Late effects of total body irradiation on hematopoietic recovery and immune function in rhesus macaques.

While exposure to radiation can be lifesaving in certain settings, it can also potentially result in long-lasting adverse effects, particularly to hematopoietic and immune cells. This study investigated hematopoietic recovery and immune function in rhesus macaques Cross-sectionally (at a single time point) 2 to 5 years after exposure to a single large dose (6.5 to 8.4 Gray) of total body radiation (TBI) derived from linear accelerator-derived photons (2 MeV, 80 cGy/minute) or Cobalt 60-derived gamma irradiation (60 cGy/min). Hematopoietic recovery was assessed through measurement of complete blood counts, lymphocyte subpopulation analysis, and thymus function assessment. Capacity to mount specific antibody responses against rabies, Streptococcus pneumoniae, and tetanus antigens was determined 2 years after TBI. Irradiated macaques showed increased white blood cells, decreased platelets, and decreased frequencies of peripheral blood T cells. Effects of prior radiation on production and export of new T cells by the thymus was dependent on age at the time of analysis, with evidence of interaction with radiation dose for CD8+ T cells. Irradiated and control animals mounted similar mean antibody responses to proteins from tetanus and rabies and to 10 of 11 serotype-specific pneumococcal polysaccharides. However, irradiated animals uniformly failed to make antibodies against polysaccharides from serotype 5 pneumococci, in contrast to the robust responses of non-irradiated controls. Trends toward decreased serum levels of anti-tetanus IgM and slower peak antibody responses to rabies were also observed. Taken together, these data show that dose-related changes in peripheral blood cells and immune responses to both novel and recall antigens can be detected 2 to 5 years after exposure to whole body radiation. Longer term follow-up data on this cohort and independent validation will be helpful to determine whether these changes persist or whether additional changes become evident with increasing time since radiation, particularly as animals begin to develop aging-related changes in immune function.

Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens.

Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS® based data analysis program. The goodness of fit (R2) of the standard curves for five analytes was ≥0.99, LLOQ 0.04-0.15 IU or AU/mL, accuracy 1.9%-23.8% (%E), dilutional linearity slopes 0.93-1.02 and regression coefficients r2 = 0.91-0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5µL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.

Meningococcal Antigen Typing System (MATS)-Based Neisseria meningitidis Serogroup B Coverage Prediction for the MenB-4C Vaccine in the United States.

Neisseria meningitidis is the most common cause of bacterial meningitis in children and young adults worldwide. A 4-component vaccine against N. meningitidis serogroup B (MenB) disease (MenB-4C [Bexsero]; GSK) combining factor H binding protein (fHBP), neisserial heparin binding protein (NHBA), neisserial adhesin A (NadA), and PorA-containing outer membrane vesicles was recently approved for use in the United States and other countries worldwide. Because the public health impact of MenB-4C in the United States is unclear, we used the meningococcal antigen typing system (MATS) to assess the strain coverage in a panel of strains representative of serogroup B (NmB) disease in the United States. MATS data correlate with killing in the human complement serum bactericidal assay (hSBA) and predict the susceptibility of NmB strains to killing in the hSBA, the accepted correlate of protection for MenB-4C vaccine. A panel of 442 NmB United States clinical isolates (collected in 2000 to 2008) whose data were down weighted with respect to the Oregon outbreak was selected from the Active Bacterial Core Surveillance (ABCs; CDC, Atlanta, GA) laboratory. MATS results examined to determine strain coverage were linked to multilocus sequence typing and antigen sequence data. MATS predicted that 91% (95% confidence interval [CI95], 72% to 96%) of the NmB strains causing disease in the United States would be covered by the MenB-4C vaccine, with the estimated coverage ranging from 88% to 97% by year with no detectable temporal trend. More than half of the covered strains could be targeted by two or more antigens. NHBA conferred coverage to 83% (CI95, 45% to 93%) of the strains, followed by factor H-binding protein (fHbp), which conferred coverage to 53% (CI95, 46% to 57%); PorA, which conferred coverage to 5.9%; and NadA, which conferred coverage to 2.5% (CI95, 1.1% to 5.2%). Two major clonal complexes (CC32 and CC41/44) had 99% strain coverage. The most frequent MATS phenotypes (39%) were fHbp and NHBA double positives. MATS predicts over 90% MenB-4C strain coverage in the United States, and the prediction is stable in time and consistent among bacterial genotypes. IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA)-based system that assesses the levels of expression and immune reactivity of the three recombinant MenB-4C antigens and, in conjunction with PorA variable 2 (VR2) sequencing, provides an estimate of the susceptibility of NmB isolates to killing by MenB-4C-induced antibodies. MATS assays or similar antigen phenotype analyses assume importance under conditions in which analyses of vaccine coverage predictions are not feasible with existing strategies, including large efficacy trials or functional antibody screening of an exhaustive strain panel. MATS screening of a panel of NmB U.S. isolates (n = 442) predicts high MenB-4C vaccine coverage in the United States.

