Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Abstract

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.

Fig 1.

Reactivity of each MAb against rfIFN-γ using Western immunoblotting. M, marker; lane 1, Noko 1-12.3; lane 2, Noko 3-31.1; lane 3, Noko 5-23.1; lane 4, Ame 4-12.4; lane 5, Ame 6-31.1; lane 6, Ame 7-19.1. The arrow shows rfIFN-γ-specific band which is observed at around 18 kDa.

Fig 2.

Measurement of the concentration of fIFN-γ in the PBMCs culture supernatants using sandwich ELISA. PBMCs were derived from 10 FIPV-infected cats without FIP (Ia–Ie and IIa–IIe were inoculated with the type I FIPV KU-2 and type II FIPV 79-1146 strains, respectively), three FIP cats (FIPa–FIPc) and six SPF cats (SPFa–SPFf). The cells were cultured with the heat-inactivated FIPV KU-2 strain (grey bar; A), FIPV 79-1146 strain (solid bar; B), or culture medium alone (open bar). Each experiment was performed in quadruplicate. The results are expressed as means±SEM. ∗ indicates a significant difference by the t-test (P<0.01).

Fig 3.

Count of spot-forming fIFN-γ-secreting cells using ELISpot assay. PBMCs were obtained from five FIPV-infected cats without FIP (IIa–IIe were inoculated with the type II FIPV 79-1146 strain), three FIP cats (FIPa–FIPc), and three SPF cats (SPFa, SPFe, and SPFf). The cells were cultured with the heat-inactivated type II FIPV 79-1146 strain (solid bar) or culture medium alone (open bar). Each experiment was performed in quadruplicate. The results are expressed as means±SEM. ∗ and ∗∗, P<0.05 and P<0.01, respectively.

Fig 4.

Calculation of the percentage of CD4⁺fIFN-γ ⁺ and CD8⁺fIFN-γ ⁺ cells in small lymphocyte and lymphoblast populations using two-colour flow cytometry. PBMCs were derived from five FIPV-infected cats without FIP (IIa–IIe were inoculated with the type II FIPV 79-1146 strain), three FIP cats (FIPa–FIPc), and three SPF cats (SPFa, SPFb, and SPFe). The cells were cultured with the heat-inactivated type II FIPV 79-1146 strain (solid bar) or culture medium alone (open bar). A and B represent the percentage of CD4⁺fIFN-γ ⁺ and CD8⁺fIFN-γ ⁺ cells, respectively.