Towards an improved Neisseria meningitidis B vaccine: vesicular PorA formulations

There is a great need for vaccines against Neisseria meningitidis serogroup B. This is especially important in Western European countries, were approximately two thirds of the cases of meningococcal disease can be attributed to serogroup B strains. Against this serogroup, traditional vaccines based on capsular polysaccharides are not effective. The immunogenicity of the capsular polysaccharide of group B meningococci is low, due to structural similarity with epitopes expressed by host neural tissue. During the search for vaccine candidates against serogroup B meningococci, a great interest has been focused on PorA, a porin located in the outer membrane of the bacteria. PorA is one of the immunodominant antigens expressed by Neisseria meningitidis. Monoclonal antibodies directed against PorA have shown to have a high bactericidal activity and are protective in an infant rat meningitis model. The vaccine potential of PorA embedded in outer membrane vesicles (OMV) developed by different groups has been studied in various clinical trials. The vaccine presently being developed by the Netherlands Vaccine Institute (NVI) is based on OMV purified from genetically modified meningococcal strains expressing three different PorA serosubtypes (trivalent OMV). The vaccine contains a mixture of two different trivalent OMV are mixed, which together represent the majority of PorA serosubtypes circulating in the Netherlands and other European countries. Among the PorA serosubtypes included in the NVI vaccine, the serosubtype P1.7-2,4 is present in up to 40% of the serogroup B strains causing bacterial meningitis. Thus, a PorA-based vaccine should cover at least this subtype. On the other hand, the immunogenicity of PorA P1.7-2,4 is not yet optimal in its present formulation as OMV vaccine. For these reasons, PorA P1.7-2,4 was chosen as a model protein for our studies. The objective this thesis was to improve the immunogenicity of PorA. The research was exclusively focused on the study of the effect of the presentation form on the immunogenicity of PorA P1.7-2,4. For this purpose, three lines of investigation were followed: ? Detailed study of physicochemical and immunochemical characteristics of PorA and its presentation form. Also, study of the effects of different storage conditions on the physicochemical stability and immunogenicity of PorA formulated in different types of OMV. ? Preparation of well-defined liposomal PorA formulations and study of the effect of various targeting strategies for dendritic cells (DC) on the immunogenicity of PorA. ? Addition of adjuvants to liposomal PorA formulations and study of their effect on the PorA-specific immune response

Application of Machine Learning to Arterial Spin Labeling in Mild Cognitive Impairment and Alzheimer Disease

PURPOSE: To investigate whether multivariate pattern recognition analysis of arterial spin labeling (ASL) perfusion maps can be used for classification and single-subject prediction of patients with Alzheimer disease (AD) and mild cognitive impairment (MCI) and subjects with subjective cognitive decline (SCD) after using the W score method to remove confounding effects of sex and age. MATERIALS AND METHODS: Pseudocontinuous 3.0-T ASL images were acquired in 100 patients with probable AD; 60 patients with MCI, of whom 12 remained stable, 12 were converted to a diagnosis of AD, and 36 had no follow-up; 100 subjects with SCD; and 26 healthy control subjects. The AD, MCI, and SCD groups were divided into a sex- and age-matched training set (n = 130) and an independent prediction set (n = 130). Standardized perfusion scores adjusted for age and sex (W scores) were computed per voxel for each participant. Training of a support vector machine classifier was performed with diagnostic status and perfusion maps. Discrimination maps were extracted and used for single-subject classification in the prediction set. Prediction performance was assessed with receiver operating characteristic (ROC) analysis to generate an area under the ROC curve (AUC) and sensitivity and specificity distribution. RESULTS: Single-subject diagnosis in the prediction set by using the discrimination maps yielded excellent performance for AD versus SCD (AUC, 0.96; P .05). CONCLUSION: With automated methods, age- and sex-adjusted ASL perfusion maps can be used to classify and predict diagnosis of AD, conversion of MCI to AD, stable MCI, and SCD with good to excellent accuracy and AUC values

