Feline leukemia virus (FeLV) is an oncogenic retrovirus

J Vet Intern Med 2004;1:4 42 Therapeutic Effects of Recombinant Feline Interferon- on Feline Leukemia Virus (FeLV)-Infected and FeLV/Feline Immunodeficiency Virus (FIV)-Coinfected Symptomatic Cats Karine de Mari, Laurence Maynard, Annaelle Sanquer, Bernard Lebreux, and Hyone-Myong Eun The clinical efficacy of a recombinant feline interferon, rfeifn-, was evaluated for the treatment of cats presented with clinical signs associated with feline leukemia virus (FeLV) infection and FeLV/feline immunodeficiency virus (FIV) coinfection in the field. In this multicentric, double-blind, placebo-controlled trial, 1 cats meeting the inclusion criteria were randomly placed into 2 groups and treated subcutaneously with rfeifn- (1 million [M]U/kg per day) or placebo once daily for consecutive days in 3 series (day 0, 14, 60). The cats were monitored for up to 1 year for clinical signs and mortality. During the initial 4-month period, interferon (IFN)-treated cats (n 3) had significantly reduced clinical scores compared with placebo (n 42), with all cats having received concomitant supportive therapies. Compared with the control, the IFN-treated group showed significantly lower rates of mortality: 3% versus % (1.-fold higher risk of death for controls) at the -month time point and 4% versus % (1.4-fold higher risk of death for controls) at the 12-month time point. The IFN treatment was associated with minor but consistent improvement in abnormal hematologic parameters (red blood cell count, packed cell volume, and white blood cell count), apparently underlying the positive effects of IFN on clinical parameters. These data demonstrate that rfeifn- initially has statistically significant therapeutic effects on clinical signs and later on survival of cats with clinical signs associated with FeLV infection and FeLV/FIV coinfection. Key words: Feline leukemia; Interferon therapy; Recombinant feline interferon. Feline leukemia virus (FeLV) is an oncogenic retrovirus belonging to the genus Gammaretrovirus of the family Retroviridae. In addition to neoplasia, FeLV causes a variety of degenerative and immunosuppressive disorders. Since its discovery in 164 in a multiple-cat household in which several cats had developed leukemia, 1 FeLV has been shown to occur worldwide, infecting % to 20% of cats depending on the country. Susceptibility is age related, and, in general, kittens are more likely to become persistently infected than are adults. Once infected with FeLV, 30% to 0% of cats become persistently viremic and develop clinical disease after an asymptomatic period of 0 to 3 days (mean 11 days). 2 Once clinical signs develop, 0% of infected cats die within 2. to 3. years, as compared with % of uninfected cats of similar ages. 3 FeLV infects all hematopoietic cells in bone marrow and lymphoid tissues, commonly resulting in aplastic anemia, leukopenia, or thrombocytopenia. 4 6 FeLV-infected cats thus experience severe anorexia, cachexia, progressive weakness, and a multitude of opportunistic infections as a consequence of FeLV-induced immunodeficiency. Among the opportunistic infections, the deadliest is perhaps coinfection of FeLV-infected cats with feline immunodeficiency virus (FIV), leading to more severe immunodeficiency. According to a recent survey, FIV coinfection was found to occur in % of the FeLV-infected cats. Since its first description in 1, FIV, a feline lentivirus, has become a subject of intense studies for its complex genomic and immunopathogenic features closely resembling those of human immunodeficiency virus., FIV infection in suscep- From the R&D Department, Virbac S.A., Carros Cedex, France. Reprint requests: H.M. Thomas Eun, PhD, R&D Department, Virbac S.A., BP 2, 06 Carros Cedex, France; e-mail: teun@virbac.fr. Submitted July 16, 2003; Revised December 16, 2003; Accepted February 13, 2004. Copyright 2004 by the American College of Veterinary Internal Medicine 01-6640/04/104-0004/$3.00/0 tible cats often takes a subclinical course similar to that of FeLV infection. Cats coinfected with FeLV/FIV have a poor prognosis as a result of infection by 2 retroviruses that infect all lineages of hematopoietic and lymphoid cells with partially overlapping tropisms. 