidelines. The questions were tick box or scales with minimal free text except for a final open ended question. Cases and contacts were identified from the case management database. The questionnaire was administered by telephone to contacts by an experienced health protection nurse (AH). Epidata was used for data entry and STATA 11.2 for analysis.

Results

There were 130 contacts of 28 cases with an average number of 4.6 contacts per case. Analysis of factors impacting on compliance was not possible as compliance was 99.23%.

Key findings were:
  • 100% of contacts received advice, 75% within 24 hours
  • 34% received written information
  • 100% reported advice as “clear”
  • 48% of contacts were issued antibiotics on the hospital ward
  • Of the 52% issued with a prescription, 75% received this from a GP and 23% from a hospital doctor
  • Of those given a prescription, 28% had to go to more than one pharmacy and 22% reported obtaining the antibiotic from a pharmacy as “difficult”.
Review of the responses to the open ended question suggested high levels of overall satisfaction with the advice given but difficulty in obtaining antibiotics from community pharmacies. Further analysis of the open ended question will be provided in the poster.

Conclusions

The national guidance on chemoprophylaxis changed in March 2011, recommending a single dose of ciprofloxacin in preference to a two day course of rifampicin. This may resolve a number of the problems identified in obtaining the chemoprophylaxis (which was usually rifampicin) in this study.

Compliance was unexpectedly high in this audit but access to antibiotics was reported as easier when these were directly issued on the ward rather than by prescription.

Efforts will be made by the HPU to improve the provision of written materials and there will be further dialogue with clinicians in relation to directly providing chemoprophylaxis to family contacts whenever feasible.

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Vaccine Discovery and Vaccinology

Correlation of a high-throughput flow cytometric antibody-mediated membrane attack complex assay with the serum bactericidal assay

L.Allen, C.Brookes, S. Taylor, A.Gorringe

Microbiological Services Porton, Health Protection Agency, Porton Down, Salisbury, UK

Keywords: Neisseria meningitidis, complement, correlation, SBA, flow cytometry

Serum bactericidal activity (SBA) has long been established as a correlate of protection for meningococcal vaccines with SBA titres of ≥1:4 measured using human complement established as providing a protective response. Thus potential vaccine efficacy is widely assessed by the measurement of serum bactericidal antibodies and this method is currently a requirement for vaccine licensure. However, the serum bactericidal assay requires large volumes of sera and can be laborious to perform. Development of high-throughput assays which require very low sera volumes is important in order to enable the assessment of bactericidal responses against large strain panels.

We have developed a high-throughput flow cytometric assay measuring antibody mediated complement deposition. The assay uses fixed meningococci, IgG-depleted human plasma as the complement source and fluorescent anti C3c and C5b-9 antibodies to measure deposition of C3b/iC3b and C5b-9 (membrane attack complex) on the surface of bacteria. We have previously demonstrated good correlation between the deposition of C5b-9 and bactericidal titres using meningococcal strain M01-240149 (R=0.87, p<0.01).

We have analysed antibody-mediated complement deposition and SBA with a panel of 40 human sera and a number of diverse meningococcal strains. We have confirmed the correlation between antibody-mediated C5b-9 deposition and bactericidal response. This data will be used to establish a cut-off value for C5b-9 deposition that will predict SBA.

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Pfizer strategy for prevention of meningococcal B disease

Annaliesa S. Anderson1, Shannon Harris1, Ellen Murphy1, Neil Oldfield2, Dlawer Ala’Aldeen2 and Kathrin U. Jansen1

1Pfizer Vaccine Research, Pearl River New York, USA
2Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

Neisseria meningitidis serogroup B (MnB) is the last of the invasive disease causing N. meningitidis serogroups for which no broadly effective vaccine is licensed. Current vaccines in late stage development for the prevention of invasive meningococcal serogroup B disease (IMBD) include meningococcal factor H binding protein (fHBP) as the most important antigen to generate serum bactericidal antibodies. fHBPs are common to all N. meningitidis serogroups and can be categorized into two distinct subfamilies (A and B) on the basis of sequence diversity and serological response. The distribution of subfamily A and subfamily B is 30% and 70% respectively from subjects with IMBD. The vaccines in clinical development have differing degrees of cross-reactivity across fHBP variants.

N. meningitidis polysaccharide conjugate vaccines for other serogroups are effective both at preventing invasive disease and interrupting oral-pharyngeal carriage, the primary mechanism for disease transmission. It is expected that a vaccine that targets IMBD isolates should also be effective at interrupting carriage. We evaluated the fHBPs of common carriage isolates, which are also associated with disease, to assess whether fHBP based vaccines have the potential to be effective against carriage-associated fHBP variants.

Analysis of fHBP variants associated with carriage in the UK and US reveals that there is a higher proportion of subfamily A variants associated with carriage than with IMBD, increasing to 50% (UK) and 73% (US). The majority of carriage isolate variants are associated with IMBD, 95% (UK) and 85% (US), and are responsible for 84% to 92% of IMBD in these regions. The Pfizer bivalent fHBP vaccine was evaluated for its ability to generate serum bactericidal antibodies to kill IMBD isolates with fHBP variants associated with carriage. Isolates with variants that represented over 75% of fHBP carriage variants were tested. Carriage associated variants could be killed by the human bivalent fHBP immune serum. The coverage for IMBD isolates associated with carriage translated to >81% (US) and >85% (UK). Testing of additional variants is ongoing. These studies demonstrate that a bivalent lipidated vaccine has the potential to be effective against most fHBP variants that are associated with IMBD and meningococcal carriage isolates.

Examination of two meningococcal surface proteins as potential vaccine targets

F. Bidmos1*, H. Chan2, E. Kaczmarski3, I. Feavers2 and C. D. Bayliss1

1Department of Genetics, University of Leicester, LE1 7RH, UK
2National Institute of Biological Standards and Control, Blanche Lane, Potters Bar, EN6 3QG, UK
3Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK
* Corresponding author. Email: fab5@le.ac.uk;

Keywords: menB, phase variation, haemoglobin receptors, antibodies, vaccine

Recombinant protein preparations are currently being employed as alternatives to capsular polysaccharide conjugates in the production of effective vaccines against meningitis due to menB strains. While one of these vaccines (Bexsero® by Novartis), due for licensing later this year, has a strain coverage of about 80%1, there is a need for vaccines against other OMP targets that will provide broader protection. In this study, two phase-variable haemoglobin receptors that are believed to be crucial to the survival of the meningococcus in the blood, HpuAB and HmbR, are being considered as targets for the production of bactericidal antibodies. Previous studies in our lab have shown that one or both receptors are present and expressed in >95% of disease isolates tested2, indicative of the importance of the expression (phase variation ON-state) of these receptors during infection. A total of 5 variants of genes encoding these proteins were cloned from 1 carriage and 2 disease isolates and expressed in the E. coli expression strain BL21. HpuA was purified by binding to a nickel affinity column before elution with 120 mM or 150 mM imidazole. Inclusion bodies of HpuB and HmbR were solubilised in 6 M guanidine hydrochloride and refolded in the presence of 50 M hemin. Refolded proteins were subsequently purified using an ion exchange column. Mice were immunized subcutaneously with 20g of each protein at weeks 0, 3 and 5. Terminal bleeds and spleens were taken at week 7. Sera were tested for reactivity with the relevant immunogens and also for cross-reactivity with antigenic variants and unrelated proteins in ELISAs and western blots. ⍺-HpuA antibodies reacted against antigenic variants of HpuA but not HpuB or HmbR. A degree of cross-reactivity was observed between ⍺-HmbR antibodies and the HpuB protein. Similarly, ⍺-HpuB antibodies reacted with HmbR. Epitopes shared between both proteins could be responsible for this cross-reactivity. Ongoing and future work includes the construction of deletion and complementation mutants, which will be used in whole blood assays to investigate the ability of meningococci lacking HpuAB and HmbR to grow in blood. Immunodetection assays to probe for antibodies against these receptors in carrier sera will be performed in addition to serum bactericidal assays to ascertain the ability of ⍺-HpuA, ⍺-HpuB and ⍺-HmbR antibodies to kill meningococci.

References

1. Donnelly, J., Medini, D., Boccadifuoco, G., Biolchi, A., Ward, J., Frasch, C., Moxon, E. R., Stella, M., Comanducci, M., Bambini, S., Muzzi, A., Andrews, W., Chen, J., Santos, G., Santini, L., Boucher, P., Serruto, D., Pizza, M., Rappuoli, R. and Giuliani, M. M. 2010. Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines. Proceedings of the National Academy of Science. 107 (45), 19490-19495.
2. Tauseef, I., Harrison, O. B., Wooldridge, K. G., Feavers, I. M., Neal, K. R., Gray, S. J., Kriz, P., Turner, D. P., Ala'Aldeen, D. A., Maiden, M. C., Bayliss, C. D. 2011. Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence. Microbiology. 157, 1446-1456.

