ELSEVIER Veterinary Immunology and Immunopathology

46(1995)181-194

Veterinary immunology

and InunuIlopathoIogy

Therapeutic effects of diethylcarbamazine and 3 ‘-azido-3 ‘-deoxythymidine on feline leukemia

virus lymphoma formation

Phillip Nelson”, Rance Sellon”, Carol Novotneya, Cristina Devera”, Marie Davidianb, Robert English”, Mary Tompkins”,

Wayne Tompkins”** “Department of Microbiology, Pathology. and Parasitology, College of Veterinary Medicine,

North Carolina State University. 4700 Hillsborough Street, Raleigh, NC 27606. USA “Department of Statistics, North Carolina State University. 4700 Hillsborough Street, Raleigh. NC 27695-8401.

USA

Abstract

Twenty-four specific pathogen-free kittens were infected with the Rickard strain of feline leukemia

virus (FeLV,). The kittens were divided into four equal groups and were orally administered either a high dose of diethylcarbamazine (DECH, 12 mg kg- ’ ) , a low dose of diethylcarbamazine ( DECL, 3 mg kg- ‘), 3’-azido-3’-deoxythymidine (AZT, 15 mg kg-‘, b.i.d.), or a placebo (250 mg granular

dextrose) daily for 10 weeks. Blood was collected at 2-week intervals for complete blood counts (CBC) and flow cytometric analysis (FACS) of peripheral blood lymphocytes (PBL). Plasma was assayed for antibodies to FeLV gp70 and for FCLV p27 antigen using ELISA assays. For FACS analysis, lymphocytes were incubated with monoclonal antibodies to feline Pan T, CD8’, CD4+,

and B cell ( Anti-Ig) antigens. In the placebo treated cats, FeLV, infection caused an early (2 weeks p.i. ) and persistent decrease in leukocyte numbers attributable primarily to a decrease in neutrophil numbers and a secondary lesser decrease in B and CD4+ lymphocyte numbers. The DEC-treated groups showed a delayed but similar leukopenia by 4 weeks p.i. The lymphopenia in the DEC groups (primarily B cells and CD4+ cells) was reversed by 10 weeks p.i., but the neutropenia persisted. AZT treatment inhibited FeLV,-induced lymphopenia but did not prevent a reduction in neutrophil numbers. A marked p27 antigenemia that peaked at 4 weeks p.i. was noted in the placebo treated cats and in most cats ( 1 1 / 12) treated with either dose of DEC. However, AZT significantly inhibited the p27 antigenemia and all cats were negative for p27 antigen between 6 and 10 weeks of treatment. In general, placebo treated cats as well as DECH and DECL cats had low levels of antibody to gp70 throughout the study, suggesting FeLV,-induced immunosuppression. In contrast, significantly higher titers of anti-gp70 antibodies were seen in AZT-treated cats at 6 weeks p.i., and were maintained

* Corresponding author. Tel: 919-515-7394; Fax: 919-515-4237

01652427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO165-2427(94)07017-2

182 P. Nelson et al. / Veterinary Immunology and Immunopathology 46 (1995) 181-194

throughout treatment. Eighteen month survival rates provide efficacy data for AZT as well as both DEC treatment groups. While all placebo treated cats were euthanized by 52 weeks p.i. due to FeLV associated lymphomas with a mean survival time of 35.5 weeks p.i., median survival time of the AZT treated group was 2 102 weeks p.i., while that of the DECH and DECL groups was 69.7 and 72 weeks p.i., respectively. Thus, DEC as well as AZT therapy delays the development of lymphomas associated with FeLV infection and significantly improves survival.

Abbreviations

AIDS, acquired immune deficiency syndrome; AZT, 3’-azido-3’-deoxythymidine; CBC, complete blood counts; DEC, diethylcarbamazine (N,N-diethyl-4-methyl- 1 -piperazine carboxymide) citrate; DECH, high dose DEC; DECL, low dose DEC; FACS, flow cytometric analysis; FeLV, feline leukemia virus; FeLV,, Rickard strain of FeLV; FOCMA, feline oncoma cell membrane antigen; LSA, lymphosarcomas; PBS, phosphate buffered saline; RBC, red blood cell; SPF, specific pathogen free; WBC, white blood cell.

