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

BLG-derived peptides recognized by the BLG-specific chimeric human IgE monoclonal antibodies and 10 individual CMA serum samples (i.e., peptide recognition by ≥3/10 sera).

<p>BLG-derived peptides recognized by the BLG-specific chimeric human IgE monoclonal antibodies and 10 individual CMA serum samples (i.e., peptide recognition by ≥3/10 sera).</p

Orientation and comparison of the regions recognized by BLG-specific chimeric IgE monoclonal antibodies (A: BLG regions in red) or CMA serum (B: BLG regions in blue) in a three-dimensional ribbon structure of BLG.

<p>Pictures of the three-dimensional ribbon structure of the BLG protein created by using PyMol molecular visualization system version 1.5.</p

RBL-huFcεRI assay with the pool of BLG-specific chimeric human IgE monoclonal antibodies.

<p>Human FcεRI α chain expressing RBL cells were incubated with the serially-diluted pool of chimeric huIgE anti-BLG antibodies (ranging from 0.008 to 8 µg/ml of each individual huIgE; X-axis), and subsequently cross-linked with anti-huIgE antibodies (A; 5 µg/ml), BLG (B: 1 µg/ml), whey (C; (1 µg/ml), and milk powder (D; 1 µg/ml). The percentage of BLG content in each examined preparation is indicated. Results (n = 1) are representative of 1–4 independent experiments.</p

RBL-huFcεRI assay with whey hydrolysates with different peptide size patterns.

<p>Human FcεRI α chain expressing RBL cells were incubated with the pool of chimeric huIgE anti-BLG antibodies (1 µg/ml of each individual huIgE; grey bars), and subsequently cross-linked with anti-huIgE antibodies (5 µg/ml), BLG (1 µg/ml) or whey hydrolysates (1 µg/ml) with different peptide size patterns (HA<10 kDa, HA<5 kDa or HA<3 kDa). Controls: spontaneous degranulation (white bars), i.e., FcεRI α chain expressing RBL cells alone or incubated with pool of chimeric huIgE anti-BLG antibodies (Mab pool) w/o cross-linking, and maximum degranulation (black bar), i.e., FcεRI α chain expressing RBL cells treated with commercial huIgE and anti-huIgE antibodies (both at 5 µg/ml). Results are expressed as mean + SD (n = 2–6); **<i>P</i><0.001 when compared to MAb pool w/o cross-linker.</p

Overview of PCR primer sets for each antibody to amplify VL and VH regions.

<p>Overview of PCR primer sets for each antibody to amplify VL and VH regions.</p