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.

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 (MenACWY) administered alone (n = 41) or concomitantly with other vaccines (n = 279) to United States 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, various influenza vaccines, among others; two vaccine groups excluded Tdap and IPV. Immune responses were evaluated in baseline and post-vaccination sera for Neisseria meningitidis serogroups C and Y 1-12 months (mean = 4.96) 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 ≥2 mug/mL; rSBA titer ≥8) corresponding to an immunologic response was higher post-vaccination 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 (odds ratios [ORs] range = 1.5 to 21.7) and more of them seroconverted (ORs 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 United States military personnel.

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.

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 0.14mug/mL at baseline, 1.07mug/mL at 5-7 months, and 0.66mug/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.

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.

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 (1.2x10(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.

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.

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.

We measured anti-Hia capsular polysaccharide serum immunoglobulin G (IgG) antibodies in cord blood sera from Mexican (n=68) and Chilean mothers (n=72) by ELISA. Measurable antibodies were found in 79.3% of samples. IgG antibodies correlated with serum bactericidal activity (r=0.66). This ELISA can be used for the evaluation of adaptive immune responses to Hia and sero-surveillance studies in populations at risk.

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

Lot 89SF has been the reference standard serum pool used in pneumococcal enzyme-linked immunosorbent assays (ELISAs) since 1990. In 2005, it was estimated that there remained between 2 and 5 years' supply of lot 89SF. Since lot 89SF was the reference standard used in the evaluation of the seven-valent pneumococcal conjugate vaccine Prevnar (PCV7), the link to clinical efficacy would be severed if stocks became completely depleted. Furthermore, demonstration of immune responses comparable to those elicited by PCV7 is a licensure approach used for new pneumococcal conjugate vaccines, so a replacement reference standard was required. A total of 278 volunteers were immunized with the 23-valent unconjugated polysaccharide vaccine Pneumovax II, and a unit of blood was obtained twice within 120 days following immunization. Plasma was prepared, pooled, and confirmed to be free from hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV. The pooled serum was poured at 6 ml per vial into 15,333 vials and lyophilized. Immunological bridging of 007sp to 89SF was used to establish equivalent reference values for 13 pneumococcal capsular serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) by five independent laboratories. Antibody concentrations in 007sp were established relative to the lot 89SF reference preparation using the WHO reference ELISA. Subsequently, 12 existing WHO calibration sera had concentrations reassigned for 13 pneumococcal serotypes using new serum 007sp as the reference, and these were compared to concentrations relative to the original reference serum. Agreement was excellent for the 12 WHO calibration sera. The 007sp preparation has replaced 89SF as the pneumococcal reference standard. Sufficient quantity of this new preparation is available such that, with judicious use, it should be available for at least 25 years.

Haemophilus influenzae type a (Hia) is an important pathogen for some American Indian, Alaskan natives and Northern Canada aboriginal populations. Assays to measure serum bactericidal activity (SBA) to Hia have not been developed or validated. Here we describe two methods for the measurement of SBA: SBA with a viability end-point (CFU counts) and SBA with a fluorometric end-point using alamarBlue as metabolic indicator. Both SBA assays measure Hia-specific functional antibody and correlate with anti-Hia IgG ELISA concentration of naturally-acquired antibodies.

Antibody mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies using a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and a requirement for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis of variance (ANOVA) mixed-effects model, provided a common reference to assess agreement among these laboratories. Agreement was evaluated using assay accuracy, reproducibility, repeatability, precision and bias. We also reviewed four acceptance criteria intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy and concordance results among laboratories and the consensus titers revealed acceptable agreement. Results of this study indicate that the bioassays evaluated in this study are robust and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template to evaluate future multilaboratory studies.

Many studies suggest that with aging, immune capabilities gradually diminish, leading to a decrease in antibody production, cytokines, and various effector cells (1-4). In this study, we examined the effects of an immune-enhancing peptide on aged mice. P4, a 28-amino-acid cationic peptide derived from pneumococcal surface adhesin A (PsaA), is a eukaryotic cellular activator (10). Previously, we demonstrated that the cellular activation properties of P4 can be utilized to rescue severely ill young mice from fatal pneumococcal infection in the presence of pathogen-specific antibodies and active complement (8, 12). While P4 therapy was used to rescue young Swiss Webster mice (6 to 10 weeks old), we questioned its effectiveness in aged mice (11 and 15 months old). | Intranasal inoculation of mice with Streptococcus pneumoniae WU2 (serotype 3) and P4 therapy were done using protocols previously described, with minor modifications (12). Eleven-month-old BALB/c (n = 20) and 15-month-old Swiss Webster mice (n = 20) were infected intranasally with S. pneumoniae WU2 (∼2.1 × 107 cells/mouse). Mice were monitored and visually scored twice daily for moribund characteristics as previously described (12). At 48 h postchallenge, 80% (16/20) were moribund. Moribund mice were divided into a control (n = 8) and a treatment group (n = 8). Two doses of P4 therapy with pathogen-specific antibody (intravenous immunoglobulin [IVIG]; Gamunex, Telecris, NC) and P4 were administered intravenously (postinfection) in the treatment group. Treated and untreated animals were monitored for 166 h, and the data computed for significant differences among various groups using a t test for paired samples for the means (MS Excel 2007).