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Estimating the Potential Strain Coverage in Europe of a Multicomponent Vaccine Targeting Serogroup B Meningococci

Giuseppe Boccadifuoco1, John Donnelly1, Duccio Medini1, Marzia Giuliani1, Maria Stella1, Giacomo Frosi1, Maurizio Comanducci1, Stefania Bambini1, Alessandro Muzzi1, Mariagrazia Pizza1, Rino Rappuoli1, Jamie Findlow2, Ray Borrow2, Stefanie Gilchrist2, Danielle Thompson2, Morgan Ledroit3, Eva Hong3, Muhamed-Kheir Taha3, Raquel Abad4, Julio Vazquez4, Paola Mastrantonio5, Paola Stefanelli5, Cecilia Fazio5, Anna Carannante5, Jan Oksnes6, Dominique A. Caugant6, Heike Claus7, Ulrich Vogel7

1Novartis Vaccines and Diagnostics, Siena, Italy;
2Health Protection Agency, Manchester, UK;
3Institut Pasteur, Paris, France;
4Instituto de Salud Carlos III, Madrid, Spain;
5Istituto Superiore di Sanita, Rome, Italy;
6Norway Institute of Public Health, Oslo, Norway;
7University of Wurzburg, Wurzburg, Germany

Keywords: Serogroup B, multicomponent, vaccine, coverage, Europe

Background

A multicomponent vaccine (4CMenB) is proposed for prevention of invasive serogroup B (MenB) meningococcal disease. Because MenB clinical isolates are diverse, it is necessary to assess the potential public health impact of 4CMenB. We defined a target population of MenB strains in Europe to estimate strain coverage by 4CMenB.

Methods

To evaluate strain coverage by 4CMenB we used the Meningococcal Antigen Typing System (MATS), which predicts the potential for bactericidal activity of sera from immunized 13-month-olds, based on quantity and crossreactivity with the vaccine-induced immune response of three antigens (factor H binding protein, Neisserial Heparin Binding Antigen, and Neisserial Adhesin A), and the genotype of a fourth antigen, PorA. As a recent and representative target strain population, we evaluated invasive MenB strains isolated mainly in a single epidemiologic year (July 2007-June 2008) by the national reference laboratories of England and Wales, France, Germany, Norway, and Italy, a total of 1052 strains. Valid MATS results were obtained for 1011/1052 strains and were used to estimate coverage, and were linked to MLST and antigen sequence data.

Results/Conclusion

Using MATS, we estimated that 78% of the strains (95% confidence interval 66-91%) would be covered by 4CMenB. Half of the total strains (64% of the covered strains) potentially could be targeted by bactericidal antibodies against more than one antigen. Coverage estimates varied by country and ranged from 73% to 87%. 4CMenB has the potential to protect against a significant proportion of the MenB strains that have caused invasive disease recently in Europe.

The application of Structural Vaccinology in the development of a meningococcal antigen inducing broad protective immunity

MJ Bottomley1, M Scarselli1, B Aricò1, B Brunelli1, S Savino1, F Di Marcello1, E Palumbo1, D Veggi1, L Ciucchi1, E Cartocci1, E Malito1, P Lo Surdo1, M Comanducci1, MM Giuliani1, F Cantini2, S Dragonetti2, A Colaprico1, F Doro1, P Giannetti1, M Pallaoro1, B Brogioni1, M Tontini1, M Hilleringmann1, V Nardi-Dei1, L Banci2, M Pizza1 & R Rappuoli1

1Novartis Vaccines & Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.
2CERM Magnetic resonance Center, University of Florence, 50019 Sesto Fiorentino, Italy
Contact email: matthew.bottomley@novartis.com

Keywords: vaccine, antigenic variability, structure, fHBP, MenB

The development of vaccines against many pathogens is hampered by the sequence variability of their protective antigens. Where antigenic variability is limited (e.g. S. pneumoniae), vaccines have been developed by including several antigenic variants in a single vaccine. However, when antigenic variability is very high, as for Neisseria meningitidis serogroup B (MenB), the development of a successful vaccine has been problematic. To solve this problem, we have developed a Structural Vaccinology approach, to design a single antigen capable of inducing broad protective immunity against all natural variants of MenB.

The factor H binding protein (fHBP) has been identified as a protective antigen of MenB. However, >300 variants of fHBP have been described, making it difficult to select a single variant to be included in a vaccine. Therefore, we analyzed all known fHBP variant sequences and were able to classify them into three groups (variant groups 1, 2 and 3). Using the NMR structure of variant 1 fHBP as a scaffold, we designed 54 different chimeric variant 1 proteins, each engineered to display a portion of a variant 2 or 3 protein on its surface. The structural integrity of each lead candidate eliciting bactericidal antibodies was verified by several biophysical techniques. Ultimately, by ‘transplanting’ patches of residues from variant 2 and 3 proteins onto the variant 1 scaffold, we created a single fHBP protein (named ‘G1’) bearingcharacteristics of variants 1, 2 and 3, which, when combined with novel adjuvants (see poster by M Pallaoro et al.) was able to induce antibodies able to kill all strains of MenB tested. We determined the crystal structure of the efficacious G1 protein, revealing at atomic resolution how the multiple immuno-dominant epitopes have been successfully grafted onto a single molecule§. We believe that the Structural Vaccinology approach may be suitable to develop antigens against other pathogens which, like MenB, show a high degree of antigenic variation.

§ Reference: M Scarselli et al., Science Translational Medicine (July 2011), vol 3 issue 91.

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Potential strain coverage of a multi-component meningococcus B vaccine in Germany and the Czech Republic

Heike Claus1, Pavla Krizova2, Martin Musilek2, Giuseppe Boccadifuoco3, Maria Stella3, Giacomo Frosi3, Duccio Medini3, Maurizio Comanducci3, Mariagrazia Pizza3, John Donnelly3 and Ulrich Vogel1

1University of Würzburg, Institute for Hygiene and Microbiology and German Reference Laboratory for Meningococci, Würzburg, Germany;
2National Reference Laboratory for Meningococcal Infections, National Institute of Public Health, Prague, Czech Republic;
3Novartis Vaccines and Diagnostics, Siena, Italy
Contact email: hclaus@hygiene.uni-wuerzburg.de

Keywords: meningococcus, serogroup B, vaccine, antigen expression, strain coverage

Object of the study

The multi-component vaccine 4CMenB has been developed to target the antigenically variable population of serogroup B meningococci. The immune-dominant components of the vaccine are: FHbp (factor H binding protein), NHBA (Neisserial heparin binding antigen), NadA (Neisserial adhesion A), and outer membrane vesicles. Aim of the study was to estimate the strain coverage of 4CMenB in serogroup B meningococci in Germany and the Czech Republic.

Material and methods

Measurement of the relative antigen potencies of FHbp, NHBA, and NadA by the MATS ELISA (Donnelly et al. PNAS 2010) as well as antigen sequence typing of the variable region 2 of porin A (PorA) and multi-locus sequence typing were performed on 222 German serogroup B strains isolated 2007/2008 and 108 Czech serogroup B strains isolated 2007-2010.

Results and conclusions

The strain population differed with regard to the ST-41/44 clonal complex, which was more prevalent in Germany, and the ST-18 and ST-35 clonal complexes which were more prevalent in the Czech Republic. Relative potencies above the positive bactericidal threshold were observed for FHbp in 69% of the German and 67% of the Czech isolates, for NHBA in 63% of the German and 42% of the Czech isolates and for NadA in 3% of the German and 4% of the Czech isolates. Antigen sequence typing revealed the presence of PorA P1.4 in 21% of the German and 1% of the Czech isolates. In total, the strain coverage of 4CMenB was estimated to be 81% (95% confidence interval 71-93%) for the German and 74% (95% CI 59-87%) for the Czech isolates.

Antigen diversity of the 4CMenB vaccine components in serogroup B meningococcal patient isolates from five European countries

S. Bambini1, J. Findlow2, H. Klaus3, M.K. Taha4, P. Stefanelli5, D.A. Caugant6, J. Lucidarme2, S. Gilchrist2, R. Borrow2, U. Vogel3, P. Mastrantonio5, R. Rappuoli1, J. Donnelly1, M. Pizza1, A. Muzzi1, M. Comanducci1

1Novartis Vaccines and Diagnostics, Siena, Italy
2Health Protection Agency, Manchester, UK
3University of Wurzburg, Germany
4Institut Pasteur, Paris, France
5Istituto Superiore di Sanità, Rome, Italy
6Norwegian Institute of Public Health, Oslo, Norway

Keywords: MenB, 4CMenB, NadA, fHbp, NHBA

Background

To evaluate presence and degree of conservation of the main antigens of 4CMenB vaccine in the epidemiological background of some European countries, we analysed molecular diversity of the vaccine components in patient isolates. Molecular data were used with Meningococcal Antigen Typing System (MATS) results, for an estimation of 4CMenB coverage.