1. Introduction

Cats experimentally infected with various isolates of feline leukemia virus (FeLV) show

several important immunopathological abnormalities also characteristic of HIV- 1 infection

in humans (Gardner and Luciw, 1990). The Rickard strain of FeLV (FeLVn) causes an early panlymphopenia followed by indications of functional immune suppression. Infected cats eventually develop thymic and/or mesenteric lymphomas approximately 1 year post

infection (Essex et al., 1975). Hoover et al. ( 1987) described an isolate of FeLV (FeLV- FAIDS) that induces a fatal immunodeficiency syndrome without concomitant leukemo-

genesis. Both the FeLVn and FeLV-FAIDS isolates have been used effectively to evaluate potential therapies for HIV-l infection. Tavares et al. ( 1987) reported that 3’-azido-3’-

deoxythymidine (AZT) was a potent inhibitor of FeLVn infection in cats when given early during infection, but was less effective as a therapy for established infections. Zeidner et al. ( 1990b) reported that AZT in combination with alpha interferon (1lW-c~) was prophy- lactically effective in preventing FeLV-FAIDS induced immunodeficiency syndrome in cats. IFN-a, but not AZT, was effective as a therapy for established FeLV-FAIDS infections

(Zeidner et al., 1990a). A number of recent studies with the FeLV, model have suggested that the anti-filarial

drug, and purported immunomodulator, diethylcarbamazine (DEC), may be a useful ther- apy for retroviral infections (Kitchen, 1987, 1988; Kitchen and Cotter, 1988; Kitchen et al., 1988; Kitchen and Mather, 1989). In one study, oral administration of DEC increased the serum antibody titers to feline oncorna cell membrane antigen (FOCMA) in cats infected with FeLV, (Kitchen et al., 1988). The same authors reported that chronic oral adminis- tration of DEC prevented or delayed the lymphopenia associated with FeLV, infection (Kitchen et al., 1988). In a follow-up study with a larger group of cats, data showed that oral administration of DEC to chronically FeLV, infected adult cats resulted in a marked decrease in lymphocyte counts (Kitchen and Mather, 1989). The authors speculated that DEC may be therapeutic by reducing the number of circulating FeLV-infected lymphocytes.

P. Nelson et al. / Veterinary Immunology and Immunopathology 46 (1995) 181-194 183

While the mechanisms by which DEC modulates FeLV infection are unknown, the

observations that DEC reduces circulating microfilaria only in animals that have antibodies

to the parasite (Piessens et al., 1981)) and that DEC promotes adherence of microfilaria to leukocytes in the presence of serum containing antibodies to the microfilaria, allow the speculation that DEC provides some positive immunomodulatory signal via increased opsonization. Cesbron et al. ( 1987) have presented data to suggest that platelets mediate the action of DEC on microfilariae. These authors reported that DEC triggers release of

tilarial excretory antigens that in turn stimulate platelet aggregation and the release of cytotoxic oxygen free radicals. They also reported that platelet killing was antibody inde- pendent. If these data are correct, one could predict that a similar mechanism may occur in

the case of FeLV infected cells and therefore could explain the results of Kitchen and co- workers (Kitchen, 1987, 1988; Kitchen and Cotter, 1988; Kitchen et al., 1988; Kitchen and

Mather, 1989). Because of the potential value of DEC as an effective, non-toxic therapeutic agent for

human acquired immune deficiency syndrome (AIDS), the present study was designed to further evaluate antiviral and immunomodulatory effects of DEC in the experimental FeLV, model. AZT was used as a known antiviral agent for FeLV, infection. This study was also designed to evaluate the early effects of FeLV, on circulating lymphocyte subsets (Pan T, CD4 +, CD8 + and B cells) and virus-specific antibodies.

2. Materials and methods

2. I. Virus isolate

The virus isolate utilized in this investigation was the FeLV,, subgroups A and B, originally obtained from Keith Haffer of SmithKline Beecham Animal Health (Lincoln,

NE). This strain consistently induces thymic and/or mesenteric lymphomas by 1 year post inoculation when inoculated into kittens less than 10 weeks of age (Essex et al., 1975). The virus was obtained from three Liberty Laboratories specific pathogen free (SPF) cats that had been experimentally infected with the FeLV, virus for approximately 1 year.