In the early 1900s, passive immunization/antibody therapy was used to treat a variety of human ailments such as hypoimmunoglobulinemia, cancer and infectious disease. The advent of antibiotic therapy had relegated this type of therapy obsolete for treatment of infectious diseases. Emergence of multi-drug resistant pathogens along with novel monoclonal antibody production techniques has rekindled the interest in passive immunization (PI). An increase in the number of monoclonal antibody patent applications in the recent past suggests a renewed commercial interest in PI. Despite these developments, antibody therapy for infectious diseases has limitations including the need for large or frequent dosages. P4, a 28-amino acid peptide is a multi-lineage cellular activator. P4, along with infectious disease (i.e. Pathogen) specific immunoglobulin, has been shown in vitro and in vivo in mice to potentiate innate immunity. This review will discuss the progress made in passive antibody therapy, the challenges still to be surmounted, and the potential expanded role of an immune-potentiating peptide (bio-molecule) in the quest to utilize and revitalize passive immunization.

Although two centuries have passed since Vieusseux described epidemic meningococcal disease, (1) Neisseria meningitidis remains a leading cause of meningitis and sepsis. Overwhelming meningococcal disease can develop rapidly and is associated with mortality rates exceeding 20%. (2)Thus, efforts to control the disease have focused on vaccination. In the past, vaccines against meningococcal disease have failed to provide immunogenicity and long-term protection in infants, who are at greatest risk. Although recent vaccines have improved coverage for this age group, there is still no broadly effective vaccine against N. meningitidis group B (NMB), now the predominant disease-causing isolate in industrialized countries.

A murine colonization model was used to determine the effect of co-administering 7-valent polysaccharide-protein conjugate vaccine and pneumococcal surface adhesin A. Mice were challenged intranasally with either PCV7 serotypes, 4 or 14, or a non-PCV7 serotype, 19A. Post-challenge samples were evaluated for IgG antibody levels, opsonophagocytic activity, and nasopharyngeal colonization. No interference was observed between immune responses from the concomitant and individual immunizations. Concomitant immunizations reduced carriage for tested serotypes; largest reduction was observed for 19A. From these mouse studies, co-administering pneumococcal antigens appear to expand coverage and reduce colonization against a non-PCV7 serotype without inhibiting immunogenicity to other serotypes.

Serotype-specific IgG, as quantified by a standardized WHO ELISA, is a serologic end-point used to evaluate pneumococcal polysaccharide-based vaccine immunogenicity. Antibodies to each vaccine polysaccharide in licensed multivalent vaccines are quantified separately; this is laborious and consumes serum. We compared three bead-based immunoassays, a commercial assay (xMAP(R)Pneumo14, Luminex) and two in-house assays (Health Protection Agency [HPA] and Centers for Disease Control and Prevention [CDC]) using WHO recommended standard reference and reference sera (n=11) from vaccinated adults. Multiple comparisons of the IgG concentrations for seven conjugate vaccine serotypes were performed by sample (percent error), serotype (equivalency testing), and laboratory (concordance correlation coefficient [CCC]). When comparing concentrations by sample, bead-based immunoassays generally yielded higher antibody concentrations than ELISA and had higher variability for serotypes 6B, 18C, and 23F. None of the three assays met the current WHO recommendation of 75% of sera falling within +/-40% of the assigned antibody concentrations for all seven serotypes. When compared by serotype, CDC and HPA were equivalent for 5 of 7 serotypes, whereas Luminex was equivalent for 4 of 7 serotypes. When overall mean IgG concentrations were compared by laboratories, a higher level of agreement, CCC close to 1, was found among bead-based immunoassays than between the assays and WHO assignments. When compared to WHO assignments, the HPA assay out performed (r = 0.920, rc = 0.894, Ca = 0.972) the other assays. Additional testing with sera from immunogenicity studies should demonstrate the applicability of this methodology for vaccine evaluation.

BACKGROUND: Older adults are at high risk of developing invasive pneumococcal disease, but the optimal timing and number of vaccine doses needed to prevent disease among this group are unknown. We compared revaccination with 23-valent pneumococcal polysaccharide vaccine (PN23) with primary vaccination for eliciting initial and persistent functional antibody responses. METHODS: Subjects aged > or = 65 years were enrolled. Functional (opsonic) and total immunoglobulin (Ig) G antibody levels were measured following either PN23 primary vaccination (n = 60) or revaccination 3-5 years after receiving a first PN23 vaccination (n = 60). Antibody against vaccine serotypes 4, 14, and 23F was measured at prevaccination (day 0), 30 days after vaccination, and 5 years after vaccination. RESULTS: By day 30, both primary vaccination and revaccination induced significant increases in opsonic and IgG antibody levels. Day 30 levels following revaccination were slightly lower but not significantly different than those after primary vaccination. Year 5 levels were similar in both groups and remained significantly higher than prevaccination levels for primary vaccination subjects. There was good agreement between postvaccination opsonic and IgG antibody levels. CONCLUSIONS: Revaccination of older adults with PN23 was comparable to primary vaccination for inducing elevated and persistent functional and IgG antibody responses.