Immune Responses in U.S. Military Personnel Who Received Meningococcal Conjugate Vaccine (MenACWY) Concomitantly with Other Vaccines Were Higher than in Personnel Who Received MenACWY Alone.

Immunological responses to vaccination can differ depending on whether the vaccine is given alone or with other vaccines. This study was a retrospective evaluation of the immunogenicity of a tetravalent meningococcal conjugate vaccine for serogroups A, C, W, and Y (MenACWY) administered alone (n = 41) or concomitantly with other vaccines (n = 279) to U.S. military personnel (mean age, 21.6 years) entering the military between 2006 and 2008. Concomitant vaccines included tetanus/diphtheria (Td), inactivated polio vaccine (IPV), hepatitis vaccines, and various influenza vaccines, among others; two vaccine groups excluded Tdap and IPV. Immune responses were evaluated in baseline and postvaccination sera for Neisseria meningitidis serogroups C and Y 1 to 12 months (mean, 4.96 months) following vaccination. Functional antibodies were measured by using a serum bactericidal antibody assay with rabbit complement (rSBA) and by measurement of serogroup-specific immunoglobulin G (IgG) antibodies. The percentage of vaccinees reaching threshold levels (IgG concentration in serum, ≥2 µg/ml; rSBA titer, ≥8) corresponding to an immunologic response was higher postvaccination than at baseline (P < 0.001). Administration of MenACWY along with other vaccines was associated with higher geometric means of IgG concentrations and rSBA titers than those measured 4.60 months after a single dose of MenACWY. In addition, higher percentages of vaccinees reached the immunological threshold (range of odds ratios [ORs], 1.5 to 21.7) and more of them seroconverted (OR range, 1.8 to 4.8) when MenACWY was administered with any other vaccine than when administered alone. Additional prospective randomized clinical trials are needed to confirm the observed differences among groups in the immune response to MenACWY when given concomitantly with other vaccines to U.S. military personnel.

Antibody Persistence 1-5 Years Following Vaccination With MenAfriVac in African Children Vaccinated at 12-23 Months of Age.

BACKGROUND: Following mass vaccination campaigns in the African meningitis belt with group A meningococcal conjugate vaccine, MenAfriVac (PsA-TT), disease due to group A meningococci has nearly disappeared. Antibody persistence in healthy African toddlers was investigated. METHODS: African children vaccinated at 12-23 months of age with PsA-TT were followed for evaluation of antibody persistence up to 5 years after primary vaccination. Antibody persistence was evaluated by measuring group A serum bactericidal antibody (SBA) with rabbit complement and by a group A-specific IgG enzyme-linked immunosorbent assay (ELISA). RESULTS: Group A antibodies measured by SBA and ELISA were shown to decline in the year following vaccination and plateaued at levels significantly above baseline for up to 5 years following primary vaccination. CONCLUSIONS: A single dose of PsA-TT induces long-term sustained levels of group A meningococcal antibodies for up to 5 years after vaccination. CLINICAL TRIALS REGISTRATION: ISRTCN78147026.

