Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk

<div><p>Background</p><p>Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.</p><p>Objective</p><p>To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV.</p><p>Methods</p><p>ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. <i>S. Epidermidis</i> and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.</p><p>Results</p><p>bIgG recognised human RSV, influenza haemagglutinin and <i>Haemophilus influenza</i>. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.</p><p>Conclusions</p><p>The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.</p></div

Bovine Ig enhances internalisation of hRSV by hPMN.

<p>GFP-renilla expressing RSV was pre-incubated with medium in the presence or absence of IVIg or bIgG and allowed to bind to PMN at 4°C. Subsequently, cells were incubated at 37°C and thereafter treated with trypsin and acid to remove extracellular RSV. Cells were then washed and analysed by flow cytometry for the percentage of GFP+ cells. GFP+ cells were tested in the absence of RSV (–), in the presence of RSV but absence of Ig (0) and in the presence of IVIg or bIgG (µg/ml). Mean and S.E.M. of triplicate measurements of one out of five donors tested are shown.</p

bIg-mediated binding and phagocytosis of <i>S. epidermidis</i> by IFN-γ-stimulated monocytes and GM-CSF-differentiated moDCs.

<p>FITC-labelled bacteria were opsonised or not with human (IVIg) or bovine (bIgG) IgG. Subsequently cells were allowed to bind to opsonised bacteria and incubated at 4°C (negative control) or 37°C degrees and stained with APC-conjugated antibodies recognizing FITC. Extracellular bacteria were defined as FITC+APC+ and intracellular bacteria as FITC+APC−. Extracellular bacteria can be observed at both 4°C and 37°C incubated cells, whereas intracellular bacteria are only present in cells incubated at 37°C. A) Example of FACS dot plot and gating strategy. B and C) Percentage of IFN-γ conditioned monocytes (B) and moDCs (C) with extracellular (left) and intracellular (right) bacteria of IVIg (top) and bIgG (bottom) incubated at 4°C or 37°C (indicated at x-axes). Black bars indicate medium (–) or bacteria alone without Ig (0). X-axes show µg/ml Ig used for opsonisation of bacteria. Mean and S.E.M. of triplicate measurements are shown of one out of three donors tested.</p

bIgG binds to human airway pathogens.

<p><b>A</b>) RSV, influenza or <i>Haemophilus influenzae</i> type b was coated in ELISA plates and human (IVIg) or bovine (bIg) IgG was added in different concentrations (x-axes in µg/ml). Mean OD or delta OD (RSV) values and S.E.M. are shown of triplicate measurements. <b>B</b>) Inhibition of binding of IVIg (left) or bIg (right) to vaccines by pre-incubating the Ig-samples (167 µg/ml) with the antigen. Horizontal text below graphs indicates vaccine used for coating, whereas diagonal text indicates the vaccine used for pre-incubation; ‘−’ indicates pre-incubation with medium. Mean and S.E.M. of triplicate measurements are shown.</p

Neutralisation of infection of HEp-2 cells by human RSV.

<p><b>A</b>) 5*10∧4 HEp-2 cells were seeded overnight in flat bottom 96-wells plates and infected the next day with 1*10∧5 PFU RSV-GFP which was pre-incubated with different concentrations of bovine (bIgG) or human (IVIg) IgG or monoclonal F-protein-specific palivizumab. GFP intensity was determined by flow cytometry as a measure for HEp2 cell infection by RSV-GFP. For the calculation of the inhibition percentage the MFI of uninfected cells was set to 100% and the MFI of infected cells without Ab incubation to 0%. Mean and S.E.M. of three independent experiments is shown. <b>B</b>) IC50 values for neutralisation of RSV-GFP by palivizumab, IVIg and bIgG are shown.</p

Targeted Delivery of a Sialic Acid-Blocking Glycomimetic to Cancer Cells Inhibits Metastatic Spread

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F_ax-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F_ax-Neu5Ac into poly(lactic-<i>co</i>-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F_ax-Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F_ax-Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation

Overview of antigenic sites on pre- and postfusion F.

<p>(A) prefusion [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref008" target="_blank">8</a>] and (B) postfusion [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref010" target="_blank">10</a>] structures of RSV F. Antigenic sites recognized by antibodies used in this study are indicated (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref008" target="_blank">8</a>]): site I (green; recognized by MAb 131-2a), site II (blue; recognized by MAb Palivizumab), and site Ø (red; recognized by MAbs D25 and AM22). The region to which the α6HB PAb binds is indicated by the dotted orange circle. Site Ø is disrupted in postfusion F, while site I appears to be shielded in the prefusion conformation.</p

Schematic representation of the different recombinant soluble RSV F protein constructs.

<p>(A) RSV F ectodomains (amino acid 26–513) lacking the transmembrane domain (TM) and cytoplasmic tail (CT) were genetically fused to a CD5 signal peptide (CD5) and to a carboxy-terminal triple Strep tag II (tag). When indicated a GCN4 trimerization motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref034" target="_blank">34</a>] and a LysM linker [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref035" target="_blank">35</a>] immediately downstream of the RSV F ectodomain were added (indicated by GCN). The F2 and F1 subunits of F are indicated, as well as the p27 peptide (P27) that is released after furin cleavage. Protease cleavage sites are indicated by black arrows. Grey arrows indicate mutated furin cleavage sites. The approximate location of the fusion peptide (FP), heptad repeat A (HRA) and B (HRB) is also shown. The residues substituted in HRB by alanines or cysteines in Flys.HRBala-GCN and Flys.HRBcys-GCN, respectively, are indicated. The amino acids deleted in the F proteins that lack HRB are also indicated. (B) SDS-PAGE and Western blot analysis of recombinant soluble F proteins. Purified F proteins Fwt (lane 1), Flys-GCN (lane 2), Flys.∆HRB-GCN (lane 3), Flys.∆HRB (lane 4), Flys.HRBala-GCN (lane 5), Flys.HRBcys-GCN (lane 6) were analyzed by SDS-PAGE followed by Western blotting. Prior to SDS-PAGE analysis, the samples were resuspended in LSB containing 5% ME and heated at 96°C for 5 minutes. The size of the molecular mass markers (in kDa) is shown on the left side.</p

Reactivity of full length RSV F protein lacking HRB with pre- and postfusion-specific antibodies.

<p>(A) Cells transfected with expression plasmids for full length wild-type (Fwt) or full length proteins lacking HRB (Fwt.ΔHRB) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI.</p

Reactivity of full length RSV F proteins with pre- and postfusion-specific antibodies.

<p>(A) Cells transfected with full length F protein expression plasmids or (B) infected with RSV (strain Long) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI. Wild type F protein (Fwt) or F proteins containing cysteine pairs in HRB (Fcys; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130829#pone.0130829.ref017" target="_blank">17</a>]) were expressed. (C) Sandwich ELISA of RSV virus particles. RSV particles (+RSV) were captured using 131-2a or AM22. Capture of virus particles was detected using a PAb against RSV. As controls the experiment was performed without adding RSV particles (-RSV) or after heating of the particles (+heated RSV).</p