4 FeLV- and FIV-related diseases have been treated in various ways and with varying efficacy. The principal agents evaluated during the past 20 years include interferons (IFNs), other immunomodulators (eg, staphylococcal protein A, Serratia marcescens membrane/ribosome preparation, inactivated parapox ovis virus, and acemannan), and antiretroviral agents (eg, azidodideoxythymidine, dideoxycytidine, phosphomethoxyethyl-adenine, and suramin), either alone or in combination. Type I IFNs, comprising IFN-, IFN-, and the recently characterized IFN-, display antiviral, antiproliferative, and immunomodulatory activities. As such, human interferons (HuIFNs) as well as veterinary IFNs, whether recombinant or natural, have been studied for their use in veterinary medicine. Regarding the use of IFNs in FeLV- and FIV-associated diseases, some studies showed encouraging and sometimes dramatic therapeutic effects on symptomatic as well as asymptomatic cats, 1 whereas other more recent studies failed to confirm previous observations. 16,1 The discrepancies may be attributed, at least in part, to different study designs, notably regarding (sub)types of IFN, species of origin, purity of preparations, treatment route, dosage, and schedule, as well as the age and pathological stage of FeLVinfected cats. Recently, a recombinant feline IFN (rfeifn) has been produced in silkworm larvae (Bombyx mori) by using a baculovirus vector. 1 The rfeifn has been characterized as type I IFN on the basis of a sequence homology of 60% with HuIFN- 1 or HuIFN- 1 but classified as an type considering the processing pattern of the N-terminal sequence. 1,1 The purified rfeifn- then was shown in vitro to have antiviral activities on various enteroviruses of dogs and cats 20,21 and also antitumor activities on a wide range

4 de Mari et al Table 1. Characteristics of cats on the first day of treatment. a,b Parameters IFN (n 3) Placbo (n 42) ELISA test FeLV ( ) FeLV ( ) FIV ( ) 26 13 31 Clinical Parameters Rectal temperature ( C) Behavior Appetite Thirst Dehydration Aspect of mucosa Stomatitis Severity Score (3 3 C) 1 increased ( 3 C) 2 decreased ( 3 C) 1 weak 2 very weak 3 prostrated 1 slightly reduced 2 reduced 3 anorexia 1 increased 2 very increased 3 suppressed 0 absence 1 slight (%) 2 moderate (%) 3 severe (12%) 1 colorless 2 very colorless 3 white 0 absence 1 redness, vesicles 2 several ulcerations 3 ulcerous erosion 21 1 1 1 13 4 21 0 1 12 1 16 22 1 6 21 1 6 1 14 2 23 3 16 13 Death 20 0 0 Total clinical score (mean SD).4 4.6.4 4.4 Hematologic parameters WBC ( 6 /ml) (mean SD) 12 1 Normal (. 1.) Leukocytosis ( 1.) Leukopenia (.0) 24 24 RBC ( /ml) (mean SD) Normal (. ) 6. 2.4 6.3 2.4 PCV (%) (mean SD) 33 12 32 12 Normal ( 30) Anemia ( 30) a IFN, interferon; ELISA, enzyme-linked immunosorbent assay; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; SD, standard deviation; WBC, white blood cell; RBC, red blood cell; PCB, packed cell volume. b The IFN-treated and placebo groups had no significant differences (P.0) in any of the parameters. 24 1 2 1 of tumor cell lines of dogs and cats. 22,23 In addition, rfeifn- was shown to have therapeutic effects in vivo on canine parvoviral enteritis. 24 26 The recent development of an rfeifn- and its commercial availability provided us an opportunity to evaluate the therapeutic potential of IFN on cats presented with clinical signs associated with natural infection with FeLV or FeLV/FIV. Our study demonstrates that rfeifn- can provide important therapeutic benefits to symptomatic cats infected with FeLV and those coinfected with FeLV and FIV. Materials and Methods Reagents and Products Interferon- a (a lyophilized product) was dissolved in the diluent (physiological saline) at a final concentration of mu/ml immediately before use. The active ingredient is the purified rfeifn- produced in silkworm larvae via a recombinant baculovirus expression system. 1 The placebo is an excipient formulation without rfeifn. All reagents were kept at 4 C until use.