2.2. Infection of cats

Twenty-four SPF kittens, (aged 5-7 weeks), were obtained from Liberty Laboratories (Liberty Corners, NJ). Complete blood counts (CBC), and flow cytometric analysis (FACS) of peripheral lymphocyte subsets were performed prior to infection with FeLV. These kittens were housed in three communal stalls separated by chain link fencing. This allowed moderate physical interaction between adjacent stalls. Each kitten was randomly assigned to one of four experimental treatment groups. Each kitten was inoculated intra- venously with 1 ml of pooled whole blood obtained from the three virus source cats described above. This dose proved to be 100% effective in producing p27 antigenemia in the FeLV infected, non-treated, control group.

2.3. Treatment groups

Treatments consistedof DEC (N,JV-diethyl-4-methyl-1-piperazinecarboxymide) citrate; (DEC; The Wellcome Foundation, London, UK) and AZT (Zidovudine; Burroughs Well-

184 P. Nelson et al. /Veterinary Immunology and Immunopathology 46 (1995) 181-194

come Co., Research Triangle Park, NC). The placebo group received encapsulated dextrose. AZT and DEC were provided through the Developmental Therapeutics Branch, AIDS Program, NIAID, Bethesda, MD, USA.

Each treatment group contained six randomly assigned kittens. The treatment groups were designated as fellows: ( 1) DEC high dose (DECH; 12 mg kgg ’ day-‘, once a day

peros); (2) DEClowdose (DECL;3mgkgg’day-‘,onceadayperos); (3) AZT(l5 mg kg-’ twice a day per OS) ; (4) placebo (FeLV control, 250 mg dextrose once a day per OS). DEC doses of 3 mg kg-’ day-’ and 12 mg kgg’ day-’ flanked the dose of 10 mg

kg-’ day-’ shown to have therapeutic activity in FeLV, infection (Kitchen and Cotter, 1988; Kitchen et al., 1988). Each drug was mixed with 2.50 mg granular dextrose and placed

in gelatin capsules at the appropriate concentration based on the average weight of the

kittens in each group. Total daily dosages were adjusted every two weeks to compensate for mean weight changes in each treatment group.

The DEC and placebo groups were initially treated at the time of infection with FeLV, and at 24-h intervals thereafter for 10 weeks. Treatment of the AZT group began 24 h prior

to inoculation of FeLVR, and was continued at the aforementioned dose for 4 weeks.

Prophylactic treatment with DEC and AZT was chosen in an effort to optimize the oppor- tunity of demonstrating a positive response by DEC and the positive control AZT. It has previously been demonstrated that AZT is effective against FeLV when used prophylacti-

cally, but not against established infection (Haschek et al., 1990). The development of Heinz body anemia and neutropenia in two of the six AZT treated cats required lowering

the total daily dose for the entire group to 15-20 mg kg- ’ during week 5 post infection (p.i.). After resolution of the anemia and neutropenia (week 6 pi.), the original AZT

dosage was re-instituted and continued through week 10 p.i.

2.4. Blood collection, fractionation, and lymphocyte subset analysis

Three to five milliliters of whole blood was collected from each kitten 1 week before and

at 2,4,6,8, and 10 weeks after infection. Whole blood was collected viajugular venipuncture in EDTA tubes/syringes after sedation with 20 mg ketamine HCl given intravenously. Approximately 0.5 ml of whole blood was submitted for CBC and differential leukocyte

counts. The remainder of the samples were centrifuged, then the plasma was removed and

was stored at - 70°C in three equal aliquots for later testing. The cells were then resuspended in Hank’s balanced salt solution to twice the original volume and separated over Histopaque, density 1.083 (Sigma Chemical Company, St. Louis, MO). Histopaque separated cells were prepared for flow cytometric analysis as previously described using a panel of monoclonal antibodies developed in our laboratory (Tompkins et al., 1990, 1991). Briefly, cells were incubated at a concentration of 5 X lo” cells in 100 ,ul of monoclonal antibody ( 1.572: Pan T; 3.357: CDS+; CAT30A: CD4+; aIg: B cell) overnight at 4°C. The cells were then washed three times with phosphate buffered saline (PBS) and incubated for 30 min at 4°C with a FITC-conjugated goat anti-mouse antibody that had been pre-absorbed with normal cat serum. The percent positively stained lymphocytes was determined by flow cytometric analysis using a Becton Dickinson FACScan. Total leukocyte counts were determined on a Coulter counter by standard procedure, and absolute lymphocyte counts were obtained from differential leukocyte determinations.

