Poster presentations

Pathogenesis

P1Transcriptional response of pathogenic Neisseria meningitidis with non-phagocytic cells

Hajah Mohd Afsar, Tom Mendum, Jane Newcombe, Kikki Bodman-Smith and Johnjoe McFadden

Faculty of Health and Medical Sciences, University of Surrey, Guildford UK

Email:
hm00022@surrey.ac.uk

Neisseria meningitidis is a Gram-negative bacterial pathogen that remains a leading cause of systemic meningococcal infection ranging from bacteraemia, meningitis, and fulminant meningococcal septicaemia world-wide. The ability to adhere to and invade a range of human origin cells is an absolute necessity for N. meningitidis to colonise and disseminate inside its host and so to cause the rapid and damaging invasive form of the disease. From previous studies it has been established that two-component regulatory systems such as PhoP/PhoQ are involved in processes which are crucial for bacterial pathogenesis. We focused on investigation of the specific role played by the N. meningitidis PhoP regulator in the interaction of meningococci with human epithelial cells.

In this study, to facilitate confocal microscope studies, bacteria were made fluorescent by transforming them with a Green Fluorescent Protein plasmid. A549 epithelial cells, a respiratory human cell line were challenged with the fluorescent N. meningitidis strain wild-type (L91543) and N. meningitidis strain phoP mutant (L91543/NMB0595 knockout mutant). Thus an epithelial colonisation model was developed to compare the binding and uptake ability of the phoP mutant with the parental wild-type via adhesion and invasion assays. The influence of colonisation on the host cell cytoskeleton during bacterial adhesion and invasion was also examined by confocal microscopy. Finally we analysed the transcriptome of N. meningitidis wild-type and the phoP mutant with and without epithelial cell interaction via a comparative genome hybridisation approach using microarray technology.

Our findings demonstrated that the N. meningitidis phoP mutant shows a defect in adherence to and invasion into epithelial cells and this is independent of numbers or replications in cultures. The active nature of the invasion was confirmed by inhibition by cytocholasin D and less damaging of the cell membrane by the phoP mutant was observed by confocal microscopy. Our data suggested that PhoP regulator of the two-component system is involved in the colonisation process of the N. meningitidis. This fits with our hypothesis that some of the genes controlled or influenced by the PhoP regulator are involved in the N. meningitidis virulence.

We have identified some important virulence genes including nspA, tbpA, tbpB and clpB that were differentially significantly regulated in the phoP mutant as revealed by the transcriptomic microarray data. These findings may lead to further identification and characterisation of the novel targets for drug design and future therapeutics.

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P2Temperature triggers immune evasion by Neisseria meningitidis

Edmund Loh1,2, Elisabeth Kugelberg2, Alexander Tracy1, Qian Zhang2, Bridget Gollan2, Helen Ewles2, Ronald Chalmers3, Vladimir Pelicic2, Christoph M. Tang1,2

1Sir William Dunn School of Pathology, University of Oxford, Sir Parks Road, Oxford OX1 3RE, UK; 2Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK; 3School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2NR, UK

Email: edmund.loh@path.ox.ac.uk

Neisseria meningitidis has several strategies to evade complement mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here, we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature. We have identified three independent RNA thermosensors located in the 5´-untranslated regions of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing. Therefore increased temperature (which occurs during inflammation) acts as a ‘danger signal’ for the meningococcus, enhancing its defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an increased temperature and recruitment of immune effectors. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium.

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P3Functional tspB genes are essential for meningococcal survival in human serum

Maike G. Müller, Patrick G. Wall, Gregory R. Moe

Center for Immunobiology and Vaccine Development, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609

Email: gmoe@chori.org

Keywords: T and B cell stimulating protein B (TspB), prophage, serum survival, IgG-binding protein, DNA- binding protein

Background: In developed countries meningococcal disease is relatively rare. However, nasopharyngeal carriage of meningococci without disease is relatively common, ranging from 5% to >80% depending on the population studied. A study of carriage versus disease-causing strains during an epidemic in the Czech Republic showed that prophage DNA was associated with disease-causing strains (Bille et al. 2006, J Exp Med). However, the reasons for the association of prophage DNA with pathogenicity were not determined. Recently, we showed that prophage gene ORF6, whose product, T and B cell stimulating protein B (TspB), is an IgG-binding protein specific for the Fc domain of human IgG2 (Müller et al. 2013, J Immunol). In addition, we found that bacteria grown in the presence of human serum form aggregates enveloped in a matrix of TspB, IgG and DNA that has characteristics of biofilm.

Objective: The aim of this study was to determine whether tspB expression had an effect on meningococcal survival in human serum. In addition, we investigated whether functional activities of TspB that give rise to the aggregate/biofilm phenotype can be attributed to specific subdomains of the protein.

Methods: Mutants of group B strain H44/76 in which combinations of multiple tspB genes were knocked out were tested for their ability to survive in different concentrations of normal human serum (NHS) or IgG- depleted NHS (dNHS). The effect on serum survival was compared to mutants in which other survival factors such as capsular polysaccharide and factor H binding protein had been knocked out.
To investigate the functional activities of TspB, we produced and purified recombinant proteins corresponding to highly conserved subdomains, then characterised their Ig- and DNA-binding activities by ELISA and agarose gel shift assay, respectively.

Results: Knocking out 3 of 3 tspB genes in H44/76 was equivalent to knocking out production of capsular polysaccharide, which was essential for survival. At least two functional copies of tspB were required for survival in >5% NHS or dNHS. In contrast, knocking out fHbp affected survival only at higher concentrations of NHS (>20%) and had no effect on survival in dNHS.
Binding activities of TspB to IgG2 Fc and non-specific DNA were localised within separate structural domains.

Conclusions: The IgG- and DNA-binding activities of TspB facilitate formation of a biofilm that appears to protect the bacteria from activities leading to bacteriolysis in human serum. The results suggest one possible reason why strains carrying prophage DNA are more likely to be associated with disease.

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P4Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis

Susanna Ricci1,5, Michael Wenzel2, Denis Grandgirard3,5, Tiziana Braccini1, Marco Rinaldo Oggioni4,5, Uwe Koedel2,5, Stephen Leib3,5.

1Laboratory of Molecular Microbiology and Biotechnology (LA.M.M.B.), Department of Medical Biotechnologies, University of Siena, Italy; 2Department of Neurology, Klinikum Großhadern, Ludwig-Maximilians University, 81377 Munich, Germany; 3Institute for Infectious Diseases, University of Bern, 3010 Bern, Switzerland; 4Department of Genetics, University of Leicester, Leicester LE1 7RH, UK; 5The ESCMID Study Group for Infectious Diseases of the Brain (ESGIB)

Email: susanna.ricci@unisi.it

Keywords: Neisseria meningitidis, experimental meningococcal meningitis, brain damage, matrix metalloproteinases

Background: Neisseria meningitidis is the major causative agent of acute bacterial meningitis in infants, children and young adults. Mortality due to meningococcal meningitis (MM) varies from 4 to 8%, and approximately 5-20% MM survivors suffer from neurological sequelae due to brain damage in the course of meningitis. There are several mediators of brain damage, including matrix metalloproteinases (MMPs).
Objectives: Assess the efficacy of a broad metalloproteinase inhibitor, batimastat (BB-94), in a novel model of MM-induced brain damage in the mouse.

Methods: The brain damage model is based on i.cist. infection of 6-7 week old BALB/c mice with a serogroup C N. meningitidis strain. Mice were treated with BB-94 at the time of infection and 24h post-infection. Control and treated animals were humanely sacrificed 48h after i.cist. challenge. Enzymatic activity of MMPs in cerebella of infected mice, intracerebral haemorrhages, integrity of the blood-brain barrier (BBB), and apoptosis in the hippocampus were analysed.

Results: Mice that received BB-94 presented significantly lower zymographic activity of MMP-9 compared to controls (P<0.001). Treated mice also showed reduced intracerebral bleeding (P<0.05) and diminished BBB breakdown (P<0.05) in comparison with untreated animals. In contrast, no differences in apoptosis were observed between the groups. Finally, zymography data significantly correlated with both BBB disruption (P<0.05) and intracerebral haemorrhages (P<0.001).

Conclusions: The MM-induced brain damage model established in the mouse is an effective tool to analyse several crucial readouts in the disease. By using this mouse system, we have shown that: (i) MMPs significantly contribute to experimental MM, and (ii) inhibition of MMPs reduce intracranial complications in mice suffering from MM, thereby representing a potential adjuvant molecule in MM post-infection sequelae.

P5Characterisation of pilE antisense RNA in Neisseria meningitidis

Felicia Tan, Edmund Loh, Christoph M. Tang and Rachel M. Exley
Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK

Email: felicia.tan@path.ox.ac.uk

Keywords: Neisseria meningitidis, gene regulation, type four pili, pilin, small RNA

Expression of Type four pili (Tfp) is important for virulence in Neisseria meningitidis. Pili mediate adhesion to host surfaces, twitching motility, DNA uptake, and are subject to phase and antigenic variation. Pili are important for the survival of the meningococcus during colonisation and disease. Moreover, the extensive antigenic variation of Tfp has been proposed to enable the bacterium to avoid immune surveillance. Pilin expression and antigenic variation may be modulated in response to environmental cues, however, the precise mechanisms of such regulation are as yet unclear. Previous studies have revealed that the transcription of pilE, which encodes the major pilin subunit, is influenced by the RNA chaperone Hfq, suggesting that noncoding RNAs may be involved in pilE regulation. We have identified a putative promoter for expression of a cis-encoded RNA in the antisense strand of pilE. This putative promoter is conserved across N. meningitidis isolates from different strains and clonal complexes, suggesting that it may play an important role in mediating pilE expression or pilin function.

By performing mutational analysis of this promoter along with Northern blotting and strand-specific RT-PCR, we have shown that the promoter is functional in an ectopic E. coli system. We have successfully introduced the promoter mutation into a wild type strain of N. meningitidis and are currently investigating the possible function of the pilE antisense and its mechanism of action in this important human pathogen.

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P6The role of multiple copies of the capsulation b locus of Haemophilus influenzae serotype b (Hib) in resisting antibody-dependent, complement mediated bacteriolysis using a functional serum bactericidal assay

Kelly Townsend1, Helen Findlow1, Xilian Bai1, Shamez Ladhan2, Mary Slack3, Ray Borrow1

1Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester; 2Immunisation, Hepatitis and Blood Safety Department, Public Health England, London; 3Respiratory and Systemic Infection Laboratory, Public Health England, London

Keywords: cap b copy, Hib, SBA assay, Hib vaccine failure, Hib polysaccharide


Background: The Haemophilus influenzae serotype b (Hib) polysaccharide capsule, which is encoded by the capsulation (cap) b locus, is a major virulence factor and plays an important role in evading the host immune system by resisting complement-mediated lysis and opsonisation. Up to five copies have been detected in clinical isolates from individuals with invasive disease. Such strains with multiple copies of the cap b locus have been shown to produce significantly more capsular polysaccharide than strains with fewer copies of the cap b locus. In addition, an isogenic strain containing four copies of the cap b gene was shown to be significantly more resistant to complement-mediated, antibody- dependent bacteriolysis as well as complement- mediated opsonisation by macrophages when compared to a two-copy strain.
A recent study reported that children who had been fully vaccinated against Hib were three times more likely to have been infected by a Hib isolate with more than two copies of the cap b gene than unvaccinated paediatric controls. The objective of this study was to use a newly-developed Hib serum bactericidal assay (SBA) to assess the functional relevance of multiple cap b loci in clinical isolates from patients with invasive Hib disease.

Methods: A total of 164 serum samples collected from children who developed invasive Hib disease despite having being vaccinated against Hib (vaccine failures) were tested using the Hib SBA against five Hib strains containing 1, 2, 3, 4 or 5 copies of the cap b locus. Geometric mean titres (GMTs) and proportion of children achieving a Hib SBA titre of =8 were calculated for samples assayed against each strain at T0 and T60 time points.

Results: Of the 164 serum samples, 127 had sufficient serum for testing against all Hib strains. Geometric mean SBA titres for strains expressing 1, 2, 3, 4 and 5 copies of the cap b locus at time T60 were 46 (95% CI, 29-73), 24 (95% CI, 15-38), 43 (95% CI, 27-69), 55 (95% CI, 33-90), and 20 (95% CI, 13-31), respectively. Overall, there was no association between the number of cap b copies and either Hib SBA GMT or the proportion of children achieving a Hib SBA titre of =8.

Conclusions: The lack of association between cap b copy numbers and Hib SBA titres suggests that, although increased capsule production may be advantageous in evading complement killing, antibodies against other Hib surface proteins may play an important role inducing bactericidal activity.

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P7Antibody-mediated complement C3b binding to Group B Streptococcus in paired mother and baby serum samples in a refugee population at the Thai-Myanmar border

Stephen Thomas1, Jenny Herbert1, Charlotte Brookes1, Claudia Turner2,3,4, Paul Turner2,3,4, Paul T. Heath5, Andrew Gorringe1, Stephen Taylor1

1Public Health England, Porton Down, Salisbury; 2Shoklo Malaria Research Unit, Mae Sot; 3Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand; 4Centre for Tropical Medicine, University of Oxford, Oxford; 5Paediatric Infectious Diseases Research Group, Clinical Sciences, St George’s, University of London, UK

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

Streptococcus agalactiae (group B Streptococcus; GBS) is currently the leading cause of neonatal sepsis and meningitis. Previous studies have shown that opsonophagocytosis determined in a bacterial killing assay may correlate with protection provided by serotype-specific polysaccharide conjugate vaccines. This opsonophagocytosis assay requires a large serum volume and only small number of samples can be evaluated at one time. Thus we have developed a flow cytometry assay to quantify antibody-mediated complement C3b deposition onto GBS bacteria representing the five most prevalent GBS. We have used this assay to determine antibody- mediated C3b binding to GBS serotypes 1a, 1b, II, III and V using 1068 sera taken from mothers and
Pathogenesis

cord pairs obtained at the time of birth at the Thai- Myanmar border (Turner et al., 2012, BMC Infect Dis). The initial aim of the study was to determine the level of functional antibody in neonates required to prevent disease. However, there were no cases of GBS disease during the sampling period. The distribution of antibody-mediated C3b binding determined in the sample cohort corresponded with the carriage data, with the highest levels of C3C deposition observed to the most prevalent serotype in this region (serotype II) followed by serotypes 1a, III, V and 1b. Neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody- mediated C3b deposition against serotype II GBS than neonates born to mothers with no carriage. Thus these neonates at risk were likely to be protected from disease by maternal antibody. The trend of higher antibody-mediated C3b deposition in neonates born to carriage positive mothers was also observed for serotype 1a, and likely was not observed for the other three serotypes tested due to small numbers of carriage positive mothers in the study. A maternal GBS carriage rate of 12% was observed in this population, yet detectable antibody-mediated C3b deposition was detected to at least one serotype in 91% of mothers, suggesting carriage may be underestimated in this region or that carriage occurs transiently.

P8The choroid plexus epithelia as an alternative bacterial entry point into the brain in E. coli K1- associated neonatal meningitis

Andrea Zelmer & Theresa H. Ward
 
Infection and Immunology Department, London School of Hygiene and Tropical Medicine, London, UK
Email: andrea.zelmer@lshtm.ac.uk


Keywords: neonatal bacterial meningitis, E. coli K1, choroid plexus, blood-brain-barrier

Neonatal bacterial meningitis (NBM) is a devastating disease, which often leaves affected newborns with serious neurological sequelae. The Gram-negative bacterium E. coli is a major cause of NBM, and accounts for approximately 20% of cases of NBM in the UK and Ireland. The K1 polysaccharide capsule is a major virulence factor, and 80-85% of E. coli isolates from NBM cases carry the K1 capsular antigen on their surface. Upon infection, the bacteria colonise the intestine and cross the intestinal barrier into the blood stream, before entering the central nervous system (CNS). It has been widely suggested that bacteria cross the blood-brain-barrier by invading and traversing the cells of the cerebral microvascular endothelium. Some studies showed that the epithelial lining of the choroid plexus, the part of the brain where the cerebrospinal fluid is produced, may be an alternative port of entry into the CNS; however this notion is poorly studied, and the potential mechanisms are incompletely understood. In this study we aim to investigate the possibility of E. coli K1 invasion and traversal of the choroid plexus epithelium by utilising an in vitro model of the choroid plexus epithelial barrier. To test our hypothesis, a choroid plexus derived epithelial cell line, Z310, was infected with pathogenic E. coli K1 and non- pathogenic E. coli K12 as a control. A gentamicin exclusion assay followed by a colony forming units assay was performed to assess invasion efficiency of E. coli K1 compared to the nonpathogenic E. coli K12 strain. Intra- or extracellular location of the bacteria was confirmed by confocal microscopy. We found that Z310 cells were efficiently invaded by E. coli K1 but not by E. coli K12.

