Veterinary Microbiology 175 (2015) 167–178

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Evaluation of the effect of short-term treatment with the

integrase inhibitor raltegravir (IsentressTM) on the course of
progressive feline leukemia virus infection
Andrea Boesch a, Valentino Cattori a, Barbara Riond a, Barbara Willi a,b,
Marina L. Meli a, Katharina M. Rentsch c, Margaret J. Hosie d,
Regina Hofmann-Lehmann a, Hans Lutz a,*
a
Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
b
Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
c
Institute of Clinical Chemistry, University Hospital, Raemistrasse 100, 8091 Zurich, Switzerland
d
Medical Research Council, University of Glasgow Centre for Virus Research, Henry Wellcome Building for Comparative Medical Sciences,
464 Bearsden Road, Glasgow G61 1QH, UK

A R T I C L E I N F O A B S T R A C T

Article history: Cats persistently infected with the gammaretrovirus feline leukemia virus (FeLV) are at
Received 29 January 2014 risk to die within months to years from FeLV-associated disease, such as immunosup-
Received in revised form 27 October 2014 pression, anemia or lymphoma/leukemia. The integrase inhibitor raltegravir has been
Accepted 28 October 2014 demonstrated to reduce FeLV replication in vitro. The aim of the present study was to
investigate raltegravir in vivo for its safety and efficacy to suppress FeLV replication. The
Keywords: safety was tested in three naı̈ve specified pathogen-free (SPF) cats during a 15 weeks
Feline leukemia virus treatment period (initially 20 mg then 40 mg orally b.i.d.). No adverse effects were noted.
Retrovirus
The efficacy was tested in seven persistently FeLV-infected SPF cats attained from 18 cats
Viral loads
experimentally exposed to FeLV-A/Glasgow-1. The seven cats were treated during nine
Antiretroviral therapy
Immune response, raltegravir
weeks (40 mg then 80 mg b.i.d.). Raltegravir was well tolerated even at the higher dose. A
significant decrease in plasma viral RNA loads (5) was found; however, after treatment
termination a rebound effect was observed. Only one cat developed anti-FeLV antibodies
and viral RNA loads remained decreased after treatment termination. Of note, one of the
untreated FeLV-A infected cats developed fatal FeLV-C associated anemia within 5 weeks
of FeLV-A infection. Moreover, progressive FeLV infection was associated with
significantly lower enFeLV loads prior to infection supporting that FeLV susceptibility
may be related to the genetic background of the cat. Overall, our data demonstrate the
ability of raltegravir to reduce viral replication also in vivo. However, no complete control
of viremia was achieved. Further investigations are needed to find an optimized treatment
against FeLV. (250 words)
ß 2014 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +41 44 635 83 11; fax: +41 44 635 89 23.
E-mail addresses: andieboesch@gmail.com (A. Boesch), valentino.cattori@gmail.com (V. Cattori), briond@vetclinics.uzh.ch (B. Riond),
bwilli@vetclinics.uzh.ch (B. Willi), mmeli@vetclinics.uzh.ch (M.L. Meli), katharina.rentsch@usb.ch (K.M. Rentsch), margaret.hosie@glasgow.ac.uk
(M.J. Hosie), rhofmann@vetclinics.uzh.ch (R. Hofmann-Lehmann), hlutz@vetclinics.uzh.ch, hanslutz@me.com (H. Lutz).

http://dx.doi.org/10.1016/j.vetmic.2014.10.031
0378-1135/ß 2014 Elsevier B.V. All rights reserved.
168 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178

