Man versus Microbe---who gets pneumococcal disease and why?

Advanced age is associated with enhanced susceptibility to severe invasive pneumococcal disease. This is exemplified by the fact that respiratory tract infections are the 4th leading cause of death for the elderly worldwide. One reason for this is the chronic low-grade inflammation that develops with advanced age (i.e. inflamm-aging). Inflamm-aging results in the expression of host-proteins co-opted by Streptococcus pneumoniae for lung cell adhesion and tissue invasion. Most recently, we have determined that inflamm-aging also contributes to alveolar macrophage dysfunction; specifically the inability of aged macrophages to respond to bacteria with a robust proinflammatory cytokines response. We tested the hypothesis that chronic inflamm-aging induces production of A20, a homeostatic suppressor of the NFκB and MAPK signaling cascades, and this is responsible for the muted cytokine response by alveolar macrophages. Comparison of tissues from young, mature, and aged C57BL/6 mice indicated that A20 was strongly elevated in lungs and alveolar macrophages from aged mice. Following co-incubation of macrophages with S. pneumoniae, TRAF6 polyubiquitination, the target of A20, was diminished in alveolar macrophages isolated from aged versus young mice. A20 production was inducible in macrophages by overnight incubation with TNFα but not IL-6; with TNFα treated macrophages having poor IL-6 production following their exposure to S. pneumoniae. Retrovirus-induced expression of A20 in macrophages resulted in their diminished production of IL-6 following their exposure to S. pneumoniae but had no effect on levels of phagocytosis. We conclude that elevated A20 due to TNFα contributes to poor macrophage function in the elderly with pneumonia.


Background and Aims:
We report the discovery of a novel cause of primary immunodeficiency, Activated PI3 Kinase Delta Syndrome, or APDS, characterised by susceptibility to Streptococcus pneumoniae. Methods: Exome sequencing of a cohort of patients with primary immunodeficiency has identified a recurrent missense mutation in the gene encoding the p110delta isoform of PI3 kinase in a number of families that was absent from over 3,000 healthy genomes -p110delta.E1021K. We show that this mutation arises spontaneously and is inherited in an autosomal dominant fashion, causing activation of this kinase leading to increased generation of the second messenger PIP3. Results: APDS patients are clinically characterised by extreme susceptibility to infection, most notably with encapsulated organisms including S. pneumoniae and Haemophilus influenzae. They suffer from recurrent sino-otopulmonary disease culminating in progressive lung damage more severe than that seen in cystic fibrosis sufferers. Subjects often die before the age of 40. They have increased circulating IgM and reduced IgG2, and a marked expansion of T cells with an exhausted phenotype. Their T cells are susceptible to activation induced cell death and have disordered cytokine secretion in response to stimulation. Conclusion: APDS is a novel cause of primary immunodeficiency that is typified by recurrent infection with S. pneumoniae. Discovery of this disease has coincided with inhibitors of p110delta being tested in phase III clinical trials for leukaemia. Mutant p110delta remains fully amenable to inhibition with small molecule inhibitors, thus these compounds may represent a viable treatment strategy for individuals with APDS.

MATHEMATICALLY MODELLING OF EARLY INTERACTIONS OF STREPTOCOCCUS PNEUMONIAE WITH THE HOST DURING THE DEVELOPMENT OF PNEUMONIA
E. Camberlein 1 , J. Cohen1, R. Jose 1 , C. Hyams 1 , R. Callard 2 , S. Chimalapati 1 , J. Yuste 1 , J. Brown 1 1 Medicine, UCL, London, United Kingdom; 2 Institute of Child Health, UCL, London, United Kingdom Background and Aims: Alveolar macrophages have an important role for controlling invading Streptococcus pneumoniae, thereby dictating whether pneumonia develops. However alveolar macrophages are only one component of lung host defences, and there are few data on bacterial factors that may affect early lung clearance. We have now investigated the effects on early lung clearance of alveolar macrophage dependent and independent immunity, S. pneumoniae replication rate, and the capsule. Methods: Mice with or without alveolar macrophages depletion were inoculated with 5 x 10 6 colony forming units (CFU) of TIGR4 wild-type, non-replicating (ΔpabB) and unencapsulated S. pneumoniae. Bronchoalveolar lavage fluid (BAL) were plated to obtain bacterial CFU up to 4 hours post-inoculation. These data were analysed by mathematical modelling to calculate t1/2s for alveolar macrophage dependent and independent clearance, and the S. pneumoniae replication rate. Results: Log 10 CFU bacterial clearance was linear for the first 2 hours post-infection. Calculated half-lives for alveolar macrophage dependent and independent bacterial clearance and the S. pneumoniae replication rate are shown in the Half-life of AM-independent clearance (mins) 24 14

