The viral protein corona directs viral pathogenesis and amyloid aggregation

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.Peer reviewe

Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins : Studies on applications and toxicity

Cell-penetrating peptides (CPPs) have for a little bit more than a decade been employed as delivery vectors for a wide range of cargoes, ranging from gold particles to entire plasmids. Although CPP are well studied and utilized in numerous publications, our knowledge about CPP mediated transport is still poor. The articles presented in this thesis all consider different aspects of CPP mediated delivery. The first two papers are evaluating and improving already known techniques. In paper I, standard polyethyleneimine (PEI) transfection is improved by conjugating the CPP TP10 to the cationic polymer. In paper II, the same CPP is employed to deliver a dsDNA decoy oligo, resulting in decreased activity of the transcription factor c-Myc. The third paper is a more general overview of the delivery efficiency of well known CPPs and how the delivered cargo influences the CPP mediated toxicity. The study shows that different CPPs are suitable for different cargos and that toxic side effects depend heavily on the cargo and coupling strategy used. In Paper IV, a novel CPP, M918, is evaluated as a delivery vector for a transposon based non-viral gene therapy system. M918 display simultaneous delivery of a plasmid carrying a selection gene and a transposase into cultured cells. This is the first study where two so vastly different molecules as a cationic protein and an anionic plasmid, are simultaneously transported into cells by a peptide vector. The method might be a first step towards a safe peptide based non-viral gene therapy platform. Taken together, the results presented in this thesis might help to improve already existing techniques, increase our understandings about CPP mediated delivery and, at the same time, develop new CPP based delivery systems

Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG₁

<div><p>Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in <i>E. coli</i> and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs). The best variant, denoted Z_F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z_F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z_F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z_F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.</p></div

Analysis of labeling and comparison of the antigen binding potency of differently biotinylated antibody samples.

<p>Two samples of a mouse anti-human interferon-gamma mIgG1 mAb were biotinylated using either a direct or a Z_{F5I-Q32C-MBP-BIO} probe-mediated strategy and the resulting preparations compared with respect for their antigen binding potency. (<b>A</b>) Western blotting analysis of the samples and the probe, using a streptavidin horse radish peroxidase (HRP) conjugate. Lane 1: blank; lane 2: a sample of the anti-human interferon-gamma mIgG1 mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent resulting in "global" biotinylation of both heavy (<i>ca.</i> 50 kDa) and light chains (<i>ca.</i> 25 kDa); lane 3: the Z_{F5I-Q32C-MBP-BIO} probe alone, biotinylated using the same amine reactive sulfo-NHS-ester-biotin reagent; lane 4: a sample of the anti-human interferon-gamma mIgG1 mAb after photoconjugation to the Z_{F5I-Q32C-MBP-BIO} probe (ca. 8 kDa) showing a selective biotinylation of only the heavy chains (<i>ca.</i> 50 + 8 kDa). A small amount of unreacted probe is also visible. (<b>B</b>) Biosensor analysis of the relative antigen binding potency of the biotinylated antibody preparations. Using a streptavidin coated biosensor chip, the biotinylated antibody proteins were selectively immobilized onto separate sensor chip surfaces, followed by injection over both surfaces of a common 7.5 nM solution of the antigen human interferon-gamma. This allowed for direct comparison of the effect on the antigen binding potency from the different biotinylation strategies. Sample (i): the anti-human interferon-gamma mIgG₁ mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent; sample (ii): the anti-human interferon-gamma mIgG₁ mAb biotinylated via photoconjugation using the Z_{F5I-Q32C-MBP-BIO} probe.</p

Photo-conjugation of Z domain probes to mouse IgG₁.

<p>(<b>A</b>): Analysis by SDS-PAGE of the mouse IgG₁ mAb photocoupling efficiency of nine different Z_WT- or Z_F5I-based probes, differing in the position of the maleimide benzophenone (MBP) group. As indicated, variants containing the MBP group at positions 2, 3, 4, 9, 11, 15, 24 or 32 were investigated. (<b>B</b>): Analysis by SDS-PAGE of the photocoupling efficiency of the Z_WT-Q32C-MBP and Z_F5I-Q32C-MBP probes, both containing the MBP group at position 32, to 19 different mouse IgG₁ mAbs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056597#s4" target="_blank">Material and Methods</a> for a list). The designations HC+P, HC, LC and P, refer to heavy chain+probe, heavy chain, light chain and probe, respectively. The M lanes refer to marker protein with molecular weights in kDa as indicated.</p

Structure and sequence data.

<p>(<b>A</b>): Computer graphic representation of a part of the complex between a single staphylococcal protein A domain (domain B, closely related to the Z domain used in the present study) (yellow) and human IgG₁ Fc (brown) (PDB file: 1FC2.pdb). The amino acid side chains corresponding to the seven positions addressed for substitutions in the Z domain are highlighted in cyan. Highlighted in purple, red and green, respectively, are three IgG1 Fc subregions in close contact with the B domain. (<b>B</b>): Alignment of amino acid sequences of Fc regions of human IgG₁, mouse IgG₁, mouse IgG_2a and mouse IgG_2b, respectively, corresponding to the three contact areas shown in (A), using the same colour code. (<b>C</b>): Amino acid sequence of the 58-residue Z domain, with the seven positions included in the engineering boxed. Indicated with red dots are the ten positions at which unique cysteine residues were introduced for site-specific labeling with a photoactivable maleimide benzophenone (MBP) group.</p

Relative binding responses for Z domain variants to mouse IgG₁.

<p>Solutions of Z domain variants (2 µM concentration) obtained after single amino acid substitutions at the seven targeted positions (K4, F5, Q9, Q10, F13, H18 and K35) were injected over a sensor chip surface containing immobilized mIgG₁ monoclonal antibody protein and the equilibrium responses (plateau values) were recorded and normalized to the response values obtained for the wild type Z domain (WT) A blank surface (activated/deactivated) was used as negative control and used for buffer effect subtraction. (<b>A</b>) A representative overlay sensorgram from injections of Z_WT, Z_F5R and Z_F5I variants over mIgG₁ monoclonal protein showing higher equilibrium response values for the two mutant variants than for the Z_WT domain. The response obtained from buffer injection only is also indicated (BUFFER). (<b>B</b>) Results from the analysis of the different variants. The horizontal line in each panel corresponds to the normalized response obtained for the wild type Z domain ( = 100).</p

A Population Genomic Investigation of Immune Cell Diversity and Phagocytic Capacity in a Butterfly

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences