Immunization with Small Amyloid-β-derived Cyclopeptide Conjugates Diminishes Amyloid-β-Induced Neurodegeneration in Mice

Background: Soluble oligomeric (misfolded) species of amyloid-beta (A beta) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against A beta is a promising disease modifying strategy. However, eliciting an immune response against A beta in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric A beta, inducing a specific antibody response. Objective: Here we investigate the protective effects of the experimental vaccine against oligomeric A beta(1-42)-induced neuronal fiber loss in vivo. Methods: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric A beta(1-42) into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled A beta(1-42) peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain. Results: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R-2 = 0.29, p = 0.02). Conclusion: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms

Identification of Formaldehyde-Induced Modifications in Diphtheria Toxin

Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH3, (2) formaldehyde, (3) formaldehyde and NaCNBH3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography–electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD+-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD+-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4+ T-cell epitopes were affected to some extent, one universal CD4+ T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine

Identification of Formaldehyde-Induced Modifications in Diphtheria Toxin

Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH3, (2) formaldehyde, (3) formaldehyde and NaCNBH3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography–electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD+-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD+-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4+ T-cell epitopes were affected to some extent, one universal CD4+ T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine

Immunological Signatures after <i>Bordetella pertussis</i> Infection Demonstrate Importance of Pulmonary Innate Immune Cells

<div><p>Effective immunity against <i>Bordetella pertussis</i> is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a <i>B</i>. <i>pertussis</i> challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against <i>B</i>. <i>pertussis</i>. These responses comprised of innate makers, such as <i>Clca3</i>, <i>Retlna</i>, <i>Glycam1</i>, <i>Gp2</i>, and <i>Umod</i>, next to adaptive markers, such as CCR6⁺ B-cells, CCR6⁺ Th17 cells and CXCR6⁺ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against <i>B</i>. <i>pertussis</i>.</p></div

Pulmonary gene expression profiles in protected and unprotected mice following a <i>B</i>. <i>pertussis</i> challenge.

<p>(A) Fold changes in gene expression of both unprotected and protected mice were calculated compared to naive mice (D0 unprotected). The expression results (FR≥1.5, p-value≤0.001) are visualized as heatmap (mean of n = 3). Genes not exceeding a fold change of 1.5 are depicted as basal level (black) at this time point. In total, 786 genes were found to be differentially regulated. Genes were divided in six clusters (I-VI) based on their expression profiles (color coding for these clusters is depicted in an additional table): cluster I (Differential expression in unprotected mice, absent in protected mice), Cluster II (Differential expression in unprotected mice and protected mice), Cluster III (Differential expression in unprotected mice and in protected mice before and after challenge), Cluster IV (Differential expression in unprotected mice and additional differential expression as result of challenge in protected mice), Cluster V (Absent in unprotected mice but differential expression in protected mice) and Cluster VI (absent in unprotected mice but differential expression pre- and post-challenge in protected mice). (B) Transcriptomic profiles obtained on 2 days p.i. in unprotected and 2 days p.c. protected mice were compared by plotting all 786 genes in a scatter plot and divide the genes in different fractions based on co-expression. The black solid lines are the thresholds for the significant FR (FR ≥ 1.5 or ≤ 0.67) compared to naive mice (D0 unprotected) for both unprotected and protected mice. Black dots represent genes that are not significantly regulated compared to naive mice (D0 unprotected) in both groups. The red solid lines represent the threshold for the significant FR (FR ≥ 1.5 or ≤ 0.67) of both unprotected 2 days p.i. and protected mice 2 days p.c. All red triangles represent genes that show significant differential expression (FR ≥ 1.5 or ≤ 0.67) between unprotected 2 days p.i. and protected mice 2 days p.c. In total, 212 genes were differentially expressed between both groups of which 108 genes were upregulated and 104 were downregulated. These genes were divided in eight fractions that are significantly up-regulated (1–4) or downregulated (5–8) in protected mice compared to unprotected mice and are further specified as heatmaps in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164027#pone.0164027.s001" target="_blank">S1B Fig</a>. Dots with other colors (orange, green, brown, and blue) represent genes that are significantly regulated in unprotected and/or protected mice compared to naive mice (D0 unprotected) but these genes are not differentially regulated between unprotected 2 days p.i. and protected mice 2 days p.c. (C-D) A selection of eight terms (KEGG-pathways and GO-BP terms) found enriched in the ORA of the 786 genes and the kinetics over time of indicated terms is depicted. (C) Kinetics was determined by averaging the FR for each term at each time point and is expressed on LN-scale. (D) For each enriched term, the Benjamini score and the number of upregulated (red) and downregulated (green) genes in the protected mice and unprotected mice is shown.</p

Systemic T-cell responses in unprotected and protected mice.

