Emerging Vaccine Informatics

Vaccine informatics is an emerging research area that focuses on development and applications of bioinformatics methods that can be used to facilitate every aspect of the preclinical, clinical, and postlicensure vaccine enterprises. Many immunoinformatics algorithms and resources have been developed to predict T- and B-cell immune epitopes for epitope vaccine development and protective immunity analysis. Vaccine protein candidates are predictable in silico from genome sequences using reverse vaccinology. Systematic transcriptomics and proteomics gene expression analyses facilitate rational vaccine design and identification of gene responses that are correlates of protection in vivo. Mathematical simulations have been used to model host-pathogen interactions and improve vaccine production and vaccination protocols. Computational methods have also been used for development of immunization registries or immunization information systems, assessment of vaccine safety and efficacy, and immunization modeling. Computational literature mining and databases effectively process, mine, and store large amounts of vaccine literature and data. Vaccine Ontology (VO) has been initiated to integrate various vaccine data and support automated reasoning

Paraneoplastic necrotizing myopathy associated with adenocarcinoma of the lung - a rare entity with atypical onset: a case report.

Introduction. Inflammatory myopathies (such as dermatomyositis and polymyositis) are well-recognized paraneoplastic syndromes. However, paraneoplastic necrotizing myopathy is a more recently defined clinical entity, characterized by rapidly progressive, symmetrical, predominantly proximal muscle weakness with severe disability, and associated with a marked increase in serum muscle enzyme levels. Paraneoplastic necrotizing myopathy requires muscle biopsy for diagnosis, which typically shows massive necrosis of muscle fibers with limited or absent inflammatory infiltrates. Case presentation. We report the case of an 82-year-old Italian-born Caucasian man who was admitted to hospital because of heart failure and two drop attacks. Over the following days, he developed progressive severe weakness, dysphagia, and dysphonia. Testing showed increasing serum muscle enzyme levels. Electromyography showed irritative myopathy of the proximal muscles and sensorimotor polyneuropathy. Muscle biopsy (left vastus lateralis) showed massive necrosis of muscle fibers with negligible inflammatory infiltrates, complement membrane attack complex deposition on endomysial capillaries, and moderate upregulation of major histocompatibility complex-I. Computed tomography of the thorax showed a nodular mass in the apex of the right lung. The patient was diagnosed with paraneoplastic necrotizing myopathy. In spite of high-dose corticoid therapy, he died 1 month later because of his aggressive cancer. Subsequent electron microscopic examination of a muscle biopsy specimen showed thickened walls and typical pipestem changes of the endomysial capillaries, with swollen endothelial cells. Poorly differentiated adenocarcinoma of the lung was confirmed on post-mortem histological examination. Conclusions: Paraneoplastic necrotizing myopathy is a rare syndrome with outcomes ranging from fast progression to complete recovery. Treatment with corticosteroids is often ineffective, and prognosis depends mainly on the characteristics of the underlying cancer. This case shows that paraneoplastic necrotizing myopathy may have an atypical appearance, and should be considered in elderly patients with neoplastic disease. In this case, the diagnosis was delayed by the unusual clinical picture that suggested heart disease rather than muscle disease

Real-time measurements of Hg0 in volcanic, geothermal and anthropogenic systems: a multi-methodological approach using Lumex® instrumentation

Mercury represents a pollutant of global concern and strong environmental impact since is highly toxic. Hg is present in air in the oxidation states of 0 and +2, the former being the dominant species with a residence time of 1-2 years due to its high volatility, relatively low solubility and chemical inertness. Both volcanic/geothermal and anthropogenic systems are crucial contributor to the release of Hg0 in the atmosphere. In this work, a Lumex® (RA-915M) was used to evaluate the environmental impact in air of Hg0 from: i) the abandoned Hg mining site and geothermal areas from Mt. Amiata (Siena, Central Italy) and ii) selected Mediterranean volcanic and geothermal systems. The Lumex® instrumentation, based on atomic absorption spectrometric technique with Zeeman effect, allows to measure Hg0 at high frequency, in real-time and at a wide range of concentrations (from 2 to 50,000 ng/m3). Hg0 measurements were coupled with those of other pollutants, such as CO2 H2S, and SO2. Carbon dioxide was measured using a Multi-GAS instrument manufactured by INGV-Palermo, whereas H2S and SO2 using Thermo Scientific® Model 450i analyzer. GPS and meteorological parameters were continuously recorded, too. The data acquisition was carried out along transects at an approximately constant speed or at selected fixed points. Wherever possible, the analytical data were then converted into a spatial interpolation providing a qualitative model for the areal dispersion of the contaminants. The Lumex® device was also applied to measure Hg0 concentrations in interstitial soil gases collected from a probe inserted into the soil at 70 cm depth, in order to produce Hg0 maximum concentration maps in Hg-polluted areas (e.g. Abbadia San Salvatore Hg mining area, Mt. Amiata). Diffuse Hg0 soil fluxes were measured using a chamber positioned above the soil from which, at periodic time intervals, gases were extracted and injected into the Lumex® device. This instrument was also applied to measure Hg0 concentrations along vertical profiles in thermal wells at Santorini (Greece) and Vulcano (Italy) by using a Rilsan® tube lowered into the wells at pre-defined depths. With this approach, a significant stratification of the air masses in terms of Hg0, strictly dependent on water temperature, air pressure and well depth, was observed. The efficiency of Lumex® for these different approaches demonstrated the reliability of this instrument to produce Hg0 data that can be used to identify gaseous Hg-emitters in natural and anthropogenic environments, especially when coupled with other physical and chemical parameters