Further, K1 bacteria exhibited cytotoxic effects on Z310 cells and infection led to damage of cellular monolayers. These results strengthen the notion that E. coli K1 can use the choroid plexus epithelium as an alternative port of entry into the CNS during neonatal bacterial meningitis. The mechanisms of entry are unclear, and we are currently investigating the molecular host-pathogen interactions. This work could potentially identify new host targets required for bacterial CNS invasion, leading to the design of new treatments for neonatal bacterial meningitis in the future.

Epidemiology & Surveillance

E9Epidemiology and molecular typing of Neisseria meningitidis capsular group W in England and Wales, 2000-2013

Kazim Beebeejaun1, Helen Campbell1, Ray Borrow2, Mary Ramsay1, Steve Gray3, Ed Kaczmarski2, Jay Lucidarme1, Shamez Ladhan1

1Public Health England Immunisation Department; 2Public Health England Vaccine Evaluation Unit, Public Health Laboratory, Manchester; 3Public Health England Meningococcal Reference Unit

Email: kazim.beebeejaun@phe.gov.uk

Keywords: N. meningitidis, Epidemiology, W-135, MenW

Introduction: Invasive meningococcal disease caused by capsular group W (MenW) incidence has been increasing in England and Wales since 2008. The study describes the epidemiology and molecular characteristics of invasive MenW disease in England and Wales during 2000-2013.

Methods: Public Health England (PHE) routinely conducts enhanced surveillance of invasive meningococcal disease through a combination of clinical reporting and laboratory confirmation and characterisation of clinical isolates submitted to the national Meningococcal Reference Unit (MRU).

Results: Following the control of national outbreaks related to Hajj pilgrimages in the early 2000s, the number of laboratory-confirmed, invasive MenW cases declined to its lowest in 2008 (19 cases). After 2008, however, MenW cases increased year-on-year to 46 in 2012. In 2013, 20 cases were identified in the first 3 calendar months, which is more than in the whole of 2008. In contrast, the total number of laboratory-confirmed, invasive meningococcal cases has declined from 1256 in 2008 to 746 in 2012. MenW was responsible for 7% of all invasive meningococcal cases in 2012, compared with 1.5% in 2008. Molecular analysis of MenW isolates revealed the increase to be associated with serotype 2a, a predictor of ST/cc11. Cases of MenW:2a increased from 0 cases in 2008 to 25 cases in 2012. In the first 3 months of 2013, MenW:2a was responsible for 14 of the 20 (70%) reported cases. All but one of the 79 non-2a cases during 2008-2013 were caused by non-typeable MenW (MenW:nt), which did not increase during the surveillance period. The age distribution of MenW:2a cases was similar to MenW:nt, with most cases occurring in children and in older adults aged =65 years. Case fatality was also comparable (5/60 [8.3%] vs. 9/79 [11.4%] cases; P=0.55).

Conclusions: An increase in invasive MenW cases has been observed in England and Wales since 2008. This increase was associated with serotype 2a, a predictor of cc11, which was previously associated with MenC disease carrying a worse than average prognosis. Although the disease profile of MenW:2a is currently comparable with MenW:nt this increase warrants careful monitoring in the coming years.

E10The epidemiology of community-acquired bacteraemia in Liverpool

Edward Bevan & Neil French

Institute of Infection & Global Health, University of Liverpool and Royal Liverpool & Broadgreen University Hospital Trust

Email: edward.bevan@rlbuht.nhs.uk; edwardbevan@me.com

Keywords: community-acquired bacteraemia, deprivation, co-morbidity

Background: Bacteraemia represents the most significant culmination of any bacterial infection and carries extremely high morbidity and mortality. Few contemporary studies describe the epidemiology of community acquired bacteraemia in the UK1.

Objectives:
  • Describe the incidence and causes of community acquired bacteraemia in adults in Liverpool: January 2010 to July 2012.
  • Link these data with population data, to provide estimates of bacteraemia incidence by age group, district of residence, social deprivation and relationship to patient co-morbidity.
Methods: This is a retrospective study, linking hospital data with local population data. Community acquired bacteraemia is defined as a positive blood culture collected within 48 hours of admission to hospital. All positive blood cultures in the study period were linked with hospital admissions data to separate community- acquired from nosocomial bacteraemias. Isolates considered to be contaminants were excluded according to CDC criteria2.

This dataset was linked with local population data from national census records3, to produce incidence rate estimates by age group, postcode district and deprivation index, and relationship to patient comorbidity.

Results: There were 748 episodes of community- acquired bacteraemia during the study period. The overall annual incidence rate of community-acquired bacteraemia was 81.5 per 100,000 (95% CI 75.7- 87.3). The most common organisms were: E. coli (32%), S. aureus (12%), S. pneumoniae (8%), K. pneumoniae (7%), and anaerobic organisms (4%). S. pyogenes accounted for 3%. The annual incidence of community-acquired bacteraemia in adults was found to increase with increasing age. In the >75 age group the incidence rate was: 196.6 per 100,000 (95% CI 173.8-219.4). The incidence rates of bacteraemia by postcode district within the catchment area of the Royal Liverpool University Hospital were also determined. Deprivation scores were determined for each postcode district. We found that the rate of community-acquired bacteraemia was directly proportional to deprivation score (R2=0.47). Many patients who developed bacteraemia had underlying co-morbidities: for S. pneumoniae, 19% had chronic pulmonary disease, 13% had ischemic heart disease and 3% had type 2 diabetes mellitus.

Conclusions: This study outlines the epidemiology of community-acquired bacteraemia in Liverpool. Unsurprisingly, we found a strong relationship between patient age and incidence of community- acquired bacteraemia. There was also a positive correlation between deprivation and incidence of community-acquired bacteraemia, and a large proportion of patients with bacteraemia had underlying co-morbidities. Understanding the epidemiology of serious community acquired bacterial infection will allow the rational implementation of novel public health interventions.

References
1. Hounsom L, Grayson K, Melzer M. Mortality and associated risk factors in consecutive patients admitted to a UK NHS trust with community acquired bacteraemia. Postgrad Med J 2011; ;87:757- 762
2. CDC, Laboratory-Confirmed Bloodstream Infection Criteria, Central Line-Associated Bloodstream Infection (CLABSI) Event, January 2013.
3. http://www.ons.gov.uk/ons/guide-method/census/2011/index.htmlhttp://www.ons.gov.uk/ons/guide-method/census/2011/index.html

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E11Antibiotic susceptibility profile of Neisseria meningitidis and Haemophilus influenzae invasive isolates in Croatia

Suzana Bukovski1,2

1University Hospital for Infectious Diseases “Dr Fran Mihaljevi”, Zagreb, Croatia; 2Faculty of Medicine Osijek, University Josip Juraj Strossmayer in Osijek, Croatia

Keywords: Neisseria meningitidis, Haemophilus influenzae, antibiotic, susceptibility, profile

Introduction: In Croatia invasive disease (ID), meningitis and/or sepsis, in children is mainly caused by Neisseria meningitidis also in recent years cases in older adults take attention too. On contrary incidence of invasive disease caused by Haemophilus influenzae (IHD) decreased after introduction of conjugate Hib vaccine in 2002. Nevertheless in last five years 10 cases of IHD are recorded and 6 of them in adults caused by H. influenzae non b type. The basis of ID treatment together with supportive care is prompt antibiotic treatment. Therefore antibiotic profile of invasive Neisseria meningitidis and Haemophilus influenzae is of utmost importance.

Material and methods: Data of antibiotic susceptibility testing of invasive isolates of Neisseria meningitidis from 2005 to March 2013 and invasive isolates of Haemophilus influenzae from 2008 to 2012 were analysed.

For Neisseria meningitidis minimal inhibitory concentration (MIC) using gradient diffusion test (E-test Bio Merieux) on Mueller-Hinton blood agar was done while for Haemophilus influenzae disk diffusion test was done and data were interpreted as susceptible (S), intermediate (I) and resistant (R). Only for 2 isolates MIC for ampicillin and ceftriaxone were done. Results were interpreted according to Clinical Laboratory Standard Institute (CLSI till 2011) and European Committee on Antimicrobial Susceptibility Testing (EUCAST from 2011) recommendations.

Results: Antibiotic sensitivity was available for 92 N. meningitidis isolates. All isolates were tested to penicillin (MIC 0,004 µg/mL - 0, 50 µg/mL). Penicillin MIC50 was 0,023 µg/mL while MIC90 was 0,094 µg/ mL. Ceftriaxone was tested for 91 isolates having MICs 0,002 µg/mL- 0,094 µg/mL and MIC90 0,002 µg/mL.

Sixty two isolates were tested to ciprofloxacin having very low MICs 0,002 µg/mL – 0,006 µg/mL and MIC90 0,003 µg/mL. All isolates were tested to rifampicin (MIC 0,002 µg/mL - 0,064 µg/mL) with MIC90 0,016 µg/mL.

All 10 H. influenzae isolates were susceptible to amoxiclav, ceftriaxone and co-trimoxazole and 9/10 were susceptible to amoxicillin. Ampicillin MIC of two isolates were 0,094 and 0,19 µg/mL and for ceftriaxone 0,002 and 0,004 µg/mL. According to EUCAST interpretation cefuroxim for per oral administration has to be interpreted only as (I) or (R) so 3 isolates were interpreted as (I) and 2 (R).

Conclusion: Antibiotic sensitivity profile of N. meningitidis is changing through time and increasing penicillin MIC having MIC90 0,094 µg/ mL were recorded. Taking into consideration that more than 50% of IMD were confirmed only by PCR introduction of penA detection routinely should be accelerated. Low ceftriaxone MIC90 and low MICs of ciprofloxacin and rifampicin provide further safe therapeutic and prophylactic use.

Between Croatian invasive H. influenzae beta- lactamase negative ampicillin resistant (BLNAR) or beta-lactamase positive amoxiclav resistant (BLPACR) isolates emerging in the world were not found. Cefuroxim resistant isolates interpreted according to EUCAST has to be taking in consideration for otitis media treatment.

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E12Development of an African adapted PCR algorithm to characterise Neisseria meningitidis from carriers, including uncommon capsular groups

Diallo K1, Coulibaly MD1, Rebbetts, LS2, Harrison OB2, Lucidarme J3, Gamougam K4, Tekletsion YK5, Bugri A6, Toure A1, Issaka B7, Dieng M8, Trotter C9, Stuart, JM10, Collard JM7, Sow SO1, Wang X11, Borrow R3, Greenwood B10, Maiden MCJ2, Manigart O10.

1Centre pour le Développement des Vaccins (CVD), Bamako, Mali; 2University of Oxford (Department of Zoology), Oxford, UK; 3Public Health England, (PHE – Vaccine Evaluation Unit), Manchester, UK; 4Centre de Support en Santé Internationale (CSSI), Ndjamena, Chad; 5Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia; 6Navrongo Health Research Centre (NHRC), Navrongo, Ghana; 7Centre de Recherche Médicale et Sanitaire (CERMES), Niamey, Niger; 8Institut de Recherche pour le Développement (IRD), Dakar, Senegal;
9University of Cambridge (Disease Dynamics Unit -Department of Veterinary Medicine), UK; 10London School of Hygiene and Tropical Medicine (LSHTM), London, UK; 11Centre for Disease Control, (CDC – Division of Bacterial diseases), Atlanta, USA

Email: Olivier.manigart@lshtm.ac.uk

Keywords: Neisseria meningitidis, Molecular characterisation, African Meningitis Belt, carriage, uncommon capsular groups

Introduction: Improvement of detection of Neisseria meningitidis (Nm) is crucial for reliable laboratory diagnosis of meningococcal carriage and better understanding of its epidemiology in the meningitis belt. The African Meningococcal Carriage Consortium (MenAfriCar) has established a simple gel-based PCR for detection and characterisation of NmA, NmW, NmX and NmY, but little is known on the epidemiology of the less common capsular groups (H, E, Z, cnl). We have developed a PCR algorithm that allows simple, rapid and systematic characterisation of all Nm capsular groups using two PCR techniques: gel-based, cheaper but labour intensive, and real-time (RT), more expensive but faster.

Samples and methods: Pre-existing primers for gel-based PCR and RT-PCR were blasted against consensus sequences obtained from the Oxford database (ODB). Primers for gel-based PCR (sodC, H, Z) and RT-PCR (porA, cnl, H, E, Z) were created or modified using Primer Express software. A total of 184 isolates from seven African countries were chosen from the first MenAfriCar cross-sectional study in parallel with a panel of 16 positive controls to optimise the PCR algorithm. All samples were characterised at the Department of Zoology, University of Oxford. Positive controls and 38 samples from Mali were utilised in monoplex and multiplex for PCR optimisation. Other isolates were characterised using optimised gel-based and RT-PCR multiplex only.

Sensitivity, Specificity, Positive Predictive value (PPV) and Negative Predictive value (NPV) were determined for each primers/probe PCR using Oxford’s results as the Gold Standard.

Results: Some existing primers and probes were modified following identification of mismatches between published and consensus sequences available in ODB.

Our newly designed algorithm allows simultaneous identification of Nm and determination of expression of capsule genes through a first porA, sodC, cnl multiplex PCR; if the isolate is sodC, porA positive and cnl negative, three sequential multiplex PCR from most common capsular groups were initially used: A, W, X, followed by B, C, Y and finally H, E and Z. Sensitivity of our pairs of primers, considered separately, in the multiplex assays range from 75% (porA) to 100%; specificity from 91% (porA) to 100% for gel-based assays; PPV ranges from 60% to 100% and NPV from 64% (porA) to 100%. For RT- PCR assays, sensitivity ranges between 50-100%, specificity 74-100%, PPV 50-100% and NPV 89- 100%. All uncommon capsular groups were detected.

Conclusions: Our results demonstrate that a systematic approach for characterisation of 10 different Nm capsular groups using four multiplexes can be used. This tool will allow better understanding of the epidemiology of rare carriage strains in the African meningitis belt.

This study was funded by Meningitis Research Foundation.

E13Genetic characterisation of invasive Neisseria meningitidis isolates in Belarus

Glazkova S, Lebedzeu F, Yanovitch O, Nosova E, Titov L

Laboratory for Clinical and Experimental Microbiology, The Republican Research and Practical Center for Epidemiology and Microbiology (RRPCEM), Minsk, Belarus

Email: slavglaz@gmail.com


Keywords: MLST, Neisseria meningitidis, sequence type

Background and aims: Incidence of meningococcal meningitidis during 2006-2012 ranged between 1.2-2.8 per 100.000 population in Belarus. Mainly three serogroups of meningococci circulate in the republic: A, B and C, the leading serogroup is B. Children have a leading role in the age structure of disease: over 70% of total cases of invasive infection came to an age group 0-14 years. The aim of the present study was to describe invasive N. meningitidis strains circulating in Belarus using multilocus sequence typing (MLST).

Material and methods: Thirty-three N. meningitidis strains, isolated from patients having meningococcal meningitidis in 2006-2012 in different regions of Belarus were analysed. DNA from N. meningitidis strains was isolated using «DNA-Sorb-B» kit (AmpliSens, Russia).

All isolates were confirmed as N. meningitidis according to growing characteristics and using PCR «50R Neisseria spp., Haemophilus spp., Streptococcus spp. Eph» kit (AmpliSens, Russia). Genogrouping was performed using PCR «N. meningitidis A, B, C» kit (Amplisens, Russia). MLST was performed according to the recommendations of the Neisseria Multi Locus Sequence Typing website (http://pubmlst.org/ neisseria/).

Results: From 33 N. meningitidis the isolates were determined as genogroup A (n= 2; 6%), B (n= 21; 64%), C (n= 6; 18%), and four isolates were not possible to genogroup (12%). A total number of 30 sequence types (STs) were determined, 21 of which were not previously reported (70%). Several new alleles of housekeeping genes were obtained: abcZ allele 451, aroE alleles 602 and 603, fumC allele 541 and gdh alleles 560 and 621. Neisseria meningitidis sequence types 18, 3346 and 9300 were represented in two isolates; the remaining STs were only represented by one isolate (27 different STs). Among all STs observed 15 belonged to next clonal complexes: ST 11/ ET 37 complex, ST-18 complex (13.3%), ST 32 complex/ET-5 complex, ST 41/44 complex Lineage 3 (20%), ST 53, ST 103 (13.3%), ST 174. The high number of unique STs (n=21) may reflect that meningococcal population circulating in Belarus was genetically highly heterogeneous.

Conclusion: According to the data obtained the meningococcal population circulating in Belarus was highly diverse: showing circulation both unique and widely spread hypervirulent clonal complexes. However, due to the few isolates examined from Belarus the data need to be interpreted with some caution. The results of the study show the need for N. meningitidis epidemiological surveillance and global monitoring of the migration of pathogenic variants through the country.