1. Introduction the immune system of the cats would be able to overcome

the viremia, turning the course of infection from progressive
Feline leukemia virus (FeLV) is a gammaretrovirus with to regressive. Indeed, complete suppression of viral
worldwide distribution, affecting domestic cats and some replication after a transient period of viremia of more than
free-living felids, among them the Iberian lynx and the a year has been documented in some cats (Lutz et al., 1980b),
Florida puma (Cunningham et al., 2008; Geret et al., 2011c; but these are very rare cases. A similar phenomenon was
Meli et al., 2009). Of the four known outcomes of infection, observed in some HIV patients, who were treated in the
progressive infection, regressive infection with or without early phase of HIV infection. Probably due to the low viral
antigenemia and abortive infection (Hofmann-Lehmann reservoir in these patients, a full recovery from infection was
et al., 2008), especially the persistently viremic cats possible with no rebound of viremia even after treatment
(progressive infection) are at risk of dying from hemato- interruption (Hocqueloux et al., 2010).
poietic disorders, fatal neoplasia or the consequences of The aims of the present study were to assess (1) the
immunodeficiency (Lutz et al., 2009). Although the FeLV tolerance to raltegravir in domestic cats and (2) the effect
prevalence seems to have decreased worldwide probably of short-term treatment with raltegravir on the course of
due to vaccination programs and a consistent management FeLV infection in persistently infected cats, by monitoring
to detect infected cats (Lutz et al., 2009), there remain the clinical outcome, as well as the FeLV antigen, proviral,
countries with high prevalences (Akhtardanesh et al., viral loads and specific antibody levels in these cats.
2010; Blanco et al., 2009; Duarte et al., 2010).
So far, there are no specific therapies to treat FeLV
2. Materials and methods
infection. In one study, feline interferon omega was shown
to inhibit FeLV in vitro and to have immunomodulatory 2.1. Animals and experimental design
activities, leading to an improvement of clinical scores and
an extended survival time in vivo (de Mari et al., 2004). All animal experiments were performed in accordance
However, there was no direct demonstration of antiviral with Swiss law and were officially approved by the
effects in vivo; no viral parameters were measured veterinary office of the Zurich canton (TVB 160/2010).
throughout the study. In a recent clinical study on three The specified pathogen-free (SPF) cats (Liberty Research,
FeLV-infected cats and seven cats infected with feline Inc., Waverly, NY, USA) were kept in groups under barrier
immunodeficiency virus, recombinant interferon omega conditions and ethologically and hygienically ideal condi-
seemed to have immunomodulatory properties, but no tions, as described (Geret et al., 2011a). The SPF status of all
antiviral effect (Domenech et al., 2011). In vivo treatment cats was verified prior to the experiment, as described
trials with the nucleoside analogue 30 -azido-20 ,30 -dideox- previously (Museux et al., 2009).
ythymidine (AZT) showed an inhibitory effect, but high In the treatment tolerance study, the feasibility of
toxicity as well resulting in non-regenerative anemia administration of the antiviral compound and potential
(Hartmann et al., 1992). In an in vivo study using AZT and/ side effects were assessed in three healthy adult SPF cats,
or human interferon alpha 2a for six weeks in cats three to five years of age and four to five kg of body
naturally infected with FeLV, no antiretroviral efficacy weight. The cats received a dosage in the range used in
could be demonstrated; nonetheless, no notable side humans, corresponding to 5 to 10 mg/kg twice daily
effects were detected (Stuetzer et al., 2013). Recent (Merck, 2007). The cats were monitored for 15 weeks and
antiretroviral agents emerging for the treatment of human blood samples were collected regularly under light
immunodeficiency virus (HIV), namely the integrase sedation (0.1 mg/kg midazolam, Dormicum1, Roche
strand transfer inhibitors raltegravir and Elvitegravir, were Pharma AG, Reinach, Switzerland; and 10 mg/kg keta-
tested in vitro against lentiviruses, alpha-, beta- and mine, Narketan1, Vétoquinol AG, Belp, Switzerland) and
gammaretroviruses of different species (Koh et al., analyzed for hematology, clinical chemistry and deter-
2011). Raltegravir is the only integrase inhibitor so far mination of the plasma concentration of the antiviral
available on the market, which exhibited good efficiency compound.
against gammaretroviruses. It showed excellent inhibitory In the FeLV treatment study, eighteen male SPF kittens
potential against the gammaretrovirus xenotropic murine were employed (BP1, BP2, BS1, BX3, CC1, CK1, CK2, CK3,
leukemia-related retrovirus XMRV, a virus closely related CK4, CK5, CL2, CN2, CN3, CN4, CP1, CP2, CP4, and CR2;
to FeLV (Paprotka et al., 2010; Singh et al., 2010; Smith Fig. 1). After an adaptation period and at the age of 17 to 20
et al., 2010). An inhibitory effect on FeLV was also verified weeks all kittens were castrated using standard proce-
in three different feline cell lines with EC50 in the low dures under general anesthesia (3 mg/kg ketamine;
nanomolar range, similar to what has previously been 0.05 mg/kg medetomidine; 0.2 mg/kg butorphanol).
observed for HIV and XMRV (Cattori et al., 2011). Thereafter, at the age of 19 to 21 weeks, each cat was
If effective, treatment of viremic domestic cats with exposed intraperitoneally to FeLV-A/Glasgow 1 as de-
raltegravir would be of enormous value to alleviate the scribed below under general anesthesia. Eleven of the
consequences of a progressive FeLV infection. However, due eighteen cats received a second virus challenge intraperi-
to financial and patient/owner compliance considerations, toneally under general anesthesia six weeks later (Fig. 1).
the goal of the use of raltegravir in veterinary medicine has This second challenge was performed with the aim of
to be a temporally limited treatment, resulting in a complete increasing the number of cats with progressive FeLV
recovery, even after treatment interruption. We speculate infection. Fifteen weeks after the initial virus challenge (at
that when achieving a significant reduction of viral loads, the age of 33 to 36 weeks), the treatment with the antiviral
 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178 169