Antibody Persistence at 1 and 4 Years Following a Single Dose of MenAfriVac or Quadrivalent Polysaccharide Vaccine in Healthy Subjects Aged 2-29 Years.

BACKGROUND: Mass vaccination campaigns of the population aged 1-29 years with 1 dose of group A meningococcal (MenA) conjugate vaccine (PsA-TT, MenAfriVac) in African meningitis belt countries has resulted in the near-disappearance of MenA. The vaccine was tested in clinical trials in Africa and in India and found to be safe and highly immunogenic compared with the group A component of the licensed quadrivalent polysaccharide vaccine (PsACWY). Antibody persistence in Africa and in India was investigated. METHODS: A total of 900 subjects aged 2-29 years were followed up for 4 years in Senegal, Mali, and The Gambia (study A). A total of 340 subjects aged 2-10 years were followed up for 1 year in India (study B). In study A, subjects were randomized in a 2:1 ratio, and in study B a 1:1 ratio to receive either PsA-TT or PsACWY. Immunogenicity was evaluated by measuring MenA serum bactericidal antibody (SBA) with rabbit complement and by a group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay. RESULTS: In both studies, substantial SBA decay was observed at 6 months postvaccination in both vaccine groups, although more marked in the PsACWY group. At 1 year and 4 years (only for study A) postvaccination, SBA titers were relatively sustained in the PsA-TT group, whereas a slight increasing trend, more pronounced among the youngest, was observed in the participants aged <18 years in the PsACWY groups. The SBA titers were significantly higher in the PsA-TT group than in the PsACWY group at any time point, and the majority of subjects in the PsA-TT group had SBA titers ≥128 and group A-specific IgG concentrations ≥2 µg/mL at any point in time in both the African and Indian study populations. CONCLUSIONS: Four years after vaccination with a single dose of PsA-TT vaccine in Africa, most subjects are considered protected from MenA disease. CLINICAL TRIALS REGISTRATION: PsA-TT-003 (ISRCTN87739946); PsA-TT-003a (ISRCTN46335400).

Human Complement Bactericidal Responses to a Group A Meningococcal Conjugate Vaccine in Africans and Comparison to Responses Measured by 2 Other Group A Immunoassays.

BACKGROUND: PsA-TT (MenAfriVac) is a conjugated polysaccharide vaccine developed to eliminate group A meningococcal disease in Africa. Vaccination of African study participants with 1 dose of PsA-TT led to the production of anti-A polysaccharide antibodies and increased serum bactericidal activity measured using rabbit complement (rSBA). Bactericidal responses measured with human complement (hSBA) are presented here. METHODS: Sera collected before and at 28 days and 1 year after vaccination with either PsA-TT or quadrivalent polysaccharide vaccine (PsACWY) from a random, age-distributed 360-subject subset of the Meningitis Vaccine Project study of PsA-TT in Africans aged 2-29 years were tested for hSBA. Geometric mean titer, fold-rise, and threshold analyses were compared between vaccine groups and age groups. hSBA, rSBA, and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) results were compared and assay correlation and agreement determined. RESULTS: hSBA responses to PsA-TT were substantially higher than those to PsACWY at 28 days and 1 year following immunization, similar to previously reported rSBA and IgG results. The hSBA and IgG ELISA results identified differences between age groups that were not evident by rSBA. The rSBA data indicated sustained high titers 1 year after immunization, whereas hSBA GMTs at 1 year approached 4 in young children. CONCLUSIONS: The high level of protection following PsA-TT immunization campaigns is consistent with the strong hSBA immune responses observed here. Future implementation decisions will likely depend on immunologic data and their long-term correlation with disease and carriage prevention. Expanded immunologic and epidemiologic surveillance may improve the interpretation of differences between these immunoassays.