P. Nelson et nl. /Veterinary Immunology and Immunopathology 46 (1995) 181-l 94 185

2.5. Assay.forp27 antigen

Plasma screening for group-specific antigen p27 was accomplished by using Dia Systems FeLV-Flex-II test kit from Tech America (Omaha, NE). The results were spectrophoto- metrically quantitated on an ELISA plate reader (VMax, Molecular Devices). An OD unit was defined as the sample reading plus the negative control divided by the sum of the

positive and negative control (OD units = [x + ( - Control) ] + [ + Control + ( -Control] ) .

2.6. Assay for arltibody to FeLV gp70

Plasma samples from each sample collection were assayed for antibody titers to FeLV

gp70 using an ELISA method developed by Dr. S. Hansen (Hansen Veterinary Laboratories, Inc., Davis, CA). FeLV was obtained from supernatants of productively infected FL-74 cells. The virus was denatured and purified by SDS-PAGE. A band corresponding to a molecular weight of 70 kDa was excised from the gel and eluted and the eluted protein

bound to microtiter 96 well plates at a concentration of 200 ng per well for use in a capture ELISA. To assay anti-gp70 antibody, plasma samples were diluted and incubated in the

coated wells for 30 min at room temperature. The plates were washed in buffered saline and captured antibody detected by a peroxidase labeled goat anti-cat IgG (heavy and light

chain specific). After incubation at room temperature for 60 min, the plates were washed in buffered saline and a TMB substrate added for development. Reactions were quantitated

by OD at 650 nm. Results were expressed as spectrophotometric OD units determined as for the p27 assay.

2.7. Statistical analysis of data

All multiple sample data were subjected to a repeated measures analysis of variance

(RMANOVA) . Contrast comparisons (post-infection to pre-infection) between treatment

groups were performed at each time point of interest to determine significant differences in treatment effects. Statistical significance for RMANOVA was defined asp < 0.05. Rejection

level for the pairwise comparisons was 0.05.

3. Results

3.1. Toxicity

Cats showed no clinical or hematological evidence of toxicity to either dose of DEC. In the case of AZT treatment, two of six cats exhibited moderate and transient anemia (HCT I 20%), depression, anorexia, and neutropenia (less than 1000 neutrophils per ~1 of blood) 2 weeks p.i., as has been reported by us previously (Haschek et al., 1990). The anemia was characterized by macrocytic RBCs containing Heinz bodies. All other RBC indices (mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration) were

186 P. Nelson et al. / Veterinary Immunology and Immunopathofogy 46 (1995) 181-194

normal. With the exception of the two AZT-treated cats, no other cats in any group showed

changes in the RBC indices measured.

3.2. Total white blood cell (WBC), neutrophil and lymphocyte population changes

The FeLV infected placebo treated control group exhibited a leukopenia that was apparent

at 2 weeks and maximal at 4 weeks. The reduction in total numbers was primarily explained

by a decrease in neutrophil numbers (Fig. I). While the DEC treatment groups and the AZT treatment group also exhibited a neutropenia of similar magnitude to the FeLV control

group by 4 weeks pi (Fig. 1 ), the onset of neutropenia was delayed as compared with the FeLV control group. All treatment groups, with the exception of the AZT group, experienced

a generally mild and transient lymphopenia (Figs. 2 and 3)) that was due predominantly to

a decrease in B cells (Fig. 2) and CD4+ T lymphocytes (Fig. 3). CD8+ T lymphocytes

were not significantly altered in any of the treatment groups (Fig. 4). After the initial

lymphopenia, both B lymphocyte and CD4+ lymphocyte numbers expanded more rapidly

in the DECL and DECH groups than in the placebo group (Figs. 2 and Fig. 3). Unlike the

other treatment groups, there was no apparent decrease in lymphocyte numbers in the AZT

treatment group throughout the IO-week treatment period (Figs. 2 and 3).

In summary, FeLV, infection caused an early (2 weeks p.i.) decrease in neutrophil

numbers and to a lesser extent a lymphopenia, specifically a B-cell and CD4+ T cell

lymphopenia. Interestingly, DEC appeared to delay the FeLV induced neutropenia and

iymphopenia and enhance a recovery of B cells and CD4+ lymphocytes after the initial

lymphopenia, as compared with placebo treated cats.

15

f

-*- DECH

-A- DECL

-.- AZT

-3- FELV CTL

, 2 4 6 8 10

WEEKS POST INFECTION

Fig. 1. Mean neutrophil values for all treatment groups (n = 6 per group). Neutrophil values were determined by

multiplying the percent of neutrophils, (determined by counting and characterizing 100 white blood cells in a

blood smear), by the total WBC. DECL treatment group was significantly different from FeLV control at O-2

weeks p.i. (P < 0.05).