Conclusion:
The results show (a) surprisingly rapid S. pneumoniae replication during early lung infection; (b) that the capsule inhibits both alveolar macrophage dependent and independent clearance; and (c) for clearance of encapsulated bacteria alveolar macrophage independent dominated alveolar-dependent clearance during early lung infection. These data will help explain why there is an increased susceptibility to pneumonia for some at risk patient populations. Background and Aims: Inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) are a result of an inappropriate immune response. Therefore, the main purpose of the medical treatment is to moderate the immune response thus reducing disease activity, leading to a theoretically increased risk of invasive pneumococcal infection (IPI). The objective of this study was to examine the impact of IBD on the risk of IPI.

No conflict of interest
Methods: Patients diagnosed with IBD from 1977 to 2013 were identified from the Danish National Patient Register. For each IBD patient, 20 individuals matched according to sex, age, and municipalities were selected from the Danish Civil Registration System. The IBD and control group data were linked with IPI data from the national laboratory surveillance. Using Cox regression with time since onset of IBD/date of matching as underlying time axis we calculated hazard rate ratios (HRRs) for IPI after IBD. Results: Among 83,358 IBD cases we found 316 IPI cases giving an incidence of 38 per 10,000, whereas the controls had an incidence of 26 per 10,000. The HRRs for CD and UC within the first 6 months after IBD diagnosis were high (>3) and then decreased to a constant level which for CD was significantly higher (approximately twofold) than for the controls and for UC non-significantly just above 1. Conclusion: We found an increased risk of IPI infections among patients with IBD, which was most pronounced in the first years after diagnosis but remained increased over time, especially in CD. Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, Netherlands Background and Aims: Young children and elderly are particularly vulnerable for invasive pneumococcal diseases. The aim of this study was to determine whether crowding factors and individual contact patterns are associated with pneumococcal antibody concentrations in young children and elderly. Methods: In 2006-2007 a population-based serum bank was set up in the Netherlands for individuals aged 0 to 79 years of age to obtain insight into seroepidemiology, in particular, of vaccine preventable diseases. Blood samples were analyzed using fluorescent-bead based multiplex assay to obtain serotype-specific pneumococcal IgG concentrations. Associations between crowding factors, individual contact patterns and pneumococcal IgG antibody concentrations of 13 serotypes were analyzed with generalized estimating equations (GEE) for children aged 2 months to 3 years (n = 642) and elderly of ≥65 years (n = 1174). Results: Young children had significantly higher pneumococcal antibody concentrations in the crude model when they attended a day care center or had contact with other young children. The adjusted model showed higher antibody concentrations when children lived in households consisting of more than 4 persons. Elderly had significantly higher concentrations of antibodies in the crude model when they had contact with 5-19 year olds. In the adjusted model, elderly who reported contact with 20-59 year olds, had lower antibody concentrations. Conclusion: Individual contact patterns and crowding factors are associated with pneumococcal antibody concentrations in young children and elderly in the Netherlands. To our knowledge, this is the first time that GEE analysis is used to analyze data of different pneumococcal serotypes. Activating transcription factor-3 (ATF3) plays a crucial role in regulation of innate immunity. However, how ATF3 regulates innate immunity against Gram-positive bacterial infection remains unknown. Here, we investigated role of ATF3 for host defense upon Streptococcus pneumoniae infection. Pneumococcal infection induced ATF3 significantly high in various cell lines in vitro and many organs in vivo. Surprisingly, pneumolysin (PLY) induced ATF3 via Toll-like receptor 4 (TLR4) signaling. Moreover, pneumococcal infection induced ATF3 resulted in positive stimulation of production cytokines (TNF-α, IL-1β, and IFN-γ). In pneumonia model infection, wild type mice are more resistant than the ATF3 knock-out mice. Thus, ATF3 induced-cytokines might protect the host from pneumococcal protection. Taken together, ATF3 regulates innate immunity positively upon pneumococcus infection via TNF-α, IL-1β, and IFN-γ secretion resulting in clearance of pneumococci. Introduction: IL-17 is a cytokine that promotes the release of anti-microbial factors and neutrophil recruitment. We investigated the role of IL-17 in a murine model of pneumococcal