Neisseria meningitidis Group A IgG1 and IgG2 Subclass Immune Response in African Children Aged 12-23 Months Following Meningococcal Vaccination.

BACKGROUND: A group A meningococcal conjugate vaccine, PsA-TT, was licensed in 2010 and was previously studied in a phase 2 clinical trial to evaluate its safety and immunogenicity in African children 12-23 months of age. METHODS: Subjects received either PsA-TT; meningococcal group A, C, W, Y polysaccharide vaccine (PsACWY); or Haemophilus influenzae type b conjugate vaccine (Hib-TT). Forty weeks following primary vaccination, the 3 groups were further randomized to receive either PsA-TT, one-fifth dose of PsACWY, or Hib-TT. Group A-specific immunoglobulin G (IgG) subclass response was characterized using an enzyme-linked immunosorbent assay. RESULTS: The predominant IgG subclass response, regardless of vaccine, was IgG1. One month following primary vaccination, the geometric mean concentrations (GMCs) of IgG1 and IgG2 in the PsA-TT group were 21.73 µg/mL and 6.27 µg/mL, whereas in the PsACWY group the mean GMCs were 2.01 µg/mL and 0.97 µg/mL, respectively (P < .0001). Group A-specific IgG1 and IgG2 GMCs remained greater in the PsA-TT group than in the PsACWY group 40 weeks following primary vaccination (P < .0001). One week following revaccination, those given 2 doses of PsA-TT had the greatest IgG1 and IgG2 GMCs of 125.23 µg/mL and 36.12 µg/mL, respectively (P = .0008), and demonstrated a significant increase in IgG1:IgG2 mean ratio, indicative of the T-cell-dependent response associated with conjugate vaccines. CONCLUSIONS: Vaccination of African children aged 12-24 months with either PsA-TT or PsACWY elicited a predominantly IgG1 response. The IgG1:IgG2 mean ratio decreased following successive vaccination with PsACWY, indicating a shift toward IgG2, suggestive of the T-cell-independent immune response commonly associated with polysaccharide antigens. CLINICAL TRIALS REGISTRATION: SRCTN78147026.

A Proteomic Characterization of Bordetella pertussis Clinical Isolates Associated with a California State Pertussis Outbreak.

Bordetella pertussis (Bp) is the etiologic agent of pertussis (whooping cough), a highly communicable infection. Although pertussis is vaccine preventable, in recent years there has been increased incidence, despite high vaccine coverage. Possible reasons for the rise in cases include the following: Bp strain adaptation, waning vaccine immunity, increased surveillance, and improved clinical diagnostics. A pertussis outbreak impacted California (USA) in 2010; children and preadolescents were the most affected but the burden of disease fell mainly on infants. To identify protein biomarkers associated with this pertussis outbreak, we report a whole cellular protein characterization of six Bp isolates plus the pertussis acellular vaccine strain Bp Tohama I (T), utilizing gel-free proteomics-based mass spectrometry (MS). MS/MS tryptic peptide detection and protein database searching combined with western blot analysis revealed three Bp isolates in this study had markedly reduced detection of pertactin (Prn), a subunit of pertussis acellular vaccines. Additionally, antibody affinity capture technologies were implemented using anti-Bp T rabbit polyclonal antisera and whole cellular proteins to identify putative immunogens. Proteome profiling could shed light on pathogenesis and potentially lay the foundation for reduced infection transmission strategies and improved clinical diagnostics.

Persistence of serogroup C antibody responses following quadrivalent meningococcal conjugate vaccination in United States military personnel.