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E14The epidemiology of invasive meningococcal disease in Belarus, 2011 and 2012

Glazkova S1, Shymanovich V 2, Titov L1

1Laboratory for Clinical and Experimental Microbiology, The Republican Research and Practical Center for Epidemiology and Microbiology (RRPCEM), Minsk, Belarus; 2Department of Immunoprophilaxy, The Republic Center for Hygiene, Epidemiology and Public Health (RCHEPH), Minsk, Belarus

Email: slavglaz@gmail.com

Keywords: Epidemiology, IMD, Neisseria meningitidis

Background and aims: Clinicians in different regions of Belarus should report about each case of invasive meningococcal disease (IMD) following EU-wide reporting standards where possible. Questionnaires are then gathered for a general report issued by RCHEPH. All viable meningococcal strains, isolated from patients with IMD were delivered to RRPCEM for further investigation. Despite IMD is a reportable condition in the republic, no N. meningitidis national AMR surveillance is performed in Belarus, as well as no vaccination against meningococcal meningitidis is used. The objective of the analysis was to describe the epidemiology and surveillance of IMD in Belarus in 2011 and 2012.

Material and methods: Neisseria meningitidis microbiological identification and serogrouping was performed using classical cultural methods, biochemical test API NH (BioMerieux, France), and PCR «NHS kit» (Amplisens, Russia). Serogroups of meningococci were determined using «Slidex meningite-Kit-5» (BioMerieux, France) and PCR «N. meningitidis A, B, C» kit (Amplisens, Russia).

Results: Invasive meningococcal disease incidence in Belarus decreased from 1.3/100.000 inhabitants in 2011 to 1.2/100.000 in 2012, a total number of 125 and 116 cases of IMD were reported in Belarus in 2011 and 2012, respectively. More than 70% of patients with IMD were under 2 years of age showing the highest incidence rates over 22.5/100.000 in both years. Annual estimate of IMD cases showed seasonal prevalence of autumn-spring period: the majority of cases (up to 70%) were registered in September - October and March. Case fatality ranged from 9.5% - 11%, children under the age of 14 were the main risk group (up to 80%).

Serogroup B is dominating in meningococcal population in Belarus and caused up to 41.9% of IMD (in 2012). IMD cases caused by meningococci of serogroup A and serogroup C were reported in 10.9% and in 12.9%, respectively, in both years. The level of nongroupable and/or polyagglutinable meningococci isolated in patients remained on a high level – up to 37% (in 2011). Meningococci of minor serogroups were isolated rarely: X and W135 - 1.6% in 2011, W135 and E29 - 3.2% in 2012.

Conclusion: In 2012 the number of IMD cases decreased in Belarus in comparison with previous year, showing however high morbidity and mortality rates and wide distribution of nongroupable meningococci among Neisseria meningitidis population. Enhanced information of the molecular epidemiology as well as presence of decreased susceptibility or resistance to antimicrobials used for treatment and chemoprophylaxis of meningococcal infection is crucial in Belarus.

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E15Epidemiology and surveillance of meningococcal disease in England and Wales

Gray SJ1, Campbell H2, Lord A1, Carr AD1, Newbold LS1, J Lucidarme1, Mallard RH1, Guiver M1, Ladhani S2, Borrow R1, Ramsay M2 and Kaczmarski EB1.

1Public Health England (PHE) Meningococcal Reference Unit (MRU), Public Health Laboratory Manchester, Manchester Royal Infirmary, Manchester M13 9WL, UK; 2Immunisation Department, PHE, Colindale, London, UK

Email: Steve.gray@phe.gov.uk


Keywords: meningococcal disease, epidemiology, surveillance, laboratory

Background and Aims: Public Health England (PHE) performs surveillance of invasive meningococcal disease for England and Wales to ascertain case numbers, characterise strains and inform vaccine policy.

Materials and Methods: Clinicians notify suspected cases of meningococcal meningitis/septicaemia to the Office for National Statistics. Hospital microbiology laboratories in England and Wales routinely submit invasive meningococcal isolates to PHE for phenotypic characterisation and, since October 2007, porA sequencing. MICs of penicillin, cefotaxime, rifampicin and ciprofloxacin are determined. PHE also routinely receives clinical samples for non-culture detection and capsular group confirmation by PCR. Characterisation of non-culture positives by porA sequencing commenced in January 2012.

Results and Conclusions: Laboratory confirmed cases rose from 1,448 (1995) to peak at 2,804 (1999) falling to 784 in 2012. The major reduction in cases has been due to the decrease in serogroup C infections, ranging 10 - 39 cases per year from 2005; the consequence of direct and indirect protection afforded by the UK serogroup C conjugate vaccine programme since November 1999. There has also been a sustained decrease in serogroup B cases from 1,710 cases (2001) to 621 (2012), in the absence of any intervention/s.
In 2012, serogroup B accounted for 79% of all confirmed cases whereas only 4% (28 cases) were confirmed as serogroup C and 6% W. Serogroup Y accounted for 10% (80) cases in 2012 a reduction from the peak of 93 cases in 2011.

During 2012, 379 cases (48%) of invasive meningococcal disease were confirmed by PCR alone. Similar proportions of group B porA strain variants were confirmed for both non-culture positives and isolates.
Phenotypic and genotypic shifts have been observed since 1999: specifically the relative contributions of serogroup B associated clonal complexes ST-41/44 (stable), ST-269 (increasing), ST-32 (reducing), ST-213 (low rise and fall) and the reduction of the previously predominant serogroup C ST-11 CCs to meningococcal epidemiology. Although low numbers, an increase in disease due to serogroup W:2a strains (a predictor of CC11) is being monitored: from 2 cases (2009) to 25 cases (2012), nationwide and across all age ranges.

E16The MRF Genome Library: Epidemiology of meningococcal disease-causing lineages in England and Wales from 2010/11 to 2011/12

Hill D. M.C.1, Lucidarme J2, Jolley K. A.1, Tang C3, Kaczmarski E2, Parkhill J4, Green J5, Maiden M.C.J.1, Borrow R2

1Department of Zoology, University of Oxford, Oxford, OX1 3PS;
2Vaccine Evaluation Unit, Public Health England, Manchester, M13 9WZ, UK; 3The Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, UK; 4Pathogen Genomics, The Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK; 5Bioinformatics Group, Public Health England Bioinformatics Unit, Colindale, London, NW9 5EQ, UK

Email: dorothea.hill@zoo.ox.ac.uk


Keywords: Meningococcal disease, England and Wales, Public access, Molecular epidemiology, Whole-genome sequencing

The Meningitis Research Foundation Meningococcus Genome Library is an open-access database containing the genomes of all invasive meningococcal disease isolates from England, Wales, and Northern Ireland from the 2010/11 and 2011/12 epidemiological years. The 2012/13 genomes will be in the database early in 2014. This project is a collaboration between MRF, Public Health England (PHE), the University of Oxford, and the Sanger Institute, to provide a dataset for all fields of meningococcal research. Its primary objective is to facilitate the design of novel interventions against invasive meningococcal disease (IMD).
DNA prepared at the PHE Meningococcal Reference Unit (MRU) is sent to the Sanger Institute for whole- genome sequencing. In Oxford, genomes are assembled and uploaded to the PubMLST database for annotation; genomes can be accessed at all stages of annotation via meningitis.org/research/ genome. PubMLST integrated BIGSdb software analysis tools such as Genome Comparator were used for the extraction of nucleotide and allelic data from loci of interest for population analyses using eBurst and SplitsTree4.

There were 899 culture-confirmed cases of IMD in England and Wales in 2010/11 and 2011/12. The most prevalent clonal complex (CC) was ST-41/44CC, comprising 26% of the total. A significant proportion of cases were caused by ST-269CC (19%), ST-23CC (13%), ST-213CC (8%), ST-11CC (7%), and ST-32CC (5%), with a further sixteen CCs causing disease rarely. Since serogroup Y is over-represented in cases confirmed by culture compared to all laboratory- confirmed cases, ST-23CC may be responsible for a larger proportion of overall IMD than reporting suggests.

Current incidence of meningococcal disease in England and Wales is low relative to previous decades (<2 cases/100,000). Cases of ST-269CC IMD have declined in recent years; this CC is diverse however, and rMLST analysis revealed the existence of a further 29 cases from this lineage that are not designated to a CC due to the limited resolution of MLST. The addition of ST-275 as a joint central genotype within ST-269CC is proposed to accurately capture the prevalence of this meningococcal lineage.

Unlike other lineages, ST-11CC and ST-23CC cases increased between 2010/11 and 2011/12, by 5% and 3% respectively. The proportion of ST-11CC increased in all age groups, with most serogroup W and serogroup C ‘ET-15’ increases in the over 25s. As expected from the association of ST-23CC with older patients, the increase in IMD caused by this CC occurred in the over 25s. Currently, this comprehensive dataset allows high-resolution surveillance and identification of emerging strains.

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E17 - Oral Presentation, day twoQuality of life changes in children diagnosed with group B meningococcal disease in months following illness

Iain Kennedy & Shamez Ladhani

Department of Immunisation, Hepatitis and Blood Safety, Centre for Disease Surveillance and Control, Public Health England, London, UK

Email: iain.kennedy@phe.gov.uk


Keywords: meningitis B, children, quality of life

Background: N. meningitidis group B is currently the most frequent causative organism for meningococcal disease in the UK. The first ever vaccine against Men B was licensed in early 2013. The Joint Committee on Vaccination and Immunisation’s draft recommendation is that the vaccine is not introduced to the routine schedule, but may be useful in other settings. Previous work has examined the long-term quality of life (QoL) impacts of Men B disease. The key objective of this project was to identify the short term impacts of Men B disease in children in the months following diagnosis.

Methods: Patients aged 15 or under in England who had confirmed N. meningitidis group B with sample dates between 1st November 2012 and 31st May 2013 and for whom sufficient demographic details had been captured were identified. The GPs of these patients were contacted to confirm patient details and diagnosis. The parents of the patients were then contacted with a letter explaining the project and a questionnaire. The questionnaire contained questions on clinical course including pre-existing and subsequent conditions, as well as the standardised EQ-5DY QoL questions. The project was performed as part of Public Health England’s responsibilities for on-going surveillance and control of communicable disease and was endorsed by meningitis charities.

Results: There was a response rate of 43% (110/254) and the gender and age profile of responders and non-responders appears similar. Preliminary results show 98% of cases required admission to hospital and 47% admission to PICU/ICU or other high care unit. Around one third of cases had at least one new health condition following their Men B illness, with hearing loss being the most common. EQ- 5DY responses indicate the majority of cases had large loss of QoL during the illness, but many had recovered at least some, or all of that loss in the months following the illness.

Discussion: This study demonstrates that short term QoL loss is a major problem for children following Men B infection, and although some of that loss is regained over time, a significant proportion of cases have sequelae that can cause long term disability. The study is limited as the QoL measure used, which is that recommended by NICE, is not validated in younger age groups and some questions are difficult to apply to the youngest cases. These results add to the available evidence on Men B disease, and may help with future policy decisions.

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E18The epidemiology of invasive meningococcal disease serogroup B in Scotland 1999-2012

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

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 serogroup B in Scotland from 1999-2012 and discusses the potential implications for meningococcal B vaccine introduction.

Laboratory confirmed cases of invasive meningococcal disease submitted to the Scottish Haemophilus, Legionella, Pneumococcal and Meningococcal Reference Laboratory (SHLMPRL) are routinely serogrouped and MLST typed. There has been an average of 70 cases of meningococcal disease serogroup B reported each year in Scotland. This has decreased steadily from 117 cases (2.3 cases per 100,000 population) in 2000 to 38 cases (0.7 cases per 100,000) in 2012 and serogroup B is now the most commonly reported serogroup in Scotland, accounting for 73% of laboratory confirmed cases in 2012. Just over half of all cases reported are in children under five years of age (535/989; 54.1%) and the most commonly reported clinical presentation for cases was meningitis (320 cases; 43%), followed by septicaemia (222 cases; 30%) and both meningitis and septicaemia (177 cases; 24%). There were 55 deaths reported in the time period equating to an overall case fatality ratio (CFR) of 5.5%. However, CFR was found to vary by age and clinical presentation. The most commonly circulating MLST types were 413 (27 cases), 213 (25 cases), 275 (19 cases) and 269 (14 cases).

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

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E19Meningococcal seroepidemiology in Burkina Faso, one year after the MenAfriVac® mass campaign

Yaro S1, Tall H2, Ouangraoua S1, Kpoda H1, Trotter C3, Njanpop Lafourcade B2, Martin C2, Findlow H4, Bosco JB5, Gessner BD2, Borrow R4, Mueller JE6

1Centre Muraz, Bobo-Dioulasso, Burkina Faso; 2Agence de Médecine Préventive, Paris, France; 3Cambridge University, Cambridge, UK; 4Vaccine Evaluation Unit, Public Health England, Manchester, UK; 5Institut de Recherche en Science et Santé, Bobo-Dioulasso, Burkina Faso; 6EHESP French School of Public Health, Paris, France

Email: Judith.mueller@ehesp.fr


Keywords: African Meningitis Belt, serogroup A conjugate vaccine (MenAfriVac®), seroepidemiology, immunisation coverage, vaccine impact assessment

Background: To eliminate meningococcal meningitis epidemics, a meningococcal serogroup A conjugate vaccine (MenAfriVac) has been introduced in Burkina Faso via mass campaigns in December 2010, targeting the 1- to 29-year-old population. This study describes specific antibody titre in the population 11 months after vaccine introduction and compares them to pre-introduction data obtained during 2008 using the same protocol (Trotter et al. 2013).

Methods: During October-November 2011, we included a representative sample of the population of urban Bobo-Dioulasso aged 6-months to 29-years. Participants were examined using standardized questionnaires and blood draws. Using rabbit complement, serum bactericidal antibody (SBA) titres were measured against two serogroup A strains: reference strain F8238 of immunotype L11 (all sera) and strain 3125 (L10; 200 randomly selected sera).

Results: Among the 562 participants, 477 were =23-months-old and had been eligible for the MenAfriVac campaign. Among them, 204 (43%) reported vaccination and 160 (34%) could provide a vaccination card. No systematic difference in SBA titre was found between persons reporting vaccination or not; therefore, overall data are reported.

GMT was 8 among the 85 children under 23 months, 1458 among vaccine-eligible children up to 4 years (2100 if vaccination confirmed), and 2355 (2494) among vaccine-eligible older persons.
Titre <128 or <1024 were found with 5% and 23%, respectively, of 2- to 4-year-old children and with 1% and 12%, respectively, of 5- to 29-year-old persons.

Compared to the 2008 pre-introduction data, GMTs against the reference strain and 3125 strain were 11 and 42 fold higher, respectively, among 2- to 4-year- old children, and 8 and 78 fold higher, respectively, among 5- to 29-year-old persons.

Discussion: Since the MenAfriVac mass campaign, serogroup A meningococcal SBA titres in the targeted population of Burkina Faso have substantially increased. The increase is more pronounced with SBA against strain 3125 than against the reference strain, possibly as the latter is sensitive to natural immunity, as suggested by high pre-vaccination titre. SBA-3125 should therefore be included in studies on antibody persistence after meningococcal vaccination in populations with substantial natural immunity. Further studies are planned to evaluate longer term antibody persistence.

This study was funded by Meningitis Research Foundation.

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E20Neisseria meningitidis: Epidemiological markers and antimicrobial susceptibility of invasive strains. Cuba, 2002-2011

Suarez E1, Martinez I2, Llanes R1, Feliciano O1, Gutierrez O1

1Pedro Kourí Institute (IPK), Havana, Cuba; 2Finlay Institute (IPK), Havana, Cuba

Email: nitzaes@infomed.sld.cu

Keywords: Epidemiology, antimicrobial susceptibility

Introduction: Neisseria meningitidis is one of the main causative agents of bacterial meningitis and sepsis. The study of epidemiological markers and antimicrobial susceptibilities of the circulating strains constitute the main activities of meningococcal disease surveillance.

Aims: To investigate epidemiological markers and the antimicrobial susceptibility patterns of invasive isolates of N. meningitidis in Cuba for ten years (2002-2011).

Methods: A transversal descriptive study was conducted to characterise invasive meningococcal isolates received at the National Reference Laboratory of Pathogenic Neisseria during 10 years. A total of 69 viable meningococcal strains were studied. Conventional laboratory methods were used to identify the species and serogroups; PCR technique was used when the latter was doubtful. Phenotypic characterisation was carried out using ELISA of whole cells with monoclonal antibodies, while antimicrobial susceptibility was determined by the E-test method.

Results: Strains of serogroup B (98.6%) were predominant, with only one strain belonging to serogroup C (1.4%); strains of serotype 4 were the most frequent (55.1%), followed by non-typable and serotype 2a (15.9% and 8.7%), respectively. The more frequent subtypes were P1.15 (42.0%), P1.19 (20.3%) and non subtypable strains (10.1%). Of serogroup B strains, phenotype B:4:P1.15 was predominant (26.1%), and phenotype C:2a:P1.5 was detected for the first time in Cuba. Susceptible meningococci strains were predominant: ceftriaxone (100.0%), rifampicin (98.2%), chloramphenicol (92.8%), ciprofloxacin (89.3%), and penicillin (87.5%), except for cotrimozaxol, to which however 92.8% of isolates were resistant.

