Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis

31.3.2020, Translation 25.4.2021
Tomoyoshi Doki, Masahiro Toda, Nobuhisa Hasegawa, Tsutomu Hohdatsu & Tomomi Takano
Original article: Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis

Abstract

Infectious feline peritonitis (FIP) is a deadly disease of wild and domestic species of cats. Although some drugs have been shown to be effective in the treatment of FIP, they are still not available in clinical practice. In this study, we evaluated the therapeutic effect of the combined use of adalimumab (anti-human TNF-alpha monoclonal antibody, ADA) and itraconazole (ICZ), which is currently available to veterinarians. ADA neutralizing activity against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. Ten specific pathogen-free (SPF) cats were inoculated intraperitoneally with FIPV KU-2 type I. Cats that developed FIP were administered ADA (10 mg / animal) twice between day 0 and day 4 after the start of treatment. ICZ (50 mg / head, SID) was administered orally every day from day 0 after the start of treatment. ADA demonstrated dose-dependent neutralizing activity against rfTNF-alpha. In an animal experiment, 2 of 3 cats showed an improvement in FIP clinical symptoms and biochemistry results, an increase in peripheral blood lymphocytes, and a decrease in plasma alpha 1-AGP levels after initiation of treatment. One of the cats did not respond to treatment and was euthanized, although the level of the viral ascites gene temporarily decreased after treatment. ADA was found to have neutralizing activity against rfTNF-alpha. The combined use of ADA and ICZ has shown a therapeutic effect on experimentally induced FIP. We consider these drugs to be a treatment option until effective anti-FIPV drugs are legally available.

Introduction

Feline Infectious Peritonitis Virus (FIPV), Feline Coronavirus (FCoV) Coronaviridae, causes a deadly disease in wild and domestic cat species called feline infectious peritonitis (FIP). In cats that develop FIP, several organs are affected, including the liver, lungs, spleen, serosis, kidneys, eyes, and central nervous system, and the formation of lesions in these organs is accompanied by necrosis and pyogenic granulomatous inflammation. Accumulation of pleural effusion and ascites fluid has been reported in some cats [1].

The FCoV virion consists mainly of nucleocapsid (N), envelope (E), membrane (M) and peplomer spike (S) proteins. FCoVs are classified into two serotypes, FCoV types I and II, based on differences in the amino acid sequence of the S protein. [2, 3]. FCoV type II was generated by genomic recombination between FCoV type I and canine coronavirus II. type (CCoV) [4,5,6]. Several serological and genetic surveys have shown that type I FCoV is dominant and that most cases of FIP are caused by FIPV type I infection [7,8,9].

We have previously shown that TNF (tumor necrosis factor) -alpha plays a crucial role in the progression of FIP. TNF-alpha is overproduced by FIPV-infected macrophages. TNF-alpha is involved in lymphopenia and elevated levels of the cellular receptor FIPV serotype II, aminopeptidase N (APN) [10, 11]. Neutrophil apoptosis in cats with FIP is also reported to be inhibited by TNF-alpha [12]. This finding suggests that neutrophilia in cats with FIP is due to TNF-alpha-induced neutrophil survival. We have found that monoclonal antibody (mAb) 2-4 with high neutralizing activity against feline TNF-alpha (fTNF-alpha) is useful as a treatment for FIP [13, 14]. However, the anti-fTNF-alpha mAb has never been marketed and cannot be used clinically in animal hospitals. In human medicine, the anti-human TNF-alpha mAb is used to treat inflammatory diseases such as rheumatoid arthritis and Crohn's disease. [15]. It is commercially available and can also be used in veterinary practice. However, it is not known whether this human antibody is effective against FIP, feline disease.

Several antiviral agents have been described for the treatment of FIPV [16,17,18,19,20]. These drugs have been shown to reduce FIPV proliferation, but are not commercially distributed as veterinary drugs. We have previously found that itraconazole (ICZ), which is used in veterinary medicine, reduces the multiplication of FIPV type I [21,22,23]. However, the therapeutic effects of ICZ in cats with FIP remain unclear.