Serogroup C meningococcal (MenC) disease accounts for one-third of all meningococcal cases and causes meningococcal outbreaks in the U.S. Quadrivalent meningococcal vaccine conjugated to diphtheria toxoid (MenACYWD) was recommended in 2005 for adolescents and high risk groups such as military recruits. We evaluated anti-MenC antibody persistence in U.S. military personnel vaccinated with either MenACYWD or meningococcal polysaccharide vaccine (MPSV4). Twelve hundred subjects vaccinated with MenACYWD from 2006 to 2008 or MPSV4 from 2002 to 2004 were randomly selected from the Defense Medical Surveillance System. Baseline serologic responses to MenC were assessed in all subjects; 100 subjects per vaccine group were tested during one of the following six post-vaccination time-points: 5-7, 11-13, 17-19, 23-25, 29-31, or 35-37 months. Anti-MenC geometric mean titers (GMT) were measured by rabbit complement serum bactericidal assay (rSBA) and geometric mean concentrations (GMC) by enzyme-linked immunosorbent assay (ELISA). Continuous variables were compared using the Wilcoxon rank sum test and the proportion of subjects with an rSBA titer ≥ 8 by chi-square. Pre-vaccination rSBA GMT was <8 for the MenACWYD group. rSBA GMT increased to 703 at 5-7 months post-vaccination and decreased by 94% to 43 at 3 years post-vaccination. GMT was significantly lower in the MenACWYD group at 5-7 months post-vaccination compared to the MPSV4 group. The percentage of MenACWYD recipients achieving an rSBA titer of ≥ 8 decreased from 87% at 5-7 months to 54% at 3 years. There were no significant differences between vaccine groups in the proportion of subjects with a titer of ≥ 8 at any time-point. GMC for the MenACWYD group was 0.14 µg/mL at baseline, 1.07 µg/mL at 5-7 months, and 0.66 µg/mL at 3 years, and significantly lower than the MPSV4 group at all time-points. Anti-MenC responses wane following vaccination with MenACYWD; a booster dose is needed to maintain protective levels of circulating antibody.

The impact of pre-existing antibody on subsequent immune responses to meningococcal A-containing vaccines.

Major epidemics of serogroup A meningococcal meningitis continue to affect the African meningitis belt. The development of an affordable conjugate vaccine against the disease became a priority for World Health Organization (WHO) in the late 1990s. Licensing of meningococcal vaccines has been based on serological correlates of protection alone, but such correlates might differ in different geographical regions. If high pre-vaccination antibody concentrations/titers impacts on the response to vaccination and possibly vaccine efficacy, is not clearly understood. We set out to define the pre-vaccination Meningococcal group A (Men A) antibody concentrations/titers in The Gambia and study their impact on the immunogenicity of Men A containing vaccines. Data from subjects originally enrolled in studies to test the safety and immunogenicity of the MenA vaccine recently developed for Africa meningococcal A polysaccharide conjugated to tetanus toxoid, MenAfriVac(®) (PsA-TT) were analyzed. Participants had been randomized to receive either the study vaccine PsA-TT or the reference quadrivalent plain polysaccharide vaccine containing meningococcal groups A, C, W, and Y, Mencevax(®) ACWY, GlaxoSmithKline (PsACWY) in a 2:1 ratio. Venous blood samples were collected before and 28 days after vaccination. Antibodies were assayed by enzyme-linked immunosorbent assay (ELISA) for geometric mean concentrations and serum bactericidal antibody (SBA) for functional antibody. The inter age group differences were compared using ANOVA and the pre and post-vaccination differences by t test. Over 80% of the ≥19 year olds had pre-vaccination antibody concentrations above putatively protective concentrations as compared to only 10% of 1-2 year olds. Ninety-five percent of those who received the study vaccine had ≥4-fold antibody responses if they had low pre-vaccination concentrations compared to 76% of those with high pre-vaccination concentrations. All subjects with low pre-vaccination titers attained ≥4-fold responses as compared to 76% with high titers where study vaccine was received. Our data confirm the presence of high pre-vaccination Men A antibody concentrations/titers within the African meningitis belt, with significantly higher concentrations in older individuals. Although all participants had significant increase in antibody levels following vaccination, the four-fold or greater response in antibody titers were significantly higher in individuals with lower pre-existing antibody titers, especially after receiving PsA-TT. This finding may have some implications for vaccination strategies adopted in the future.