Interferon Therapy on FeLV-Infected Cats 4 Inclusion and Exclusion Criteria for Cats The field study involved 1 cats recruited in veterinary clinics in France for a period of 4 years (from March 1 to January 2003). Cats were enrolled if they met all the following inclusion criteria: (1) were of any age, breed, and either sex; (2) had at least 1 clinical sign potentially related to the infection with FeLV or FeLV/FIV (eg, fever, loss of appetite, altered behavior, thirst, dehydration, pale mucous membranes, stomatitis); (3) had enzyme-linked immunosorbent assay (ELISA)-positive blood tests for FeLV or FeLV/FIV; and (4) had owners who provided informed consent and were willing to participate in the full course of the treatment. The cats included in the trial were 3 months to 16 years old and weighed from 1. to. kg on day 0 (Table 1). Thirty (3%) of the cats were female and 1 (63%) were male. Five breeds were represented; the majority (6%) were mixed breed. Cats were randomly placed into 2 groups (IFN treated and placebo), and the attending veterinarian gave treatments according to preset schedules and protocols when the cats visited the clinics for health checks. Otherwise, the owners cared for their cats throughout the trial period. Cats were not included in the trial if they were FeLV positive but also had a malignancy (eg, lymphosarcoma, lymphoid leukemia) or were in terminal condition. Other cats were excluded at the beginning or during the course of the trial when (1) they were treated with an immunomodulating drug (eg, corticosteroid), (2) no blood samples had been collected on day 0, (3) they received fewer than injections (either IFN or placebo), (4) they were lost to follow-up before day 120, and () owners withdrew their pets from the trial. Treatment Cats were treated subcutaneously with rfeifn- (1.0 million [M]U/ kg per day) or placebo once daily for consecutive days in 3 courses: first (day 0 to 4), second (day 14 to 1), and third (day 60 to 64). Neither the pet owner nor the investigator were made aware of the nature of the treatment administered (double-blinded experimental design). Animal handling, treatment, reagent manipulations, and data collection all were conducted in compliance with the guidelines of Good Clinical Practice (GCP). Supportive and Symptomatic Therapy All cats received concomitant individualized supportive therapies at the discretion of the attending veterinarian. These included rehydration (eg, Ringer solution), vitamins (B12, C, K, or oral polyvitamin solutions), and, in the case of suspected bacterial infection, antibiotics (enrofloxacin, amoxicillin, and cephalexin). Other treatments included nonsteroidal anti-inflammatory drugs (tolfenamic acid, nimesulide, ketoprofen), spasmolytic drugs, liver-protective agents, parasiticides, diuretics, anesthetics, antidiarrheal agents, and antifungal agents. The use of steroidal anti-inflammatory drugs (corticosteroids) was not permitted during the course of the trial. Observation of Clinical Signs and Scoring Cats were monitored by the participating veterinarians once daily on days 0, 14, 30, 60, and 120 upon follow-up visits of the owner to the clinics. The veterinarians then contacted the owners at 6,, and 12 months. Eight categories of clinical signs were monitored and severity scores were assigned (Table 1): rectal temperature, general behavior, appetite, thirst, dehydration, mucous membrane appearance, stomatitis, and death. The clinical score (CS) of an animal was defined as the sum of the scores for the indicated signs at each examination. For cats that died or were euthanized, a CS of 20 corresponding to the maximal sum was assigned for the purpose of statistical analysis. If a category Fig 1. Survival probabilities of the cats. Interferon-treated group (, n 3) and placebo group (, n 42). of clinical signs was missing, the CS of the cat was considered missing. Adverse Events The veterinarian recorded all clinical manifestations (abnormal or new, local or systemic, transient or long-duration) that could be attributed to the administration of the product during the observation period (day 0 to 120). The recorded events were evaluated at the conclusion of the trial following the adverse events guidelines in the GCP. Blood Sample Collection and Treatment Blood samples were collected according to the schedule (days 0, 14, 30, 60, and 120) and submitted for white blood cell (WBC) count, red blood cell (RBC) count, and packed cell volume (PCV). Diagnosis of FeLV or FeLV/FIV infection was established on day 0 at the participating clinics with a diagnosis kit b according to the protocol recommended by the supplier. The kit allows detection of anti-fiv antibodies or p2-felv antigen or both simultaneously on nonclotted blood, serum, or plasma. Statistical Analysis