Methods

A total of 1052 invasive MenB strains, isolated in five European countries mainly in a single epidemiologic year were analysed by MultiLocus Sequence Typing (MLST) and tested for PorA genotypes. Presence and diversity of the vaccine components NadA, fHbp and NHBA were assessed.

Results

The clonal complex (cc) 41/44 was prevalent in 4 of the 5 countries considered. In those countries, cc32 was the second most represented cluster. The exception to this trend was the UK, where cc269 was predominant, and exceeded cc41/44 by 1.5%. The association between vaccine antigen variability and clonal complexes was confirmed. As a consequence, subvariants associated with cc41/44 (fHbp-1.14, 1.4 and NHBA-2) were predominant. fHbp-1.1, that is associated with cc32 only, accounted for more than 20% in France, and for 19, 12 and 9 % in Germany, Norway and Italy, respectively. The occurrence of NadA reflected those of the NadA (+) clonal complexes, namely cc32, 8 and 213.

Conclusions

The distribution and the vaccine antigen repertoire of each clonal complex differed among European countries. For compiling coverage estimates, genetic data are to be linked with expression and bactericidal data. This is accomplished by MATS.

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Serogroup C invasive meningococcal disease (IMD) in Ontario, Canada, 2000-2010: vaccine impact assessment

Anne Wormsbecker1,2,3, Vica Dang1,2, Frances Jamieson1,4, Sarah Wilson1,2, Prasad Rawte1, Natasha S Crowcroft1,2, Karen Johnson1, Shelley L Deeks1,2

1Public Health Ontario, Toronto, Canada
2Dalla Lana School of Public Health, University of Toronto, Toronto, Canada
3Division of Paediatric Medicine, Hospital for Sick Children, Toronto, Canada
4Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Canada

Keywords: Invasive meningococcal disease, Serogroup C, cohort analysis, Meningococcal C conjugate vaccine, Immunisation programs

Objectives

Meningococcal C conjugate (MCC) vaccination programs were introduced in the province of Ontario at age 1 and for grade 7 students in 2004/5. We sought to assess the impact of MCC vaccine on serogroup C invasive meningococcal disease (IMD) through descriptive epidemiologic and cohort analyses.

Methods

The period under surveillance was 2000 to 2010; a comprehensive province-wide dataset was created through probabilistic record linkage of data from the integrated Public Health Information System and Public Health Ontario Laboratories (PHOL). For most isolates, serotype and serosubtype were ascertained by indirect whole-cell enzyme-linked immunosorbent assay and electrophoretic type (ET) was determined by multilocus enzyme electrophoresis. Analysis was primarily descriptive and cohorts reflected vaccine program eligibility. Incidence rates were calculated using demographic data from Statistics Canada.

Results

Of 713 cases of IMD identified over the 11 year period, 155 were serogroup C and 141 had PHOL records. Median age was 26 years (range 17 weeks to 95 years) and case-fatality ratio was 17%. Yearly rates varied from 0.30 to 0.02/100 000. Age-specific rates were highest among 20-24, 15-19, and < 1 year olds (0.23, 0.21 and 0.20/100 000, respectively). While median age was stable throughout (p>0.05), age-specific rates for those 12-18 years (i.e. group in which all members in 2010 were eligible for grade 7 program) significantly decreased (p = 0.01).Cohort analysis showed significant reductions in IMD among both those eligible for the MCC program (born since 1992) and ineligible (born up to 1991); however, the decrease was more marked in the eligible group. Of cases that occurred in program-eligible persons, 80% (n=8) occurred in children too young to have been vaccinated (age < 1 or age < 12) and were therefore not directly preventable by the province’s vaccination program. Serotype and serosubtype were available for 91% (n=128) of PHOL records; of these, 44% (n= 56) were C:2a:P1.2. Of 114 isolates with ET, 77% (n=81) were ET-15, though its rate decreased significantly over time (p<0.01). There were 4 not ET-15, not ET-37 isolates since 2005 but none prior.

Conclusions

Rates of serogroup C IMD are low in the post-MCC vaccine era and clonal groups have changed. Cohort analysis suggests direct and indirect (herd) MCC program effects but those who have not reached program age remain at continuing, albeit reduced, risk. Future analyses with a longer time horizon and including the effect of Ontario’s new quadrivalent meningococcal conjugate vaccine program will be valuable.

Immunogenicity of an Investigational Multicomponent Meningococcal Serogroup B (4CMenB) Vaccine in Healthy Adolescents Following 1, 2 or 3 Doses

Peter M Dull, MD1, Marie Elena Santolaya2, Miguel O’Ryan3, Maria Teresa Valenzuela4, Valeria Prado3, Rodrigo Vergara5, Huajung Wang1,Alan Kimura1 for the V72P10 Meningococcal B Adolescent Vaccine Study group

1Novartis Vaccines & Diagnostics, Cambridge, MA, USA
2Departamento de Pediatría, Hospital Dr Luis Calvo Mackena, Facultad de Medicina, Universidad de Chile, Santiago, Chile
3Programa de Microbiología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
4Departamento de Salud Pública y Epidemiología, Universidad de Los Andes, Santiago, Chile
5Escuela de Medicina Universidad de Valparaíso, Valparaíso, Chile
Contact email: peter.dull@novartis.com

Keywords: serogroup B; adolescents; vaccine; booster; immunity

Background

Despite the success of meningococcal serogroup C vaccination programmes, there is still a heavy burden of meningococcal disease in adolescents due to serogroup B. We assessed immunogenicity and reactogenicity of an investigational, multicomponent meningococcal serogroup B vaccine (4CMenB) administered in different schedules to adolescents.

Methods

Healthy adolescents (11-17 years) were given 0, 1, 2 or 3 doses of 4CMenB or placebo, at least 1 month apart, over a 6 month period in different schedules. Immunogenicity was assessed 1 month after each vaccination and at 7 months, as protective serum bactericidal titers (≥4) with human complement (hSBA) against reference strains specific for vaccine antigen components: factor H binding protein (fHbp), Neisserial adhesin A (NadA), and outer membrane vesicles from New Zealand outbreak strain (NZOMV). Responses to the Neisseria Heparin Binding Antigen (NHBA) component were assessed by ELISA. Reactogenicity was monitored for seven days after each vaccination for solicited reactions, and throughout the study for adverse events.

Results

Overall, 1631 adolescents (13.8 ± 1.9 years) received 4CMenB or placebo. After two or three doses over two months 99–100% of 4CMenB recipients had hSBA titres ≥ 4 against test strains, compared with 92–97% after one dose (p < 0.02) and 29–50% after placebo. Antibody titres waned two months after the first dose, and continued to decline to six months in those who did not receive a second dose, when 91–100% of subjects who received two or three doses still had titres ≥ 4 for each strain, compared with only 73–76% after one dose. Following administration of a second or third dose at 6 months, rates reached 99–100% for each strain. ELISA results for NHBA displayed a similar pattern of responses.

Local and systemic reaction rates were similar after each 4CMenB injection, did not increase with subsequent doses, but remained higher than placebo. The majority were described as mild to moderate in severity and resolved within 3 days of vaccination. No vaccine-related SAEs were reported and no significant safety signals were observed.

Discussion

One or two doses of 4CMenB induced protective immune responses in almost all subjects, which were boosted with a further dose at six months. Two doses have similar immunogenicity when given 1, 2 or 6 months apart, with similar reactogenicity for each dose.

Evaluation of antibody persistence and response to a MenACWY-CRM conjugate vaccine (Menveo®) booster in adolescents five years after receiving MenACWY-CRM or a quadrivalent meningococcal polysaccharide vaccine (Menomune®) assessed by serum bactericidal assays using rabbit complement (rSBA)

Tatjana Odrljin, MD; Allen Izu, MS; and Peter M Dull, MD

Novartis Vaccines and Diagnostics, Cambridge, MA

Contact email: peter.dull@novartis.com

Keywords: meningococcal disease, conjugate vaccine, persistence, anamnestic response, immunogenicity.

Background

A randomized phase II study demonstrated that Menveo®, a CRM-197 meningococcal conjugate vaccine against Neisseria meningitidis serogroups A, C, W-135, and Y (MenACWY-CRM) was well tolerated and immunogenic in adolescents, compared with Menomune®, a quadrivalent (A, C, W-135 and Y) meningococcal polysaccharide vaccine (MPSV4).