Conclusions: Our data provide valuable epidemiological information for a better understanding and control of meningococcal disease in Cuba.

E21Development of a multiplex assay to determine IgG binding to Group B streptococcus and evaluation of anti-Group B Streptococcus IgG in the UK

Stephen Taylor, Sophie Arthur, Jenny Herbert, Stephen Thomas and Andrew Gorringe

Public Health England, Porton Down, Salisbury SP4 0JG

Keywords: GBS, immunity, flow-cytometry, multiplex-assay, seroprevalence

Streptococcus agalactiae (Group B Streptococcus, GBS) is now the most frequent cause of sepsis and infectious death in neonates in England. Risk of GBS infection is highest during the first few days of life through intrapartum contamination of the neonate from maternal flora. The organism possesses a polysaccharide capsule that defines the serotype and serotypes III and Ia are isolated from the majority of early onset neonatal cases, with disease also caused by serotypes V, Ib and II (Lamagni at al., 2013, Clin Infect Dis). In the last 20 years the distribution of serotypes varied according to patient age and serotype III has increased among early-onset cases and decreased in adults. To facilitate understanding of immunity to GBS in the UK population we have developed a multiplexed flow cytometry assay to determine concentrations of IgG against GBS serotypes Ia, Ib, II, III and V. Formaldehyde-fixed GBS strains representing each of these serotypes were stained with a different live/dead fluorescent stain. IgG binding to the five distinct GBS populations was determined using an anti-human IgG FITC conjugate. Initial assays compared IgG binding to the bacteria determined in single and multiplexed assays with good correlation of values obtained. The multiplexed assay was then used to determine naturally-acquired antibody to GBS serotypes Ia, Ib, II, III and V in a panel of 1852 age-stratified sera collected in the year 2000 from male and female donors.

Nearly all samples (99%) had detectable IgG binding to GBS of at least one serotype, with 87% of individuals having detectable IgG that bound to GBS of all five serotypes examined. The distribution of IgG binding to GBS in the entire serum panel was serotype III > V > Ib > II > Ia. The serotype distribution of anti-GBS IgG varied with age. In children under 1 year the distribution was II > III > Ib > V > Ia and in individuals over 60 years it was III> II > Ib > V > Ia. The second most common disease serotype is Ia, whereas IgG binding to this serotype is the lowest across all ages, indicating that either it is poorly immunogenic or is more pathogenic than other serotypes. Conversely, serotype II is rarely isolated from disease but elicits the highest levels of IgG in children less than 1 year of age.

E22Bacterial load and host cytokine response in the CSF of adults with pneumococcal meningitis

Emma C Wall1,2,3, Jenna Gritzfeld2, Matthew Scarborough4, Katherine Adjukiewcz5, Mavuto Mukaka1, Caroline Corless6, David G Lalloo1,2, Stephen B Gordon1,2

1Malawi-Liverpool-Wellcome Trust clinical research programme, Malawi; 2Clinical Group, Liverpool School of Tropical Medicine, Liverpool, UK; 3College of Medicine, Department of Medicine, University of Malawi, Malawi; 4Infectious Diseases Department, John Radcliffe Hospital, Oxford, UK; 5Infectious Diseases department, North Manchester NHS Trust, Manchester, UK; 6Clinical Microbiology, Institute of Global Heal, University of Liverpool, UK

Keywords: Meningitis, pneumococcus, pneumolysin, bacterial load, cytokine

Specific objective of the study: Bacterial meningitis in sub-Saharan Africa is predominately caused by Streptococcus pneumoniae and is associated with mortality rates double those seen in well-resourced settings, which reach 50-60% in adults. We undertook a study to investigate bacteria and host response in the CSF compartment to identify potential causes of poor outcome. No published data currently quantify the host cytokine response by outcome in adults with meningitis from a high HIV prevalence country in sub-Saharan Africa, and there are no published data quantifying the CSF pneumococcal load in adult meningitis.

Methods: We quantified the number of copies of pneumococcal DNA using real time PCR and six common pro-inflammatory cytokines using bead array in the cerebrospinal fluid (CSF) of adults with culture proven pneumococcal meningitis in Malawi. We correlated the results to outcome at discharge from hospital and 6 week follow up.

Results: The median age was 32 years, 82% of patients were HIV antibody-positive and the mortality rate by six week follow up was 49%. The median CSF white cell count was 730 cells/mm3, with significantly lower WCC in non-survivors p=0.02. Bacterial loads were high (median 6.5x105 copies/ml CSF) and there was no significant variation in bacterial load by outcome at ten days or six weeks. All pro- inflammatory CSF cytokines were elevated in the CSF, with a trend towards a more intense response in non- survivors reaching marginal statistical significance for IL8 and IL10 on multivariate analysis p=0.034 and 0.029 respectively. There were no differences in the CSF bacterial load or cytokine response between adults who were HIV-infected and non HIV-infected.

Conclusion: Mortality from pneumococcal meningitis in adults in sub-Saharan Africa is not related to pneumococcal bacterial load. Anti-cytokine therapies are unlikely to be effective adjunctive treatment to improve mortality. We have previously shown that pneumolysin persists in the CSF of non-survivors of pneumococcal meningitis. This finding, with the altered WCC response presents an opportunity for exploring pathogenesis and potential anti-pneumococcal toxin adjunctive therapy further. More research is needed to understand the very high mortality from meningitis in this region.

Clinical Diagnosis, Treatment & Sequelae

C23Feverish illness in children: discordance between British, Italian and Swedish clinical guidelines

Inge Axelsson

Mid Sweden University and Östersund Hospital, Östersund, Sweden

Email: inge.axelsson@miun.se

Keywords: fever, children, meningococcal disease, Kawasaki disease, encephalitis

Object: In 2007, NICE published Feverish illness in children (CG47), a clinical guideline (CG) based on systematic review of the scientific literature. A second, revised edition, Feverish illness in children (CG160), was published in 2013, after extensive peer review. In 2013, the Swedish Institute for Communicable Disease Control (SMI) published a national CG for Signs of serious infections in children (in Swedish), also after extensive peer review. I was coeditor. The SMI paper was partly based on NICE CG160. In 2009, the Italian Pediatric Society published Management of Fever in Children: Summary of the Italian Pediatric Society Guidelines (Chiappini E et al., Clin Ther. 2009;31:1826-1843). However, these three CGs are in part discordant. I will here identify these differences and discuss their importance.

Method: The three guidelines were read and compared. The British, Italian and Swedish CGs are called UK, IT and SE, respectively. Discordance was documented in a summary of findings table.

Results: UK and SE had different definition of fever; IT had no definition. UK and IT recommended electronic measurement of fever in an axilla or an ear while SE still recommended rectal thermometer. Children should be screened for Kawasaki disease if they have fever lasting more than 5 days (UK) or 5 days or more (SE). According to British studies, pain in arm or leg is an early sign of meningococcal disease. This was mentioned in SE but not in UK or IT. Herpes simplex encephalitis should be considered in children with fever and focal neurological signs, focal seizures or decreased level of consciousness according to UK but herpes simplex encephalitis was not mentioned in IT or SE. UK and SE used ‘traffic lights’ and recommended ‘safety nets’; IT did not. UK recommended teaching parents how to detect signs of dehydration by looking for the following features: sunken fontanelle, dry mouth, sunken eyes, absence of tears and poor overall appearance. Also, UK recommended health staff to teach parents to identify a non-blanching rash. IT or SE had no such recommendations.

Conclusions: This comparison may be used to improve revisions of the clinical guidelines.

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C24The management of suspected childhood bacterial meningitis in a UK district general hospital

Doyle K1, Houseman C1, Makwana N2

1
Foundation year 2 trainee, West Midlands Deanery, UK; 2Consultant Paediatrician, Department of Child Health, Sandwell and West Birmingham NHS Trust

Keywords: Paediatrics, bacterial meningitis, antibiotics, management

Background and aims: To compare the management of suspected bacterial meningitis at the Sandwell and West Birmingham NHS Trust with that recommended by the National Institute for Health and Care Excellence (CG102 Guideline, 2010.)†

Method: Retrospective case note based analysis. Notes were requested for admissions within the trust coded as meningitis. This included all children aged between 4 weeks to 16 years admitted between 2006 and 2011. Of the 42 notes received, 34 fitted inclusion criteria. 22 were male, 6 patients were less than 3 months.

Results: 100% of patients were expected to have;
1) Initial investigations (detailed in Table).

Table: Initial investigations for suspected bacterial
meningitis
 % Investigations
Completed (N)
% Abnormal
results (N)
FBC
 100 (33)
 55 (18)
Blood culture
 97 (32)
 19 (6)
CRP
 91 (30) 73 (22)
Blood gas
 36 (12)
 NA
Coagulation screen55 (18)28 (5)
 Blood glucose67 (22)5 (1)
 Blood PCR (n=30)20 (6)0

2) A lumbar puncture (LP) if no contraindications (N=26): 26 LPs were attempted (8 excluded.) 4 attempts failed. Of those completed 86% (N=19) of results were abnormal. In 3 cases LP was contraindicated but performed.
3) A laboratory blood glucose at time of LP (N=22). 36.4% (N=8) had a laboratory glucose. Of the remaining 14, 78.6% (N=11) had a capillary glucose level. 3 cases did not have a blood glucose at the time of LP.
4) Received the recommended empirical antibiotics. Of those aged <3 months (N=6), 4 received antibiotics as recommended by NICE (cefotaxime/ ceftriaxone and amoxicillin/ampicillin). All =3 months (N=28) received recommended antibiotics (Ceftriaxone).
5) Received antibiotics for recommended duration (N=31) Out of the 31 eligible patients 45.2% received antibiotics for the duration recommended by NICE. 83.9% received antibiotics for the duration recommended by trust guidelines.
6) Follow up (N=31): 87.1% had a documented plan for audiological referral and 77.4% for paediatric referral upon discharge. There were no deaths from meningitis.

Conclusions:
The NICE guidelines were followed in the majority of cases. There are several areas that can be developed within the department including a review of departmental guidelines. Education for junior doctors about the NICE guidelines is planned and improved communication between paediatrics and microbiology needed.

References:
† National Institute for Health and Care Excellence. CG102. Bacterial meningitis and meningococcal septicaemia in children and young people younger than 16 years in primary and secondary care. 2010.

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2013 POSTER PRIZE WINNER

C25The Orthopaedic Sequelae of Childhood Meningococcal Septicaemia

Tomos Edwards1, Fiona Bintcliffe2, Lowri Bowen1, James Aird2, Fergal Monsell2

1
University of Bristol Medical School, Bristol, UK; 2Department of Paediatric Orthopaedic Surgery, Bristol Children’s Hospital, Bristol, UK
Email: fergal.monsell@btinternet.com


Keywords: Meningococcal, septicaemia, orthopaedic, sequelae, limb-reconstruction

Objective:
Meningococcal septicaemia is a potentially devastating disease that can result in a variety of disabling complications. The aim of this study is to use a defined population as a common denominator enabling correct identification of the incidence of orthopaedic sequelae as well as to investigate the specific complications developed.

Methods: Clinic letters and radiographs were analysed retrospectively of all patients admitted to the Paediatric Intensive Care Unit (PICU) of the Bristol Royal Hospital for Children from 01/01/2001 to 31/12/2012 with a primary diagnosis of meningococcal septicaemia.

Results: 138 patients with meningococcal septicaemia were identified, 130 of which were alive at PICU discharge. Of these, 10 developed orthopaedic sequelae, representing an overall incidence in this patient population of 7.7%. Those who developed orthopaedic complications were admitted to PICU at a significantly younger age (P < 0.05) and for a longer time period (P < 0.001). This study is the first to report a greater proportion of boys than girls developing orthopaedic complications (risk ratio: 3.1; 95% CI: 0.69 – 14.14).

9 patients required an amputation, and these were much more common in the lower limb, 16/22 (72.7%). Each patient requiring an amputation had multiple limb involvement. Amputations most often occurred distally, however a more proximal revision amputation was required in 2 patients.

A total of 48 growth plate abnormalities were identified in 8 patients. 39 (81.3%) of the affected growth plates were in the lower limb, and the most commonly affected growth plate in both right (40%) and left (36.8%) lower limbs was the distal tibia. Of the 6 patients with documented angular deformity, 10 ankles were identified as having a varus malalignment. Partial fusion across the distal tibia with relative fibular overgrowth was the most common causative factor. 6 patients had documented leg length discrepancy that was most commonly due to significant shortening of the tibia.

All 10 patients had documented skin scarring and had suffered skin necrosis or purpura fulminans. Additionally, 3 patients required a fasciotomy.

Conclusion: This study has identified a relatively high incidence of orthopaedic sequelae following meningococcal septicaemia. The data collected suggests the most common complications as well as those patients at greatest risk of developing them. We recommend considering the cost benefit implications of a yearly review of these patients to enable early identification of potentially treatable complications.

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Presenting author, Tomos Edwards receives the 2013 poster from MRF CEO, Chris Head

Presenting author, Tomos Edwards, receives the 2013 poster prize from MRF CEO, Chris Head, on behalf of the authors of poster C25 "The Orthopaedic Sequelae of Childhood Meningococcal Septicaemia"


C26Fluorescence immunoassay for procalcitonin hormone in whole blood

Eun Seon Yoon1, Do Won Kim2, Eui Yul Choi1,2
 
1Department of Biomedical Science, Hallym University, Chuncheon, South Korea 200-702; 2Boditech Med Inc. Chuncheon, South Korea 200-702
Email: yesseony@gmail.com


Keywords: Procalcitonin (PCT), calcitonin, katacalcin

The determination of blood level of TSH is an important biomarker for the clinical assessment of sepsis status. Here, we presented a new fluorescence (FL) immunochip PCT assay system, which was developed with a platform of point-of- care test (POCT) for clinical applications. The assay system modified a later-flow immunochromatographic technology and consisted of anti-PCT-mAb coated strip in a disposable chip, a detection buffer containing FL-labeled anti-PCT-poly Ab, a chip for the calibration curve, and a laser FL scanner. The analytical performance of FL immunochip PCT assay system was evaluated by linearity, interference, recovery, and imprecision tests. The comparability of the developed assay was examined with automated reference assay. The developed assay system exhibited an excellent linearity in working range of 0.25 –50 ng/ml. The analytical mean recovery of control was 97.6% in a dynamic working range and the imprecision of intra- and inter-assay of CVs was less than 10%. There was highly significant correlation between the developed PCT assay and automated Beckman Coulter Access 2 reference assay with r = 0.989 (p < 0.001). The developed FL immumo assay is the only method that quantifies PCT concentration in whole blood, which meets the criteria of POCT, including affordable cost, a disposable device, and requiring minimum maintenance to perform test.

C27Outbreak of Serotype W135 Neisseria meningitidis in Central River Region (CRR) of the Gambia - A Clinical perspective

Osuorah Donatus1, Bilques Shah1, Manjang Ahmed1, Ebba Secka2

1Pneumococcal Surveillance Project (Central River Region Research site), Child survival unit, Medical research council UK, The Gambia unit; 2Regional Health Authority, Central River Region, The Gambia Ministry of Health and Social Welfare The Gambia

Email: dosuorah@mrc.gm or chidi.osuorah@gmail. com

Keywords: Neisseria meningitis, W135 strain, Central river region, Bansang hospital

Background: Meningitis still accounts for many deaths in children especially during epidemics in countries within the African meningitis belt. The Gambia recently witnessed another outbreak with the W135 strain of Neisseria meningitidis. There have been studies on similar outbreaks in Africa but only a few have looked specifically into the clinical and child perspective of these outbreaks that usually and unfortunately are most affected. This study therefore presents a clinical perspective of the most recent outbreak in the Central River Region of the Gambia and evaluated predisposing and risk factors associated with acquiring the infection among children.

Methods: This is a descriptive and prospective cohort study using the nested case-control design. Suspected cases of meningitis presenting to the paediatric ward of the Bansang Hospital in the Central River Region of the Gambia during the outbreak period were employed for the study. Eighty- nine (89) confirmed cases of N. meningitidis were enrolled as cases and 108 matching controls with negative blood cultures admitted for other reason during the same period were recruited as controls.

Result: Between February and May 2012, over 250 suspected cases presented to the Bansang hospital of whom more than 95% were children. The outbreak had a double peak period in weeks 5 and 8 during which the epidemic threshold of 10 cases/100,000 per week was exceeded. The W135 strain of Neisseria meningitidis was responsible for 98.9% of meningitis infection during the 2012 outbreak in the Central River Region of the Gambia with an incidence rate of 47.9 per 100,000 and a case fatality rate of 7.9%.
Clinical Diagnosis, Treatment & Sequelae

Highest attack rate was seen in the 12-49 months age group. Atypical signs such as conjunctivitis and joint swelling were seen in cases. Aside from environmental conditions, contact with cases was the most important identified risk factor for acquiring and transmitting the organism. Adjusted regression analysis showed 0.13 less likelihood of infection without a positive contact history [RR 0.13 CI (0.06- 0.30)]. There was no significant difference in mortality rate between cases and controls [HR 0.78 CI (0.29- 2.13)].