The 2 groups were compared on day 0 by the chi-square test for qualitative parameters (eg, sex, living conditions, infection status based on ELISA, and clinical signs) and the Student s t-test for quantitative parameters (eg, age, body weight, and CS), both at a significance level of.0. Survivals were compared by a Cox model taking into account the status of anemia or nonanemia on day 0. The time course of total CS was compared between groups by an analysis of variance (ANOVA) for repeated measures with anemia on day 0 as a covariable. The time course of WBC count was analyzed separately for cats with leukopenia or leukocytosis on day 0. The time course of RBC count and PCV was analyzed only for anemic cats on day 0. These hematologic parameters were analyzed by an ANOVA for repeated measures by the last-observation-carried-forward method. The percentage of productrelated adverse effects in the 2 groups was compared by the Fishers exact test. The efficacy and safety analyses were performed by a 1- tailed test. P values.0 were considered significant. All statistical analyses were performed by NCSS 2000 software package. c Results IFN Treatment and Survival Rate When the cats were monitored up to 1 year and evaluated for survival, the IFN-treated cats showed a higher rate of survival than did the controls (Fig 1). Although supportive

40 de Mari et al Table 2. Treatment statistics for cats having received supportive therapies. Treatment Interferon (n 3) Placebo (n 42) Group Difference (P-value) Rehydration Antibiotics Vitamins Others c Overall (%) 32 2 32 (2.1) 1 3 4 3 (.1) 0.2 a 0.0 b 0.6 b 1.00 b 0.44 a a Chi-square test. b Fishers exact test. c For details of other treatments, refer to the text (Supportive and Symptomatic Therapy in Materials and Methods). Fig 2. Time course of clinical scores (CS). Interferon-treated group (, n 3) and placebo group (, n 42). The group difference of CS on day 0 was not statistically significant (Table 1). therapies were administered in a similar manner to both groups (Table 2), the placebo and IFN groups had mortality rates of % (23/3) and 3% (1/3) at the -month time point, which corresponds to a 1.-fold higher risk of death for the control cats (P.04). At the 12-month time point, the placebo group had mortality rates of % (23/3), whereas the IFN group had mortality rates of 4% (1/3) or a 1.4-fold higher risk of death for the control cats (P.). Effect of Treatments on Clinical Signs All participating cats were monitored for clinical signs according to the fixed schedule up to day 120. Subsequently, however, only a few cats were available for examinations, specifically 12% on day 10, 12% on day 20, and % on day 360, obliging us to rely on the data only up to day 120 for meaningful statistical analysis. On day 0, the CS of the IFN and placebo groups were not statistically different (Table 1). Immediately after treatment, all cats experienced rapid and general improvement of clinical signs (Fig 2). However, on day 14, for example, the reduction of CS was substantially greater in the IFN group (3%) than in the placebo group (1%). From day 30 on, the CS generally showed a rising tendency as the contribution of dead cats progressively increased (Fig 2). Overall, IFN-treated cats demonstrated CS consistently lower than those of the placebo group (P.04). Adverse Events During the course of treatment, no local adverse reactions were observed that could be attributed to rfeifn-. Including 1 cat with renal failure for which a drug-related effect has not been established, systemic adverse events were observed in 6.2% (/1) of the cats. Major clinical signs consisted of lethargy, transient vomiting, and diarrhea with no statistically significant differences between the IFN-treated group and the placebo group. Effect of IFN Treatment on Hematologic Parameters At the beginning of the treatment (day 0), IFN and placebo groups showed no statistically significant differences in any of the hematologic parameters (Table 1). The time course of WBC count was analyzed according to the day- 0 status of the cats: normal WBC count (% cats), leukopenia (1% cats), and leukocytosis (22% cats). The cohorts with normal WBC count on day 0 exhibited a profile that remained virtually constant as a function of time and also indistinguishable between the IFN-treated group and the placebo group. In contrast, IFN treatment provided a Fig 3. Time courses of white blood cell (WBC) count. (a) Cats with leukopenia on day 0: interferon (IFN)-treated group (, n ) and placebo group (, n ). The middle broken line represents the lower limit of WBC count normal range in cats. (b) Cats with leukocytosis on day 0: IFN-treated group (, n ) and placebo group (, n ). The middle broken line represents the upper limit of WBC count normal range in cats.