In this study, we evaluated whether adalimumab (ADA), an anti-human TNF-alpha mAb, has neutralizing activity against fTNF-alpha. We further evaluated the therapeutic effects of ADA and ICZ on FIP by administering them to cats with experimentally induced FIP.

Materials and methods

Cell cultures and viruses

WEHI-164 mouse sarcoma cells were maintained in RPMI 1640 growth medium supplemented with 10% FCS, 600 U potassium benzylpenicillin per ml, 240 μg streptomycin sulfate per ml and 50 μM 2-mercaptoethanol. WEHI-164 mouse sarcoma cells were from the American Type Culture Collection (ATCC CRL1751).

FIPV KU-2 type I was grown in whole Felis catus fetal (fcwf) -4 cells at 37 ° C. FIPV type I KU-2 type I was isolated in our laboratory.

Monoclonal antibody

Anti-fTNF-alpha mAbs 2-4 have been described in [13] and have been shown to have neutralizing activity for recombinant fTNF-alpha (rfTNF-alpha) and native fTNF-alpha. mAbs 2-4 were purified from the hybridoma culture supernatant using Protein G Sepharose (GE Healthcare Life Sciences, Marlborough, MA, USA). The anti-human TNF-alpha mAb, adalimumab (ADA, Humira®), was purchased from Eisai Co., Ltd. (Tokyo, Japan) as a 20 mg / 0.2 ml solution. ADA was diluted to a concentration of 2 mg / ml in saline.

Itraconazole

Itraconazole (ICZ) was purchased from NICHI-IKO (Toyama, Japan) and provided as a tablet (100 mg). The cats were given half a tablet, ground to a powder and mixed with food.

Neutralization of feline TNF-alpha by ADA in WEHI-164 cells

TNF-alpha neutralization in WEHI-164 cells was assayed as described by Doki et al. [13]. Briefly, WEHI-164 cells were suspended at a density of 1 × 106 cells / ml in dilution medium containing 1 μg actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) per ml and preincubated at 37 ° C for 3 hours. Serially diluted mAbs were mixed with 40ng rfTNF-alpha per ml. The mixture was incubated at 37 ° C for 1 hour. Preincubated cells were seeded in a volume of 50 μl in wells of a 96-well plate. Then 50 microliters of the mixture was added to each well. After incubation at 37 ° C for 24 hours, 10 μl of WST-8 solution (WST-8 Cell Proliferation Assay Kit; Kishida Chemical Co., Ltd., Osaka, Japan) was added, and the absorbance of the formazan produced was measured at 450 nm. The percent neutralization was calculated using the following formula: Neutralization (%) = (OD wells containing mAb and rfTNF-alpha - OD wells containing rfTNF-alpha without mAb) / OD wells without mAb and rfTNF-alpha × 100.

Experiment on animals

All relevant national and institutional guidelines for the care and use of animals have been complied with. The protocol on animal experimentation was approved by the President of Kitasato University based on the decision of the Institutional Committee on the Care and Use of Animals at Kitasato University (Approval No. 18-152). Specific pathogen-free (SPF) cats were bred in our own laboratory and maintained in an isolated temperature-controlled facility.

Experimental schedule

According to the experimental schedule shown in FIG. 1, 10 SPF cats (9- or 10-month-old domestic shorthair cats) were inoculated intraperitoneally with FIPV KU-2 type I (103.6 TCID50 / ml / animal). The cats were then examined daily for clinical signs and their body temperature and weight were measured. Cats that developed FIP were administered ADA (10 mg / animal) twice intravenously between day 0 and day 4 after the start of treatment. ICZ (50 mg / animal, SID) was administered orally every day from day 0 after the start of treatment. Blood was collected from cats at weekly intervals after virus inoculation and at the time of ADA administration. Heparinized blood was used to measure complete and differential cell numbers and to isolate plasma. Plasma samples were stored at -30 ° C until the day of analysis. Cats were euthanized after reaching the human endpoint or 60 days after infection. The diagnoses of FIP were confirmed by post-mortem examination with the detection of peritoneal and pleural effusions and pyogranuloma in the main organs.