pneumonia. Methods: IL-17RA-/-or wild-type mice were infected via the intra-nasal route with either TIGR4 (serotype 4) or strain SRL1 (serotype 3). Results: TIGR4 produced high-level bacteremia in infected animals whereas the serotype 3 strain SRL1 caused dense consolidative pneumonia and pleural infection, but only invaded the blood stream late in the course of infection. IL-17A was present in the lungs of mice infected with both strains of pneumococci within 6 hours of infection. IL-17RA-/-mice had decreased expression of IL-17A target genes and delayed recruitment of neutrophils to blood and lung following infection with TIGR4 and SRL1. In TIGR4 infection, IL-17RA-/-mice had increased bacteremia and increased mortality compared to wild-type mice. In contrast, IL-17RA-/-mice had decreased mortality compared to ISPPD-9 / pneumonia 2014 Mar 9-13;3:  wild-type when infected with SRL1. In vitro studies confirmed that neutrophils effectively phagocytosed and killed TIGR4 whereas SRL1 was highly resistant to phagocytosis. When mice were treated with a neutrophil depleting monoclonal antibody, neutrophil recruitment was delayed and a similar trend in mortality was seen. Conclusion: IL-17 signaling has a variable effect on outcome in this model, which depends on pneumococcal strain. IL-17 recruitment of neutrophils is crucial to host defence against an invasive serotype 4 pneumococcal strain but worsens outcome in infection with a heavily encapsulated serotype 3 organism. Thus, Th17 immunity may not always be protective in pneumococcal infection. Background and Aims: Excessive oxidative stress is a pathological feature of chronic lung diseases including COPD. As a large proportion of COPD patients are chronically colonized with respiratory pathogens, including Streptococcus pneumoniae (the pneumococcus), our aim is to identify the cellular and molecular mechanisms that underlie susceptibility to pneumococcal infections in the airways under high oxidant burden. Methods: An experimental mouse model was used in which C57BL/6 mice were intranasally infected with Stretococcus pneumoniae EF3030 and coinfected with influenza A virus (IAV) to trigger pneumococcal disease. To model the increased levels of extracellular superoxide radicals detected in COPD airways, mice lacking SOD3 (superoxide dismutase 3) were used. Results: Infection of SOD3-/-mice with pneumococci resulted in substantial weight loss (>15%) over a seven day period compared with infected wildtype mice. The pneumococcal load in the lung and nasal tissues was also increased in SOD3-/-mice compared to wildtype mice. Co-infection wth IAV resulted in dramatically increased pneumococcal numbers in the lungs and the nose, with more pneumococci in co-infected SOD3-/-mice compared with co-infected wildtype mice. Flow cytometry analysis of bronchoalveolar lavagefound that co-infected SOD3-/mice had two-fold higher numbers of recruited inflammatory cells, in particular neutrophils. SOD3-/-mice show increased susceptibility to infection with pneumococci, suggesting that pathogenesis of S. pneumoniae may be altered under oxidative stress. We are currently exploring differences in pneumococcal and host gene expression that may explain these changes insusceptibility to pneumococcal infection. Background and aims: Streptococcus pneumoniae causes meningitis with high mortality rate and 50% of survivors present sequelae including learning impairment, deafness, mental retardation and hydrocephalus. We have previously identified several putative bacterial adhesin and their respective receptors on epithelial cells in the host that are proteins known to function in brain development or affect neural cell function. We hypothesize that the very same adhesins are involved in the interaction of the bacteria with neural cells and may affect neural cell function and survival. Method: To study alterations in the functional state of the U251 cells following infection with the pneumococci we tested DNA Topoisomerase I (Topo I) activity. Topo I is an essential nuclear enzyme that participates in all DNA transaction processes and is important for gene expression. Results: We demonstrate a significant reduction in pneumococcal adhesion to glioblastoma cells (U251) in the presence of the recombinant adhesins (rPtsA and rGtS). Moreover, bacteria lacking the putative adhesins demonstrate reduced adhesion to U251 cells in comparison to the wild type bacteria. Infection of U251 with S. pneumonia inhibited Topo I activity significantly, albeit the mutated bacteria inhibited Topo I activity to a significantly lesser extent then the wild type bacterial strain. Conclusion: The bacterial adhesins previously found to mediate bacterial epithelial cells interactions were currently found to mediate adhesion of pneumococci to glial cells and alter Topo I activity in these cells. Thus, these adhesins may be involved in pneumococcal meningitis development.