Week after initial FeLV challenge Switzerland); these capsules were administered using a
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pet pill dispenser.
BP1 x
BP2 2.3. Treatment tolerance study
BS1 x
BX3 x Three adult SPF cats (R4, B6 and J6) were treated with
CC1 raltegravir for 15 weeks. They received 20 mg of raltegravir
CK1 b.i.d. during the first eight weeks followed by 40 mg b.i.d.
CK2 x for another seven weeks. To assess the elimination rates of
CK3 y raltegravir in the cats and compare them to those reported
CK4 for humans (Kassahun et al., 2007), the administration of
CK5 yy the antiviral compound was stopped for 24 hours in week
CL2 x 10 and for 36 hours in week 11 prior to blood collection,
CN2 x respectively. Blood samples were collected prior to the
CN3 x start of the treatment (week 0) and at weeks 1, 2, 4, 6, 8, 9,
CN4 x 10 and 11, 13 and 15. In weeks 10 and 11 the samples were
CP1 x collected after the drug withdrawal.
CP2 x Complete hemograms were performed from ethylene-
CP4 x diaminetetraacetic acid (EDTA) anticoagulated blood using
CR2 a Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan)
(Weissenbacher et al., 2011). White blood cell differentials
FeLV p27 negative FeLV provirus & viral RNA negative were performed microscopically using blood smears that
FeLV p27 positive FeLV provirus positive, viral RNA negative
were prepared manually and Wright stained using a
Hema-Tek 1000 (Bayer AG, Zurich, Switzerland). The
x FeLV re-challenge FeLV provirus & viral RNA positive
hematological reference ranges were determined using
identical methods and blood samples from 63 clinically
Fig. 1. Experimental setup and outcome of FeLV challenge in the
raltegravir treatment study. Detection of plasma FeLV p27 antigen, viral healthy cats. All reference ranges in this study are given as
RNA and proviral DNA in the 18 SPF kittens exposed to FeLV. Symbols are 5% and 95% quantiles.
explained in the figure. Cat CK4 had to be euthanized 4.3 weeks after the Serum chemistry was performed on heparinized
initial FeLV challenge (y). No samples were collected in week 5 after the plasma using a Cobas Integra 800 system (Roche
initial FeLV challenge. In week 6, eleven cats were re-exposed to FeLV
(indicated with ‘‘x’’). Cats with persistent antigenemia were included in
Diagnostics, Rotkreuz, Switzerland) and included total
the raltegravir treatment study. bilirubin, glucose, fructosamine, blood urea nitrogen,
creatinine, protein, albumin, cholesterol, triglyceride,
alkaline phosphatase, amylase, aspartate aminotrans-
ferase, alanine aminotransferase, lipase, sodium, chlo-
compound was started in the seven available persistently ride, potassium, calcium, and phosphate. Reference
infected cats (Fig. 1). The cats were treated for a total of ranges for clinical chemistry were determined using
nine weeks. The cats were monitored throughout infection identical methods and blood samples from 59 clinically
and treatment and blood samples were collected regularly healthy cats.
under light sedation as described above for hematology, Raltegravir plasma concentrations were measured by
clinical chemistry, and characterization of FeLV infection high performance liquid chromatography-mass spectrom-
and determination of the plasma concentration of the etry, under comparable conditions described previously
antiviral compound. (Rentsch, 2003). Heparin-anticoagulated plasma from
untreated cats was used as negative controls.
2.2. Preparation and administration of the antiviral
compound 2.4. FeLV infection

The antiviral component tested in this study was For the initial FeLV challenge in the raltegravir
IsentressTM (raltegravir 400 mg film tabs, MSD Merck treatment study, four aliquots of cell culture superna-
Sharp & Dohme AG, Luzern, Switzerland). Tablets were tant (a total of 17 ml) containing FeLV-A/Glasgow 1
ground and re-encapsulated into smaller portions in (Jarrett et al., 1973) were thawed at 37 8C and pooled.
gelatin capsules to facilitate the uptake by the cats. For the They had been stored at 80 8C before use. The
treatment tolerance study, capsules (size 14.4  5.1 mm; supernatant contained 1.1  10 6 focus forming units
Inter Delta SA, Givisiez, Switzerland) contained 20 or (FFU) per mL. The virus was diluted on ice to a final
40 mg of raltegravir, respectively (Kantonsapotheke volume of 23 ml and a final concentration of 8  10 5
Zurich, Zurich, Switzerland). These capsules were admin- FFU FeLV per ml using cell culture medium (RPMI,
istered with the food twice daily. For the FeLV treatment Invitrogen AG, Basel, Switzerland). Subsequently, ali-
experiment, the remaining capsules from the treatment quots of 1 ml were drawn up into syringes and stored on
tolerance study were used. In addition, larger capsules ice until use. Each cat was injected intraperitoneally
(19.0  6.6 mm) were used containing 40 mg raltegravir using one syringe and thus received 8  10 5 FFU in a
and 320 mg of lactose (Apotheke zum Rebstock, Stäfa, volume of 1 ml intraperitoneally.
170 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178

Six weeks after the initial FeLV challenge a second virus The extracted TNA was analyzed by real-time PCR for
challenge with 1.7  106 FFU (in 1.8 ml) was performed FeLV proviral loads or by reverse-transcriptase (RT) PCR for
intraperitoneally in eleven cats (BP1, BS1, BX3, CK2, CL2, FeLV plasma viral RNA loads, as described (Tandon et al.,
CN2, CN3, CN4, CP1, CP2, and CP4; Fig. 1), which had 2005), using a ABI PRISM 7700 or 7500 sequence detection
plasma p27 levels of <10% of the positive control 4 weeks system (Applied Biosystems, Rotkreuz, Switzerland). All
after the first challenge. The origin and virus preparation PCR runs were performed with positive and negative
for the second challenge was identical to the first controls.
challenge.
2.7. Detection of FeLV p27 virus antigen
2.5. FeLV treatment study
FeLV viremia was determined by p27 sandwich
Out of the 18 SPF cats exposed to FeLV, seven cats (BP2, enzyme-limked immunosorbent assay (ELISA), as de-
CC1, CK1, CK3, CK5, CP1 and CR2; Fig. 1), developed a scribed (Lutz et al., 1983). All samples were tested in
progressive infection. Because of the small number of duplicates. Results were calculated as the percentage of a
progressively infected cats, it was decided to treat all seven positive control (culture supernatant of FL-74 feline
cats rather than subdivide the group into treatment and lymphoblastoid cell line persistently infected with FeLV),
control groups. The antiviral treatment was started 15 which was assayed with every plate. Values 4% were
weeks after the initial FeLV challenge (week 0 of considered to be positive. In agreement with the European
treatment). Each cat received 40 mg of raltegravir b.i.d. Pharmacopoeia (2005), a cat was designated as progres-
(corresponding to 10–15 mg/kg b.i.d.) for 6.5 weeks. sively infected when FeLV p27 antigen was positive for
Because none of the cats had cleared antigenemia by three consecutive weeks or on five occasions between
then, the dose was increased to 80 mg b.i.d. (20–25 mg/kg weeks 3 and 15 post infection.
b.i.d.) for another 2.5 weeks.
Blood samples were collected at weeks 0, 1, 2, 3, 4, 6, 7, 2.8. Detection of anti FeLV antibodies
8, 9, 10, 11, 12, 13, 14, and 15 after the initial FeLV
challenge, at weeks 1, 2, 3, 4, 5, 6, 7, 8, and 9 during the Plasma samples were analyzed for the presence of
treatment (corresponding to weeks 16 to 24 after FeLV antibodies to FeLV p45 and to FeLV whole virus by ELISA,
challenge) and at weeks 1, 2, 4, 6 and 8 post-treatment using 100 ng of p45 antigen and 100 ng of gradient purified
(corresponding to weeks 25 to 32 after FeLV challenge). FL-74 FeLV per well and serum dilutions of 1:200 and
Hematological and clinical chemical parameters were 1:100, respectively, as described (Lehmann et al., 1991;
assessed as described above. The raltegravir plasma Lutz et al., 1980a). In each assay pooled SPF sera (=negative
concentration was measured as described above at weeks control) and pooled sera collected from FeLV immune cats
0, 3, 6, and 9 of the treatment and one week after the (=100% positive control) were tested. Values 25% of the
treatment had been stopped. positive control were considered to be positive.
One cat (CK4) developed FeLV associated disease and
had to be euthanized for humane reasons. The cat received 2.9. Detection of FeLV subgroups
a light intramuscular anesthetic injection (20 mg/kg
ketamine and 0.1 mg/kg midazolam; Dr. E. Graeub AG, FeLV-subgroups were investigated in the plasma and
Bern, Switzerland) followed by a pentobarbital adminis- whole blood of cat CK4 at the time of necropsy by
tration via intravenous catheter (Esconarkon, 150 mg/kg; conventional PCR using the FeLV-A specific primers RB59
Streuli Pharma SA, Uznach, Switzerland). and RB17, the FeLV-B specific primers RB53 and RB17 and
the FeLV-C specific primers RB58 and RB47, as described
2.6. Nucleic acid extractions, detection of FeLV proviral and (Helfer-Hungerbuehler et al., 2010; Mathes et al., 1994;
plasma viral RNA loads Sheets et al., 1993)

For the quantification of FeLV proviral blood loads, total 2.10. Quantification of enFeLV proviral loads
nucleic acid (TNA) was extracted from selected samples
collected during the FeLV challenge (weeks 0, 3, 9 and 15 Provirus loads of endogenous FeLV-like sequences
after the initial challenge) and all samples collected during (enFeLV) and loads of feline glyceraldehyde-3-phosphate
the FeLV treatment study. TNA was extracted from a blood dehydrogenase (fGAPDH) were determined in the samples
volume containing 106 white blood cells, or from a of the 18 SPF cats enrolled in the FeLV treatment study—
maximum volume of 100 ml, using the MagNa Pure LC loads were measured in peripheral blood samples collect-
Total Nucleic Acid Isolation Kit (Roche diagnostics AG, ed prior to the initial FeLV challenge applying quantitative
Rotkreuz, Switzerland) according to manufacturer’s real-time TaqMan PCR assays on an ABI 7500 FAST Real-
instructions. All volumes were adjusted to 100 ml with Time PCR System (Applied Biosystems), as reported earlier
phosphate-buffered saline (PBS). For plasma viral RNA (Tandon et al., 2007, 2008a). The following enFeLV targets
loads, TNA was extracted from 200 ml of plasma using the were measured enFeLV-U3-1, enFeLV-U3-2 and enFeLV-
MagNa Pure LC Total Nucleic Isolation Kit according to env. For absolute quantification, enFeLV and fGAPDH DNA
manufacturer’s instructions. Negative controls containing standards were used. Total enFeLV-U3 copy numbers were
PBS were concurrently prepared with each batch of calculated by addition of enFeLV-U3-1 and enFeLV-U3-2
samples to monitor for cross-contamination. copy numbers. Copy numbers of enFeLV were divided by
 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178 171

fGAPDH copy numbers to calculate the amount of enFeLV a)

per cell. 20 mg bid 40 mg bid

Raltegravir plasma conc. (nM)

1600
2.11. Statistics
withdrawal
1200 R4
Statistical analyses were carried out using R software
version 2.14.0 (the R Foundation for Statistical Computing, B6
800
Vienna, Austria). Historical data from previous experi- J6
ments was used to compare the course of infection of
400
untreated progressively infected cats with the seven
treated cats in the present FeLV treatment study; data
0
from weeks 11, 15 (comparison to baseline), 17, 21 and 24 0 2 4 6 8 10 12 14
(comparison to treatment) post-infection were available
Treatment week
(Gomes-Keller, 2011). Longitudinal effects of the treat-
ment over time on the viral RNA, viral DNA and p27 loads b)
40 mg bid 80 mg

Raltegravir plasma conc. (nM)

were compared by multivariate analysis of variance
bid
(pMANOVA). For the correlation analysis, the Spearman 1600 BP2
rank correlation test was used (pS), performed with
CC1
Graph-Pad Prism (GraphPad Software, Version 3.0. San 1200
Diego, CA, USA). For comparison of enFeLV proviral loads, CK1
the Mann–Whitney U-test (pMWU) was used (Graph-Pad CK3
800
Prism). A p-value <0.05 was considered to be statistically CK5
significant. CP1
400
CR2
3. Results 0
0 2 4 6 8 10 12 14
3.1. Treatment tolerance study Treatment week