Methods

This open-label extension study was conducted to assess long-term persistence of bactericidal antibodies after vaccination with MenACWYCRM (n = 50) or MPSV4 (n = 51), and response to a MenACWY-CRM booster given 5 years post-primary immunization in all subjects. Booster results were compared with a control group of 54 age-matched naive subjects. Immunogenicity was assessed by serum bactericidal activity assay using human complement (hSBA) and rabbit complement (rSBA). Here, we present the results of the rSBA analysis.Immunogenicity was assessed as the percentage of subjects with rSBA titers ≥8 and ≥128, and by rSBA geometric mean titers (GMTs).

Results

Over 75% of subjects vaccinated 5 years previously with MenACWY-CRM maintained rSBA titers ≥8 against serogroups A (98%), W-135 (89%) and Y (78%); 56% maintained rSBA titers ≥8 against serogroup C. In contrast, 84%, 36%, 60% and 34% of MPSV4 vaccinated subjects maintained rSBA titers ≥8 against serogroups A, C, W-135 and Y, respectively, 5 years later. Using a more stringent measure, over 75% of Menveo pre-vaccinated subjects had rSBA titers ≥128 against serogroups A (96%), W-135 (85%), Y (76%), with 40% of MenACWY-CRM pre-vaccinated subjects maintaining rSBA titers ≥128 against serogroup C. A MenACWY-CRM booster administered 5 years post-prime elicited an anamnestic booster response that peaked at 1 week post-booster in Menveo pre-vaccinated subjects (GMT range, 5368–31002), but responses in MPSV4 pre-vaccinated subjects peaked at 1 month post-booster and were similar in magnitude to the response of subjects not previously vaccinated (GMT range: Menomune, 363–5366; previously Naive, 906–8670).

Conclusions

MenACWY-CRM provided persistence of bactericidal antibodies against all vaccine antigens for at least 5 years after vaccination, and immune memory as evidenced by an anamnestic response to a MenACWY-CRM booster in MenACWY-CRM pre-vaccinated subjects.Responses in MPSV4 pre-vaccinated subjects resembled a primary immune response, but the booster dose of MenACWY-CRM elicited a robust antibody response, regardless of vaccine history. These results confirm previously reported conclusions based on hSBA data.

Pivotal Safety and Immunogenicity of an Investigational Quadrivalent Meningococcal Conjugate Vaccine (MenACWY-CRM; Menveo®) in Infants in the United States and Latin America

Nicola P. Klein, MD, PhD1; Miguel W. Tregnaghi, MD2; Maria-Gabriela Graña, MD3; Lisa Bedell, MA3; Tatjana Odrlijn, MD3; Peter M Dull, MD3

1Kaiser Permanente, Oakland, CA;
2CEDEPAP, Córdoba, Argentina;
3Novartis Vaccines and Diagnostics, Cambridge, MA.
Contact email: peter.dull@novartis.com

Keywords: Meningococcal disease, conjugate vaccine, immunogenicity, safety, infants

Background

Infants are the group at highest risk of invasive meningococcal disease. MenACWY-CRM, a quadrivalent conjugate meningococcal vaccine, elicited robust immunologic responses in an earlier small-scale study in young infants.

Methods

In this Phase III study performed in the United States (US) and Latin America (LA), 4545 healthy infants were randomized 2:1 to receive routine vaccines along with MenACWY-CRM administered in a 3 or 4-dose schedule [2, 6, and 12 months (LA); or 2, 4, 6 and 12 or 16 months (US)], or routine vaccines alone followed by 1 or 2 MenACWY-CRM toddler doses in the second year of life. All subjects were monitored for local and systemic reactions and adverse events after each dose. Immunogenicity was assessed in subsets of subjects by serum bactericidal assay using human complement (hSBA).

Results

In both US and LA subjects, after 2 or 3 doses during the first year of life, 94-99% of subjects achieved an hSBA titer ≥ 8 for serogroups C, W-135 and Y and 67-89% for serogroup A (n = 182 to 274). After a 4-dose series, the % of subjects with hSBA titers ≥8 against the 4 serogroups was 94–100% in US subjects (n ~ 85). The percentage with hSBA titers ≥ 8 was 95–100% against all 4 serogroups after the final toddler dose of the 3-dose (n ~ 100) and 4-dose (n~115) series in LA subjects. In MenACWY-CRM naive toddlers, administration of 1 versus 2 toddler doses of MenACWY-CRM (n ~ 74, n ~ 99, respectively) the % of subjects with hSBA titers ≥ 8 was 72–91% and 97–100%, respectively. Responses to concomitant routine vaccines were non-inferior to routine vaccinations alone, except for slightly reduced responses to pertactin and PnC serotype 6B in US subjects. Safety profiles were similar in those who received routine vaccinations with and without MenACWY-CRM, with comparable rates of solicited local and systemic reactions typical of routine infant vaccines, and no evidence of any increase in unsolicited or vaccine-related adverse events.

Conclusion

MenACWY-CRM was shown to be highly immunogenic and well tolerated when administered as a four dose series with no evidence of clinically significant interference when co-administered with routine vaccines.

Immunogenicity of Meningococcal Quadrivalent (MenACWY-CRM197) Conjugate and Measles-Mumps-Rubella-Varicella (MMRV) Vaccines when Administered Concomitantly in 12 Month-Old Toddlers

Nicola P.Klein, MD, PhD1; Julie S. Shepard, MD2; Matthew Hohenboken, MD3; Christopher Gill, MD3; Peter M. Dull, MD3

1Kaiser Permanente Vaccine Study Center, Oakland, CA;
2Ohio Pediatric Research Association, Huber Heights, OH;
3Novartis Vaccines & Diagnostics, Cambridge, MA.
Contact email: peter.dull@novartis.com

Keywords: Meningococcal disease, conjugate vaccine, immunogenicity, safety, infants

Background

A quadrivalent polysaccharide-protein conjugate vaccine (MenACWY-CRM197, Menveo®) is currently licensed in the USA for use from 2 years of age. To assess immune responses during the first year of life, we gave two doses of MenACWY at 7–9 and 12 months of age, with the second dose being given separately or concomitantly with measles, mumps, rubella, varicella (MMRV, ProQuad®) vaccine.

Methods

This phase 3, open-label, randomized, multi-center study enrolled and randomized 1630 healthy toddlers to three groups: Group A (n = 504) received MenACWY at 7– 9 months and MenACWY+MMRV at 12 months; Group B (n = 510) MenACWY at 7–9 and 12 months and MMRV at 13.5 months; Group C (n = 616) MMRV only at 12 months. Sera drawn 6 weeks later were tested for serum bactericidal activity with human complement (hSBA) against serogroups A, C, W and Y, expressed as geometric mean titers (GMT) and % with titers ≥ 8. Antibodies to MMRV were measured by standard ELISA methods.

Results

One month after a first MenACWY dose at 7-9 months, percentages of infants with hSBA ≥ 1:8 were 50% (45-56), 88% (83-92), 37% (30-44) and 31% (24-38) for serogroups A, C, W and Y, respectively. Six weeks after a second dose of MenACWY, regardless of concomitant MMRV, there were robust responses against all meningococcal serogroups expressed as proportions with hSBA titers ≥ 1:8 (Table), or as GMTs (not shown). Similarly, concomitant Menveo did not affect rates of seroconversion to MMRV components (Table), or as GMTs (not shown).

   % hSBA ≥ 8 (95% CI)
    Seroconversion rate (95% CI)
Vaccine(s)   Group A
MenACWY + MMRV
Group B
MenACWY 
   Group A
MenACWY + MMRV
Group C
MMRV
 Serogroup  N = 384/~203*  N = 379/~199*  Antigen  N = 337–370  N = 459–515
A  88% (84-91) 88% (84-91)  Measles  98% (96-99)  99% (98-100)
C
100% (98-100)
100% (98-100)
 Mumps  98% (96-99)  96% (94-98)
W 100% (97-100)  98% (96-100)  Rubella  95% (93-97)  97% (95-98)
 Y  98% (95-99)  96% (93-99) Varicella
 99% (97-100)  99% (98-100)
    * N = 384 & 379 for Serogroup A, 201-205 & 198-199 for C, W, Y, in Groups A & B, respectively.

Conclusion

Co-administration of a second dose of Menveo with MMRV did not affect immune responses elicited against either vaccine. Two doses of Menveo generated protective immune responses against all four serogroups in the majority of toddlers.

