Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Jean N Manirarora,Gourapura J Renukaradhya,Joanna Labresh,Yvonne Sullivan,Kristen E Walker,Ore Francis,Joan K Lunney,Veerupaxagouda Patil

doi:10.3389/fimmu.2022.786396

Abstract

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Highlights

Immunological research in pigs remains hindered by limited reagent availability [1,2,3,4]
To define individual antigenic determinants that are recognized by each of the 10 aPoIL-17A monoclonal antibodies (mAbs), a competition ELISA was used to measure the ability of excess unlabeled mAb to inhibit the binding of each biotin-labeled mAb to the target rPoIL-17A
Based on the cross-reactivity patterns shown in Figure 1, and the inhibition patterns shown in Table 2, eight antigenic determinants (A-H) were proposed for the 10 aPoIL17A mAbs: Group A: Represented by aPoIL-17A-1.1, this mAb cross-reacted with human, dolphin, and monkey rIL-17A proteins and was not inhibited by any of the other mAbs

Summary

Introduction

Immunological research in pigs remains hindered by limited reagent availability [1,2,3,4]. Two cytokines of interest are interleukin-17A (IL-17A) and interferon-gamma (IFNg). The IL-17 family is best known for its important role in host defense and immune pathology [5,6,7]. Six members of the IL-17 family, annotated as IL-17A through IL-17F, have been identified [8, 9]. IL-17A is an essential player in host disease defense; aberrant expression of IL-17A can lead to many autoimmune diseases and cancers. IL-17A signaling enhances production of proinflammatory molecules in multiple cell types. T helper 17 (Th17) cells (a subset of CD4+ T cells) and gdT cells are major producers of IL-17A other cell subsets have been indicated [10]

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