In the treatment tolerance study, none of the three cats Fig. 2. Raltegravir plasma concentration (nM). Raltegravir plasma
showed any behavioral changes or clinical signs during the concentration in the treatment tolerance study (a). The three adult SPF
cats received 20 mg raltegravir b.i.d. in weeks 1 to 8 and 40 mg raltegravir
15 weeks of antiviral treatment. The hematological and b.i.d. in weeks 9 to 15, respectively (see also gray boxes). Data points in
clinical–chemical parameters remained unchanged and weeks 10 and 11 represent plasma values after 24 h and 36 h of raltegravir
within the reference range. Raltegravir plasma concentra- withdrawal, respectively (arrows ‘‘withdrawal’’). Missing data in week 2
tions ranged from 46 to 1040 nM at 20 mg b.i.d., and from (cat B6) are due to the collection of high blood volumes in the beginning of
the experiment and therefore a treatment-unrelated excessive decrease
151 to 1057 nM at 40 mg b.i.d. (Fig. 2a). Plasma concen-
of the hematocrit and in week 8 (cat J6) to anesthesia failure and therefore
trations of raltegravir were almost undetectable after inability to collect blood from this cat, respectively. raltegravir plasma
24 hours (week 10) and 36 hours (week 11) of drug concentration in the FeLV treatment study (b). The seven persistently
withdrawal, respectively (Fig. 2a). Both, 20 mg and 40 mg FeLV infected SPF kittens (see Fig. 1) received 40 mg raltegravir b.i.d. in
dosages, resulted in sufficient plasma concentrations to weeks 1 to 6.5 and 80 mg raltegravir b.i.d. in weeks 6.5 to 9, respectively
(see also gray boxes). Data points in week 10 represent plasma values
guarantee efficacy of at least 99% inhibition in vitro (Cattori after the end of the therapy.
et al., 2011). Because of the excellent tolerance, we opted to
use the 40 mg regimen in the FeLV treatment study.
cats showed a regressive infection with transient (n = 3) or
3.2. FeLV infection undetectable antigenemia (n = 7) (Fig. 1).
In search of an explanation for the low number of
All cats exposed to FeLV were antigen, provirus and kittens with progressive infection, enFeLV loads were
viral RNA negative prior to challenge. Three weeks after the determined in the blood samples collected from the 18
initial FeLV challenge all cats were provirus positive and all kittens prior to FeLV challenge. When the kittens were
but one cat (BP1) became viral RNA positive throughout grouped according to the infection outcome (progressive
the observation period indicating that that FeLV infection versus regressive infection), the fatally infected cat was
had become established in all of the cats (Fig. 1). A second categorized as having progressive infection), and the cats
FeLV challenge was performed 6 weeks after the initial with progressive infections had significantly lower enFeLV
FeLV challenge in eleven cats (Fig. 1). In spite of this second loads than cats with regressive infection (Fig. 3). This was
challenge, ten of the eleven cats (91%) became and/or the case for enFeLV-U3-1 (pMWU = 0.0152), enFeLV-U3-2
remained FeLV p27-negative for the remainder of the (pMWU = 0.0274), enFeLV-U3-total and enFeLV-env
study. Only one cat (CP1) remained positive and developed (pMWU = 0.0055).
persistent antigenemia; CP1 had the highest p27 load
among the re-challenged cats of 9.8% of the positive 3.3. FeLV treatment study
control. Overall after two FeLV challenges, seven cats
developed persistent antigenemia, one cat (CK4) suc- In the FeLV treatment study, none of the seven
cumbed due to FeLV associated disease (see below) and ten progressively FeLV-infected cats showed any behavioral
172 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178

Fig. 3. enFeLV proviral loads of the kittens in the FeLV treatment study. enFeLV loads were determined as described (Tandon et al., 2007, 2008a). The cats
were grouped according to the infection outcome (progressive versus regressive infection). The one cat undergoing fatal FeLV-associated disease, CK4, was
grouped as having progressive infection. a) enFeLV-U3-1; b) enFeLV-U3-2; c) enFeLV-U3-total; d) enFeLV-env. enFeLV loads are given as copies per cell, as
described earlier (Tandon et al., 2007, 2008a). Significant differences between the two groups of cats as calculated by the Mann-Whitney U-test are
indicated.

changes or clinical signs during the nine weeks of antiviral for p27 antigen: pMANOVA = 0.087 for weeks 11, 15
treatment. Raltegravir plasma concentration ranged from (baseline), 17, 21 and 24 (treatment) after the initial FeLV
122 to 1413 nM at 40 mg b.i.d. and from 655 to 1704 nM at challenge. However, two cats (CK3 and CK5) turned to
80 mg b.i.d. (Fig. 2b). By week 10, one week after nearly negative p27-values within eight to nine weeks of
termination of the treatment raltegravir was barely treatment (Fig. 4a).
detectable (Fig. 2b). No effect of raltegravir was observed on provirus loads
In order to assess the inhibitory potential of the (pMANOVA = 0.645) (Fig. 4c). None of the cats developed
treatment FeLV p27 antigen loads, anti-FeLV whole virus significant antibodies to FeLV p45 (data not shown) or FeLV
and p45 antibodies, as well as FeLV provirus and plasma whole virus (Fig. 4d) during raltegravir therapy.
viral RNA loads were determined. The raltegravir treat- Raltegravir treatment was interrupted after nine weeks
ment led to an approximately fivefold reduction in the (at week 24 after the initial FeLV challenge) and the course
plasma viral RNA load, which first became evident five of infection was monitored for a further eight weeks.
weeks into the treatment (Fig. 4b; Table 1). In the absence Plasma viral RNA loads started to increase again, returning
of an untreated control group of persistently infected cats, to the levels prior to treatment within four weeks after the
the results were compared with data from previous end of the treatment in all cats with the exception of cat
experiments using a similar challenge dose (5  105 FFU) BP2 (Fig. 4b; Table 1). In the latter cat, the FeLV RNA load
of the same virus in experimentally infected SPF cats reduction was still approximately fivefold at the end of the
(Gomes-Keller, 2011). In comparison with these controls, study. Moreover, cat BP2 was the only cat that developed
the seven raltegravir treated cats had significantly lower antibodies to FeLV, but only after the treatment was
viral RNA loads (pMANOVA = 0.048). stopped (week 2 to 8 post-treatment; Fig. 4d). The two cats
In all seven cats, a significant correlation was observed that had transiently shown some antibodies to FeLV four
between the FeLV p27 antigen loads and the plasma viral weeks after FeLV infection (CK5 and CR5) did not develop
RNA loads (p < 0.0001; Fig. 5). Nonetheless, the effect of any antibodies during the treatment period or thereafter
the raltegravir treatment was only statistically marginal (Fig. 4d).
 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178 173