Potential Coverage of English and Welsh Capsular Group B Isolates by an Investigational Meningococcal Group B Vaccine (4CMenB)

J. Findlow1, S. Gilchrist1, D. Thompson1, M. Stella2, G. Frosi2, R. Borrow1

1Vaccine Evaluation Unit, Health Protection Agency, Manchester Royal Infirmary, Manchester, UK;
2Novartis Vaccines, Siena, Italy
*Corresponding author - Vaccine Evaluation Unit, Health Protection Agency North West, 2nd Floor CSB II, Manchester Royal Infirmary, Manchester, M13 9WZ, UK.
E:mail: jamie.findlow@hpa.org.uk

Keywords: Meningococcal, group B, vaccine, coverage, England & Wales

An investigational multicomponent meningococcal group B vaccine (4CMenB) has proven safe and immunogenic in Phase I to III trials. The vaccine contains three recombinant proteins; factor H binding protein (fHBP), Neisserial Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) formulated with Outer Membrane Vesicles containing the immunodominant PorA protein. Protection afforded by 4CMenB will depend upon antigen expression and cross-reactivity of induced antibody to antigen variants. To address this issue, a meningococcal antigen typing system (MATS) was developed to predict whether 4CMenB covers individual isolates. As group B currently accounts for approximately 90% of meningococcal disease in England and Wales, we investigated the potential coverage of 4CMenB using MATS.

All 535 capsular group B isolates all from cases of invasive disease received at the Health Protection Agency Meningococcal Reference Unit from the epidemiological year 2007/2008 were characterised genetically for PorA and by MATS for recombinant antigens.Of the isolates, 63.3%, 54.7% and 0.6% had positive MATS phenotype for fHBP, NHBA and NadA, respectively. Additionally, 20.3% harboured the homologous PorA (P1.4). Coverage (determined as >1 antigen with positive phenotype/homologous PorA genotype) was estimated at 72.9%. Among covered strains, 69% were positive for more than one antigen. To conclude, 4CMenB has the potential to protect against a significant proportion of MenB disease in England and Wales.

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The Neisseria meningitidis-macrophage infectivity potentiator (MIP) protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate

Miao-Chiu Hung, Omar Salim, Jeannette N. Williams, John E. Heckels, and Myron Christodoulides

Molecular Microbiology, Division of Infection, Inflammation and Immunity, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, Southampton, UK

Keywords: Neisseria meningitides, macrophage infectivity potentiator; meningococcal vaccine; liposome, monophosphoryl lipid A

Objective

Abundance of a ~29kDa protein - Macrophage Infectivity Potentiator (MIP) was observed in prior proteomic analysis of the meningococcal outer membrane (OM). Our objective was to investigate the potential of MIP as a vaccine candidate by preparing a recombinant protein and testing its ability to induce functional bactericidal antibodies.

Materials and Methods

The mip gene from MC58 was cloned into pRSETA system, propagated in E.coli DH5⍺ and expressed in E.coli BL21(DE3)pLysS. Recombinant MIP (rMIP) protein was purified by nickel column chromatography under native conditions to high yield and purity. Several preparations containing rMIP protein were used for animal immunization. Serological studies were performed to investigate the functional immunogenicity
of rMIP.

Results

ELISA showed high antibody titres against rMIP and OM of MC58 (homologous strain). The surface location of MIP protein was demonstrated by Immunofluorescence. Several strains isolated either from patients or carriers were sequenced to survey the conservation of mip gene. Only 3 amino acid sequence types (type I, II and III) were identified with 98-100% similarity. Western blot showed the similar expression levels of MIP protein amongst all the surveyed strains. Serum bactericidal assay (SBA) showed that antisera raised against rMIP (type I) elicited antibodies with high bactericidal titres (up to 1/1,024) against MC58 (type I). Antisera were then tested against heterologous sequence type II and III strains and showed cross protection with high killing titres (1/256).

Conclusion

Meningococcal MIP protein is highly conserved amongst meningococci, surface-located, and functionally immunogenic. Therefore, it can be considered as a novel vaccine candidate against meningococcal B infection.

Reactogenicity and Safety of Multicomponent Meningococcal Serogroup B Vaccine (4CMenB) Administered with or without Routine Infant Vaccinations in Different Schedules

Nicoletta Gossger1, Ina Beeretz1, Matthew Snape1, Adam Finn2, Paul Heath3, Andrew Collinson4, Gianni Bona5, Susanna Esposito6, Peter Dull7, Ellen Ypma7, Daniela Toneatto7, Alan Kimura7, Clarissa Oeser3, Maggie West2 , Tessa John1, Andrew J Pollard1 and the European Men B Vaccine Study Group.

1Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, UK
2Bristol Children’s Vaccine Centre, University of Bristol, Bristol, UK
3St Georges Vaccine Institute, St George’s, University of London, London, UK
4Royal Exeter and Devon Hospital, University of Exeter, Exeter, UK
5Ospedale Maggiore della Carità - Clinica Pediatrica Boni, Novara, Italy
6Fondazione IRCCS dell'Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena di Milano, Milano, Italy
7Novartis Vaccines and Diagnostics Cambridge, MA and Siena, Italy
Contact email: nicoletta.gossger@paediatrics.ox.ac.uk

Keywords: Meningococcus serogroup B, vaccine, clinical trial, reactognicity, routine infant vaccines

Background

An investigational serogroup B meningococcal vaccine containing 3 recombinant-proteins plus outer-membrane-vesicles (4CMenB) is in latestage development. We studied the reactogenicity of this vaccine when administered at 2, 4 and 6 months of age (concomitantly with and separately from routine-vaccines) and at 2, 3 and 4 months of age (concomitantly with routine-vaccines).

Methods

An open-label, parallel-group, multi-centre study was conducted with randomisation of 1885 participants 2:2:1:1 to receive i) 4CMenB at age 2, 4 and 6 months concomitantly with routine-vaccines (7-valent pneumococcal-vaccine and a combined DTaP/HepB/IPV/Hib-vaccine) ii) 4CMenB at 2, 4 and 6 months administered separately from routine-vaccines (given at 3, 5 and 7 months) iii) 4CMenB with routine vaccines at 2, 3 and 4 months or iv) routine-vaccines alone. Local and systemic reactions were recorded for 7 days after each vaccination.

Results

Fever (≥38°C) was found in 44%-61% of participants after each dose of 4CMenB given concomitantly with routine-vaccines, in 26%-41% receiving 4CMenB alone and in 23%-36% with routine-vaccines alone. A similar pattern was seen for the percentage of participants with irritability (66%-79% 4CMenB and routine-vaccines, 53%-63% 4CMenB alone, 44%-57% routine-vaccines only). Local reactogenicity was similar between all groups. Four seizures (none febrile) were reported within 48 hours after vaccination; one after 4CMenB and routine vaccines, one after 4CMenB alone and two after routine-vaccines alone.

Conclusion

4CMenB when administered alone had a reactogenicity profile which was comparable to that produced by routine-vaccines, but systemic reactogenicity was increased when the study-vaccine was combined with routine-vaccination. No major safety concerns were raised.

Using lipooligosaccharide modified natural outer membrane vesicles to target meningococcal antigens to human dendritic cells

Hannah Jones1, Hendrik-Jan Hamstra3, Alastair Copland1, Jeremy Brown2, Nigel Klein1, Peter van der Ley3, Garth Dixon1.

1Infectious Diseases and Microbiology Unit, Institute of Child Health, UCL, London, UK
2Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, Rayne Institute, UK.
3Unit Vaccinology, National Institute of Public Health and the Environment (RIVM), Bilthoven, Netherlands

Keywords: dendritic cells, lipooligosaccharide, natural outer membrane vesicles, DC-SIGN, vaccine

Neisseria meningitidis (Nm) is a major cause of meningitis and septicaemia worldwide. Effective vaccines have been developed against several serogroups but no broad coverage vaccine is currently licensed against serogroup B. Several minor outer membrane proteins (OMP) have been identified as potential vaccine targets and these are currently being explored. One way of presenting these minor OMPs in a vaccine is to use natural outer membrane vesicles (NOMV). Here we describe structure modification of lipooligosaccharide (LOS) to reduce toxicity and target NOMV to dendritic cells (DC), which have been widely considered to play a key role in the induction of protective immunity.

We engineered an unencapsulated strain of Nm that expresses pentacylated lipid A (lpxL1), conferring reduced toxicity, and lgtB oligosaccharide structure that targets meningococcal OMV to DC via DC-SIGN (lgtB/lpxL1). Single deletion mutations lpxL1 and lgtB, were used as comparators. Human DC were stimulated with NOMV and DC responses were assessed using the following parameters 1) cytokine production, 2) antigen uptake, 3) DC maturation and 4) T cell stimulatory capacity.

All NOMV induced DC maturation and production of TNFα, IL-1β, IL-6, IL-23 and IL-10, although those expressing lpxL1 were less potent stimulators than NOMV expressing wild type lipid A. NOMV with the lgtB/lpxL1 structure were significantly more potent stimulators of DC maturation and IL-23 and IL-10 production than NOMV with lpxL1 lipid A and wild type oligosaccharide. DC phagocytosed NOMV expressing lgtB/lpxL1, but not the single mutant lpxL1, and this was shown to be exclusively through DC-SIGN.

