Production of IFN-γ in feline whole blood after incubation with potential T-cell epitopes of the nucleocapsid protein of feline coronavirus

Abstract

Interferon gamma (IFN-γ) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-γ in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-γ concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-γ mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-γ in samples incubated with peptides decreased and the expression of IFN-γmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a "pre-activated" immune response). These cats responded to "avirulent" or "mixed" peptides by increasing the concentration of IFN-γ and the expression of IFN-γ mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-γ especially in cats with a "pre-activated" cell mediated response.

Characterization of peritoneal cells from cats with experimentally-induced feline infectious peritonitis (FIP) using RNA-seq

“…However, evidence that cellular immunity is crucial for preventing FIP is sparse and largely indirect. PBMC or whole blood cultures from FIPV immune cats proliferate in response to live virus exposure in vitro [ 57 ] and produce IFNγ in response to T cell epitopes of the viral N protein [ 61 ]. Delayed-type hypersensitivity responses can be elicited in the palpebral mucosa [ 58 ] or skin [ 62 ] with FIPV antigen in immune cats.…”

An update on feline infectious peritonitis: Virology and immunopathogenesis

“…A test to measure the ability of leukocytes in whole blood to produce IFN-γ (protein and mRNA) in vitro in response to eight immunogenic peptides derived from the N protein of virulent (FIPVs) and avirulent (FECVs) feline coronaviruses has been developed for cats (Rossi et al, 2011). The sequences used were obtained from an earlier study by Battilani et al (2010) that compared a small number of N gene sequences from lesions of cats with FIP and feces of clinically healthy cats, and purported to show N epitopes that would differentiate the two.…”

Leishmania infantum-specific IFN-γ production in stimulated blood from cats living in areas where canine leishmaniosis is endemic

“…However, detection of IFN-γ in antigen stimulated whole blood or peripheral blood mononuclear cells (PBMCs) was used in cats to evaluate cellular immune response mechanisms to other pathogens (e.g. feline coronavirus or Toxoplasma gondii ) [ 16 – 18 ].…”