Conclusion: Effective district level surveillance and contact tracing should be put in communities and residents should be educated before or around outbreak seasons to isolated suspected cases as soon as possible to the nearest health facility. Vaccination with meningococcal vaccine for epidemic strain should be incorporated into the EPI schedule of the Gambia and yearly booster dose given to children especially prior or during the epidemic seasons.

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C28A review of the management of paediatric sepsis on the University Paediatric Ward in the Karapitiya Teaching Hospital, Galle, Sri Lanka

Quicke E1, Last A2, Jayantha UK3

1University of Birmingham Medical School, Birmingham, UK; 2London School of Hygiene & Tropical Medicine, Clinical Research Department, London, UK; 3Department of Paediatrics, Karapitiya Teaching Hospital, Galle, Sri Lanka

Email: exq890@bham.ac.uk


Keywords: Sepsis, Paediatric, Audit, Resource- limited, Management

Background: Sepsis is a leading cause of death in both the developed and developing world. In 2008, 70% of the 9 million paediatric deaths worldwide were from sepsis. Following the Surviving Sepsis campaign, guidelines were independently written in 2012 by the World Federation of Paediatric Intensive and Critical Care Societies which were adjusted for low/middle income countries. These guidelines provide a modified, evidence based system for management of sepsis, severe sepsis and septic shock in resource limited settings. This audit aimed to compare the management of cases of paediatric sepsis to these guidelines.

Methods: A prospective audit of 45 cases of confirmed/suspected paediatric sepsis admitted to the University Paediatric Ward of the Karapitiya Teaching Hospital in 10 days of May 2013. Several aspects of the management of each case including fluid management, oxygen administration, antimicrobial administration, and the use of appropriate microbiological investigations were compared to the guidelines written by the World Federation of Paediatric Intensive and Critical Care Societies.

Results: The most common infection was a lower respiratory tract infection (60%, 27/45), with other sources of infection being meningitis or suspected meningitis (6.7%, 3/45), abscesses (8.9%, 4/45) and urinary tract infections (8.9%, 4/45). Fluid management: 24% (11/45) cases received fluids. The rate at which fluids were administered varied between cases. Oxygen: 13% (6/45) were given oxygen on admission. Of the 42 cases where oxygen saturations were recorded, 95% (40/42) had saturations >90%. Antibiotics: 91% cases (41/45) received empirical antibiotic therapy, 43.8% (14/32) were within one hour. Appropriate Microbiological Investigations: 40% (18/45) cases had appropriate cultures taken, 55.5% (10/18) were taken before the administration of antibiotics.

Conclusion: This audit has highlighted that many of the recommended standards are not being met. Despite 91% receiving antibiotics, in only 43.8% cases was this within one hour. This is likely to be a factor that could significantly affect patient outcomes. Similarly, only 40% had cultures taken which can affect the ability to identify and later treat the causative organism. Both these issues could be addressed by introducing a policy of giving antibiotics at the time of taking cultures thereby prompting the clinician to undertake both management steps quickly. In addition, awareness should be raised amongst clinical staff about the guidelines to ensure that they are aware of the current problems and the appropriate management steps. A plan to re-audit should be implemented to ensure that any measures introduced are effective.

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C29Audit of UHCW Paediatric Department’s management of meningitis against the NICE quality standards

Tan Y, Jorge P, Wallace C, Manias K

Department of Paediatrics, University Hospitals Coventry
Warwickshire (UHCW) NHS Trust, Coventry

Email: yok.tan@uhcw.nhs.uk


Keywords: Meningitis, meningococcal, children, NICE, guidelines

Background: Bacterial meningitis is an infection of meninges by bacteria that travel from mucosal surfaces via the bloodstream. Invasive meningococcal disease describes meningococcal meningitis and meningococcal septicaemia, which can lead to multiorgan failure and death. The incidence of meningococcal disease and bacterial meningitis has decreased over the last 20 years, primarily due to meningitis C vaccination. Meningococcal disease remains the leading cause of death from infection in early childhood in the UK (mortality rate 10%). Prompt recognition of symptoms and signs is key to preventing death or disability.
In 2012, 41/443 of patients with invasive meningococcal infection were from the West Midlands, accounting for 9% of the UK population.

In June 2012 NICE published a quality standard QS19 for bacterial meningitis and meningococcal septicaemia in children and young people.

Aim: The aim of this audit was to assess UHCW Paediatric Department’s management of meningococcal disease against the NICE QS19.

Methods: A proforma listing the 14 quality standards was created. Prospective data was collected from May 1st-June 12th 2013.

Results: Seventeen patients were suspected of having bacterial meningitis or meningococcal sepsis. The average age was 12 months. All patients were admitted, with an average inpatient stay of 5 days. One patient required intubation & ventilation and transfer to PICU. Four patients had confirmed bacterial meningitis or meningococcal sepsis.

All patients with petechial rash received appropriate antibiotics. The average time before administering the first antibiotic dose was 3 hours. All patients with suspected bacterial meningitis, unless clinically unstable, had an LP with CSF results available within 4 hours. Intubation & transfer were performed by adequately trained healthcare professionals. All confirmed cases of meningococcal disease were discharged with appropriate information and follow up.

The following recommendations were made. An advice sheet (including a picture of a petechial rash) should be given to parents sent home with febrile children. Temperature, respiratory rate, pulse, blood pressure, urine output, oxygen saturation and neurological condition should be monitored hourly until stable. Patients should receive intravenous or intraosseous antibiotics within an hour of arrival. Meningococcal PCR testing should be performed. Children should have an audiological assessment before discharge.

Conclusions: The majority of patients with suspected meningococcal disease in UHCW were managed in accordance with the NICE QS19. There were, however, areas for improvement, particularly in terms of advice sheets given to parents of febrile children and time taken to commence antibiotics.

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Public Health Management

PH30A cluster of four cases of meningococcal disease in a single nuclear family

Peter Acheson1, Ruth Barron2, Ray Borrow3, Steve Gray3, Csaba Marodi4, Mary Ramsay5, Julia Waller1, Terry Flood6

1North East Public Health England Centre, Newcastle upon Tyne, UK; 2Department of Paediatrics, South Tees Hospitals NHS Foundation Trust, Middlesborough, UK; 3Meningococcal Reference Unit, Public Health England, Manchester, UK; 4Department of Clinical Microbiology, South Tees Hospitals NHS Foundation Trust, Middlesborough, UK; 5Public Health England, London, UK; 6Department of Paediatric Immunology and Infectious Diseases, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK

Email: peter.acheson@phe.gov.uk

Keywords: meningococcal disease, family, cluster, prophylaxis

A cluster of four confirmed cases of meningococcal disease was seen in the same nuclear family in the North East of England across a 15 week period in 2010. The first two cases presented within 48 hours of each other, followed by a gap of 40 days to the next case and a further 63 days to the fourth. The cases were three siblings and a parent. All cases recovered well from their illness.

The first case was confirmed by meningococcal PCR only (serogroup not determined) but the subsequent three cases were due to indistinguishable strains of Group B disease (B:NT:P1.19-1,15-11). Contact tracing was initially undertaken and reviewed in detail after each subsequent case. Antibiotic prophylaxis was administered to close family contacts on three separate occasions, including switching of antibiotic agents. The family reported having taken full courses of prophylaxis each time this was prescribed. Throat swabs were undertaken after the final round of prophylaxis to try to establish that carriage had been eliminated from the family group. No throat swab sample grew Neisseria meningitidis.

All family members were subsequently investigated for complement deficiency. Despite this, no obvious abnormality in the family group has been identified to date and no further significant infections have been recognised.

A cluster of meningococcal disease of this nature and timescale is highly unusual. Details of the cluster, investigation and implications for health protection practice are discussed.

Vaccinology

V31Physico-chemical and immunological investigation into the thermal stability of meningococcal MenACWY-TT conjugate vaccine

Nicola Beresford & Barbara Bolgiano

Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK

Keywords: Conjugate, vaccine, stability, physico- chemical, immunogenicity

The thermal stability of a meningococcal serogroup ACWY tetanus toxoid (TT) conjugate vaccine was analysed following exposure to four storage temperatures (-20°C, +4°C, +37°C, +56°C) and repeated freeze-thawing cycles (FT) for 28 days. Both the bulk conjugates and the final fill were analysed for their molecular integrity, free saccharide content and carrier protein folding by size exclusion chromatography (HPLC-SEC), high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and fluorescence spectroscopy. The data shows that the molecular integrity of the bulk conjugates is significantly altered at high temperatures leading to degradation into small molecular weight material. This observation is most profound in MenA bulk conjugate above +37°C. The MenY bulk conjugate was most resistant with degradation only observed at +56°C. Following +56°C incubation the percentage free saccharide content in the bulk conjugates also increased for MenACW ranging from 38% to complete depolymerisation (100%). Only minor changes were observed in the pH of the bulk conjugates and final fill with the exception of MenA which resulted in a significant drop in the pH associated with depolymerisation and instability. Neither the molecular integrity nor the percentage free saccharide of the lyophilised final fill was significantly affected by FT or by storage at high temperatures. Correlates between physico-chemical characterisation and immunogenicity measuring the total IgG responses against serogroup specific polysaccharides were analysed. A reduction in the immunogenicity was observed in the IgG response to all polysaccharides at high temperatures. There was a significant drop in the anti-MenA IgG response when incubated above +37°C and a similar response was seen for anti-MenC IgG when incubated at +56°C.

Although the same trend extends to anti-MenW and anti-MenY IgG responses stored at high temperatures these were not significant. In conclusion the immunogenicity correlated with the physico- chemical analysis of the bulk conjugates with MenA and MenC being the most prone to instability at high temperatures and MenY the most stable in adverse storage conditions.

V32Serum Bactericidal Antibody Levels Following Quadrivalent Conjugate (MenACWY-CRM) or Serogroup B (4CMenB) Meningococcal Vaccines in a Phase 3 Study to Evaluate the Effect of Vaccination on Pharyngeal Carriage of N. meningitidis in Young Adults

Peter Dull1, Xilian Bai2, Rohit Bazaz3, Kate Nolan2, Abida Nazir3, Annette Karsten1, Ellen Ypma1, Daniela Toneatto1, Robert Read3, Ray Borrow2

1
Novartis Vaccines and Diagnostics, Cambridge, Inc., Massachusetts, USA; 2Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK; 3Sheffield University, and Royal Hallamshire Hospital Sheffield, Department of Infection & Immunity, Sheffield School of Medicine and Biomedical Science, Sheffield, UK

Email: peter.dull@novartis.com

Keywords: vaccine, meningococcal, serogroup B, quadrivalent, immunogenicity

Background: Immunogenicity of a quadrivalent meningococcal conjugate vaccine, MenACWY- CRM (Menveo®) and a serogroup B meningococcal vaccine, 4CMenB (Bexsero®) were assessed in a subset of subjects in a carriage study among English university students (NCT01214850).

Methods: There were 2968 subjects enrolled to receive either MenACWY-CRM/placebo, 4CMenB or Japanese encephalitis vaccine (Ixiaro®). At the Sheffield site, immunogenicity of MenACWY-CRM and 4CMenB was evaluated in a subject (n~200 per group) by serum bactericidal antibody using human complement (hSBA) response at time points up to one year after initial vaccination. Subjects from the immunogenicity subset were tested by hSBA against serogroups C and Y, and MenB strains 44/76-SL, 5/99, and NZ98/254.

Results: At baseline across all vaccine groups, 38–71% of subjects had hSBA titres = 4 against 44/76-SL, 5/99, and NZ98/254. One month after the second dose of 4CMenB, 99–100% of subjects had hSBA titres = 4 against each of the MenB strains. Eleven months after the second vaccination, 85–97% of these subjects maintained hSBA titres = 4. The MenACWY-CRM and control groups showed no substantial increase in hSBA titres = 4 against MenB strains.

At baseline, 80–90% and 72–78% of subjects across all vaccine groups showed hSBA titres = 8 against serogroups C and Y, respectively. Against serogroup C, most subjects maintained hSBA titres = 8 at all post-vaccination time points across all groups. Against serogroup Y, there were further increases in subjects with hSBA titres = 8 from baseline in the MenACWY-CRM group, while in the 4CMenB and control groups the percentage of subjects with hSBA titres = 8 was similar to baseline.

The carriage rate in seropositive subjects was generally higher than in seronegative subjects at baseline; however, no clear association between carriage rates and post-vaccination hSBA levels was shown against MenB or serogroup C or Y strains. In subjects with documented prior vaccination with a serogroup C conjugate vaccine, evidence of an anamnestic response was demonstrated among MenACWY-CRM recipients with a mean serogroup C hSBA GMT of 1905 two months after vaccination (baseline GMT 91)

Conclusion: High percentages of MenACWY-CRM and 4CMenB recipients maintained bactericidal antibodies 11-12 months post-vaccination. No clear association between carriage rates and post- vaccination hSBA levels was observed.

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V33Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: A new perspective for vaccine development

Timm Fiebig1, Francesco Berti2, Friedrich Freiberger1, Vittoria Pinto2, Heike Claus3, Maria Rosaria Romano2, Daniela Proietti2, Barbara Brogioni2, Katharina Stummeyer4, Monika Berger1,, Ulrich Vogel3, Paolo Costantino2, Rita Gerardy-Schahn1

1Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany; 2Novartis Vaccines and Diagnostics, Research, Via Fiorentina 1, 53100 Siena, Italy; 3Institute for Hygiene and Microbiology, University of Würzburg, 97080 Würzburg, Germany; 4Present address: GRS - Gesellschaft für Anlagen- und Reaktorsicherheit, Schwertnergasse 1, 50667 Köln, Germany

Email: fiebig.timm@mh-hannover.de


Keywords: capsule polymerases, Neisseria meningitidis serogroup X, NMR, recombinant protein production, vaccine development

Neisseria meningitidis
(Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that negatively impacts complement activation and thus supports bacterial survival in the host. Twelve serogroups characterised by immunologically and structurally different CPS have been identified. Meningococcal CPS elicit bactericidal antibodies and consequently are used for the development of meningococcal vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here we describe the molecular cloning, recombinant expression, and purification of the capsule polymerase of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide, recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides demonstration that the previously identified gene csxA encodes the NmX capsule polymerase CsxA, the data presented in this study pave the way for the use of the recombinant capsule polymerase as a safe and economic way to generate the NmX capsular polysaccharide in vaccine developmental programs.

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V34The MenC content of meningococcal conjugate vaccines using the WHO 1st International Standard for Serogroup C Polysaccharide does not necessarily correlate with their immunogenicity

Fang Gao, Nicola Beresford, Kay Lockyer, Karena Burkin, Angela Martino and Barbara Bolgiano

Division of Bacteriology, National Institute for Biological Standards and Control, Medicines and Healthcare products Regulatory Agency, South Mimms, Potters Bar, Herts, EN6 3QG, UK

Keywords: meningococcal serogroup C, polysaccharide, conjugate vaccine, HPAEC-PAD, resorcinol

Introduction: The meningococcal serogroup C (MenC) saccharide content in monovalent and multivalent meningococcal conjugate vaccines was determined using various standards for their licensure and batch release control testing before the establishment of an international standard. The WHO 1st International Standard for Meningococcal Serogroup C Polysaccharide (NIBSC code 08/214) was adopted in 2011, based on the MenC content as determined by eight laboratories participating in a collaborative study (Vipond et. al., 2012). At NIBSC, it has been used in this study as the ‘gold standard’ to determine MenC content in meningococcal vaccines including a MenC-Hib combination, three monovalent MenC conjugates, and three MenACWY tetravalent vaccines. The aim of this study was to compare the content of MenC saccharide in currently licensed conjugate vaccines. The immunogenicity of the vaccines was also evaluated in a mouse model.

Method: The MenC content was determined by either high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) or for the MenC-TT vaccine, the resorcinol assay, using the MenC PS 1st International Standard. The MenC-specific IgG of the vaccines following 2 doses of 1/10 SHD in Balb/C mice was measured by ELISA titre, using an anti-MenACWY-DT reference sera.

Results: When the WHO 1st International Standard for MenC polysaccharide was applied as the quantitative standard, varying amounts of MenC content were found in the different vaccines, which did not precisely correspond with their labelled contents. The highest MenC content was in a monovalent MenC-TT conjugate with 12.1 µg/dose. The lowest contents were found from the MenC-TT components in a MenC-Hib and a MenACWY combination, with 2.9 and 3.0 µg/dose, respectively. The MenC content of all other tested vaccines were as expected based on their labelled contents. The post-2 geometric mean titres of MenC PS-specific IgG were not significantly different from each other.