Molecular signatures of antibody responses derived from a systems biology study of five human vaccines.

Many vaccines induce protective immunity via antibodies. Systems biology approaches have been used to determine signatures that can be used to predict vaccine-induced immunity in humans, but whether there is a 'universal signature' that can be used to predict antibody responses to any vaccine is unknown. Here we did systems analyses of immune responses to the polysaccharide and conjugate vaccines against meningococcus in healthy adults, in the broader context of published studies of vaccines against yellow fever virus and influenza virus. To achieve this, we did a large-scale network integration of publicly available human blood transcriptomes and systems-scale databases in specific biological contexts and deduced a set of transcription modules in blood. Those modules revealed distinct transcriptional signatures of antibody responses to different classes of vaccines, which provided key insights into primary viral, protein recall and anti-polysaccharide responses. Our results elucidate the early transcriptional programs that orchestrate vaccine immunity in humans and demonstrate the power of integrative network modeling.

Absence of high molecular weight proteins 1 and/or 2 is associated with decreased adherence among non-typeable Haemophilus influenzae clinical isolates.

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×10(5) cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 10(2), 10(3) or 10(4) c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

Immunoactivating peptide p4 augments alveolar macrophage phagocytosis in two diverse human populations.

New treatment strategies are urgently needed to overcome early mortality in acute bacterial infections. Previous studies have shown that administration of a novel immunoactivating peptide (P4) alongside passive immunotherapy prevents the onset of septicemia and rescues mice from lethal invasive disease models of pneumococcal pneumonia and sepsis. In this study, using two diverse populations of adult volunteers, we determined whether P4 treatment of human alveolar macrophages would upregulate phagocytic killing of Streptococcus pneumoniae ex vivo. We also measured macrophage intracellular oxidation, cytokine secretion, and surface marker expression following stimulation. Peptide treatment showed enhanced bacterial killing in the absence of nonspecific inflammation, consistent with therapeutic potential. This is the first demonstration of P4 efficacy on ex vivo-derived human lung cells.

Phenotypic, genomic, and transcriptional characterization of Streptococcus pneumoniae interacting with human pharyngeal cells.

BACKGROUND: Streptococcus pneumoniae is a leading cause of childhood morbidity and mortality worldwide, despite the availability of effective pneumococcal vaccines. Understanding the molecular interactions between the bacterium and the host will contribute to the control and prevention of pneumococcal disease. RESULTS: We used a combination of adherence assays, mutagenesis and functional genomics to identify novel factors involved in adherence. By contrasting these processes in two pneumococcal strains, TIGR4 and G54, we showed that adherence and invasion capacities vary markedly by strain. Electron microscopy showed more adherent bacteria in association with membranous pseudopodia in the TIGR4 strain. Operons for cell wall phosphorylcholine incorporation (lic), manganese transport (psa) and phosphate utilization (phn) were up-regulated in both strains on exposure to epithelial cells. Pneumolysin, pili, stress protection genes (adhC-czcD) and genes of the type II fatty acid synthesis pathway were highly expressed in the naturally more invasive strain, TIGR4. Deletion mutagenesis of five gene regions identified as regulated in this study revealed attenuation in adherence. Most strikingly, ∆SP_1922 which was predicted to contain a B-cell epitope and revealed significant attenuation in adherence, appeared to be expressed as a part of an operon that includes the gene encoding the cytoplasmic pore-forming toxin and vaccine candidate, pneumolysin. CONCLUSION: This work identifies a list of novel potential pneumococcal adherence determinants.

Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection.

