Fc-Mediated Functions of Porcine IgG Subclasses

Abstract

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

Keywords: ADCC; ADCP; CDCC; Fc functions; influenza monoclonal antibodies; porcine IgG subclasses.

Figure 1

HA binding sites of pb27 and pb39. The receptor binding site is shown as a yellow circle and amino acid substitutions selected by antibodies in escape variants are shown as red. Mab pb27 recognized residues in antigenic Ca site surrounding the receptor binding site, namely K130E, G155E, D187E, and Q223R. Whereas, mAb pb39 selected substitution G53D in a Cb site. Substitutions were mapped with Pymol version 1.7 onto PDM:4M4Y.

Figure 2

Genomic context and amino acid alignment of porcine IgG subclasses. The genomic context of the porcine IGH constant region is shown at left. The genes encoding the IgG2 and IgG5 subclasses regions are shaded gray, as these are variable in copy number and/or may not be present depending on an individual’s haplotype. Where applicable, old IgG subclass nomenclature is parenthesized and shown in smaller font below current nomenclature, which is based on Zhang et al., 2020 (10). The flanking genes that encode IgD, IgM, IgE, and IgA are in grey and shown for context. Presently used nomenclature is based on. Gene sizes and intergenic distances are not to scale. The amino acid alignments include the construct sequences used in the present study (“PneoSec”), and genomic sequences from the reference assembly, Sscrofa11 (AEMK02000452), and a previous Yorkshire bacterial artificial chromosome assembly (AB699686 and AB699687) (11). For IgG2b, supporting sequences are from a Xiang pig (XP_IGHG2B) as described by Zhang et al., 2020 and a partial Yorkshire cDNA sequence (U03782). Asterisks (*) at the end of the alignments indicate the termination codons.

Figure 3

Binding and neutralizing activity of pb27 and pb39 IgG subclasses. (A) Binding activity of pb27 and pb39 IgG isotypes. (B) Neutralizing activity of pb27 and pb39 IgG subclasses. Anti-fluorescein human IgG1 (Absolute Antibody) was used as a control. The taller bars represent poorly neutralising antibodies. Representative data from 3 independent experiments shown.

Figure 4

Binding of pb27 IgG subclasses to leucocytes. Binding of free (blue) and H1N1pdm09 virus immune complexed pb27 IgG subclasses (red) to NK cells, monocytes and macrophages. Anti-Nipah virus G protein IgG1 mAb was used as a control. Representative profiles from 3 experiments are shown.

Figure 5

Complement dependent cytotoxicity mediated by pig IgG subclasses. (A) CDCC activity of IgG subclasses were measured on MDCK-HA incubated with a serial dilution of pb27 and pb39 IgG subclasses in the presence of rabbit complement and (B) pig complement. Error bar represents the SD of 3 independent experiments. (C) CDCC activity of immune sera was measured in the presence of rabbit complement, error bar represents SEM, n=6.

Figure 6

NK cell degranulation activity of pb27 and pb39 IgG subclasses. (A) Schematic of the assay setup (figure was created with Biorender.com) and flow cytometry gating strategy for NK cells defined as CD3^-CD8α⁺. (B) Histogram normalized to mode showing the degranulation by pb27 and pb39 subclasses complexed with HA (red) and IgG only (blue). Anti-Nipah virus G protein IgG1 mAb was used as a control. (C) Bar chart showing degranulation by IgG complex with HA (red) or IgG only (blue). The error bars represent the SEM, n=4. (D) Assessment of degranulation titer of immune and naïve sera. Error bar represents the SEM, n=6.

Figure 7

Phagocytic activity induced by pb27 and pb39 IgG subclasses. (A) Gating strategy for flow cytometry analysis. MDCK-HA cells and differentiated macrophages were stained with CFSE and CellTrace Violet respectively and near IR live/dead fixable stain. Macrophages positive for both CFSE and CellTrace Violet were gated to analyse phagocytosis. (B) Phagocytosis of MDCK-HA cells complexed with different pig IgG isotypes, pb27 isotypes (left) and pb39 isotypes (right). Anti-Nipah virus G protein IgG1 mAb was used as a control. Representative data from 3 independent experiments are shown.