Figure 1
Experimental schedule of treatment and vaccination of FIPV cats

Measurement of plasma alpha1-glycoprotein (AGP)

AGP plasma concentrations were determined using an alpha1 AG cat plate (The Institute for Metabolic Ecosystem Lab., Osaki, Japan) according to the manufacturer's protocol.

Measurement of plasma vascular endothelial growth factor (VEGF) concentration

Plasma VEGF concentrations were determined using a human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. The ELISA kit primarily detects feline VEGF isoform 164 [24].

Real-time quantitative RT-PCR (qRT-PCR)

To quantify viral RNA in supernatant and ascites cells, qRT-PCR was performed using a primer targeting the 3'-UTR [17]. One milliliter of ascites was collected from cat no. 6 and after centrifugation, the supernatant and cells were stored separately. Total RNA was isolated from the supernatant and ascites cells using a high purity RNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. RNA was reverse transcribed and amplified using RNA-direct Realtime PCR Master Mix (TOYOBO, Osaka, Japan) with specific primers 3′-UTR-F (5′-GGAGGTACAAGCAACCCTATT-3 ') and 3′-UTR-R (5' -GATCCAGACGTTAGCTCTTCC-3 ') and probe (FAM-5′-AGATCCGCTATGACGAGCCAACAA-3'-BHQ1). The reaction was performed in a total volume of 20 μl / well in 48-well PCR plates using the StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) at 90 ° C for 30 s, 60 ° C for 20 minutes and at 95 ° C for 1 minute, followed by 45 cycles at 90 ° C for 15s and 60 ° C for 1 minute. To obtain control RNA for quantification, cDNA fragments amplified with primers 3'-UTR-F and 3'-UTR-R were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). The linearized and purified plasmid was transcribed using the RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, WI, USA) according to the manufacturer's instructions. The concentration of transcribed RNA was measured using a spectrophotometer. Ten-fold dilutions of RNA transcript ranging from 1 × 10 were prepared1 up to 1 × 109 copies / μl with 10ng MS2 RNA per ml. RNA copy number was calculated according to the procedure described in Fronhoffs et al. [25]. In vitro stock solutions of transcribed RNA were stored at -80 ° C and diluted working solutions were stored at -30 ° C.

Protein sequence

The deduced amino acid sequences of feline and human TNF-alpha were obtained from RefSeq (accession numbers NP_001009835.1 and NP_000585.2). Comparison of the deduced amino acid sequences of feline and human TNF-alpha was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

The results

Neutralizing activity of ADA against rfTNF-alpha

A comparison of the deduced amino acid sequences of feline and human TNF-alpha is shown in FIG. 2. The sequences were found to be identical to 89.7%.

Figure 2.
Amino acid sequence comparison of feline and human TNF-alpha. *: residues that interact with adalimumab

The neutralizing activity of ADA against rfTNF-alpha was measured using the WEHI-164 cytotoxicity assay. ADA neutralized rfTNF-alpha activity in a dose-dependent manner, similar to anti-fTNF-alpha mAb 2-4 (Fig. 3). The dose of mAb2-4 and ADA required to inhibit 50% cell death (IC50) was 18.1 ± 4.9 ng / ml and 36.9 ± 23.4 ng / ml, respectively.

Figure 3.
Neutralization dose-response curve against recombinant fTNF-alpha. ADA neutralizing activity against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. WEHI-164 cells were treated with mixtures of serial dilutions of ADA and fTNF-alpha and the level of TNF-alpha-induced cytotoxicity was measured after 24 hours.

Changes in body temperature and body weight

Ten SPF cats were inoculated intraperitoneally with 103.6 TCID50 FIPV KU-2 type I. Three cats (2, 3 and 6) showed fever, anorexia, lethargy and jaundice. Abdominal distension caused by ascites was observed in cat no. 6. These 3 cats were used in the following experiments as FIP cats. Treatment for cats 2, 3 and 6 was started on days 34, 32 and 21 after virus vaccination.

The changes in body temperature and body weight of the three cats are shown in FIG. 4. All developed fever ≥ 39 ° C several days before treatment (Fig. 4A). In cats 2 and 3, body temperature decreased to a pre-FIP range on day 7 after the start of treatment. Then the body temperature of cat no. 3 increased to 38.7 ° C on day 31 after the start of treatment. Cat body temperature no. 6 remained at ≥ 39.0 ° C even after the start of treatment.

Figure 4.
Changes in body temperature and weight of cats. †: The cat was euthanized because its clinical condition reached a human endpoint

In cats 2 and 3, no weight loss was observed compared to body weight on day 0 of treatment (Fig. 4B). Body weight of cat no. 6 continued to decrease from day 0 to day 15, resulting in a 8% loss compared to day 0.

Changes in WBC and lymphocyte counts

Total white blood cell and lymphocyte counts were measured in FIPV-infected cats. Cats 2 and 3 showed a small change in total white blood cell count throughout the experiment (Fig. 5A). In cat no. 6, the total white blood cell count increased after the start of treatment and remained at ≥ 20,000 cells / μl after day 6.

Figure 5.
Changes in WBC and lymphocyte counts in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

In cats 2 and 3, the number of lymphocytes increased slightly after the start of treatment (Fig. 5B). In cat no. 6, the lymphocyte count remained low throughout the experiment. It decreased to approximately 800 cells / μl on day 6 after the start of treatment, but recovered to near pre-FIP levels on day 13 and did not show a significant change up to the human endpoint.

Changes in alpha1-acid glycoprotein (AGP) plasma concentrations

Plasma alpha 1-AGP levels were measured sequentially in three cats. In all three cats, it increased to a high level ≥ 750 μg / ml at the start of treatment (Fig. 6). In cats 2 and 3, plasma alpha 1-AGP levels decreased to near pre-FIP levels on days 14 and 16 after treatment. However, in cat no. 6, the increase in plasma alpha 1-AGP levels was suppressed until day 6 after the start of treatment, but increased to approximately 1,900 μg / ml on day 13.

Figure 6.
Changes in plasma alpha1-acid glycoprotein (AGP) levels in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

Changes in plasma vascular endothelial growth factor (VEGF) concentration

Plasma VEGF levels were measured sequentially in three cats. In cat no. 2 remained low (FIG. 7). In cat no. 3, increased temporarily to approximately 200 pg / ml on day 5 before treatment, but decreased to near pre-FIP levels on day 0 of treatment and showed no increase after treatment. In cat no. 6, plasma VEGF levels increased to approximately 170 pg / ml after FIPV inoculation on day 0 of treatment. It then decreased from day 6 of treatment and was similar to the level before the onset of FIP on day 13.

Figure 7.
Changes in plasma vascular endothelial growth factor (VEGF) levels in cats. †: The cat was euthanized because its clinical condition reached a human endpoint

Levels of viral RNA in supernatant and ascites cells in cat no. 6

Levels of viral RNA in the supernatant and ascites cells collected from cat no. 6 were quantified by qRT-PCR. The level of viral RNA temporarily decreased on day 4 after the start of treatment in the supernatant and in the ascites cells (Fig. 8). However, on day 20 after the start of treatment, viral RNA levels in the supernatant and ascites cells doubled on day 0 of treatment.