Poster Abstracts
No conflict of interest ISPPD-9 / pneumonia 2014 Mar 9-13;3:1-286 A mutation in the gene encoding Bruton's tyrosine kinase enzyme leads to impaired B cell receptor signaling in Xid mouse. Xid mouse response to T cell independent type 2 (TI-2) antigens such pneumococcal vaccines is severely impaired. The B cell molecule, TACI is critical in the development of antibody response against TI-2 antigens. Here, we found that Xid B cells had significantly reduced levels of TACI as compared to wild type mouse. More importantly, Xid B cells did not secrete immunoglobulins after stimulation with BAFF or APRIL, the two ligands of TACI. Analysis of the signaling cascade induced by BAFF and APRIL showed that while canonical NFkB pathway was blocked, non-canonical NFkB pathway was intact in Xid B cells. These data suggested that reduced TACI expression may be responsible for impaired Ig secretion because TACI mediates signals that activate the canonical NFkB pathway. Since Xid mouse responds poorly to polysaccharide vaccines, it is likely that reduced TACI expression plays an important role in this outcome. The dependence of BAFF and APRIL functions on the expression levels of TACI was further demonstrated in experiments where Xid B cells were pre-stimulated with CpG. Incubation of Xid B cells with CpG increased the expression of TACI and rendered them susceptible to BAFF or APRIL induced Ig secretion. Moreover, BAFF and APRIL induced canonical NFkB pathway activation was restored in CpG pre-stimulated Xid B cells. Finally, immunization of Xid mouse with a prototype TI-2, NP-Ficoll led to the generation of antibodies against NP.

PNEUMOCOCCAL LIPOPROTEINS MODULATE INNATE EFFECTOR FUNCTIONS OF ALVEOLAR MACROPHAGES
J. Bhushan 1 , P. Arya 1 , D. Sehgal 1 1 Molecular Immunology Laboratory, National Institute of Immunology, NEW DELHI, India Lipoproteins form a significant proportion of surface exposed proteins of Streptococcus pneumoniae. Given their surface localisation, and importance in pneumococcal fitness and virulence we focussed our study on functional characterization of pneumococcal lipoproteins. A pneumococcal strain deficient in lipoprotein diacylglyceryl transferase (lgt), a key enzyme in the lipoprotein biogenesis, was constructed by inframe gene replacement mutagenesis. Deletion of lgt abrogated adhesion to the murine alveolar macrophage cell line MH-S by >50% suggesting the involvement of lipoproteins in pneumococcal adherence. Further the effect of Triton X-114 extracted lipoprotein fraction on innate effector functions of the macrophage was studied. Treatment of MH-S cells with the extracted lipoproteins resulted in 1.5 fold higher reactive oxygen species (ROS) production compared to untreated sample as determined by flow cytometry. A significant increase in nitric oxide (NO) production was observed 24 hours following stimulation of MH-S cells with pneumococcal lipoproteins. The NO production diminished in the presence of inducible nitric oxide synthase (iNOS) inhibitor L-NAME suggesting the involvement of iNOS in pneumococcal lipoprotein mediated NO production. Our data suggests that pneumococcal lipoproteins have important implication in adhesion and modulation of innate effector functions of alveolar macrophage. Background and Aims: Naturally acquired adaptive immunity to Streptococcus pneumoniae is partially mediated by antibody, but whether the target antigens are the capsule or sub-capsular protein antigens is not known. We have used human intravenous immunoglobulin preparation (IVIG) to assess the comparative importance of anti-capsule or anti-protein IgG for immunity to S. pneumoniae. Methods: The effects of IVIG were assessed using IgG binding, growth inhibition, agglutination, and phagocytosis assays, and in mouse infection models. The relative contribution of anti-capsule or anti-protein IgG in these assays was assessed using encapsulated and unencapsulated strains, pronase degradation of surface proteins, and IVIG depleted of anti-capsular antibody. Results: Incubation of live S. pneumoniae with IVIG resulted in surface deposition of IgG on the bacteria, increased phagocytosis, growth inhibition, and bacterial agglutination. Results with unencapsulated mutants from 3 different ISPPD-9 / pneumonia 2014 Mar 9-13;3:1-286 serotypes showed increased IgG binding, phagocytosis, growth impairment and bacterial agglutination compared to encapsulated strains, indicating these phenotypes are independent of capsule expression. IgG binding to a capsular serotype 4 strain was not affected by depletion of anti-capsular serotype 4 IgG from IVIG, but was reduced on pronase treated S. pneumoniae. Passive treatment with IVIG preparations protected mice against septicaemia and pneumonia caused by a serotype 4 strain even after depletion of anti-capsular serotype 4 IgG. Conclusions: These data suggest that naturally acquired adaptive immunity to S. pneumoniae is largely dependent on IgG that recognises protein antigens rather than the capsule, which has important implications for preventative strategies versus S. pneumoniae infections.