3.4. FeLV associated disease

Cat CK4 became p27-positive (12%) in week 3 after the

initial FeLV challenge (Fig. 1). In week 4, the cat had
increased body temperature (>40 8C), and showed depres-
sion, anorexia, developed decreased body weight, and pale
and icteric mucous membranes. Hematology and clinical
chemistry revealed normochromic, microcytic anemia
(hematocrit 22%; 5% quantile: 33%), leukopenia (1400
cells/ml; 5% quantile 4600 cells/ml) with neutropenia (20
cells/ml; 5% quantile 2320 cells/ml), thrombocytopenia,
bilirubinemia (61.5 mmol/l; 95% quantile: 3.5 mmol/l),
mild hypoproteinemia (62 g/l; 5% quantile: 64 g/l) and
hypoalbuminemia (22 g/l; 5% quantile: 30 g/l). The cat was
euthanized for humane reasons 4.3 weeks after FeLV
infection. At that time point the cat was p27-positive (10%)
and had high plasma viral RNA loads (4  108 copies/ml of
plasma) and proviral DNA copy numbers (5 proviral copies
per cell) compatible with progressive infection (Hofmann-
Lehmann et al., 2001; Hofmann-Lehmann et al., 2006). In
the FeLV subgroup PCR, whole blood was positive for FeLV-
A and -B and the blood plasma was positive for FeLV-C
(Fig. 6).

4. Discussion

The aim of the present study was to investigate whether

the integrase inhibitor raltegravir has an inhibitory effect
on FeLV in vivo, and if the magnitude of the inhibition
enables cats to mount an adequate immune response, thus
overcoming the progressive infection. For this purpose we
first tested the drug in three healthy SPF cats, to ensure
that raltegravir treatment does not cause severe side
effects. The main metabolic pathway of raltegravir is by
glucuronidation, well-known to be poor in domestic cats
(Court and Greenblatt, 2000; Shrestha et al., 2011). Hence,
it was expected that tolerance to this drug may be worse
Fig. 4. Influence of the raltegravir treatment on the FeLV infection. than in humans or other species. However, the defective
FeLV p27 antigen loads (a), plasma viral RNA loads (b), blood proviral DNA enzyme of glucuronidation in the cat (related to the
loads (c), and anti FeLV whole virus antibody loads (d) in the seven
absence of expression of UGT1A6) (Court and Greenblatt,
persistently infected and raltegravir treated kittens. The dashed lines
represent the threshold for p27-positivivity at 4% (a) and antibody 2000) is dissimilar to the enzyme mainly responsible for
positive results at 25% (d) of the positive control, respectively. The the glucuronidation of raltegravir (UGTA1) (Kassahun
treatment period is indicated by a gray box labeled ‘‘treatment’’. Each cat et al., 2007). Therefore, this metabolic defect in cats may
received 40 mg of raltegravir b.i.d. from week 15 to 21.5 after infection not play a very important role for the elimination of
and 80 mg b.i.d. from week 21.5 to 24 after infection.
raltegravir. Observations in other species confirmed an

Table 1
Plasma viral RNA load reduction compared to baseline (fold) of the seven progressively infected cats, during and after treatment. Gray shaded areas
highlight values > 3.0. WPI: weeks post-infection. BL: baseline.

BL Treatment week Interruption week

1 2 3 4 5 6 7 8 9 1 2 4 6 8

WPI 15 16 17 18 19 20 21 22 23 24 25 26 28 30 32
BP2 1.0 1.1 1.8 2.1 3.5 3.4 15.2 6.3 13.3 7.6 6.8 13.1 8.8 5.1 4.7
CC1 1.0 0.5 0.5 0.7 1.2 1.2 3.8 3.5 0.9 1.9 2.4 1.3 2.9 1.3 2.6
CK1 1.0 0.5 0.6 0.4 1.2 1.2 2.0 3.1 0.8 1.6 2.1 1.5 0.7 0.2 1.4
CK3 1.0 0.4 0.7 1.1 1.1 5.2 4.4 5.1 5.6 3.4 6.9 2.4 2.4 0.5 3.0
CK5 1.0 0.7 0.8 0.9 1.9 8.3 4.1 3.7 5.2 4.9 6.4 1.7 1.5 0.9 0.7
CP1 1.0 0.3 0.2 0.4 0.6 2.6 0.9 2.1 1.1 1.9 2.4 0.8 0.5 0.4 0.2
CR2 1.0 0.7 1.6 1.9 1.9 13.4 5.7 26.2 6.8 5.6 7.6 2.4 0.7 0.8 3.1
Mean 1.0 0.6 0.9 1.1 1.6 5.1 5.2 7.2 4.8 3.8 4.9 3.3 2.5 1.3 2.2
St Dev 0.0 0.3 0.6 0.7 0.9 4.5 4.7 8.5 4.5 2.3 2.5 4.3 2.9 1.7 1.6
174 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178