MOLECULAR CHARACTERISATION OF A NOVEL ADP-RIBOSYLATING PUTATIVE TOXIN OF NEISSERIA MENINGITIDIS

Molecular characterisation of a novel ADP-ribosylating putative toxin of Neisseria meningitidis VEGGIi D, *BALDUCCI E, MASIGNANI V, DI MARCELLO F, SAVINO S, ARICO’ B, COMANDUCCI M, PIZZA M, RAPPUOLI R IRIS, Chiron SpA, Via Fiorentina 1, 53100 Siena Italy; *Dipartimento Scienze morfologiche e Biochimiche Comparate, Università degli Studi di Camerino, Camerino, Italy Session: Surface antigens Introduction: By computer analysis on the Neisseria meningitidis (serogroup B, MC 58 strain) genome sequence, a protein with a feature similar to known bacterial ADP-ribosylating toxins (CT produced by Vibrio cholerae, LT by Escherichia coli and PT by Bordetella pertussis) has been identified. Enzymatic assay has shown that this protein (NM-ADPRT) possesses both NAD glycohydrolase and ADP-ribosyltransferase activity. In this study we describe the identification of the putative catalytic residues, their site-directed mutagenesis, and the resulting activity of the mutants. Materials and methods: The novel NM-ADPRT and the correspondent mutants, were expressed in E. coli as C-terminus His-tag protein fusions. Site-directed mutagenesis was performed using the Multi Site-Directed Mutagenesis Kit (QuikChange). Recombinant NM-ADPRT forms were purified from E. coli in their soluble form by metal chelate affinity chromatography. Both the wild-type and the mutants were assayed for their ADP-ribosylation and NAD-glycohydolase activites, using [adenine –U-14C] NAD and agmatine as ADP-ribose acceptor. Antisera against NM-ADPRT and the mutant derivatives were obtained by immunization of CD1 mice. 20μg of each recombinant protein were given i.p. together with CFA for the first dose and IFA for the second (day 21) and the third (day 35) booster doses. Blood sample were taken on days 34 and 49. Immune sera were used in western blot and tested in a bactericidal assay. Results and discussion: On the basis of sequence homology of NM-ADPRT with LT, CT and PT we have identified the putative residues involved in enzymatic activity. These residues have been changed by site-directed mutagenesis and the purified mutant toxins have been tested for both ADP-ribosylating and NAD-glycohydrolase activities. Interestingly, some of the mutants show reduced or abolished enzymatic activity indicating that the identified residues play a role in catalysis. Antisera against the wild-type and mutant toxins have bactericidal activity. The titers induced by two mutants were higher than those induced by the wild-type form. These data suggest that the mutations introduced could influence not only the enzymatic activity but also the in vivo stability of the toxin. Conclusion: A novel ADP-ribosyltransferase has been identified in meningococcus B. Catalytic residues have been predicted by sequence homology and their role in catalysis has been confirmed by site-directed mutagenesis. These molecules are also able to induce a bactericidal response

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ߠbarrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.Full Tex

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis.

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production

HCV E1E2-MF59 vaccine in chronic hepatitis C patients treated with PEG-IFNα2a and Ribavirin: a randomized controlled trial.

Hepatitis C virus (HCV) vaccines may be able to increase viral clearance in combination with antiviral therapy. We analysed viral dynamics and HCV-specific immune response during retreatment for experienced patients in a phase Ib study with E1E2MF59 vaccine. Seventy-eight genotype 1a/1b patients [relapsers (30), partial responders (16) and nonresponders (32) to interferon-(IFN)/ribavirin-(RBV)] were randomly assigned to vaccine (V:23), Peg-IFNα2a-180-ug/qw and ribavirin 1000-1200-mg/qd for 48 weeks (P/R:25), or their combination (P/R + V:30). Vaccine (100 μg/0.5 mL) was administered intramuscularly at week 0-4-8-12-24-28-32-36. Neutralizing of binding (NOB) antibodies and lymphocyte proliferation assay (LPA) for E1E2-specific-CD4 + T cells were performed at week 0-12-16-48. Viral kinetics were analysed up to week 16. The vaccine was safe, and a sustained virological response (SVR) was achieved in 4 P/R + V and 2 P/R patients. Higher SVR rates were observed in prior relapsers (P/R + V = 27.3%; P/R = 12.5%). Higher NOB titres and LPA indexes were found at week 12 and 16 in P/R + V as compared to P/R patients (P = 0.023 and 0.025, P = 0.019 and <0.001, respectively). Among the 22 patients with the strongest direct antiviral effects of IFN (ε ≥ 0.800), those treated with P/R + V (10) reached lower HCV-RNA levels (P = 0.026) at week 16. HCV E1E2MF59 vaccine in combination with Peg-IFNα2a + RBV was safe and elicited E1E2 neutralizing antibodies and specific CD4 + T cell proliferation. Upon early response to IFN, vaccinations were associated with an enhanced second phase viral load decline. These results prompt phase II trials in combination with new antiviral therapies

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed