FcαRI Dynamics Are Regulated by GSK-3 and PKCζ During Cytokine Mediated Inside-Out Signaling

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics

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

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

Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression

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

Bovine IgG Prevents Experimental Infection With RSV and Facilitates Human T Cell Responses to RSV

Respiratory syncytial virus (RSV) infections represent a major burden of disease in infants and are the second most prevalent cause of death worldwide. Human milk immunoglobulins provide protection against RSV. However, many infants depend on processed bovine milk-based nutrition, which lacks intact immunoglobulins. We investigated the potential of bovine antibodies to neutralize human RSV and facilitate-cell immune activation. We show cow's milk IgG (bIgG) and Intravenous Immunoglobulin (IVIG) have a similar RSV neutralization capacity, even though bIgG has a lower pre-F to post-F binding ratio compared to human IVIG, with the majority of bIgG binding to pre-F. RSV is better neutralized with human IVIG. Consequently, we enriched RSV specific T cells by culturing human PBMC with a mixture of RSV peptides, and used these T cells to study the effect of bIgG and IVIG on the activation of pre-F-pecific T cells. bIgG facilitated in vitro T cell activation in a similar manner as IVIG. Moreover, bIgG was able to mediate T cell activation and internalization of pathogens, which are prerequisites for inducing an adaptive viral response. Using in vivo mouse experiments, we showed that bIgG is able to bind the murine activating IgG Fc Receptors (FcγR), but not the inhibiting FcγRII. Intranasal administration of the monoclonal antibody palivizumab, but also of bIgG and IVIG prevented RSV infection in mice. The concentration of bIgG needed to prevent infection was ~5-fold higher compared to IVIG. In conclusion, the data presented here indicate that functionally active bIgG facilitates adaptive antiviral T cell responses and prevents RSV infection in vitro and in vivo

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HCABD/LCABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LCABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life