key: cord-287157-6rwevq39 authors: Kiss, I.; Poland, A.M.; Pedersen, N.C. title: Disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (FIPV)-UCD1 and challenge-exposed with virulent FIPV-UCD8 date: 2004-02-25 journal: J Feline Med Surg DOI: 10.1016/j.jfms.2003.08.009 sha: doc_id: 287157 cord_uid: 6rwevq39 Eight cats were immunized with an avirulent strain of feline infectious peritonitis virus (FIPV)-UCD1, then challenge-exposed to a highly virulent cat passaged strain (FIPV-UCD8). Th1 and Th2 cytokine profiles in the peripheral blood mononuclear cells (PBMCs) were measured throughout in the experiment. No clinical signs of FIP were evident in the experimental cats after immunization. After challenge, the immunized cats demonstrated one of four clinical outcomes: (1) classical effusive FIP; (2) accelerated FIP; (3) non-effusive FIP, or (4) resistance to challenge. Only minor cytokine changes were observed following immunization, however, several cytokine changes occurred following challenge-exposure. The most noteworthy changes were in tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) levels. Our preliminary findings suggest that immunity against FIP is associated with TNF-α and IFN-γ response imbalance, with high TNF-α/low IFN-γ mRNA responses favouring disease and low TNF-α/high IFN-γ mRNA responses being indicative of immunity. Feline infectious peritonitis (FIP) is a highly fatal disease in Felidae caused by a coronavirus and usually affects cats between 6 months and 3 years of age (reviewed by Pedersen, 1995) . The FIPV is a common mutant of the far more ubiquitous and largely non-pathogenic feline enteric coronavirus (FECV) (Pedersen, 1987a; Vennema et al., 1998) . The enteric virus is highly tropic to the mature epithelium of the small intestine, whereas the mutant FIPV gains a tropism for macrophages. This macrophage tropism allows the virus to disseminate throughout the body and is responsible for pathogenicity (Stoddart and Scott, 1989 ). The disease presents in two major forms: (1) an effusive form associated with peritonitis and/or pleuritis and vasculitis, and (2) a non-effusive form characterized by more classical granulomatous disease of major abdominal organs, uveal tract, and meninges (reviewed by Pedersen, 1995) . Both forms of the disease are uniformly lethal, with affected cats dying over several days to many months. Immunity to FIPV is presumed to be cellular (Pedersen, 1987b) , as is typical of highly cell-associated pathogens (Tizard, 2000) . Antibody responses, by themselves, are actually harmful by facilitating virus uptake by macrophages and participating in Arthus like reactions (Pedersen and Boyle, 1980) . Therefore, whether a cat develops wet or dry FIP is thought to depend on the strength of the cat's cell-mediated immune response. Cats producing a negligible cell-mediated response and a strong antibody response rapidly develop acute wet FIP. Cats producing a partial cell-mediated response develop the chronic form of the disease. There is strong circumstantial evidence that susceptibility to FIP has a significant genetic component (Foley et al., 1997) . The goal of developing effective FIPV vaccines has been elusive. Most vaccines either fail to protect or enhance the infection (Pedersen, 1989) . Partial success has been obtained by using temperature sensitive mutants of a type II strain of FIPV (Gerber et al., 1990; Hoskins et al., 1995; Reeves et al., 1992) . We were fortunate to create another avirulent FIPV, but to a preferred type I rather than type II strain (FIPV-UCD1). Type II strains are recombinants with canine coronavirus, while type I strains are uniquely cat. Type I strains cause 70-95% or more of disease (Hohdatsu et al., 1992) . We decided to test our avirulent strain of FIPV-UCD-1 as a vaccine. After primary vaccination cats were then challenge-exposed to a highly virulent type I strain of FIPV (FIPV-UCD8). In order to see whether there was a relationship between vaccine immunity and challenge-exposure outcome, we also analysed Th1 and Th2 cytokine profiles during immunization and following challenge-exposure. Ten 5-month-old male cats were obtained from the specific pathogen free breeding colony of the Feline Nutrition Laboratory, School of Veterinary Medicine, UC Davis, USA. Cats were housed in the facilities of the Center for Companion Animal Health, School of Veterinary Medicine, UC Davis, USA under the supervision of the Center for Laboratory Animal Sciences. Eight cats were inoculated intraperitoneally, first with a non-pathogenic strain of FIPV-UCD1, then 32 days later with pathogenic FIPV-UCD8. Attenuated FIPV-UCD1 was passaged on Fcwf-4 cells and animals inoculated with 1 ml of infectious tissue culture fluid. FIPV-UCD8 was serially passaged in laboratory cats and infectious material was in the form of 1 ml of pooled ascitic fluid. This fluid had been harvested from cats dying of experimentally induced effusive FIP. Two control animals were mock vaccinated and challenge-exposed with PBS. The clinical status of the cats was monitored throughout the experiment. Three millilitres of heparinized blood were collected at the following time points: day −3, day 0 (time of experimental infection with UCD-1), day 4, day 7, day 11, day 14, day 18, day 21, day 28, day 32 (time of experimental infection with UCD-8), day 35, day 39, day 42, day 46, and day 49. All animals were euthanased on day 49. A complete necropsy was performed on each animal, including histologic