<p>(A) Study design is depicted for the collection of splenocytes in two different experiments (-D46 and D10). The first experiment (-D46) included unprotected mice 10 days p.i. and naive mice. The second experiment (D10) included unprotected mice 66 days p.i., protected mice 10 days post-secondary challenge, and naive mice. Splenocytes were <i>in vitro</i> restimulated with Prn, FHA or Ptx for 8 days. (B) The percentages of IFNγ-, IL-5-, and IL-17A-producing CD4⁺CD44⁺ T-cells were determined using ICS (n = 6). (C) Cytokine levels of IL-4, IL-5, IL-10, IL-13, IL-17A, IFNγ, and TNFα in supernatant after 7 days of stimulation were determined by using a MIA. Results (mean of n = 6) are corrected for the background level in the presence of medium as control. Statistical differences between the groups were detected for the ICS with a non-parametric Mann-Whitney test and for the MIA with a Student t-test on the log-transformed data. * = <i>p</i><0.05 experimental group versus naive group, # = <i>p</i><0.05 protected group (D10) versus unprotected group (D10).</p

Pulmonary gene expression profiles of genes related to T-cells, B-cells, MHC-I and II and novel genes in protected and unprotected mice following <i>B</i>. <i>pertussis</i> challenge.

<p>Expression profiles of genes in the lungs related to (A) T-cells, (B) B-cells, (C) and antigen presentation by MHC-I and MHC-II were selected according to GO-BP terms, KEGG pathways and text mining. In addition, genes with unknown or poorly understood function that showed interesting gene expression profiles in protected mice were listed as (D) novel genes. Additionally, the color codes of the six clusters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164027#pone.0164027.g002" target="_blank">Fig 2</a> are added.</p

Serum and pulmonary antibody profiles in unprotected and protected mice following <i>B</i>. <i>pertussis</i> challenge.

<p>Serum IgA, IgG, and IgG subclass responses specific for (A) OMV, (B) Ptx, (C) Prn, (D) FHA, and (E) Fim2/3 were determined by using a MIA. Data were obtained in naive mice and protected mice prior to challenge (D0) and 14 days post infection (p.i.) or 14 days post-challenge (p.c.) (n = 3/time point). (F) Pulmonary IgA responses against these antigens were determined on the same time points. * = p<0.05 experimental group versus unprotected group (D0), + = p<0.05 protected group (D0) versus protected group (14 days p.c.). (G) The kinetics of the anti-OMV IgA antibody formation in lung lysates were analyzed at more time points (n = 3/time point) and expressed in fluorescence intensity (F.I.). **** = p<0.0001, challenged unprotected or protected group versus unprotected or protected group (day 0), ++ and +++ = p<0.01 and p<0.001 unprotected group versus protected group (for each time point). (H) Western blot on separated <i>B</i>. <i>pertussis</i> B1917 proteins was performed with pooled lung lysates (1:50) of unprotected and protected mice prior to challenge (D0), and of protected mice 14 days p.c. with IR800-labeled secondary antibody. Left panel shows whole protein range (260kDa-<10kDa) of <i>B</i>. <i>pertussis</i> lysate. Right panel shows more detailed separation of the 110-60kDa protein range. Antigen identification for Vag8 and LPS is depicted.</p

Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity

The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate

Serum cytokine profiles and percentage of splenic neutrophils following <i>B</i>. <i>pertussis</i> challenge in unprotected and protected mice.

<p>(A) The serum concentrations of 33 cytokines were analyzed before and after a <i>B</i>. <i>pertussis</i> challenge in unprotected (lighter blue bars) and protected (dark blue and red bars) mice, as indicated. Concentrations of CXCL13, CCL11, CXCL1, G-CSF, IL-6, CXCL10, and IL-13 serum were significantly altered and represented as mean concentrations of individual values (n = 3). Significant values were calculated by one-way ANOVA with multiple comparison compared to the pre-challenge level (D0) of unprotected mice or protected mice (* = <i>p</i><0.05, ** = <i>p</i><0.01, and *** = <i>p</i><0.001). (B) The percentage of Gr1⁺ cells (neutrophils) was determined over time in the spleen of unprotected and protected mice by using Flow cytometry (* = <i>p</i><0.05).</p