ISPPD-0386
Man versus Microbe---who gets pneumococcal disease and why? Background and Aim: Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases. The knowledge of serotypes actually responsible for disease is essential for effective disease management and vaccine implementation. Hence, we have assessed the pneumococcus serotypes accountable for disease and their virulence pattern in comparison to carrier strains. Methods: Clinical strains (n = 20) were obtained from (OPD, PGIMER) sputum, nasopharyngeal and blood samples of patient's suffering from respiratory diseases. Carrier strains were already collected from community based surveillance. The isolates were serotyped by multiplex PCR and further confirmed by sequencing. The virulence was determined in terms of adherence pattern and cytopathic effect in cell lines. Results: Clinical strains from nasopharyngeal and sputum samples possessed mostly 19F serotype whereas isolates from blood samples belonged to serotypes 1, 7C/B, 22F/A, 10A and 23F. In our previous study, we have shown the presence of serotypes 7C/B (2.9%), 22F/A (1.0%), 10A (4.8%) and 23F (3.8%) in asymptomatic healthy children along with 6A/B/C, 11A/D, 15B/C, 10F/C, 34, 10A being the most prevalent. The adherence capability of clinical strains was almost 2 fold higher as compare to carrier strain with blood isolates being most virulent. The carrier strain or sputum isolates did not show any significant cytotoxic effects on human cell lines. Conclusions: We found that disease causing serotypes parallelly exist in asymptomatically healthy children and serotype variation among strains reflected in their virulence capabilities. We are further characterizing the pneumococcal surface proteins which could explain the differential adherence properties of carrier and clinical strains.

Institute of Child Health, University College London, London, United Kingdom
Background and Aims: With the increasing number of vaccines being delivered in infancy, clinical trials of new vaccines designed for the infant immunisation programme require the immune response to both the investigational and concomitant vaccines to be measured. With serum volumes limited in infant trials, samples may require recurrent freeze thaw cycles during the testing phase as assays are prioritised. Historical concerns about the deterioration in the quality of sub-optimally stored serum and samples undergoing repeated freeze thaw cycles has led to limits being imposed. We sought to test the impact of repeated freezing and thawing of serum on pneumococcal IgG quantitation and opsonophagocytic killing to establish whether such concerns are justified. Methods: A set of 12 pneumococcal Quality Control samples stored at -80 o C were thawed and frozen 20 times. Sera were assayed after 0, 1,2,4,8,12,16 and 20 freeze/thaw cycles for serotype specific IgG (13 serotypes by ELISA) and functional killing ability (4 serotypes by an Opsonophagocytic Assay). Results: Analysis of the IgG values for the 13 serotypes revealed remarkable stability. The mean ratio of IgG after 20 FT cycles compared to the starting value ranged from 0.97 (18C) -1.1 (6A). For the OPA, results after 20 cycles were within a 3-fold titer compared to the original for all 11 sera and 4 serotypes tested. A 3 fold variation is within the acceptance criteria of the assay for a given serum. Conclusion: Immune responses specific for the pneumococcus appear robust and able to withstand repeated freeze thaw cycles.

