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.

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.

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.

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.

Revaccination with a 23-valent pneumococcal polysaccharide vaccine induces elevated and persistent functional antibody responses in adults aged 65 > or = years.

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.

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.

Differentiation of Streptococcus pneumoniae conjunctivitis outbreak isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.

Pneumococcal surface adhesin A (PsaA): a review.

Pneumococcal surface adhesin A (PsaA) is a surface-exposed common 37-kilodalton multi-functional lipoprotein detected on all known serotypes of Streptococcus pneumoniae. This lipoprotein belongs to the ABC-type transport protein complex that transports Mn2+; it is also an adhesin that plays a major role in pneumococcal attachment to the host cell and virulence. PsaA is immunogenic and natural nasopharyngeal colonization of pneumococci elicits an increase in antibody towards PsaA. Hence, PsaA is being actively evaluated as a component of a vaccine in formulations composed of pneumococcal common proteins. PsaA has been expressed as an E. coli recombinant protein, purified, and evaluated in a phase one clinical trial. This article reviews PsaA, its structure and role in pneumococcal virulence, immunogenicity, and potential to reduce nasopharyngeal colonization (a major prerequisite for pneumococcal pathogenesis) as a component of a common pneumococcal protein vaccine.

Pneumococcal surface adhesin A (PsaA): a review.

Pneumococcal surface adhesin A (PsaA) is a surface-exposed common 37-kilodalton multi-functional lipoprotein detected on all known serotypes of Streptococcus pneumoniae. This lipoprotein belongs to the ABC-type transport protein complex that transports Mn(2+); it is also an adhesin that plays a major role in pneumococcal attachment to the host cell and virulence. PsaA is immunogenic and natural nasopharyngeal colonization of pneumococci elicits an increase in antibody towards PsaA. Hence, PsaA is being actively evaluated as a component of a vaccine in formulations composed of pneumococcal common proteins. PsaA has been expressed as an E. coli recombinant protein, purified, and evaluated in a phase one clinical trial. This article reviews PsaA, its structure and role in pneumococcal virulence, immunogenicity, and potential to reduce nasopharyngeal colonization (a major prerequisite for pneumococcal pathogenesis) as a component of a common pneumococcal protein vaccine.

A real-time polymerase chain reaction for the detection of Streptococcus pneumoniae in blood using a mouse model: a potential new "gold standard".

Better diagnostics for pneumococcal disease are urgently needed. In a murine model, real-time polymerase chain reaction was superior to conventional culture in detecting pneumococcus in blood, particularly in early disease and after antibiotic administration, and could distinguish between commensalism and infection.

An in vitro model to assess pneumococcal adherence to nasopharyngeal cells under competition conditions.

Pneumococcal conjugate vaccine (PCV7) reduces invasive disease and carriage caused by vaccine serotypes (VS). An increase in carriage and disease with non-vaccine serotypes (NVS) has been observed. We have developed an in vitro model with human nasopharyngeal (NP) epithelial cells (Detroit 562) to assess the adherence capacity of Streptococcus pneumoniae to NP cells in the presence or absence of a competing Pnc strain. Two hundred and fifty pneumococcal (Pnc) strains (10 strains per serotype for 7 VS and 18 NVS) were tested for their opacity phenotype. Strains exhibiting (> or =50%) the transparent phenotype (n=72) were evaluated for their adherence capacity to Detroit 562 cells. Mean adherence capacity (> or =129 CFU/well) to NP cells was high for VS 18C, 4, and 9V and for NVS 16F, 10A, and 6A. In the in vitro competition experiments, VS strains out-competed (42/108) or co-existed (43/108) with NVS strains for adherence to NP cells in most co-inoculations. By contrast, NVS (15C, 16F, 31, and 35B) out-competed with VS in only 9 of 108 co-inoculations. Serotype 16F out-competed or co-existed with some VS and NVS strains. This model may be used to identify Pnc strains of a given serotype with competitive potentials for replacement of VS in the nasopharynx and to screen Pnc strains for animal colonization models.

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.

Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I. Antibody concentrations, avidity and functional activity.

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults. These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides.

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.

Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assays.

Neisseria meningitidis remains the leading cause of fatal sepsis. Cultures may not be available in fulminant fatal cases. An immunohistochemical assay for N meningitidis was applied to formalin-fixed samples from 14 patients with meningococcal disease. Histopathologic findings in 12 fatal cases included interstitial pneumonitis, hemorrhagic adrenal glands, myocarditis, meningitis, and thrombi in the glomeruli and choroid plexus. Meningeal inflammation was observed in 6 patients. Skin biopsies of 2 surviving patients showed leukocytoclastic vasculitis and cellulitis. By using immunohistochemical analysis, meningococci and granular meningococcal antigens were observed inside monocytes, neutrophils, and endothelial cells or extracellularly. By using real-time polymerase chain reaction (PCR) on formalin-fixed tissue samples, meningococcal serogroup determination was possible in 11 of 14 cases (8 serogroup C, 2 Y, and 1 B). Diagnosis and serogrouping of N meningitidis can be performed using immunohistochemical analysis and PCR on formalin-fixed tissue samples. Immunohistochemical analysis determined the distribution of meningococci and meningococcal antigens in tissue samples, allowing better insights into N meningitidis pathogenesis.

Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae.

We determined the usefulness of 4 conventional polymerase chain reaction (PCR) assays, lytA, psaA, and two primer sets from the ply gene, for accuracy in the discrimination of nontypeable (NT) Streptococcus pneumoniae from closely related atypical streptococci. The study used 100 strains. We compared the PCR results with laboratory tests that included optochin (ethylhydrocupreine hydrochloride) sensitivity, bile solubility, the Quellung reaction, and AccuProbe (Gen-Probe Inc., San Diego, CA). These latter tests did not discriminate the atypical streptococci from the NT pneumococci. All PCR primer sets amplified the NT pneumococcal isolates in agreement with the other laboratory tests. However, the IA and IB ply primers were positive for 8 of the 16 atypical streptococcal isolates, and the IIA and IIB ply primers amplified all atypical isolates. The psaA primers had only one discrepant result, a positive among the atypical streptococci. The lytA primers were the most specific with 100% specificity for all strains tested.

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.

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.

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.