BACKGROUND: Viral upper respiratory tract infections are associated with increased colonization by Streptococcus pneumoniae but the mechanisms underlying this relationship are unclear. The objective of this study is to describe a comprehensive picture of the cellular interaction between the adhering bacteria and host cells in the presence or absence of a viral co-infection. RESULTS: Gene expression profiles of Detroit-562 pharyngeal cells, which were either mock-infected or infected with human respiratory syncytial virus (RSV) or human parainfluenza virus 3 (HPIV3), were analyzed using human microarrays. Transcription response of S. pneumoniae strain TIGR4 (serotype 4) in the presence of either mock- or viral-infected cells was analyzed by pneumococcal microarray. Significantly regulated genes were identified by both significance analysis of microarray (SAM) and a ≥ 2-fold change ratio cut-off. The adherence of S. pneumoniae to human pharyngeal cells was significantly augmented in the presence of RSV or HPIV3 infection. Global gene expression profiling of the host cells during infection with RSV or HPIV3 revealed increased transcription of carcinoembryonic antigen-related cell adhesion molecules (CEACAM1), CD47, fibronectin, interferon-stimulated genes and many other host cell adhesion molecules. Pneumococci increased transcription of several genes involved in adhesive functions (psaA, pilus islet), choline uptake and incorporation (lic operon), as well as transport and binding. CONCLUSIONS: We have identified a core transcriptome that represents the basic machinery required for adherence of pneumococci to D562 cells infected or not infected with a virus. These bacterial genes and cell adhesion molecules can potentially be used to control pneumococcal adherence occurring secondary to a viral infection.

Immunogenicity and safety of a new meningococcal A conjugate vaccine in Indian children aged 2-10 years: a phase II/III double-blind randomized controlled trial.

This study compares the immunogenicity and safety of a single dose of a new meningococcal A conjugate vaccine (PsA-TT, MenAfriVac™, Serum Institute of India Ltd., Pune) against the meningococcal group A component of a licensed quadrivalent meningococcal polysaccharide vaccine (PsACWY, Mencevax ACWY(®), GSK, Belgium) 28 days after vaccination in Indian children. This double-blind, randomized, controlled study included 340 Indian children aged 2-10 years enrolled from August to October 2007; 169 children received a dose of PsA-TT while 171 children received a dose of PsACWY. Intention-to-treat analysis showed that 95.2% of children in PsA-TT group had a ≥4-fold response in serum bactericidal titers (rSBA) 28 days post vaccination as compared to 78.2% in the PsACWY group. A significantly higher rSBA GMT (11,209, 95%CI 9708-12,942) was noted in the PsA-TT group when compared to PsACWY group (2838, 95%CI 2368-3401). Almost all children in both vaccine groups had a ≥4-fold response in group A-specific IgG concentration but the IgG GMC was significantly greater in the PsA-TT group (89.1 µg/ml, 95%CI 75.5-105.0) when compared to the PsACWY group (15.3 µg/ml, 95%CI 12.3-19.2). Local and systemic reactions during the 4 days after immunization were similar for both vaccine groups except for tenderness (30.2% in PsA-TT group vs 12.3% in PsACWY group). None of the adverse events or serious adverse events was related to the study vaccines. We conclude that MenAfriVac™ is well tolerated and significantly more immunogenic when compared to a licensed polysaccharide vaccine, in 2-to-10-year-old Indian children.

Interlaboratory standardization of the sandwich enzyme-linked immunosorbent assay designed for MATS, a rapid, reproducible method for estimating the strain coverage of investigational vaccines.

The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or "relative potency" (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

Measurement of Haemophilus influenzae type a capsular polysaccharide antibodies in cord blood sera.

We measured anti-Haemophilus influenzae type a capsular polysaccharide serum immunoglobulin G antibodies in cord blood sera from Mexican (n = 68) and Chilean mothers (n = 72) by enzyme-linked immunosorbent assay. Measurable antibodies were found in 79.3% of samples. Immunoglobulin G antibodies correlated with serum bactericidal activity (r = 0.66). This enzyme-linked immunosorbent assay can be used for the evaluation of adaptive immune responses to Haemophilus influenzae type a and serosurveillance studies in populations at risk.