Conclusions: The 1st International Standard for MenC PS provided a powerful tool to compare the ‘true’ MenC content in different vaccines. The three MenC- TT conjugates were found containing variant MenC contents. The GMTs from a 2-dose mouse model showed statistically-comparable MenC responses from all the vaccines tested, suggesting that the MenC response did not necessarily correlate with the dosage given.

V35Vaccines against MenB disease: over-expression of factor H-binding protein (fHbp) in native outer membrane vesicles elicits broader strain-coverage than recombinant fHbp

Johan Holst1, Peter T. Beernink2,Rolando Pajón2

1
Department of Bacteriology and Immunology, Division of Infectious Diseases Control, Norwegian Institute of Public Health, Oslo, Norway; 2Center for Immunobiology and Vaccine Development, Children’s Hospital Oakland Research Institute, Oakland, California, USA

Email: johan.holst@fhi.no; pbeernink@chori.org; rpajon@chori.org

One of the most promising vaccine candidates against serogroup B (MenB) meningococcal disease is factor H-binding protein (fHbp). At present this antigen is a key component in two different vaccines; one with a recent marketing authorisation in Europe and the other in late-stage clinical trials. These vaccine formulations are based on recombinant fHbp molecules with differences in sequence and lipidation state. Another vaccine strategy is to use native outer membrane vesicles (nOMV) prepared from strains with genetically over-expressed fHbp. In the present study, we immunised mice with a nOMV vaccine with over-expressed fHbp ID 9 from sub- family B (OE-nOMV-fHbp; ID 9). We measured human complement-mediated bactericidal activity against 12 invasive case isolates from Norway with different sub-family B fHbp sequence variants. As controls, we used sera raised to non-lipidated recombinant fHbp vaccines, whose sequences were matched to each of the test strains (identical fHbp sequence identification
Vaccinology

(ID) numbers. Antibodies to the recombinant fHbp vaccines elicited bactericidal activity against most of the strains. However, three strains with moderate to high expression of different fHbp sequences, as measured by flow cytometry, were resistant to these antisera. Antibodies to the OE-nOMV-fHbp ID 9 vaccine elicited high bactericidal titres against 11 of the 12 strains tested. The cross-protective responses were primarily elicited by anti-fHbp antibodies, since a control vaccine prepared from an isogenic fHbp knock-out strain (KO-nOMV) elicited no bactericidal activity against any of the strains. Thus, antibodies elicited by the nOMV-fHbp vaccine had broader functional reactivity against the strains overall than antibodies elicited by the matched recombinant fHbp vaccines. Our findings illustrate the promise of using an OE-nOMV-fHbp formulation as an improved next generation MenB vaccine, for achieving a better functional immune response, with broader strain-coverage, than by using recombinant fHbp in various vaccine formulations.

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V36Addressing knowledge gaps around Serogroup B Invasive Meningococcal Disease Vaccination: Development of an Evidence-Based Reference document for use by Canadian Health Care Professionals

Tajdin Jadavji1, Ronald Gold2, Gregory Tyrrell3,4, Marc Lebel5,6, Simon Dobson7, Ian Gemmill8, Allan Ronald9, James A.Mansi10

1Alberta’s Children’s Hospital, Calgary, AB; 2University of Toronto, Toronto, ON; 3University of Alberta, Edmonton, AB;4Division of Medical Microbiology , Edmonton, AB; 5Centre Hospitalier Universitaire Sainte-Justine, Montreal, QC; 6Université de Montréal, Montreal, QC; 7British Columbia Children’s Hospital, Vancouver, BC; 8Kingston, Frontenac and Lennox & Addington Public Health Unit, Kingston, ON; 9University of Manitoba, Winnipeg, MB; 10Novartis Vaccines & Diagnostics, Dorval, QC

Email: kidsmd@ucalgary.ca


Keywords: Invasive Meningococcal Disease (IMD), Meningitis B, MenB Vaccine, Immunisation Practice, Knowledge Translation

Background: Since the implementation of Meningococcal C (MenC) vaccine programs in Canada, significant reductions in MenC disease have been seen. With the introduction of quadrivalent meningococcal conjugate A, C, W-135, Y (MenACWY) vaccines, we hope to observe similar reductions in invasive meningococcal disease (IMD) due to serogroups Y and W. In Canada Meningococcal B (MenB) accounts for the majority of invasive meningococcal disease (IMD). A MenB vaccine will soon be available. In order to facilitate the introduction and use of MenB vaccines, existing gaps in understanding of these vaccines and immunisation recommendations will need to be addressed. We developed an evidence-based reference document to bridge to guidelines and address the immediate need for answers to commonly asked questions.

Methods: A Steering Committee of experts in IMD, infectious diseases and vaccinology was established to provide oversight and governance. Questions were collected from accredited continuing medical education (CME) events and through the Novartis Medical Information Centre support line. An Advisory Committee was convened to obtain consensus on reference based responses and their relevance from an immunisation perspective. Consensus was obtained on Frequently Asked Questions (FAQs) and evidence based answers, generating an open source document.

Results: Over an 18 month period, 8 different CMEs occurred. Of these 2 were focused on MenACWY vaccines and 6 on MenB vaccines. Questions posed during these CMEs, including those sent through the Medical Information Centre, were grouped according to the following categories: epidemiology and burden of IMD (35%), IMD pathophysiology and vaccine clinical data (45%) and IMD immunisation recommendations (20%).

Conclusion: There remain gaps in understanding of current IMD epidemiology, vaccine clinical data and vaccine guidelines. This evidence-based reference document will help bridge to upcoming guidelines and recommendations, addressing the immediate need for answers to commonly asked questions from a scientific, clinical and practical perspective.

Conflict of Interest:
The development of this document was made possible through funding provided by Novartis Vaccines & Diagnostics, a business unit of Novartis Pharmaceuticals Canada Inc. (‘’Novartis’’), and was conducted under a collaborative agreement between Novartis and the project’s scientific steering committee, which comprised Drs Tajdin Jadavji, Ron Gold, Gregg Tyrell, Marc Lebel, Simon Dobson, Ian Gemmill, Alan Ronald and James A. Mansi. The scientific steering committee was responsible for the development of the document and for the direction of the project. Novartis does not have any ownership rights to the project. The scientific steering committee has absolute discretion and the final decision over the contents, educational materials and presentations.

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V37The successful development of an efficient OMV vaccine process with the potential for producing a broad protective immune response against Neisseria meningitidis

Michael Joachim, Claire Entwisle, Ann McIlgorm, Sue Hill, Sally Harris, Lucy Blandford, Paola Cecchini, Yin Pang, Chris Bailey, Camilo Colaco

ImmunoBiology Ltd, Cambridge, UK
Email: Michael.joachim@immbio.com


Keywords: Neisseria meningitidis, OMV, Fermentation, Process, Immunogenicity

Outer membrane vesicles (OMVs) are naturally produced by Neisseria meningitidis bacteria by vesiculation or ‘blebbing’ from the bacterial membrane. The composition of OMVs makes them significant activators of host innate and acquired immune response pathways. In addition to the potent immunomodulatory molecule LPS, vesicles contain porins and other important innate immune-activating ligands. Together, vesicle components appear to act synergistically to modulate the host response in ways that can either stimulate the clearance of the pathogen, enhance the virulence of the infection, or both.
 
The current method of OMV production involves the use of detergents to extract the OMVs which gives a good yield but can change the conformation and immunogenicity from the native OMV. Whilst these detergent-extracted OMVs have been used as vaccines to control epidemics they are only protective against the homologous strain, although they have also been used in vaccines to increase the breadth of the immune response.

ImmBio has investigated the use of a non-detergent method of OMV production using a 1L fermentation scale process. The aim of the study was to produce spontaneously released OMVs (sOMV) which are released into the culture supernatant and an extracted OMV (eOMV).

OMVs were produced from wild type N. meningitidis strains 44/76-SL and M01-240149 which were grown in 1L fermenters and the effect of different culture conditions on the production process was assessed. Following downstream processing, the resultant eOMV and sOMV were assessed by SDS PAGE gels, Western blot, ELISAs and electron microscopy and compared with OMVs produced by detergent extraction.

The sOMV and eOMV were also used in an in vivo study to assess immunogenicity.

Following analysis it was found that there was a high yield of sOMVs derived from the 44/76-SL strain compared with that derived from the M01-240149 strain and that the yield of eOMVs was similar for both strains. Using SDS-PAGE and Western blot analysis both eOMV and sOMV were found to have similar protein profiles and in vivo, both the eOMV and sOMV were immunogenic and induced a good antibody response.

This study shows that this 1L scale fermentation of N. meningitidis can successfully and efficiently produce both soluble and extracted OMVs which are immunogenic in vivo.

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V38Structure-antibody recognition studies of currently licensed tetanus toxoid-conjugated bacterial polysaccharide vaccines

Kay Lockyer, Fang Gao, Laura Coombes, Paul Stickings, Barbara Bolgiano

Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG, UK

Email: Kay.Lockyer@nibsc.org

Keywords: conjugate, SEC-MALLS, epitope recognition, tetanus toxoid, monoclonal antibody

A comparison of structural features of nine different currently licensed polysaccharide-tetanus toxoid (TT) conjugate vaccines was made to determine if the accessibility of neutralising epitopes on the conjugated carrier protein was affected by the degree of polysaccharide (PS) loading or size of the conjugate. T-cell epitope accessibility and integrity is critical in conjugate vaccines so that glyco-peptides can be processed and presented to antigen- presenting cells for an effective immune response. The conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b (Hib), Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi- angle light scattering (SEC-MALS) to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies, 2 localised to the Hc (cell-binding) subunit, and 2 which
were conformation-dependent (and not localised to a particular subunit).

All TT conjugates were found to be well-folded, and did not have spectral features typical of aggregated TT. They ranged in size from 1.5 to > 10 MDa. The panel of bulk conjugates showed a correlation between PS loading (mol PS: mol protein) and the recognition of neutralising Hc epitopes (antibodies TT04 and TT10); somewhat surprisingly, increased protein epitope recognition was observed in conjugates which had higher PS loading/mol protein. In general, Trp residues were also more surface exposed and quenched by acrylamide in the larger conjugates. Low or no correlation was found for the conformational, holotoxin epitopes. Although manufactured by different conjugation chemistries, this panel of TT conjugates has been useful in establishing the concept that high PS conjugation to carrier proteins need not necessarily mask or prevent immune system recognition of key vaccine epitopes.

V39Attitudes towards vaccination against Group B streptococcus in pregnancy

Fiona McQuaid1,2, Christine E Jones3, Zoe Stevens1,2, Jane Plumb4, Rhona Hughes5, Helen Bedford6, Paul T Heath3, Matthew D Snape1,2

1Department of Paediatrics, University of Oxford, UK; 2NIHR Oxford Biomedical Research Centre, Oxford, UK; 3Paediatric Infectious Diseases Research Group, St Georges, University of London, London, UK; 4Group B Strep Support, Haywards Heath, West Sussex, UK; 5Simpson Centre for Reproductive Health, Royal Infirmary, Edinburgh, UK; 6Centre for Epidemiology and Child Health, University College London Institute of Child Health, London, UK

Email: Fiona.mcquaid@paediatrics.ox.ac.uk


Keywords: Group B Streptococcus, vaccination, pregnancy, attitudes

Introduction: Group B streptococcus (GBS) remains the most common cause of serious infection, including meningitis, in infants under three months of age. GBS vaccines are currently in development, and their use in pregnancy has the potential to prevent early and late onset disease. However, women may be reluctant to accept antenatal vaccination.

The objective of this survey was to assess the knowledge and attitudes of British women regarding GBS and the acceptability of a potential GBS vaccine.

Methods: An online questionnaire distributed to 1,013 women aged 18-44 in Great Britain by a market research company (ComRes, London, 13th-17th September 2013) assessed level of awareness of, and attitudes towards vaccination in pregnancy against pertussis, influenza and GBS. Information about GBS was then presented and questions about GBS immunisation repeated with an additional question about GBS vaccine research. Respondents were also asked to rate the importance of immunisation advice from a list of sources. The survey was funded by Meningitis UK.

Results: Nearly two thirds (63%) of women aged 18-44 years reported they either had never heard of GBS or didn’t know what it was, compared to 28% for pertussis and 40% for influenza in pregnant women. However, the level of knowledge about the condition did not affect the willingness to receive a vaccine against it during pregnancy with 75% being likely to accept pertussis vaccine, 72% influenza and 72% GBS. Once additional information was provided about GBS, the latter percentage rose to 82%. Thirty-two percent responded that they would be likely to take part in GBS vaccine research during pregnancy if the vaccine had been tested in 500 pregnant women without significant safety concerns, rising to 43% if the vaccine had been tested in 5000. Seventy-nine percent reported they would be happy to receive the vaccine if it was licensed and recommended for use by the NHS. GPs were rated the most important source of advice on maternal immunisation (considered important by 87% of women aged 18-44 years) followed by midwives (84%).

Conclusions: Despite the low level of knowledge about GBS, those completing the survey were open to the idea of immunisation during pregnancy and over a third showed interest in being involved in research. While these results are encouraging in terms of the feasibility of conducting future GBS vaccine trials in pregnancy, more work is required to establish the specific concerns of pregnant women and how these may be overcome.

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V40Knowledge and attitudes towards meningitis and vaccination: a survey of parents and health professionals in Ireland

Andy Cochrane, Diane McConnell, Caroline O’Connor

Meningitis Research Foundation Dublin

Email: Caroline@meningitis-ireland.org


Keywords: knowledge and attitudes; questionnaires; parents; GPs and practice nurses

Aims: In the past two decades the introduction of vaccines to control Hib disease, meningococcus C and some types of pneumococcus has resulted in a dramatic reduction in the number of cases each year. For example, cases of invasive meningococcal disease (IMD) caused by serogroup C has reduced from 135 in 1999 to just 2 in 2010. Nevertheless, meningitis has not ‘gone away’ and Ireland still has the highest rates of confirmed cases of IMD in Europe for both of the age groups most at risk (under 5 years: 21 per 100,000; 15-24 year olds: 5.7: 100,000). A vaccine uptake rate of = 95% is needed to achieve herd immunity. However, following the change to the immunisation schedule in July 2008, the uptake of the Men C and Hib vaccines due at 13 months fell to as low as 80% in some parts of the country. While there is some evidence of improved uptake rates there is a need to understand attitudes towards vaccination and knowledge about meningitis to inform health promotion initiatives.

Method: Telephone surveys were conducted with (1) a nationally representative sample of parents (n = 350) with one or more child under the age of two years; and (2) GPs and practice nurses (n = 150) with direct involvement in vaccination.

Results: The findings indicate that meningitis creates a high level of concern for both parents and health professionals, yet some parents are delaying completion of the vaccination schedule without realising that this leaves their child unprotected at a period when they are most at risk from the disease. In addition, over a third of parents mistakenly believed that the current vaccination schedule protects their child against all forms of meningitis.

Conclusions: Recognition and treatment of meningitis and septicaemia has improved but prevention remains critical in reducing the burden of the disease. There is an ongoing need for information about vaccination and meningitis at a national level, and for more tailored individualised information. GPs and Public Health nurses remain the key people that parents turn to for advice and information, and these professionals will be critical in ensuring the uptake rates of the current immunisation schedule continue to improve and to promote the successful introduction of any changes to the childhood vaccination schedule.

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V41Safety and Immunogenicity of a 2-Dose Schedule (12 Months and 18 Months of Age) of a Quadrivalent Meningococcal Conjugate Vaccine

Noya F1, McCormack D1, Reynolds DL2, Neame D2, Oster P3

1McGill University Health Centre, Montreal, QC Canada; 2Sanofi Pasteur Ltd., Toronto, ON, Canada; 3Sanofi Pasteur, Lyon, France

Email: philipp.oster@sanofipasteur.com


Keywords: Neisseria meningitidis; conjugate vaccine; meningococcal disease; safety; immunogenicity

Background: Monovalent serogroup C meningococcal conjugate vaccine (MCC) has been provided through Quebec vaccination programs since 2001. A 2-dose schedule of a quadrivalent meningococcal conjugate vaccine (MenACYW-D) at 12 and 18 months of age would fit current programmes and broaden coverage.

Methods: Eligible toddlers received MMRV and PCV13 at 12 months and MMR and DTaP-IPV-Hib at 18 months in a 2-armed, open-label, parallel descriptive study. Participants were randomised to receive a 2-dose schedule of MenACYW-D at 12 and 18 months of age (MenACYW-D arm) or one dose of MCC at 12 months of age (MCC arm). Blood samples were collected pre-Dose 2 at 18 months and 1 month post-Dose 2 in the MenACYW-D arm and at 1 and 7 months post-MCC in the MCC arm. Immune responses to meningococcal vaccines (via baby rabbit serum bactericidal assay [SBA-BR]) and DTaP-IPV-Hib vaccine were analysed and safety data collected.