All NOMV induced T helper (Th) 1, 2 and 17 responses in an in vitro DC-T cell co-culture model. The subtle differences in the magnitude of the T cell response induced by different NOMV may be explained by the differences in DC maturation and cytokines profiles.

NOMV with the lgtB/lpxL1 structure were significantly more potent stimulators of DC than NOMV with lpxL1 and wild type oligosaccharide, suggesting that the lgtB modification enhances the stimulatory capacity of pentacylated lipid A. lgtB/lpxL1 NOMVs were shown to target the NOMV to DC but did not induce a pronounced proinflammatory cytokine response as observed with NOMV expressing wild type LOS. The lpxL1/lgtB NOMV shows potential for use as a meningococcal vaccine vector that could be further modified to over express OMP important for induction of cross-protective immunity.

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Preclinical safety and immunogenicity evaluation of a new generation nonavalent PorA OMV vaccine against serogroup B meningococcus

Patricia Kaaijk1, Ineke van Straaten2, Elmieke Boot2, Harry van Dijken1, Germie van den Dobbelsteen1

1National Institute for Public Health and the Environment (RIVM), CIb/Vaccinology, Bilthoven, The Netherlands;
2Netherlands Vaccine Institute, Quality Control Department, Bilthoven, The Netherlands

Keywords: meningococcus serogroup B; vaccine; lpxL-1; preclinical; SBA

Background

Until now, only MenB vaccines based on outer membrane vesicles (OMV) have been proven successful in controlling MenB epidemics. The use of vaccine strains with inactivated lpxL1 gene, i.e. less toxic LPS while maintaining its adjuvant activity, enabled the use of a simple detergent-free process to obtain high yields of native OMVs with good batch-to-batch consistency. Consequently, an improved nonavalent PorA OMV vaccine (Nonamen) prepared from genetically engineered strains with attenuated endotoxin (lpxL1 LPS) was developed.

In the present study, safety and immunogenicity of this new generation Nonamen was evaluated following repeated vaccination in rabbits and mice.

Methods

A formal GLP repeated-dose toxicology study including local tolerance was performed in New Zealand white rabbits. Rabbits were immunised (i.m.) five times with either adjuvanted placebo, plain (7.5 μg/PorA type or 15 μg/PorA type per dose of 0.5 mL) or aluminiumphosphate adjuvanted Nonamen (15 μg/PorA type per dose of 0.5 mL). Serum bactericidal antibody (SBA) titers against bacteria expressing one of the 9 PorAs were determined in blood samples from New Zealand white rabbits and NIH mice taken after four immunisations with the three vaccine formulations.

Results

No toxicologically relevant findings in local and clinical signs were noted in the rabbits after vaccination. Slight increases in rectal temperature (0.1°C–0.9°C) were found compared to placebo control at 4 hours after dosing, while at 24 hours after treatment no difference in body temperature was observed in any animal. Inflammations at the injection sites were comparable to placebo control, except for the group receiving adjuvanted vaccine where more often granulomatous inflammation areas were observed.

In rabbits, Nonamen induced high SBA titers against all MenB strains expressing the 9 PorAs, i.e., P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1 and P1.18-1,3,6, present in the vaccine. In mice, also good SBA titers were observed, although SBA titers varied more between the different PorA types. No statistically significant potentiating effect of aluminium phosphate adjuvant was observed, with the exception of a slightly higher SBA response against P1.5-1,2-2 in rabbits.

Conclusions

Nonamen is shown to have an acceptable toxicity safety profile in rabbits. In addition, Nonamen induced high SBA titers in both rabbits and mice against various MenB strains expressing PorA proteins that are present in the vaccine. These preclinical results support the start of the first clinical studies with this new generation Nonamen with lpxL-1 LPS.

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Immunogenicity of an Investigational Multicomponent Meningococcal Serogroup B Vaccine (4CMenB) Administered with or without Routine Infant Vaccinations in Different Schedules

Nicoletta Gossger1, Matthew Snape1, Adam Finn2, Paul Heath3, Andrew Collinson4, Gianni Bona5, Susanna Esposito6, Peter Dull7, Ellen Ypma7, Daniela Toneatto7, Alan Kimura7, Sandra Dymond2, Su Wilkins4, Tessa John1, Sarah Kelly1, Andrew J Pollard1 and the European Men B Vaccine Study Group.

1Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, UK
2Bristol Children’s Vaccine Centre, University of Bristol, Bristol, UK
3St Georges Vaccine Institute, St George’s, University of London, London, UK
4Royal Exeter and Devon Hospital, University of Exeter, Exeter, UK
5Ospedale Maggiore della Carità - Clinica Pediatrica Boni, Novara, Italy
6Fondazione IRCCS dell'Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena di Milano, Milano, Italy
7Novartis Vaccines and Diagnostics Cambridge, MA and Siena, Italy
Contact email: nicoletta.gossger@paediatrics.ox.ac.uk

Keywords: Meningococcus serogroup B, vaccine, clinical trial, immunogenicity, routine infant vaccines

Background


When administered at 2, 4 and 6 months of age an investigational multicomponent meningococcal serogroup B vaccine (4CMenB) elicits bactericidal antibodies against reference MenB strains containing the vaccine-antigens. We studied the immunogenicity of 4CMenB when administered concomitantly or without routine-vaccines and in a 2, 3 and 4 month schedule.

Methods

An open-label, parallel-group, multi-centre study was conducted with randomisation of participants 2:2:1:1 to receive i) 4CMenB at age 2, 4 and 6 months concomitantly with routine-vaccines (7-valent pneumococcal glyco-conjugate-vaccine and a combined diphtheria-toxoid, tetanus-toxoid, inactivated-polio, acellular-pertussis, hepatitis B and Haemophilus influenzae type b-vaccine) ii) 4CMenB at 2, 4 and 6 months separately from routine-vaccines (given at 3, 5 and 7 months) iii) 4CMenB and routine-vaccines at 2, 3 and 4 months iv) routine-vaccines alone. The proportion of participants with human-complement serum bactericidal antibody (hSBA) titre ≥1:5 (serological correlate of protection) was calculated.

Results

At least 99% of participants receiving 4CMenB at 2, 4, 6 months (concomitantly or without routine-vaccines) or at 2, 3, 4 months (with routine-vaccines) developed hSBA titres ≥1:5 against strains 44/76 and 5/99. For NZ98/254 the correlate was reached or exceeded in 79% (2, 4, 6 months with routine-vaccines), 87% (2, 4, 6 months without routine-vaccines) and 81% (2, 3, 4 months with routine-vaccines). Percentages of participants responding to routine-vaccines given concomitantly with 4CMenB were non-inferior to routine-vaccines alone for all antigens except pneumococcal-serotype 6B.

Conclusion

4CMenB is immunogenic against reference strains when administered concomitantly with routine-vaccines at 2, 4, 6 or 2, 3, 4 months.

Impact of a New Serogroup A Meningococcal Conjugate Vaccine, MenAfriVacTM, on Carriage of Serogroup A Neisseria meningitidis: Preliminary results from Burkina Faso

Paul A. Kristiansen1, Fabien Diomandé2,3, Lassana Sangaré4,5, Rasmata Ouédraogo5,6, Idrissa Sanou5,7, Abdoul Salam Ouédraogo7, Ba Ki Absatou8, Denis Kandolo3, Pascal Kaboré9, Musa Hassan-King10, Jennifer D. Thomas2, Thomas Clark2, Nancy Messonnier2, Marie-Pierre Préziosi11, Marc LaForce10 and Dominique A. Caugant1,12

1Norwegian Institute of Public Health (NIPH), Oslo, Norway
2Centers for Disease Control and Prevention, Atlanta, USA
3WHO Inter Country Support Team, Ouagadougou, Burkina Faso
4CHU Yalgado, Ouagadougou, Burkina Faso
5University of Ouagadougou, Burkina Faso
6CHUP Charles de Gaulle, Ouagadougou, Burkina Faso
7CHU Souro Sanou, Bobo-Dioulasso, Burkina Faso
8Laboratoire National de Santé Publique, Ouagadougou, Burkina Faso
9Direction de la lutte contre la maladie, Ministry of Health, Burkina Faso
10Meningitis Vaccine Project, Ferney, France
11WHO Initiative for Vaccine Research, Geneva, Switzerland
12Faculty of Medicine, University of Oslo, Norway

Keywords: MenAfriVac, serogroup A carriage, Burkina Faso, herd immunity

Objective

To investigate the potential herd immunity effect of MenAfriVac™, a new conjugate vaccine against serogroup A Neisseria meningitidis (NmA), by assessing its ability to reduce NmA carriage prevalence.