Fig. 5. Course of viral RNA and p27 loads for all cats. (a) to (g): cats BP2, CC1, CK1, CK3, CK5, CP1, CR2, respectively. The treatment period is indicated by a
gray box labeled ‘‘treatment’’. Each cat received 40 mg of raltegravir b.i.d. from week 15 to 21.5 after infection and 80 mg b.i.d. from week 21.5 to 24 after
infection.
 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178 175

Fig. 6. FeLV subgroup PCR of cat CK4 at the time of necropsy. FeLV subgroups were investigated in the plasma (cDNA plasma) and peripheral white blood
cells (cDNA pWBC) of cat CK4 at the time of necropsy. The expected sizes of the positive controls are 1071 bp for system A (FeLV-A), 857 bp for system B
(FeLV-V) and 1754 bp for system C (FeLV-C). Positive and negative controls for each system were run in parallel. PCR products were analyzed on a 1.5%
agarose gel. Marker: Fermentas SM0333 (Thermo Scientific, Waltham, Massachusetts).

overall high tolerance to raltegravir: A study showed that the challenge virus. However, the virus, shipped on dry ice
raltegravir is well tolerated by dogs that received up to 9- directly from the University of Glasgow a few days before
fold the 360 mg/kg b.i.d. used in humans, by rats receiving experimental infection, was harvested, stored and the
4.8-fold of the above dosage and humans receiving the quality assessed under the same conditions as in previous
double of the recommended dose of 12 mg/kg (Merck, experiments (Gomes-Keller, 2011; Hofmann-Lehmann
2007). These observations are consistent with our findings et al., 1995, 2001, 2006; Lehmann et al., 1991). Therefore,
in cats, where no side effects were observed during the poor virus quality is unlikely, especially since already
whole period of 15 weeks, although a 2-fold higher dosage 5  104 FFU were sufficient to cause a progressive infection
of 40 mg and a 4-fold higher dosage were used. In addition, in 8 of 10 cats, infected at the age of 17-19 weeks (Gomes-
a recent in vitro study showed, that raltegravir failed to Keller, 2011). In addition, one cat had to be euthanized
induce toxicity even at concentrations 280-fold higher because of FeLV associated disease within a few weeks
than the IC50 for antiviral activity in Crandell Reese feline after infection, consistent with the virus stock having good
kidney cells (CrFK) (Greggs et al., 2012), confirming the infectivity. Remarkably, FeLV-B and FeLV-C were detect-
results obtained previously in our laboratory (Cattori et al., able by subgroup PCR by 4 weeks after infection. To the
2011). best of our knowledge this is the first documentation of the
To determine the effect of raltegravir in progressively development of FeLV-C in a FeLV-A/Glasgow-1 infected
infected cats, 18 kittens were challenged intraperitoneally cat.
with 8  105 FFU FeLV-A/Glasgow-1. According to previous Alternatively, the enFeLV sequences and/or loads in the
observations, 14 to 17 cats were expected to show a cats may have been responsible for the poor progressive
progressive infection (Gomes-Keller, 2011; Hofmann- infection rate. We have highlighted previously that enFeLV
Lehmann et al., 1995, 2001, 2006; Lehmann et al., 1991). should be considered as an important confounder in
Remarkably, only seven cats developed a progressive experimental FeLV infection or vaccination studies (Tan-
infection in the current study. An explanation for this don et al., 2008a, b). Previous studies demonstrated that
unanticipated development may be a reduced quality of high enFeLV loads were associated with higher viral
176 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178