PERSISTENCE OF ANTIBODY LEVELS FOLLOWING ROUTINE INFANT IMMUNIZATION WITH THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE
L. Grant 1 , E. Millar 1 , P. Burbidge 2 , E. Pearce 2 , R. Weatherholtz 1 , R. Reid 1 , M. Santosham 1 , D. Goldblatt 2 , K. O'Brien 1 1 International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA; 2 Institute Child Health, University College London, London, United Kingdom Background and Aims: Determining the longevity of serotype-specific antibodies induced by pneumococcal conjugate vaccines (PCVs) is important for understanding the potential long-term effectiveness of routine PCV use. Methods: An observational, prospective, longitudinal study of nasopharyngeal carriage consisting of monthly visits for 6 months was conducted among American Indian households in the Southwest United States from 2006-2008 to evaluate the impact of long-term PCV7 routine use. Unimmunized children were age-matched to those immunized with PCV7 at least four years prior (ratio 1:4). Blood collected at the final study visit was analyzed by ELISA for PCV7 serotype IgG. Geometric mean concentrations (GMCs) and the odds of having ≥ 0.35 μg/mL of serotype specific antibody were compared according to immunization status using a matched regression approach. Results: Eight unimmunized children (mean age: 7.9 years) were age-matched to 28 immunized children at the time of serum collection (mean age: 7.9 years). Serotype specific GMCs were comparable for six of the seven serotypes between immunized and unimmunized subjects; the serotype 14 GMC was significantly lower for unimmunized (immunized: 0.7 vs. unimmunized: 0.2; p = 0.02). There was no difference in the odds of the immunized and unimmunized children reaching or exceeding 0.35 μg/mL, the vaccine licensure correlate threshold. Conclusions: Four years following routine infant immunization, serotype-specific IgG levels were similar to those of age-matched unimmunized children. Natural exposure to pneumococcus may be critical in inducing or maintaining persistent antibody and thus the elimination of circulating pneumococci by PCV may have profound effects on longterm immunity. Streptococcus pneumoniae remains a leading cause of serious illness, including bacteremia, meningitis, and pneumonia among children. In Japan, 7-valent pneumococcal conjugate vaccine (PCV7) was first approved in 2009, and was introduced to routine immunization program for infant in 2013. Thus, significant decrease of invasive pneumococcal diseases (IPD) has been expected, however, the serosurveillance data before the introduction of PCV7 in children is lacking, and it is difficult to evaluate the impact of PCV in Japan. In this study, serosurveillance was conducted by measuring serotype-specific antibody and opsonophagocytic index (OI) of sera collected from children before the introduction of PCV7 in Japan. Serotype-specific antibodies in each age-group were enough high as the infection prevention threshold (0.2 µg/ml) against most of serotypes, however, OI in each age-group were separated into 2 groups (high/low OI groups). High groups contained OI more than 8, which is comparable to the infection prevention threshold of serotype-specific antibody concentration, and low groups contained relatively low OI (OI<8). This trend was rather evident in samples of 1-and 3-year-old children. Amount of serotype-specific antibodies did not correlate with OI, and older children naturally possess higher OI than younger children without PCV vaccination. This serosurveillance suggested that pneumococcal vaccination program, which has been launched in Japan, is reasonable and strongly required in children, mainly for low OI group, to induce protective immunity against pneumococcal infections. Now, serosurveillance after the introduction of PCV in Japan is evaluating its impact in terms of immune response for the protection against IPD.

No conflict of interest
Background and Aims: To investigate the impact of IgM on functional immune responses to vaccine-related serotypes 6A, 6C and 19A as well as vaccine serotype 6B and 19F in children after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). Methods: Eighteen immune sera were obtained from children aged 12-23 months after complete immunization (3+1) with PCV7. The serum opsonic indices (OI) to serotypes 6B and 19F and serotypes 6A, 6C and 19A were studied by opsonophagocytic killing assay (OPA). The role of IgM antibodies on opsonic indices of immunized sera were determined by measuring OI after depletion of IgM antibody of immune sera. The specificity of antibodies (IgG or IgM) to each serotypes was studied with competitive OPA to 6B, 6A, 6C, 19F and 19A pneumococci with 6B polysaccharide or 19F polysaccharide, respectively. Results: Compared to control sera, OI of IgM-depleted immune sera to 6B and 19F were decreased in 94% or 71% of subjects, respectively. OI to 6A, 6C, and 19A were decreased in 92%, 100% or 91% of sera after IgM depletion, too. In competitive OPA, free 6B or 19F polysaccharide completely inhibited the immune protection to serotypes 6A, 6C, and 19A as well as serotypes 6B and 19F in most samples.