Methods

Study design: A multicentre repeated cross-sectional study of meningococcal carriage prevalence in the 1- to 29-year-old population in Burkina Faso, before and after mass vaccination.

Oropharyngeal swabs were obtained from a representative portion of the target population. Standard laboratory analysis was conducted in Burkina Faso. Meningococcal isolates were confirmed at NIPH/Oslo and further characterized using molecular methods.

Baseline meningococcal carriage prevalence was determined from four sampling campaigns performed every 3 months during the year 2009, yielding a total of 20 326 samples. In September 2010 MenAfriVacTM was introduced as part of a pharmacovigilance study in Kaya, one of the three study sites, while the rest of the country was vaccinated in December 2010.

A carriage study campaign was conducted in October-November 2010, representing the first post-vaccination carriage assessment for the district of Kaya, and the last pre-vaccination campaign for the districts of Bogodogo and Dandé. Post-vaccination sampling campaigns are continuing every three months in 2011, in the same way as before vaccination.

Results

Overall baseline meningococcal carriage was 3.98% while NmA prevalence was 0.39%. NmA carriage varied by season and district with higher prevalence in the dry season and in rural areas. The highest prevalence was found in the rural district of Kaya (0.93%).

The campaign in October-November 2010 showed no NmA carriage in the vaccinated district of Kaya, while it was still present in the nonvaccinated district Dandé, at the same level as in 2009. Two additional sampling campaigns in 2011 showed that NmA was still absent in Kaya, 8 months after vaccination, and was undetected in Bogodogo and Dandé, 6 months after vaccination.

Conclusion

MenAfriVac vaccination induced a 100% reduction of NmA carriage prevalence (statistically significant, p<0.001, Chi-square test), 6 to 8 months after mass vaccination.

The dramatic impact of MenAfriVac on NmA carriage is a first strong indication of its ability to confer herd immunity by preventing colonisation and transmission of NmA.

Meningococcal Antigen Typing System (MATS) was a conservative estimate of killing when confirmed in a serum bactericidal assay using human complement (hSBA)

A. Biolchi1*, G. Frosi1*,S. Gilchrist2, M. Stella1, B. Brunelli1, M. Comanducci1, S. Bambini1, S. Comandi1, F. Rigat1, J. J. Donnelly1, M. Pizza1, J. Findlow2, R. Borrow2, M. Giuliani1, D.Medini1

1Novartis Vaccines & Diagnostics, Via Fiorentina 1, Siena, Italy
2HealthProtection Agency, Manchester RoyalInfirmary, Manchester, UK
*These authors contributed equally to the work
duccio.medini@novartis.com

Keywords: MATS, representative strain panel, hSBA, 4CMenB, vaccine strain coverage

Background

The meningococcal antigen typing system (MATS) predicts the strain coverage of a multicomponent meningococcal serogroup B vaccine (4CMenB) based on quantity of and cross reactivity with vaccine-induced immune responses of factor H binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA), and Neisserial Adhesin A (NadA), and on the genotype of PorA. MATS estimated that 4CMenB would cover 78% (95%CI: 66-91%) of 1052 isolates collected in five European countries (73-87% by country) during a specified time period. We compared MATS predictions with actual hSBA results for an unbiased representative set of isolated selected from the previously presented panel.

Methods

All 528 serogroup B strains isolated between July 2007-June 2008 by the Health Protection Agency had been evaluated by MLST and MATS, and genotyped for fHbp, NadA, and NHBA. A panel of 40 isolates was selected using stratified proportional sampling to account for coverage distribution and epidemiologically relevant strains by controlling for MATS scores and relevant genetic characteristics: MLST type and
genotype of vaccine antigens. The 40 strains were then tested in hSBA using pooled post vaccination sera from infants or adolescents. Results were compared with MATS data.

Results/Conclusion

The panel selected for hSBA testing provided an unbiased sample of strains proportionally representative of 98% of the MATS phenotypes and >80% of MLST and vaccine antigen genotypes. MATS predicted 73% (95%CI: 58-87%) killing in the hSBA. The current analysis revealed that 85% (95%CI: 70-93%) of strains were killed by pooled infant sera as were 86% (95%CI: 70-94%) of strains by adolescent sera. These results confirm that MATS is a conservative measure of killing in the hSBA.

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Inducing cross protection within fHBP variants

Michele Pallaoro1, Maria Scarselli1, Beatrice Aricò1, Brunella Brunelli1, Silvana Savino1, Federica Di Marcello1, Emmanuelle Palumbo1, Daniele Veggi1, Laura Ciucchi1, Elena Cartocci1, Matthew James Bottomley1, Enrico Malito2, Paola Lo Surdo1, Maurizio Comanducci1, Marzia Monica Giuliani1, Francesca Cantini3, Sara Dragonetti3, Annalisa Colaprico1, Francesco Doro1, Patrizia Giannetti1, Barbara Brogioni1, Marta Tontini1, Markus Hilleringmann1, Vincenzo Nardi-Dei1, Lucia Banci3, Derek O’Hagan1, Mariagrazia Pizza1, Rino Rappuoli1

1Novartis Vaccines and Diagnostics S.r.l., Via Fiorentina 1, 53100 Siena, Italy
2Genomics Institute of Novartis Research Foundation, 10672 John Jay Hopkins Drive, San Diego, CA 92121, USA
3Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy

Keywords: fHBP, formulation, adjuvants

Sequence variability of protective antigens is a major challenge to the development of vaccines; the factor H binding protein (fHBP) antigen present in different Neisseria meningitidis (MenB) vaccines under development is not different. The antigen is effective and provides excellent immune response in humans, however is extremely heterogeneous and more than 300 variants have been described to date. These variants can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. With this limitation in mind we set out to test different strategies to overcome the problem. If on one side it is possible to follow a rational design approach based on structure information to improve the antigen from a structural point of view (see Bottomley MJ et e l.), on the other side it is possible to improve the immune response with more potent adjuvants to enlarge the spectrum of coverage by enhancing immunogenicity and/or antigen presentation and processing.

Here we describe a new adjuvant combination containing a Toll Like Receptor 9 agonist (IC31) in the presence of Aluminum hydroxide and the protection elicited by several different fHBP chimeras and show that this new adjuvant combination in the presence of the final selected chimeras is able to induce significant cross protection over more distantly related variants. Our data provide a solid rationale for the use of this formulation in a vaccine that recognizes all fHBP antigenic variants and should provide efficacious and broad protection against infection with MenB.

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Development of a Multiplex Bead Based Meningococcal Antigen Typing System (BMATS)

Gowrisankar Rajam1, Ellie Kim1, Marzia Giuliani2, John Donnelly2, George Carlone1

1Division of Bacterial Diseases, CDC, Atlanta, GA 30333, USA
2Novartis Vaccines, Siena, Italy

Keywords: Neisseria meningitidis, MenB, MenB vaccine, immune-phenotype, MATS, BMATS.

Background

Meningococcal Antigen Typing System (MATS) is an ELISA based immune-phenotyping technique used for Neisseria meningitidis B (MenB) strains. Novartis MenB vaccine, 4CMenB, has 4 major antigens. MATS is used to detect 3 of these antigens in MenB bacterial lysate. To test a single strain, 3 individual MATS ELISAs have to be performed. We have developed a multiplexed bead based meningococcal antigen typing system, BMATS that offers the capacity to measure three or more antigens simultaneously.

Materials and methods

A multiplex IgG capture assay was developed to quantify anti-4CMenB IgG. BMATS detects the homologous competition between the surface expressed/exposed antigens in the bacterial lysate and antigen conjugated fluorescent beads for antigen specific antibody (IgG) in the capture assay. Using xMAP technology, fluorescent beads are screened for captured antigen specific IgG and expressed as median fluorescence index (MFI). A strain with high density and/or cross-reactivity of antigen results in specific reduction in MFI. This inverse relationship establishes the MenB antigen phenotype. Eighteen MenB strains were tested by both methods.

Results and conclusions

Assay sensitivity, specificity, reproducibility and robustness are presented. BMATS successfully detected antigens on all MenB strains tested. Reference strains for fHbp and NHBA yielded 100% reduction in MFI, and NadA yielded 70% reduction. BMATS was in agreement with MATS ELISA for all 18 strains tested. Validation of BMATS with additional MenB strains and development of data reduction software to calculate antigen specific relative potency for each isolate is in progress.