replication and loads during early infection in persistently persistently FeLV-infected cats are in the range of 108–109
infected cats (Tandon et al., 2008a). No significant copies/ml, while for HIV and SIV the range is much lower,
association could be demonstrated between the enFeLV 103–105 copies/ml. Thus, viral reservoirs seem to be more
loads and the FeLV infection outcome in that study active for FeLV than for HIV/SIV, and this may partly
probably due to the small numbers of cats, but it seems explain the lower magnitude of the reduction in RNA
noteworthy that only five out of ten cats with high enFeLV loads.
load but nine out of ten cats with low enFeLV became As observed e.g. for SIV in macaques (Lewis et al.,
persistently infected (Tandon et al., 2008a). Thus, the 2010), proviral DNA levels remained unchanged during
present data would confirm these observations: to the best treatment. This does not necessarily mean that the viral
of our knowledge this study proves the first results to reservoir was unaffected by the treatment. The fivefold
indicate that low enFeLV loads prior to FeLV exposure may decrease in RNA loads shows that new cells are steadily
be significantly associated with a worse outcome of FeLV infected in progressively infected cats even after RNA
infection, i.e. progressive infection. From the current data it loads have reached a steady state. Integrase inhibitors
cannot be concluded whether enFeLV per se could be do not prevent synthesis of DNA from viral RNA via
responsible for this phenomenon or whether the enFeLV reverse transcription, but only the integration of the
load is rather an indicator for significant differences in the proviral DNA into the host’s genome. Virus, infecting
genetic background of these cats in general. Indeed, there cells without integrating DNA, is unable to generate new
were four out of five siblings from a litter with unfortunate RNA. Unchanged DNA levels may have arisen because
infection outcome (CK1, CK3, CK4, CK5) and three out of the qPCR test for FeLV is not able to differentiate
three siblings from another litter with regressive infection. integrated (provirus) from unintegrated viral DNA.
Further studies need to be performed before any conclu- Nevertheless, once occurred, the integration process is
sion should be drawn from this observation, e.g. concern- irreversible. Therefore, treatment in the early phase of
ing breeding of cats with low FeLV susceptibility. infection is really important, as exemplified in humans,
To achieve a higher number of progressively infected where viral DNA levels were lower after treatment with
cats, the eleven cats with low antigenemia (<10% at week raltegravir in HIV primary vs. chronic infection (Koelsch
4) were again exposed to FeLV using 1.7  106 FFU FeLV-A/ et al., 2011).
Glasgow 1 six weeks after the first challenge. Remarkably, In only in one cat (BP2) a marginal anti-FeLV whole
only one out of these eleven cats (CP1), the cat with the virus antibody response was observed shortly after the end
highest p27-value (9.8%) among the eleven cats developed of the therapy, but no anti p45 antibody responses were
persistent viremia thereafter. All other re-challenged cats detected in any cat at any time point. According to our
(91%) with p27-values <8.2% did not become persistently knowledge, anti p45 antibodies are the most important to
infected. This confirms that four weeks after the initial generate an effective immune response against FeLV
FeLV challenge the p27-level is an indicator for the (Hofmann-Lehmann et al., 2007). Anti FeLV whole virus
outcome of the FeLV infection (regressive versus progres- antibodies are also associated with protection—if high
sive); in an earlier study, we have demonstrated that p27 enough and lasting for some time (Geret et al., 2011b). In
levels start to significantly differ between cats with the present study, antibody concentrations of 28% of the
progressive and regressive infection four weeks after the positive control were too low to change the course of
virus exposure (Hofmann-Lehmann et al., 2001). In infection. By week 4 after treatment interruption, plasma
addition, the results indicate that at six weeks after the viral RNA loads had increased again in all seven cats,
FeLV challenge a protective immune response had returning to the levels assessed before treatment in six of
developed in these cats; no further antigenemia was them. Although the short-term treatment was not suffi-
observed. cient to completely prevent viremia, the levels observed
Based on the low number of treatable cats, it was no after treatment interruption provided clear evidence for an
possible to include a sufficiently large untreated control inhibitory effect of raltegravir.
group in this study. Therefore, we decided to treat all Further investigations are needed to better assess the
progressively infected cats and to compare the outcome effect of raltegravir. Although – based on the values of
with baseline and historical data (Gomes-Keller, 2011). raltegravir plasma concentration – the use of a higher
During the nine weeks of treatment a decrease in the dosage is considered possible, there is no guarantee that a
plasma viral RNA load was observed, indicating the ability higher concentrations in plasma (and therefore more
of raltegravir to reduce viral replication also in vivo. The effective treatment) can be achieved. It seems that
decrease of plasma viral RNA loads was in the range of one saturation occurred in some cats after administering the
log10 for four of seven cats (Table 1). In humans, raltegravir fourfold higher dosage of 80 mg b.i.d., leading to stable
therapy achieved, in general, a higher decrease (at least plasma concentrations. Variable genetic backgrounds and
two log 10), but this was always as a combination therapy rates of metabolism of each cat are presumably responsible
(Merck, 2007), and the effect of raltegravir alone was for the none-homogeneous outcomes.
difficult to discern. In three macaques infected with simian The development of novel integrase inhibitors opens
immunodeficiency virus (SIV), the effect was of around the door to further interventions. Elvitegravir (Wills and
three log 10 reduction to undetectable levels after four Vega, 2012) and so called ‘‘new generation’’- compounds
weeks of treatment (Lewis et al., 2011). Thus, raltegravir like dolutegravir (S/GSK 1349572) (Katlama and Murphy,
seems to be less efficient in reducing replication in FeLV 2012), MK-2048 (Bar-Magen et al., 2010) or Dolutegravir
than in HIV or SIV-infected individuals. RNA levels in (Boyd, 2012) which needs lower and less frequent dosages
 A. Boesch et al. / Veterinary Microbiology 175 (2015) 167–178 177

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Survey of infectious and parasitic diseases in stray cats at the Lisbon
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BW was responsible for all clinical aspects. MM supported 2011a. Housing and care of laboratory cats: from requirements to
practice. Schweiz. Arch. Tierheilkd. 153, 157–164.
the study and performed sequence analysis. KMR performed
Geret, C.P., Cattori, V., Meli, M.L., Riond, B., Martinez, F., Lopez, G., Vargas,
the raltegravir plasma concentration measurements. MJH A., Simon, M.A., Lopez-Bao, J.V., Hofmann-Lehmann, R., Lutz, H.,
produced and provided the cell culture supernatant 2011b. Feline leukemia virus outbreak in the critically endangered
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thesis, contributed to the data analysis and drafted the Virol. 156, 839–854.
manuscript. HL designed and supported the study. All Geret, C.P., Cattori, V., Meli, M.L., Riond, B., Martinez, F., Lopez, G., Vargas,
authors read and approved the final manuscript. A., Simon, M.A., Lopez-Bao, J.V., Hofmann-Lehmann, R., Lutz, H.,
2011c. Feline leukemia virus outbreak in the critically endangered
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