Interferon Therapy on FeLV-Infected Cats 41 Fig 4. Time courses of packed cell volume (PCV). (a) Cats with anemia on day 0: interferon (IFN)-treated group (, n 1) and placebo group (, n 1). The middle broken line represents the lower limit of PCV normal range in cats. (b) Cats without anemia on day 0: IFN-treated group (, n 21) and placebo group (, n 23). The middle broken line represents the lower limit of PCV normal range in cats. noticeable improvement or correction of hematologic abnormalities in the cohorts with leukopenia or leukocytosis on days 60 and 120 (Fig 3). The time course of PCV and RBC count was analyzed according to the status of cats on day 0: anemic (3% cats) and nonanemic (63% cats). Although statistically not significant, PCV tended to be consistently higher in the IFNtreated group than in the control group in both anemic and nonanemic cohorts (Fig 4). The time course of RBC count was virtually identical to those of the PCV. Discussion Infection of cats by retroviruses such as FeLV and FIV, development of neoplastic or nonneoplastic diseases, and ultimate progression to death involve a complex interplay between viruses and host responses. Once clinical signs develop in the presence of FeLV- or FIV-induced immunosuppression, over 0% of infected cats experience progressive deterioration in health and eventually die. Whereas FeLV infection results in anemia and, to a lesser extent, leukopenia as primary clinical signs, FIV infection is associated with, among other signs, lymphopenia and a characteristic decrease of the CD4/CD T-cell ratio. Within the context of a carefully controlled study population, hematologic values can serve as valuable indicators of clinical condition and eventual mortality. Among the 1 cats ( FeLV-positive and 24 FeLV/FIVcopositive) participating in the present trial, 30 (3%) presented with anemia on day 0. Under the conditions of the current regimen, IFN treatment offered marginal but consistent beneficial effects on PCV and RBC count in day-0 anemic cohorts, but the effect was inconsequential in day- 0 nonanemic cohorts. Regarding WBC count for cats with leukopenia (1/1 cats on day 0) and leukocytosis (1/1 cats on day 0), IFN treatment produced a generally positive effect within the 120-day observation period by bringing the abnormal WBC count within the normal range. The return to normal of WBC count, which was apparent on day 60, suggests that the therapeutic effect of IFN was operational after the second series of treatments and that the protective effects could be derived at least in part from immunostimulatory activity. When the survival at or 12 months was taken as the endpoint, IFN-treated cats had a significant survival advantage over control cats at the -month time point (1.-fold) and a somewhat lesser advantage at the 12-month time point (1.4-fold). During the initial 4-month period, however, the survival advantage was not obvious, with only a 1.2- fold higher risk of death for the controls. Nonetheless, the overall CS of the IFN-treated group was lower by 2% to 4% than that of the placebo group in the same 4-month period. This tangible reduction of CS thus is presumed to reflect the superior survival of the IFN-treated cats at later time points. The therapeutic effects of IFN were observed beyond what would have been expected on the basis of supportive therapy alone. During the course of treatment, no serious adverse reactions were observed that could be attributed to rfeifn-. The present data demonstrate that rfeifn- is therapeutically active in cats with clinical signs associated with FeLV infection and FeLV/FIV coinfection and provides affected cats with significant improvements in clinical signs and survival rates. Whether or not and how rfeifn- could be therapeutically useful in another group of cats presented with FeLV-associated neoplastic diseases (T-cell or myeloid leukemia, lymphoma, and lymphosarcoma) remain to be determined. Footnotes a Virbagen Omega, Virbac S.A., Carros, France b Snap Combo FeLV/FIV kit, IDEXX S.A., Paris, France c NCSS 2000, NCSS, Kayville, UT Acknowledgment The authors thank all the veterinarians who participated in the study. References 1. Jarrett WFH, Crawford EM, Martin WB, Davie FA. A virus-like particle associated with leukemia (lymphosarcoma). Nature 164;202: 6 6.

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