Results: Participants in the MenACYW-D arm (n=61) achieved high rates of seroprotective levels (SBA-BR =8 1/dil) post-Dose 2: A (100% [95% CI, 93.0%– 100%]), C (96.1% [95% CI, 86.5%–99.5%]), Y (100% [95% CI, 93.0%–100%]), W (98.0% [95% CI, 89.6%– 100%]). In the MCC arm (only serogroup C response was expected; n=62), 66.7% [95% CI, 53.3%–78.3%] of participants achieved seroprotective levels 1 month postvaccination, declining to 25.9% (95% CI, 15.3%–39.0%) 7 months postvaccination. In the MenACYW-D arm, geometric mean titres for all serogroups were high post-Dose 2: 1740 (A), 957 (C), 719 (Y) to 970 (W). All titre increased from the 18-month pre-Dose 2 vaccination. Seroprotection rates for diphtheria, tetanus, PRP and polio types 1, 2, and 3 were 100%.
Pertussis booster response rates in the MenACYW-D and MCC arms were 100% versus 94.3% (PT), 92.7% versus 78.8% (PRN), 97.7% versus 88.0% (FHA), and 100% versus 95.8% (FIM), and were within the range of historical rates. Both vaccines were well tolerated. Three SAEs were reported. None were related to vaccination, and no participants withdrew due to AEs.

Conclusion: Two doses of MenACYW-D resulted in higher serogroup C immune responses at 19 months than a single dose of MCC and broadened serogroup coverage. MenACYW-D administered as a 2-dose series, concomitantly with a booster dose of DTaP- IPV-Hib at 18 months of age demonstrated good immunogenicity and safety profiles. MenACYW-D can be used as an alternative to MCC in vaccination programs. Study funded by Sanofi Pasteur (NCT01359449).

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V42Single Priming Dose of NeisVac-C® in Infants

Eva Maria Poellabauer, Borislava G. Pavlova, Sandor Fritsch, Julia Singer, Christian Neubauer, Jennifer Doralt, Barbara Valenta-Singer, Robert Petermann, Hartmut J. Ehrlich

Global R&D, Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria

Keywords: meningitis, vaccination, infants, single dose, schedule

The currently licensed infant immunisation schedule for meningococcal serogroup C conjugated vaccines requires two doses, followed by a booster dose in the second year of life. Several clinical studies with NeisVac-C® demonstrated high seroprotection rates in infants after a single priming dose.

The aim of the present study was to assess the feasibility of a single priming dose of NeisVac-C® given at 4 months or 6 months of age compared to a two-dose priming schedule.

956 subjects were randomly assigned to receive a single dose of NeisVac-C® at 4 or 6 months of age, or two doses at 2 and 4 months of age. All subjects received a booster between 12 and 13 months of age. Concomitant vaccinations with Infanrix® hexa and Prevenar 13® were administered to all subjects at all timepoints.

Primary endpoint of the study was to demonstrate non-inferiority of seroprotection rates following a single priming dose compared to a two-dose priming one month after the primary vaccination (rSBA =8), prior to the booster (rSBA =8), and one month after the booster (rSBA =128). The non-inferiority margin was set to 10% and 5%, for the first two and the third time points, respectively.

Rates of subjects with rSBA = 8 one month after primary vaccination were 99.6% in the 4 month dose group, 99.2% in the 6 month dose group, and 99.6% in the two-dose group. Prior to the booster, 78.0% and 90.7% of subjects had seroprotective antibody titre in the single dose groups (month 4 or month 6, respectively), compared to 67.8% in the two dose group.

One month after the booster, > 98.5% of subjects in all three dose groups showed rSBA titre = 128, with no differences between the groups. Non-inferiority of the single dose regimen vs. two doses could be demonstrated.

Thus, a single priming dose given at age = 4 months, followed by a booster early in the second year of life, is expected to offer protection, which is comparable to that of a two-dose priming schedule. This finding is in line with several previous studies which demonstrated seroprotection rates of 92%-100% after one dose of NeisVac-C® given at two - or three months of age.

Reducing the number of doses in infancy from two doses to one provides greater flexibility in the already crowded infant vaccination schedules, and allows transfer of one MCC vaccine dose into adolescence in a cost neutral way.

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V43 Impact of Quadrivalent Conjugate (MenACWY- CRM) and Serogroup B (4CMenB) Meningococcal Vaccines on Meningococcal Carriage in English University Students

Robert C. Read1, David Baxter2, David R. Chadwick3, Saul N. Faust4, Adam Finn5, Stephen B. Gordon6, Paul T. Heath7, David J.M. Lewis8, Andrew J. Pollard9, David P.J. Turner10, Rohit Bazaz1, Amitava Ganguli11, Tom Havelock4, Keith R. Neal10, Ifeanyichukwu O. Okike7, Begonia Morales-Aza5, Kamlesh Patel12, Matthew D. Snape9, John Williams3, Stefanie Gilchrist13, Stephen J. Gray13, Huajun Wang14, Maggie McCarthy14, Daniela Toneatto14, Peter M Dull14, Ray Borrow13

1Sheffield University, and Royal Hallamshire Hospital Sheffield, Department of Infection & Immunity, Sheffield School of Medicine and Biomedical Science, Sheffield, UK; 2Division of Epidemiology and Health Sciences, Medical School, The University of Manchester, UK;3The James Cook University Hospital, Middlesborough, UK; 4NIHR Wellcome Trust Clinical Research Facility, Faculty of Medicine, University Hospital Southampton NHS Foundation Trust, University of Southampton, UK; 5University of Bristol & Bristol Royal Hospital for Children, University Hospitals, Bristol NHS Foundation Trust, Bristol, UK; 6Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK; 7Vaccine Institute & Paediatric Infectious Diseases Research Group, Division of Clinical Sciences, St. Georges, University of London, London, UK; 8Clinical Research Centre, Institute of Biosciences and Medicine, FHMS, University of Surrey, Guildford, UK; 9Oxford Vaccine Group, Department of Paediatrics, University of Oxford, UK; 10University of Nottingham, and Nottingham University Hospitals NHS Trust, Derby Road, Nottingham, UK; 11Royal Liverpool University Hospital and Institute of Translational Medicine, University of Liverpool, UK; 12NIHR/Wellcome Trust Clinical Research Facility, Manchester Royal Infirmary, Manchester, UK; 13Meningococcal Reference Unit, Public Health England, Manchester Public Health Laboratory, Manchester Royal Infirmary, Manchester, UK; 14Novartis Vaccines and Diagnostics, Inc., Cambridge, Massachusetts, USA

Email: r.c.read@soton.ac.uk


Keywords: vaccine, meningococcal, serogroup B, carriage, quadrivalent

Background: Successful introduction of serogroup C meningococcal conjugate vaccination was underpinned by a significant impact on oropharyngeal carriage. With the recent introduction of ACWY meningococcal conjugate vaccines and EMA approval of a serogroup B meningococcal vaccine, we investigated the effect of these two vaccines on carriage in university students,
a potential target population for meningococcal vaccination (NCT01214850).

Methods: From September–December 2010, 2968 students (mean age ± SD, 19.9 ± 1.6 years) from ten English universities were enrolled into a phase III observer-blind, randomised, controlled study. Three equal groups received two vaccinations one month apart: a MenACWY-CRM group (n=956) given one dose of quadrivalent meningococcal conjugate vaccine (Menveo®) followed by saline placebo; a 4CMenB group (n=932) who received two doses of meningococcal serogroup B vaccine (Bexsero®); and controls (n=947) given two doses of Japanese Encephalitis vaccine (Ixiaro®). Oropharyngeal samples were taken before vaccination and at five subsequent visits over one year. Primary analysis compared cross-sectional carriage one month after the vaccine course; secondary analysis compared cumulative carriage in the period commencing after primary analysis until study termination.

Results: Before vaccination, 981 (33%) of 2941 evaluable samples yielded Neisseria cultures, mostly (98%; n=930) N. meningitidis, mainly capsular groups B and Y. The primary analysis showed no significant differences in carriage between controls and MenACWY-CRM (p=0.593) or 4CMenB (p=0.393) groups. In secondary analyses from three months after dose two 4CMenB showed significantly lower carriage of any N. meningitidis than controls at any timepoint; efficacy 18.2% (95% CI: 3.4–30.8), and lower carriage of genogroups B, C, W and Y combined: efficacy 26.6% (95% CI: 10.5–39.9). Genogroups B, C, W, and Y carriage was significantly lower in 4CMenB subgroups associated with increased susceptibility to de novo acquisition: subjects enrolled within 30 days of semester start (p=0.012), smokers (p=0.009), and subjects < 21 years-old (p=0.004). In further secondary analysis from two months after first dose, the MenACWY- CRM group showed significantly lower carriage of genogroups and serogroups A, C, W, and Y combined (p=0.011 and p=0.002, respectively) compared with controls, largely attributable to a carriage-reduction efficacy of 39% (95% CI: 17.3– 55.0) against serogroup Y strains.

Conclusion:
MenACWY-CRM and 4CMenB both show impact on carriage during 12 months post- vaccination, particularly in sub-groups associated with increased risk of meningococcal disease, raising the possibility of an impact on transmission, which may translate into herd protection in settings where the vaccines are implemented broadly.



V44Five-Year Persistence of Immune Responses to Two Licensed Quadrivalent (MenACWY) Conjugate Meningococcal Vaccines in Adolescents

Roger Baxter1, Keith Reisinger2, Stan L Block3, Sandra Percell4, Tatjana Odrljin4, Peter M Dull4, Igor Smolenov4
 
1Kaiser Permanente Vaccine Study Center, Oakland, CA, USA; 2Primary Physicians Research, Pittsburgh, PA, USA; 3Kentucky Pediatric and Adult Research, Bardstown, KY, USA; 4Novartis Vaccines and Diagnostics, Inc., Cambridge, MA, USA

Email: igor.smolenov@novartis.com


Keywords: vaccine, meningococcal, persistence, booster, quadrivalent

Background: Meningococcal conjugate ACWY vaccines are recommended for all US adolescents, with one of two currently licensed vaccines, Menveo® (MenACWY-CRM, Novartis Vaccines) and Menactra® (MenACWY-D, Sanofi Pasteur). Having previously compared immune responses to these two vaccines in adolescents, we assessed antibody persistence at five years post-vaccination (clinicaltrials.gov NCT00856297).

Methods: In this open-label, multi-center, US-based study, antibody persistence was assessed in healthy 11-18 year-old adolescents five years after vaccination (March 2007) with one dose of either MenACWY-CRM or MenACWY-D; some subjects also received a booster dose of MenACWY-CRM three years after primary vaccination. Age-matched vaccine-naïve subjects were recruited as controls. Immune responses, assessed by serum bactericidal activity with human complement (hSBA), were expressed as percentages of seropositive subjects (hSBA titer = 8) and geometric mean titre (GMT) against the four serogroups.

Results: A total of 389 subjects participated in five groups: MenACWY-CRM (n=131); MenACWYD (n=76); MenACWY-CRM + MenACWY-CRM booster (n=44); MenACWY-D + MenACWY-CRM booster (n=31); vaccine-naïve (n=107). Mean ages were 18.8–19.7 years across all groups. Five years after primary vaccination, the percentages of seropositive subjects against serogroup A in the MenACWY-CRM and MenACWY-D groups were both significantly (p < 0.001) higher than in vaccine-naive subjects (32%, 34% and 8%, respectively).
For serogroups C, W and Y, the respective seropositivity rates in the MenACWY-CRM, Men- ACWY-D and vaccine-naïve groups were: serogroup C (59% vs 60% vs 38%), serogroup W (82% vs 73% vs 66%) and serogroup Y (64% vs 54% vs 39%). All comparisons between MenACWY-CRM and control groups were statistically significant (p = 0.004). For Men-ACWY-D, only the serogroup C rate was significantly higher than controls (p = 0.004). MenACWY-CRM vaccinees had significantly higher GMTs against all four serogroups than controls, but in the MenACWY-D group only serogroup A and C GMTs were significantly higher than controls. Two years after a booster dose of MenACWY-CRM, 77–79% of subjects had hSBA titre = 8 against serogroup A, 87–95% against serogroup C, 97–100% against serogroup W, and 93–95% against serogroup Y, after primary vaccination with MenACWY-CRM or MenACWY-D.

Conclusion: Both MenACWY-CRM and MenACWY-D showed evidence of substantial seropositivity rates 5 years after a single vaccination, although only the MenACWY-CRM group had significantly higher rates than controls for all serogroups. A booster dose of MenACWY-CRM three years after either vaccine maintained high rates of seropositive subjects two years later.

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V45Development of a serum bactericidal antibody (SBA) assay for Haemophilus influenzae serotype b

Kelly Townsend1, Helen Findlow1, Xilian Bai1, Shamez Ladhani2, Mary Slack3, Ray Borrow1

1Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK; 2Immunisation, Hepatitis and Blood Safety Department, Public Health England, London, UK; 3Respiratory and Systemic Infection Laboratory, Public Health England, London, UK

Keywords: Hib, SBA assay, anti-PRP IgG, glycoconjugate vaccines, assay validation

Background: Prior to routine conjugate vaccination, Haemophilus influenzae serotype b (Hib) was a major cause of serious bacterial infections, particularly in young children. The introduction of the Hib conjugate vaccine into national childhood immunisation programmes resulted in a rapid and sustained reduction in invasive Hib disease across all age groups. Evaluation of the immune response to Hib conjugate vaccines includes the measurement of serum antibodies against the Hib capsular polysaccharide, polyribosyl-ribitol-phosphate (PRP), by ELISA, with accepted short term and long term levels of =0.15 µg/mL and =1.0 µg/mL, respectively. The relevance for protection in children who have been primed with glycoconjugate vaccines remains unclear, as these levels were derived from passive immunisation, or immunisation with pure polysaccharide.

The serum bactericidal antibody (SBA) assay measures functional antibodies that bind to a specific target strain and fix complement onto the bacterial surface, initiating complement mediated lysis. Previous SBA methodologies have been published for Hib which have shown good correlation between IgG antibody concentrations against the Hib capsular polysaccharide (PRP) with SBA titres in adult sera. This correlation, however, has not been established and evaluated in vaccinated UK infants. The objective of this study, therefore, was to develop an SBA assay to assess the functional activity of Hib antibodies induced by Hib conjugate vaccines.

Methods: A Hib SBA assay was developed and optimised using previous knowledge and understanding of the N. meningitidis SBA, and subsequently tested on sera from vaccinated adults (n=39), and from infants immunised under the current UK accelerated immunisation schedule at two, three and four months (n = 486). SBA titres were compared to previously determined anti-PRP IgG concentrations determined by a Hib Bioplex assay for correlation and the Pearson`s correlation coefficients (r value) were calculated.
Results: Validation of the Hib SBA assay was deemed acceptable in all assay parameters tested. In vaccinated adults, a strong correlation (r=0.81) between anti-PRP IgG concentrations and SBA titres were shown. In vaccinated infants, too, good correlations between anti-PRP IgG concentration and SBA titres were observed (r=0.64 post primary, r=0.75 post-booster). A predictive SBA titre of 8 was calculated using the established long-term correlate of protection (1.0 µg/mL).

Conclusions: The Hib SBA assay was shown to be reliable and reproducible when evaluating the humoral response following Hib vaccination. An SBA titre of 8 was estimated to confer long-term protection against invasive Hib disease.

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V46Assessment of non-functional variant 3 factor H binding proteins as meningococcal vaccine candidates

Stijn van der Veen, Christoph M. Tang

Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford, OX1 3RE, UK

Email: stijn.vanderveen@path.ox.ac.uk


Keywords: Neisseria, fHbp, vaccine, factor H, immunogenicity

Neisseria meningitidis
is a human specific pathogen and the leading cause of meningitis and septicaemia. Capsule-based vaccines are available against several serogroups of N. meningitidis, but not against serogroup B strains because its capsule mimics a human molecule, affecting immunogenicity and raising concerns of autoimmunity. Consequently, N. meningitidis serogroup B is the main cause of disease in developed countries. Therefore, vaccines containing outer-membrane proteins are currently being developed. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against meningococcus, and is currently being assessed in several phase II/III clinical trials. However, the interaction of fHbp with the host complement regulator factor H (fH) could impair the immunogenicity of this antigen and has led to the development of non-functional fHbps as vaccine candidates. Furthermore, fHbp can be divided into three variant groups (V1, 2 and 3) based on its amino-acid sequence and immunological cross- reactivity between these variant groups is limited. So far, nothing is known about the immunogenicity of non-functional V3 fHbps, while this variant group is expressed by approximately 10% of the clinical isolates.