examination of a range of potential target tissues. Necropsies were done to confirm the form of disease and to rule out the presence of subclinical infections in animals that appeared outwardly normal (Hoskins et al., 1995) . Blood samples were immediately centrifuged in order to separate the buffy coat and plasma for cytokine analysis and serology, respectively. All fractions were stored at −80°C until further processing. Three time points (day 21, day 42, and day 49) were selected for the serological investigations of the plasma samples by IFA technique using serum dilutions of 1:25 and 1:100 (Pedersen, 1976) . IFA substrate slides were made from Fcwf-4 cells infected with FIPV-UCD1. Cytokine mRNA measurements from unstimulated peripheral blood mononuclear cells (PBMC) were performed for the following cytokines: IL4, IL6, IL10, IL12 p40, IL18, IFN-, and TNF-as described previously (Kipar et al., 2001; Leutenegger et al., 2000) . Relative cytokine mRNA levels were determined at the time points indicated above and calibrated against cytokine mRNA levels measured 3 days prior to immunization and from the two control animals. Experimentally infected cats were euthanased and necropsied when it became obvious that their disease was terminal or at the completion of the study (day 49). The form of FIP was determined grossly and histologically. No clinical signs of illness were observed in the eight experimental cats after immunization with FIPV strain UCD1, except for slight rise in rectal temperatures lasting 1 to 3 days (data not shown). All but one (cat 622) vaccinated cats seroconverted, but to low titer (Table 1) . Three of eight vaccinated cats (nos 522, 616, 622) developed effusive FIP within 2 weeks of challengeexposure to FIPV-UCD8, typical of classical nonenhanced disease (Pedersen and Boyle, 1980) ( Table 1) . One of the eight cats (no. 527) developed FIP within 4 days, characteristic of enhanced disease (Pedersen and Boyle, 1980) . Two cats (nos 623, 624) developed non-effusive FIP. Two of eight cats (nos 524, 625) showed no signs of illness, as did the two control animals. Secondary antibody responses appeared to reflect disease outcome (Table 1 ). The three cats (nos 522, 616, 622) dying of classical effusive FIP had a rise in antibody titer post-challenge-exposure. Cats nos 623 and 624, which developed non-effusive FIP, had low primary antibody responses at day 21 postimmunization and with the same or decreased antibody titers 10 days post-challenge-exposure (day 42). The antibody titers of the two cats (nos 524 and 625) that resisted challenge-exposure declined to negligible levels even after challengeexposure with FIPV-UCD8. No major changes in cytokine mRNA levels were detected following the initial non-pathogenic FIPV-UCD1 immunization (Figs. 1-4 ) when compared to individual pre-infection values and to parallel cytokine responses in the two control cats (data not shown). A moderate elevation of TNF-mRNA occurred between the second and third week after vaccination in cat 527. A small post-immunization increase of the IFN-mRNA level was seen in cat 524, which was one of the ultimate survivors. Various cytokine mRNA changes were observed following challenge-exposure with FIPV-UCD8 (Figs. 1-4) . IL-4 and IL-6 mRNA levels did not change from pre-infection or control cat levels in any of the eight infected animals. Slight to low increases in IL-10 mRNA were seen following FIPV-UCD8 infection in all cats, while IL-18 mRNA levels increases were negligible to low following challengeexposure and bore no relationship to disease outcome (data not shown). Changes, or lack thereof, were deemed significant for three cytokines mRNAs, IFN-, TNF-and IL-12p40. The level of IFN-mRNA was strongly elevated in one of the surviving two cats; in the other one it remained unaltered. Cats that developed FIP had negligible or below normal IFNresponses, save cat 527 that showed slightly elevated levels of this cytokine on the day of challenge-exposure. All of the cats that developed FIP, regardless of form, had elevated postchallenge-exposure levels of TNF-mRNA. IL-12 mRNA responses were increased following infection with FIPV-UCD8, but did not appear to relate to disease outcome or IFN-/TNF-mRNA responses. FIPV-UCD1 immunization induced only partial protection at best, as gauged against historical data. Animal-passaged FIPV-UCD8 usually kills from 90-100% of inoculated cats, almost always from effusive FIP (NC Pedersen, UC Davis, unpublished information). In this study, two of eight vaccinated cats (nos 524 and 625) appeared immune to challenge-exposure with virulent FIPV-UCD8 and two (nos 623 and 624) developed non-effusive FIP (indicative of partial immunity; Pedersen, 1995) . Three immunized cats (nos 522, 616, and 622) died of the classical form of effusive FIP, indicating a predominance of non-protective humoral immunity. One cat (no. 527) developed a highly accelerated form of effusive FIP, indicating that immunization had elicited enhancing type antibodies (Pedersen and Boyle, 1980) . Therefore, immunization induced a spectrum of immune effects, ranging from protection (two cats), partial immunity (two cats), typical humoral immunity (three cats), to antibody enhancement (one cat). This reflects past experiences with attenuated live FIPV vaccines (McArdle et al., 1995; Pedersen and Black, 1983; Scott et al., 1995) . Although this pilot study of a potential FIP vaccine was deemed largely a failure as far as protection was