Self-assembled nanogel made of mannan : synthesis and characterization

Amphiphilic mannan (mannan-C16) was synthesized by the Michael addition of hydrophobic 1-hexadecanethiol (C16) to hydroxyethyl methacrylated mannan (mannan-HEMA). Mannan-C16 formed nanosized aggregates in water by selfassembly via the hydrophobic interaction among C16molecules as confirmed by hydrogen nuclearmagnetic resonance (1H NMR), fluorescence spectroscopy, cryo-field emission scanning electron microscopy (cryo-FESEM), and dynamic light scattering (DLS). The mannan-C16 critical aggregation concentration (cac), calculated by fluorescence spectroscopy with Nile red and pyrene, ranged between 0.04 and 0.02mg/mL depending on the polymer degree of substitution ofC16 relative to methacrylated groups. Cryo-FESEM micrographs revealed that mannan-C16 formed irregular spherical macromolecular micelles, in this work designated as nanogels, with diameters ranging between 100 and 500 nm. The influence of the polymer degree of substitution, DSHEMA andDSC16, on the nanogel size and zeta potential was studied byDLS at different pH values and ionic strength and as a function of mannan-C16 and urea concentrations. Under all tested conditions, the nanogel was negatively charged with a zeta potential close to zero. Mannan-C16 with higher DSHEMA and DSC16 values formed larger nanogels andwere also less stable over a 6month storage period and at concentrations close to the cac.When exposed to solutions of different pH and aggressive conditions of ionic strength and urea concentration, the size of mannan-C16 varied to some extent but was always in the nanoscale range.International Iberian Nanotechnology Laboratory (INL)Fundação para a Ciência e a Tecnologia (FCT

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

Purpose. In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. Methods. TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. Results. TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DTalum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. Conclusions. The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants. KEY WORDS: cholera toxin; CpG; diphtheria toxoid; microneedle array; transcutaneous immunization

Emergent Functional Properties of Neuronal Networks with Controlled Topology

The interplay between anatomical connectivity and dynamics in neural networks plays a key role in the functional properties of the brain and in the associated connectivity changes induced by neural diseases. However, a detailed experimental investigation of this interplay at both cellular and population scales in the living brain is limited by accessibility. Alternatively, to investigate the basic operational principles with morphological, electrophysiological and computational methods, the activity emerging from large in vitro networks of primary neurons organized with imposed topologies can be studied. Here, we validated the use of a new bio-printing approach, which effectively maintains the topology of hippocampal cultures in vitro and investigated, by patch-clamp and MEA electrophysiology, the emerging functional properties of these grid-confined networks. In spite of differences in the organization of physical connectivity, our bio-patterned grid networks retained the key properties of synaptic transmission, short-term plasticity and overall network activity with respect to random networks. Interestingly, the imposed grid topology resulted in a reinforcement of functional connections along orthogonal directions, shorter connectivity links and a greatly increased spiking probability in response to focal stimulation. These results clearly demonstrate that reliable functional studies can nowadays be performed on large neuronal networks in the presence of sustained changes in the physical network connectivity

Disrupted Small-World Brain Networks in Moderate Alzheimer's Disease: A Resting-State fMRI Study

The small-world organization has been hypothesized to reflect a balance between local processing and global integration in the human brain. Previous multimodal imaging studies have consistently demonstrated that the topological architecture of the brain network is disrupted in Alzheimer's disease (AD). However, these studies have reported inconsistent results regarding the topological properties of brain alterations in AD. One potential explanation for these inconsistent results lies with the diverse homogeneity and distinct progressive stages of the AD involved in these studies, which are thought to be critical factors that might affect the results. We investigated the topological properties of brain functional networks derived from resting functional magnetic resonance imaging (fMRI) of carefully selected moderate AD patients and normal controls (NCs). Our results showed that the topological properties were found to be disrupted in AD patients, which showing increased local efficiency but decreased global efficiency. We found that the altered brain regions are mainly located in the default mode network, the temporal lobe and certain subcortical regions that are closely associated with the neuropathological changes in AD. Of note, our exploratory study revealed that the ApoE genotype modulates brain network properties, especially in AD patients