In this study, we investigated the non-functional V3 fHbp mutants fHbpT228A and fHbpE255A, to establish their suitability for an fHbp-based vaccine. The binding affinity of the wild-type and mutant proteins for fH was determined by surface plasmon resonance, ELISA and far-Western and both mutants showed a marked reduction in affinity. Reduced binding by the non-functional fHbps could be the consequence of misfolding or reduced stability and unstable proteins are undesirable for vaccine development. Therefore, we investigated the structural integrity and stability of the non-functional fHbps using circular dichroism spectroscopy (overall folding), differential scanning calorimetry (thermal stability) and trypsin digestion assays (local folding disturbances) and confirmed that both non- functional fHbps are stable fully folded proteins. The immunogenicity of our non-functional fHbps was assessed by immunising transgenic mice in which the mouse fH domains involved in binding to fHbp were replaced by human fH domains. No differences in anti-fHbp antibody titre were observed between wild-type or non-functional fHbps. To determine whether these antibodies are able to elicit protective immunity, the serum bactericidal activity of the antibodies was investigated against N. meningitidis M1239, which naturally expresses the homologous 3.P28 fHbp. Equivalent or improved bactericidal activity was observed after immunisation with non- functional fHbps. These findings provide the basis for the rational design of next generation vaccines containing non-functional V3 fHbps.

V47Antibody persistence 12 months following booster vaccination with a quadrivalent meningococcal ACWY tetanus toxoid conjugate vaccine in healthy children

Timo Vesikari1, Aino Forsten1, Veronique Bianco2, Marie Van der Wielen2, Jacqueline M Miller3
1Vaccine Research Center, University of Tampere Medical School, Tampere, Finland; 2Vaccine Discovery and Development, GlaxoSmithKline Vaccines, Wavre, Belgium; 3Vaccine Discovery and Development,

GlaxoSmithKline Vaccines, King of Prussia, PA, USA

Email: marie.x.van-der-wielen@gsk.com


Keywords: MenACWY-TT, MenC-CRM197, persistence, SBA, 1-year post-booster follow-up
Background and aims: This study evaluated antibody persistence 12 months after booster vaccination with a meningococcal serogroups A, C, W-135, Y conjugate vaccine (MenACWY-TT, GlaxoSmithKline Vaccines) compared to a meningococcal serogroup C conjugate vaccine (MenC-CRM197, Wyeth LLC), in healthy children.

Methods: In this phase III, open-label, controlled, multi-centre study in Finland (NCT00955682), children previously randomised (3:1) and primed with a single dose of MenACWY-TT or MenC-CRM197 at age 12–23 months (NCT00474266) received a booster dose of the same vaccines 48 months post- priming. Immunogenicity was evaluated at month (M) 60 (12 months post-booster) with serum bactericidal antibody assays using rabbit (rSBA; cut-off 1:8) and human (hSBA; cut-off 1:4) complement. Vaccine- related serious adverse events (SAEs) were recorded until M60.

Results: Of 293 boosted children, 286 returned at M60, with 277 included in the according-to-protocol cohort for persistence at M60 (MenACWY-TT: N=231; MenC-CRM197: N=46). At M60, all MenACWY-TT recipients retained rSBA titres =1:8 (except for MenC, 97.4%) and hSBA titres =1:4 (except for MenA, 95.5%) (Table). hSBA geometric mean antibody titres (GMTs) at M60 declined compared to M49 (1 month post-booster), but were higher than after primary vaccination. MenC seropositivity rates and GMTs (rSBA, hSBA) were comparable between groups. No vaccine-related SAEs were reported.

Conclusion: Antibodies evaluated by rSBA and hSBA assays persisted for each serogroup in >95% of children 12 months after MenACWY-TT booster vaccination. These data indicate that additional booster doses of MenACWY-TT could extend the duration of vaccine-induced protection.

Funding: GlaxoSmithKline Biologicals SA

 Table: Percentage of children with rSBA titres =1:8 and hSBA titres =1:4 and corresponding GMTs (ATP cohort for persistence at M60)
 AntibodyGroup
rSBA
 
hSBA
N % =1:8
(95% CI)
 GMT
(95% CI)
N % =1:4 GMT
(95% CI)
 GMT
(95% CI)
MenA
 MenACWY-TT 231100
(98.4-100)
978.9
(860.2-1114.0)

221
95.5
(91.8-97.8)
88.0
(73.6-105.1)
MenC
 
 MenACWY-TT 23197.4
(94.4-99.0)
226.4
(98.3-100)
228100
(98.4-100)
1342.3
(1134.6-1588.1)
 MenC-CRM197 46 97.8
(88.5-99.9)
320.9
(201.1-512.2)
33100
(89.4-100)
931.1
(572.8-1513.4)
MenW-135 MenACWY-TT 231100
(98.4-100)
1390.7
(1203.2-1607.3)
218
100
(98.3-100)
2196.6
(1955.7-2467.2)
MenY MenACWY-TT 231100
(98.4-100)
1071.1
(924.9-1240.5)
206 100
(98.2-100)
1110.8
(987.5-1249.6)

GMT = geometric mean antibody titre; ATP = according-to-protocol; M60 = Month 60, 12 months post-booster; N = number of subjects with available results; 95% CI = 95% confidence interval; rSBA assay performed at Public Health England; hSBA assay at GlaxoSmithKline Vaccines

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V48 - Oral Presentation, day twoSafety, Tolerability, and Immunogenicity of an Investigational Meningococcal Serogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents When Administered in Regimens of 2 or 3 Doses in Healthy Subjects aged 11 to 18 Years

Timo Vesikari, MD, PhD1 Javier Diez-Domingo, MD, PhD,2 Lars Ostergaard, MD, PhD3, Johannes Beeslaar, MD4, Joseph Eiden, MD, PhD4, Qin Jiang, PhD5, Kathrin U. Jansen, PhD4, Thomas R. Jones, PhD4, Laura J. York, PhD4, and John L. Perez, MD4

1University of Tampere Medical School, Tampere, Finland; 2Área de Investigación en Vacunas, Centro Superior de Investigación en Salud Pública (CSISP), Universidad Católica de Valencia, Valencia, Spain; 3Arhus Universitetshospital, Skejby, Arhus, Denmark; 4Pfizer Vaccine Research, Collegeville, PA, USA

Email: timo.vesikari@uta.fi


Keywords: Vaccine, Meningitis B, rLP2086, fHBP, adolescents

Background: Neisseria meningitidis serogroup B (MnB) is a major cause of invasive disease in infants, adolescents, and young adults. A conserved, surface-exposed lipoprotein, LP2086 (a factor H binding protein [fHBP]), is a promising MnB vaccine target. Safety, tolerability, and immunogenicity of an investigational bivalent, recombinant vaccine (rLP2086) were studied in healthy adolescents 11–18 years of age using 5 dose regimens comprising 2 or 3 vaccinations (Table). Each 120-µg dose contained 60 µg of LP2086 from 1 subfamily A and 1 subfamily B variant.

Methods: All subjects in this phase 2, randomised, placebo-controlled, single-blind study attended vaccination visits at months 0, 1, 2 and 6. For blinding, a saline control was given when vaccine was not scheduled. Serum bactericidal assays using human complement (hSBA) were performed with 4 MnB test strains expressing fHBP variants A22, A56, B24 and B44, all of which are different from the variants in the vaccine. Unsolicited adverse events (AE), solicited local and systemic reactions, and antipyretic use were assessed.

Results: 1 month after the last vaccine dose, 86–99% subjects (after 3 doses; P<0.001) and 69–100% of subjects (after 2 doses) had hSBA titre =8 to each MnB test strain. After study dose 1, 19–27% (1.1–4.3% severe) and 23–27% (0.0–1.0% severe) of rLP2086 recipients experienced redness and swelling, respectively, by group. Injection site pain was the most common local reaction after study dose 1 (7.6–13.1% severe). Fever =38ºC after the first study dose of rLP2086 was experienced in 3.3–6.5% by group compared to 2.1% in saline recipients. Local and systemic reactions were generally more frequent after dose 1 than after subsequent doses. 43 of 1712 subjects (2.5%) reported 51 serious AEs; 2 cases were considered related (1 case of vertigo, chills and headache and 1 case of fever and vomiting). No deaths were reported.

Conclusions: Bivalent rLP2086 had an acceptable safety profile. All 5 dosing regimens yielded hSBA titre =8 against all 4 test strains in a high proportion of subjects.

Table: Statistical Analysis on Proportion of Evaluable Study Subjects Achieving hSBA Titer =8 for Each Primary Strain 1 Month After Last Dose of Bivalent rLP2086 – Evaluable Immunogenicity Population

Group 1Group 2
 Group 3  Group 4
 Group 5
  (0,1,6 mo) (0,2,6 mo)  (0,6 mo)   (0,2 mo)
 (2,6 mo)
Strain [variant] n†/N‡
%§(95% CI)¶
 n†/N‡ %§(95% CI)¶ n†/N‡ %§(95% CI)¶ n†/N‡ %§(95% CI)¶ n†/N‡%§(95% CI)¶ 
PMB80
[A22]
330/3
60
91.7¶
(88.3,94.3)
339/
357
95.0¶
(92.1,97.0)
345/
369
93.5¶
(90.5,95.8)
216/
238
90.8¶
(86.3,94.1)
102/
111
 91.9
(85.2, 96.2)
PMB2001
[A56]
360/3
62
99.4¶
(98.0,99.9)
355/
359
 98.9¶
(97.2,99.7)
364/
370
 98.4¶
(96.5,99.4)
240/
240
 100.0¶
(98.5,100.0)
112/
113
 99.1¶
(95.2, 100.0)
PMB2948
[B24]
315/
354
89.0¶
(85.2,92.0)
313/
354
 88.4¶
(84.6,91.6)
291/
359
81.1¶
(76.6,85.0)
173/
237
73.0
(66.9, 78.5)
76/
110
 69.1¶
(59.6, 77.7)
PMB2707
[B44]
315/
356
 88.5¶
(84.7,91.6)
303/
352
86.1¶
(82.0,89.5)
276/
356
77.5¶
(72.2,82.3)
164/
234
70.1¶
(63.8,75.9)
81/
111
73.0¶
(63.7, 81.0)

*Lower limit of quantification for all strains=8 †Number of subjects with hSBA titer =8. ‡Number of subjects with valid hSBA titre. §P<0.001 using one-sided exact test based on binomial distribution; values<0.0125 are considered significant. ¶ Exact 2-sided confidence interval (Clopper and Pearson) based upon the observed proportion of subjects.

The higher proportions against some test strains after 3 doses compared with 2 doses indicate that 3 doses may provide the broadest protection against diverse MnB clinical strains. Global phase 3 clinical trials are underway with the bivalent rLP2086 vaccine.

Acknowledgments: This study was sponsored by Pfizer Inc. Editorial/medical writing support was provided by Nicole Gudleski, PhD, of Complete Healthcare Communications, Inc., and was funded by Pfizer Inc.

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V49Sensitivity Analysis of a Model Used to Predict the Cost Effectiveness of Bexsero for Immunisation against Meningococcal Disease in the United Kingdom

Jasper Huels1, Jim Wassil2, Lars Bonefeld3, Phil Watson3

1Novartis Vaccines & Diagnostics AG, Basel, Switzerland; 2Novartis Vaccines, Cambridge, USA; 3Novartis Vaccines & Diagnostics Limited, Frimley, UK

Email: phil.watson@novartis.com

Keywords: JCVI, Meningococcal, cost-effectiveness, sensitivity, Bexsero

Background and aims: Neisseria meningitidis remains a major cause of bacterial meningitis and sepsis in the UK. Bexsero, a multicomponent vaccine approved by EMA for individuals >2 months, is the only licensed MenB vaccine. In July 2013, the Joint Committee on Vaccination and Immunisation (JCVI) concluded that routine infant or toddler immunisation using Bexsero is highly unlikely to be cost effective at any vaccine price, and that adolescent immunisation is highly unlikely to be cost effective if the vaccine has little or no impact on meningococcal carriage. These conclusions contradict recently published, peer-reviewed findings*. To demonstrate the impact of changing key inputs on cost effectiveness, we repeated the cost effectiveness sensitivity analysis and examined the effect of changing input parameters, in particular when multiple parameters were adjusted simultaneously.

Methods: A published mathematical and economic model estimated the impact of introducing Bexsero and determined the cost effectiveness of several vaccination strategies. Published input parameters from Christensen et al were used as a base case and varied 40% lower to represent the likely JCVI input parameters.

Results: Assuming 3.5% discounting of costs and benefits and a QALY threshold of £20,000, infant and adolescent Bexsero vaccination programmes would be cost effective with the published input parameters. Using 40% lower estimates of key input parameters, infant Bexsero programs were not cost effective at any vaccine price and adolescent programs only at a low price. Disease incidence, vaccine strain coverage, and frequency and quality of life impact of long-term sequelae among IMD survivors had the greatest impact on cost effectiveness. Adjustments to individual input assumptions had no substantive effect on cost effectiveness while maintaining low estimates for remaining parameters. Modest adjustments to two input parameters together had compounded effects and cost effectiveness markedly improved. Simultaneous adjustments to three key input assumptions, using plausible evidence-based data, had a profound effect.
Conclusion: The model used in this analysis is similar to JCVI’s, showing similar potential for cost effectiveness and sensitivity to changing inputs. Consistently populating this model using conservative parameters for all key inputs leads to Bexsero not being cost effective. Maintaining these low assumptions and increasing key parameters one at a time, leads to a similar conclusion. If the model is populated with evidence-based data which reflects the devastating impact and cyclical epidemiology of MenB disease, UK-wide immunisation of infants and adolescents with Bexsero could be cost-effective.

*Reference: Christensen H, Hickman M, Edmunds JW, Trotter C. Introducing vaccination against serogroup B meningococcal disease: An economic and mathematical modeling study of potential impact. Vaccine. 2013;31;2638-2646.

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V50Meningococcal vaccination in immunodeficiency – is a targeted approach sufficient?

Jo Anne Welsch, E. David McIntosh, William Egan, Theodore Tsai

Novartis Vaccines and Diagnostics, Cambridge MA, USA

Email: joanne.welsch@novartis.com


Keywords: meningococcal disease; immunodeficiency; meningococcal vaccine; serogroup B; complement deficiency

Objective: The UK JCVI recommends meningococcal ACWY conjugate and meningococcal B vaccines for children and adults with immunodeficiency, such as asplenia, splenic dysfunction or complement deficiency – conditions that may be inherited as well as acquired. Immunodeficiency is associated with increased risk for meningococcal disease. A hallmark of inherited deficiencies in complement pathway proteins is an increased risk for and repeated episodes of meningococcal disease. Treatment with the monoclonal antibody eculizumab, a targeted complement inhibitor (to the C5 complement protein), and illnesses such as membranous glomerulonephritis, also are associated with the development of invasive meningococcal disease.

Methods: We reviewed literature on meningococcal disease and immunodeficiency.

Results: Complement deficiency is difficult to detect and may have as its first manifestation an episode of invasive meningococcal disease caused by any serogroup, including those rarely seen in healthy individuals. Additionally, patients with acquired complement disorders, including patients treated with eculizumab, are susceptible to meningococcal infections. Although these individuals are recommended to receive meningococcal vaccine, the effectiveness of vaccination has not been demonstrated clinically and protection against disease may, to some degree, be limited by the complement deficiency.

Discussion: Protecting individuals with immunodeficiency indirectly, by herd effects achieved through universal and catch up vaccination programmes may be the most efficient means to protect these cohorts, as well as infants who cannot be vaccinated early enough in life to achieve direct protection. Further investigation is warranted into cross coverage by proteins contained in the multicomponent protein-based meningococcal B vaccine that may provide an opportunity to cover non-B serogroups including rarely occurring serogroups that threaten cohorts with primary immunodeficiencies.

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V51Novel approach towards the development of vaccine candidates against pneumococcal meningitis

Marie Yang1, Hansjuerg Engel2, Chrispin Chaguza1, Jen Corninck1, Lucy Hathaway2, Dean Everett1, Aras Kadioglu1

1Institute of Global Health, Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; 2Institute for Infectious Diseases, University of Bern, Bern, Switzerland

Keywords: pneumococcal meningitis, vaccine candidates, virulence factors

Streptococcus pneumonia
e is the most common cause of bacterial meningitis in children, the elderly and immunocompromised individuals. The propensity of pneumococci to progress to the brain and cause meningitis remains highly enigmatic, though current literature strongly suggests that pneumococcal meningitis is preceded by nasopharyngeal colonisation. This project is based on the rationale that virulence factors preferentially expressed during meningitis could serve as potential targets for vaccine development and therapeutic interventions.

The objectives of the study are three-fold: 1) to identify meningitis-specific virulence factors by analysing the differential gene expression of pneumococci in the cerebrospinal fluid relative to the blood of human diseased patients, 2) to elucidate the functional role of these virulence genes in the pathogenesis of pneumococcal meningitis by creating a series of clinical strain deletion mutants, and 3) to develop an in vivo mouse model of meningitis that closely mimics the human situation in order to dissect out immune responses and test the protective immunogenicity of the virulence gene products.

Here, we present a detailed outline of our study plan supported by promising preliminary results, and we discuss the implications of these results for the design of the subsequent stages of the study.

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