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AFCo1 provides nasal adjuvant activity against capsular polysaccharides of Neisseria meningitidis serogroup C and Salmonella Typhi

Belkis Romeu, Elizabeth González, Maribel Cuello, Osmir Cabrera, Julio Balboa, Miriam Lastre and Oliver Pérez

Immunology Department, Vice-presidency of Research and Development, Finlay Institute, P.O. Box 16017, Havana, Cuba
Contact email: bromeu@finlay.edu.cu

Keywords: adjuvant, cochleate, mucosal response, polysaccharide antigen

Increasing emphasis is being placed on the mucosal administration of vaccines in order to stimulate mucosal as well as systemic responses. Findings from our group suggest that proteoliposome-derived cochleate (AFCo1) acts as a potent mucosal adjuvant. As an alternative to chemical conjugation, the current study is aimed at determining the benefit of using AFCo1 to improve mucosal and systemic immune responses to capsular polysaccharides from Neisseria meningitidis serogroup C (PsC) and Salmonella Typhi (PsVi). Therefore, intranasal (i.n) immunisations of three doses one week apart with AFCo1 plus PsC or PsVi in adult mice were conducted. High specific anti PsC IgA responses were obtained at site of entry and distant sites such as the vagina. Higher IgG responses after i.n application of PsC or PsVi coadministered with AFCo1 were induced. Our results demonstrate a shift in the isotype pattern elicited in response to PsC and PsVi. Also, the avidities of PsVi antibodies elicited by AFCo1 were higher than those elicited by the polysaccharide alone. In summary, AFCo1 as a nasal adjuvant demonstrated the capability to elicit mucosal and systemic specific responses against thymus independent antigens.

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From Neisseria meningitidis serogroup B vaccine to Potent Adjuvants

Oliver Pérez*, Miriam Lastre, Osmir Cabrera, Caridad Zayas, Maribel Cuello, Elizabeth González, Belkis Romeu, Julio Balboa and Daniel Cardoso

Immunology Department, Research Vice-presidency, Finlay Institute, Havana, Cuba
Contact author: oliverp@finlay.edu.cu

Keywords: Adjuvant, Vaccine, Neisseria meningitidis, AFCo1, AFPL1

Introduction

Outer membrane vesicles (OMV) from microorganisms have been demonstrated to be a good target for vaccine development. Ameningococcal VA-MENGOC-BC® vaccine has been used since 1988 in Cuba and abroad with more than 60 million doses applied without severe adverse events. Adjuvants are essential vaccine components that immunopotentiate (IP) the innate immunity, deliver antigens todesired places, and polarize (Pz) the adaptative immune response to the desired level. Adjuvants are not licensed and are mainly kept by companies to develop their own vaccines and to avoid competitors. Furthermore, there are currently no licensed adjuvants capable of enhancing immune responses at mucosal surfaces where the majority of infectious agents enter or establish in the host. Therefore, the development of adjuvants is mandatory to any pharmaceutical companies dedicated to vaccine development, particularly for mucosal applications.

Aims

To demonstrate and characterize the adjuvant effect of OMV and to develop the Finlay Adjuvant Platform (FAP).

Results

The FAP consists of a series of detergent-extracted OMV called Proteoliposome (PL) because of their high protein content in addition to lipids and their transformation into Cochleates (Co) named AFPLn and AFCon, respectively. In addition, AFCo3x not-derived from Proteoliposome was also developed. The former (i.e. AFPLn and AFCon) contain several protective proteins (which permit it to be used as a specific vaccine candidate) and several synergistic microbial-associated molecular patterns (MAMP, which permit it to be used as vaccine adjuvant). The most studied are AFPL1 and AFCo1 derived from Neisseria meningitidis serogroup B. They contain: LPS, Porins, and a trace of bacterial DNA as the main synergistic IP; non-living delivery systems based in lipids; LPS as a main Pz driving to Th1 and CTL immune responses; and particles as nano- or micro-particles. They induce long-lasting memory response and could be used with diverse antigens and by parenteral or mucosal routes. The AFCo3 contains particular MAMP, which reduces the cost and has possible uses in veterinary vaccines.

Last but not least, finding a good mucosal adjuvant will permit the development of Single Time Vaccination Strategies which combine parenteral and mucosal vaccination simultaneously.

Conclusion

FAP seems to be very promising.

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Evaluation of the immunological properties of the Neisserial Heparin Binding Antigen (NHBA)

Santini Laura, Irene Vacca, Biolchi Alessia, Elena Del Tordello, Brunelli Brunella, Giuseppe Boccadifuoco, Bambini Stefania, Maurizio Comanducci, Alessandro Muzzi, Duccio Medini, John J. Donnelly, Rino Rappuoli, Mariagrazia Pizza, Marzia M. Giuliani, Serruto Davide*

Novartis Vaccines and Diagnostics, Via Fiorentina 1, Siena, Italy
*contact email: davide.serruto@novartis.com

Keywords: vaccine, serum bactericidal activity, NHBA, competitive SBA, recombinant strains

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis which binds heparin-like molecules.NHBA is an antigen of the multicomponent 4CMenB vaccine able to induce bactericidal antibodies in laboratory animals and humans. The aim of this study is to investigate the potential cross protection of NHBA-induced bactericidal antibodies against a panel of N. meningitidis strains.

We used various approaches to investigate the level of cross protection mediated by human anti-NHBA antibodies. In order to characterize only the immunological properties of NHBA we selected N. meningitidis strains mismatched for the other vaccine antigens (fHbp, NadA and PorA1.4). These strains have been tested in a Serum Bactericidal Assay using human complement (hSBA) and human sera from different age groups vaccinated with the 4CMenB vaccine. To further prove that the immune response was directed against NHBA, we performed a competitive hSBA using the NHBA recombinant antigen and also generated NHBA deletion mutants in different genetic backgrounds.

The hSBA analysis showed that human sera raised against the 4CMenB vaccine are able to kill natural N. meningitidis strains harboring different NHBA amino acidic sequences. We also demonstrated that the addition of recombinant NHBA antigen or the deletion of nhba gene abolished or significantly decreases bactericidal titers.

To evaluate the contribution of amino acid sequence variability to vaccine coverage, we constructed a strain that is susceptible to bactericidal killing only by anti-NHBA antibodies and engineered it to express equal levels of different NHBA peptides under an inducible promoter. This ongoing approach will be useful to further evaluate the level of cross-protection of NHBA and assess the relation between level of expressionand bactericidal killing mediated by NHBA.

The results obtained so far demonstrate that NHBA is an important vaccine antigen able to induce cross-protective bactericidal antibodies against genetically different strains in different age groups vaccinated with the 4CMenB vaccine.

Development of a flow cytometric antibody-mediated complement deposition assay for Group B Streptococcus

S. Thomas, A. Gorringe, M. Hudson, S. Taylor

Microbiological Services Porton, Health Protection Agency, Porton Down, Salisbury, UK

Keywords: GBS, Flow-cytometry, complement, vaccine, immunity

Background

Group B streptococcus (GBS) is the leading cause of severe bacterial infection in newborns. Specific and functional IgG raised to GBS antigens together with opsonising complement components, have key roles in the opsonisation of bacteria during infection. Therefore, seroepidemiological and vaccine assessment studies have measured functional immunity to GBS using the opsonophagocytic killing assay (OPKA). However, higher throughput assays such as an antibody-mediated complement deposition assay (CDA) could act as a surrogate for the more technically challenging OPKA, allowing the testing of large numbers of sera from a very small volume (<20μL). The measurement of complement deposition onto GBS bacterial surfaces is a potential surrogate of protection in assessing the efficacy of candidate GBS vaccines. This study aimed to develop a novel, potentially high-throughput assay for use in the investigation of functional immunity in GBS neonatal disease and vaccine assessment.

Methods

The CDA was adapted and optimised for use with GBS from a pre-existing assay developed for Neisseria meningitidis. Strains of GBS, representing the five prevalent serotypes in the UK, were grown and killed (2% formaldehyde) and added to a 96-well plate containing IgGdepleted human plasma and test sera. An antibody (sheep anti-human) specific to C3c (binding both C3b and iC3b) conjugated to a fluorescent label (FITC) was added to the test samples. Samples were then analysed using flow cytometry.

Results

Investigation of the growth and killing parameters revealed that CDA performed with GBS harvested at OD600nm of 0.1 and killed with 2% formaldehyde gave excellent correlations to CDA performed with live mid-log cultures with an R of 0.74 and 0.96 (p=<0.01) respectively.

Optimisation assessed assay blocking buffer, complement concentration and incubation method. Serum dilution proved to be of great importance where there was a requirement for differentiating between samples with low responding sera. Following optimisation of the CDA, good serotype-specific responses for GBS were obtained with sera raised against polysaccharide antigens.

Conclusions

The CDA has been successfully optimised for use with the 5 most prevalent serotypes of GBS currently circulating in the UK. The assay was sensitive for the measurement of antibody-mediated deposition by the complement components C3b and iC3b, and is reproducible between assays. Correlations with OKPA will be determined and the CDA will then be used in large scale epidemiological studies and to assist in the assessment of immune responses to candidate vaccines.

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