Figure 8.
Levels of viral RNA in the supernatant and ascites cells collected from cat no. 6 were quantified by qRT-PCR. The level of viral RNA temporarily decreased on day 4 after the start of treatment in the supernatant and in the ascites cells (Fig. 8). However, on day 20 after the start of treatment, viral RNA levels in the supernatant and ascites cells doubled on day 0 of treatment.

Biochemical panel

After treatment, TP and A / G ratio, which are diagnostic indices of FIP, and AST, LDH, and TB, which are related to liver function, were measured (Fig. 9). TP increased slightly in cat no. 3, but remained within the normal range for all three cats. On the other hand, the A / G ratio decreased after FIPV exposure in all three cats and was ≤ 0.8 on day 0 of treatment. In cat no. 2, the A / G ratio remained similar to that after the start of treatment until the end of the experiment. In cat no. 3, decreased until day 16 of treatment, but then showed no further decrease. In cat no. 6 the decrease continued even after the start of treatment.

Figure 9.
Biochemical panel. The white areas of the graphs represent normal ranges. †: The cat was euthanized because its clinical condition reached a human endpoint

AST, LDH and TB were higher than the normal range on day 0 or 3 of treatment in all three cats. In cats 2 and 3, AST, LDH and TB decreased to near pre-FIP levels on day 7 or 9 after treatment. In cat no. 6, AST, LDH and TB were higher than the normal range on day 0 or 4 after the start of treatment and continued to increase until the end of the experiment.

Discussion

A strong therapeutic effect can be achieved when cats with FIP are administered an anti-fTNF-alpha mAb, which suppresses TNF-alpha activity, a FIP exacerbator, in combination with ICZ, which reduces FIPV proliferation. However, approving an anti-fTNF-alpha mAb as a veterinary drug in each country is costly and time consuming. Therefore, we focused on the anti-human TNF-alpha mAb, which is already used to treat human inflammatory diseases.

Prior to animal experiments, we investigated whether the anti-human TNF-alpha mAb could neutralize fTNF-alpha. ADA, an anti-human TNF-alpha mAb, demonstrated dose-dependent neutralizing activity against rfTNF-alpha, similar to anti-fTNF-alpha mAb 2-4. The nine previously reported anti-fTNF-alpha mAbs had IC50s ranging from 5 to 700 ng / ml against rfTNF-alpha at the same concentration [13]. The IC50 of ADA (36.9 ± 23.4 ng / ml) indicates that it has a high neutralizing activity against fTNF-alpha compared to these anti-fTNF-alpha mAbs. As a rule, anti-human cytokine mAbs do not show any cross-reactive neutralizing activity against animal cytokines. However, Aguirre et al. reported that human and feline TNF-alpha have similar neutralizing epitopes [26]. In addition, the deduced amino acid sequence of human TNF-alpha at 89.7% is identical to that of feline TNF-alpha, and both have 10 of the 12 residues that interact with ADA. [27]. This appears to be the reason for the strong neutralizing activity of adalimumab against feline TNF-alpha.

We administered ADA and ICZ to cats with experimentally induced FIP and investigated their therapeutic effect. Of the 10 SPF cats that received intraperitoneally administered FIPV KU-2 type I, three developed FIP. Of these three cats, cat no. 6 had apparent ascites. These three cats were considered to have developed FIP for the following reasons: (i) all three cats were infected with FIPV, (ii) clinical signs associated with FIP were observed, (iii) elevated plasma alpha-1 levels of AGP and VEGF, and (iv) as a reference data from our previous animal experiments were used. ADA (10 mg / animal) was administered intravenously to three cats on day 0 and within 4 days after the start of treatment. In addition, ICZ (50 mg / animal) was administered orally every day, starting on day 0 of treatment. In cats 2 and 3, an improvement in clinical signs and blood test results, an increase in peripheral blood lymphocytes and a decrease in plasma alpha 1-AGP levels were observed after initiation of treatment. Symptomatic improvements were observed between weeks 1 and 2 after the start of treatment. Furthermore, no ascites or pyogenic granulomas were pathologically observed in cats 2 and 3 at the end of the experiment, and no changes suggestive of recurrence of FIP were observed by the end of the treatment period. Cat no. 6 did not respond to treatment and was euthanized, although the level of the viral ascites gene temporarily decreased after the start of treatment. These results strongly suggest that anti-human TNF-alpha mAbs and ICZs are effective in the treatment of FIP.