concerned, there were interesting findings in regards to Th1 and Th2 type cytokine responses and post-challenge exposure disease course. We were fortunate in this study to have cats representing each of the four possible disease outcomes: (1) classical effusive FIP occurring about 2 weeks following infection; (2) enhanced FIP occurring almost immediately after challenge, (3) non-effusive FIP, and (4) resistance. Based on the cytokine profile analysis of the different groups, we suggest that disease, regardless of form, is associated with a strong TNF-mRNA response in PBMC and a failure to induce IFN-mRNA. In contrast, immune cats failed to upregulate TNF-mRNA and one manifested strong IFN-mRNA responses. The former profile tends to favor Th2 (humoral) immunity, while the latter favors Th1 (cell-mediated) immun-ity. FIPV is an intracellular pathogen of macrophages, and as such, cell mediated immunity would be most important. It was interesting to note the relationship between responses to avirulent or virulent virus and the magnitude and even duration of cytokine mRNA responses in PBMCs. Cats infected with the avirulent FIPV only showed transient fevers (data not shown) and low to negligible changes in cytokine mRNAs in PBMCs. The cat that had the most pronounced primary cytokine responses was also the animal that developed the most severe febrile reaction to the vaccine. This indicated that changes in cytokine mRNAs within PBMCs were only noticeable when reactions within internal lymphoid organs reached a certain threshold, thus allowing the responses to spill over into the blood. This was also observed after challenge-exposure. Cats that became very sick with FIP tended to have marked upregulation of cytokine mRNAs in their PBMCs. A dichotomy in responses was seen between the two cats that were immune to challenge exposure with virulent FIPV-UCD8. Cat no. 524 developed weak cytokine reactions during immunization and strong cytokine responses following challenge-exposure, while cat 625 showed very little changes during either immunization or challenge-exposure. This suggested that cat no. 524, while being immune, was none the less infected by the challenge virus and did mount a vigorous secondary immune response (at the cytokine, but not antibody level). In contrast, cat 625 appeared to develop exceptionally strong protective immunity from the onset, precluding the need for a systemic response. Therefore, the magnitude and duration of cytokine mRNA responses in PBMCs does not always correlate with strength of immunity. The importance of IFN-responses in FIPV immunity is strongly supported by what has been described recently for both experimental (Kyuwa et al., 1998a,b) and natural (France et al., 1999) mouse hepatitis virus (MHV) infections. MHV, like the feline coronaviruses (Horzinek et al., 1995) , exists in two biotypes, a naturally occurring and largely enterotropic biotype and a more laboratoryassociated polytropic biotype (Homberger et al., 1998) . The enterotropic biotype of MHV is analogous to FECV, while polytropic biotypes have many parallels with FIPV. The similarities between feline and murine coronaviruses and their biotype-associated diseases were quickly noted following the creation of IFN-deficient mice. IFNknockout mice developed severe peritonitis, identical to FIP, upon experimental challenge with a polytropic MHV (Kyuwa et al., 1998a (Kyuwa et al., , 2002 and this disease could be partially inhibited with exogenously administered IFN- (Kyuwa et al., 2002) . This supports our findings on the importance of IFN-mRNA responses in cats exposed to FIPV. A granulomatous peritonitis and pleuritis has also been described in a colony of IFN-knockout mice infected naturally with enterotropic MHV (France et al., 1999) . This latter observation parallels what is seen in a group of retrovirus immunocompromised cats exposed to FECV (Poland et al., 1996) . Other similarities exist between murine and feline coronavirus infections. Age and genetic factors have been shown to play a role in naturally occurring MHV infection, with 1 week olds being more susceptible than 3 and 12 weeks, and Balb and ICR mice more susceptible than SJL mice (Barthold, 1987) . Age and genetic susceptibility have been shown to be important risk factors for FIP in purebred cats (Foley et al., 1997; Foley and Pedersen, 1996) . The central question coming out of this preliminary experiment and previous MHVrelated studies concerns the role of IFN-in FIPV immunity. In the presented preliminary experiment, vaccination of cats with an attenuated live strain of FIPV-UCD1 appeared to induce a degree of protection, in that two of eight cats were immune and two more developed non-effusive FIP post challenge. A more significant finding was the possible relationship between certain cytokine mRNA responses and disease outcome. 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Kiss was provided by a fellowship from the Fulbright Foundation and laboratory costs by the Center for Companion Animal Health Center (CCAH), School of Veterinary Medicine, University of California, Davis, CA, USA, 95616. Additional thanks go to Jill Mikovich and the animal care staff at the CCAH for their care of the animals, and to Dr Árpád Bacsadi, Veterinary Institute of Debrecen, P.O. Box 51, H-4002, Debrecen, Hungary for histopathological studies. We wish to also thank Dr Christian Leutenegger and the TaqMan ® Service, UC Davis for invaluable assistance with cytokine assays.