Conclusion:
The booster immunization of PCV7 certainly induced cross-protective antibodies to vaccine-related serotypes 6A, 6C, and 19A with both IgG and IgM isotypes. The IgM antibodies in children after complete immunization of PCV was highly contributed to opsonophagocytic activity to vaccine-related serotypes as well as vaccine types. Streptococcus pneumoniae (pneumococcus) infection causes high mortality and morbidity worldwide. Capsular polysaccharides (CPS) are major virulence factors by protecting pneumococci from host. Synthesis of CPS requires energy supplied by ATP hydrolysis, and adenylate kinases (AdKs) constitute a major family of enzymes to regulate cellular ATP level. However, it remains poorly understood whether AdK acts as a virulence factor in pneumococcal diseases. Here we show that AdK from S. pneumoniae (SpAdK) is essential for growth, CPS synthesis and bacterial survival in vivo. Expression of the wild-type adk gene in fucose-inducible strains rescued growth defect. Cellular ATP and CPS levels were increased in proportion to the expression level of adk gene. Taken together, our results support that SpAdK is required for pneumococcal growth and CPS synthesis. We propose that SpAdK is a novel pneumococcal virulence factor. Background and Aims: Immune responses to pneumococcal conjugate vaccine (PCV) have not been fully investigated in children with invasive pneumococcal diseases (IPD). The aim of this study is to investigate the antibody responses to seven-valent PCV (PCV7) in children with IPD Methods: Of fifty-six children diagnosed with IPD between October 2009 and April 2013 in Japan, 17 children were examined for the geometric mean concentration of serotype-specific IgG and the geometric mean titers of opsonization indices (OIs) using paired sera obtained at the onset of IPD and after the last PCV7 dose. Results: The GMCs of serotype-specific IgG for all PCV7 serotypes other than serotype 6B were significantly increased after the last PCV7 dose compared with those at the time of IPD onset (p < 0.01), as were the GMTs of OIs for all PCV7 serotypes. In 14 children with IPD caused by PCV7 serotypes for whom both IgG and OI results were available, the OIs for the infecting serotype were undetectable, although the IgG levels varied between from <0.2 to >5.0 μg/ml at the time of IPD onset. After the last PCV7 dose, the OIs for the infecting serotype were undetectable (OI < 8) for six (43%) of 14 children. Unresponsiveness was found in children with IPD caused by serotype 6B (n = 5) and serotype 23F (n = 1). Conclusion: Immune unresponsiveness after PCV7 was specific for the infecting serotype. Careful monitoring of the OI for the infecting serotype after vaccination with PCV is required in children who experienced IPD.

PROTECTIVE EFFECTS OF ORAL ADMINISTRATION OF LACTOBACILLUS DELBRUECKII SSP: BULGARICUS OLL1073R-1 WITH EXOPOLYSACCHARIDES IN PNEUMOCOCCAL PNEUMONIA IN MICE
S. Nakamura 1 , N. Iwanaga 1 , T. Kajihara 1 , T. Takazono 1 , Y. Imamura 1 , K. Izumikawa 1 , K. Yanagihara 2 , S. Kohno 1 1 Second Department of Internal Medicine, Nagasaki University Hospital, Nagasaki, Japan; 2 Department of Laboratory Medicine, Nagasaki University Hospital, Nagasaki, Japan Background and Aims: Intestinal flora is a major modulator of systemic immunity and oral administration of probiotics has been known to improve inflammatory responses against infectious diseases. Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1) was reported to enhance the effects of the innate immune system and the exopolysaccharides (EPS) produced by 1073R-1 are thought to play an active role in enhancing immunostimulatory effects. We show the protective effect of pre-administration of 1073R-1 and EPS against pneumococcal pneumonia in mice. Methods: 1073R-1 and EPS were orally administered to C57BL/6 mice for 21 days prior to intranasal infection with Streptococcus pneumoniae (strain D39). Results: The mice treated with 1073R-1 and EPS were prone to prolonging the duration of survival and inhibiting