VEGF is produced by FIPV-infected macrophages and improves feline endothelial cell permeability [28]. In cats with FIP, plasma VEGF levels correlate with ascites. Cat no. 6, in whom ascites was evident on both preliminary and palpation examinations (160 ml of ascites was obtained by euthanasia), had a high plasma VEGF level ≥100 pg / ml for 3 weeks. However, in cats 2 and 3, which showed no signs of ascites, plasma VEGF levels remained low or increased only temporarily. Thus, plasma VEGF levels were confirmed to correlate with the amount of ascites in cats with FIP.

Azole antifungal agents, including ICZ, are known to have hepatotoxicity as a side effect and are likely to cause cholestasis and jaundice. [29,30,31,32]. Because an increase in liver enzymes is observed in cats with FIP, the development of side effects after ICZ administration would be worrying. However, in cats 2 and 3, liver marker markers, which increased during treatment, returned to normal and the plasma jaundice disappeared. Therefore, no adverse effects of ICZ have been reported. Therefore, in terms of side effects, ICZ administration is considered safe for FIP cats.

Recently, GC-376, a 3C-like protease inhibitor, and GS-441524, a nucleoside analog, have been developed as drugs to inhibit FIPV proliferation. [16,17,18,19,20]. GC-376 and GS-441524 are effective drugs that have therapeutic effects in 30-80% cats with FIP. Although these drugs are expected to be useful in the treatment of FIP, they have not yet been approved. Therefore, no drugs are available in clinical practice to treat FIP. In this study, we demonstrated that the combined use of ADA and ICZ, which is currently available to veterinarians, is effective in the treatment of FIP. We consider these drugs to be a treatment option until antiviral drugs such as GC-376 and GS-441524 become available. In addition, these antiviral drugs have therapeutic effects on FIP by mechanisms different from ADA and ICZ [16, 18, 21, 23]. In the future, evaluating the therapeutic effects of their current use may help to develop a more effective treatment for FIP.

Conclusions

The therapeutic effects of adalimumab, anti-human TNF-alpha mAb and itraconazole on FIP were investigated. Adalimumab was found to have neutralizing activity against rfTNF-alpha, similar to anti-fTNF-alpha mAbs. FIP was successfully treated in 2 of 3 cats with experimentally induced FIP by adalimumab and itraconazole.

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Thanks

We thank the members of our laboratory for supporting animal experiments. This work was partially supported by KAKENHI (Grants-in-Aid for Scientific Research (B), No. 16H05039) from the Ministry of Education, Culture, Sports, Science and Technology in Japan and the Kitasato University Research Grants for Kitasato University Young Scientists.

Ethical statements

Conflict of interests

The authors are not aware of any potential conflicts of interest in connection with the research, authorship or publication of this article.

Ethical approval

All relevant national and institutional guidelines for the care and use of animals have been complied with. The protocol on animal experimentation was approved by the President of Kitasato University based on the decision of the Institutional Committee on the Care and Use of Animals at Kitasato University (Approval No. 18-152). SPF cats were bred in our own laboratory and maintained in an isolated temperature-controlled facility. Sample sizes were determined based on our experience with FIPV infection models and a minimum number of cats were used.

Informed consent

Informed consent was obtained from the legal guardian of all experimental animals described in this work for the procedures performed. No animals or humans are identifiable in this publication and therefore no further informed consent to the publication was required.

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