P. Nelson et al. / Veterinary Immunology and lmmunopathology 46 (1995) 181-l 94 187

-*- DEW

- - DECL

“0 5

- t

-A- AZT

-7- FELV CTL x

0’ 1

0 2 4 6 a 10

WEEKS POST INFECTION

Fig. 2. Mean IgG+ lymphocyte values for all treatment groups (n = 6 per group). Cell numbers were determined

by multiplying the percent positive IgG+ cells, determined by flow cytometric analysis, by the total lymphocyte

count from a CBC (drawn at the same time as the sample for flow cytometry). RMANOVA indicates significant

differences between DECL and FeLV control at O-2 and O-14 time intervals (P < 0.0475). and between AZT and

FeLV control at 4 and 14 weeks p.i. (P < 0.0s).

3.3. FeLV, p27 antigenemia

The FeLV control cats developed detectable plasma levels of FeLV p27 at the time of the first measurement (2 weeks p.i.), and attained peak levels at 4 weeks p.i. (Fig. 5). In

-*- DECH

4r -“- DECL

.I 0 -A- AZT

-o- FELV CTL

WEEKS POST INFECTION

Fig. 3. Mean CD4+ lymphocyte values for all treatment groups. Cell numbers were determined as described in

Fig, 2. RMANOVA indicates significant differences between AZT and FeLV control treatment groups at 14 and

18 weeks p.i. (P <0.03X).

188 P. Nelson et al. /Veterinary Immunology and Immunopathology 46 (1995) 181-194

3- -*- DECH

“0 -’ ‘- DECL

-A- AZT X

-T- FELV CTL

0

0 2 4 6 8 10

WEEKS POST INFECTION

Fig. 4. Mean CD8+ lymphocyte values for all treatment groups. Cell numbers were determined as described in

Fig. 2. CD8 + numbers did not change significantly in any of the groups throughout the duration of the experiment.

this group, FeLV p27 titers remained high throughout the 10 week duration of the study. The DEC-treated groups demonstrated an antigenemia profile similar to that of the FeLV

control group. However, one cat in the DECH group became negative at 4 weeks p.i. and

remained so throughout the lo-week treatment period (data not shown). Plasma p27 levels were generally lower in the DECH group than the DECL and FeLV control groups through-

out the study and became significantly (P<O.O075) lower than the placebo group by

2.0 - -*- DECH

z t

-o- DECL

5 -A- AZT

6 1.5 -

g -Q- FeLV CTL

r:

5 1.0 -

Y

ii

," 0.5

H

2 P

0.0

0 2 4 6 8 10

WEEKS POST INFECTION

Fig. 5. Mean plasma soluble p27 values for all treatment groups. p27 values were determined by assaying plasma

at 2 week intervals with an ELISA kit for group specific antigen (Tech America). Color changes were then

quantitated on an ELISA plate reader (VMAX. Molecular Devices). Pairwise comparisons to the FeLV control

treatment group indicates a significant difference in antigen levels when compared with the AZT treatment group

at O-4 through O-10 weeks p.i. and the DECH treatment group at O-10 weeks p.i. (rejection level 0.05).

P. Nelson et al. /Veterinary Immunology and Immunopathology 46 (1995) 181-194 189

-*- DECH 0.24 T

-o- DECL

ul iii -.- AZT

0.16 -v- FeLV CTL

,” & 0.12 - I

5 6 0.06 -

<

a’ o.ooff

0 2 4 6 6 10

WEEKS POST INFECTION

Fig. 6. Mean plasma values for antibody to gp70. All treatment groups are represented. Plasma antibody levels

were determined by an ELISA technique developed and performed by Dr. S. Hansen (Hansen Veterinary Labo-

ratories Inc.). Color changes were quantitated by an ELISA plate reader and expressed as optical density units

(ODIJ). Antibody titers were characterized as follows: low titer (0.06-0.08 ODIJ), median titer (0.09-0.19

ODU), and high titer (0.20-0.40 ODU). Pairwise comparisons with the FeLV control treatment group indicates

a significant difference in titers when compared with the AZT treatment group at O-8 and O-10 weeks p.i. (rejection

level 0.05).

analysis of the cumulative O-10 weeks p.i. data points (Fig. 5). Removal of the cat in the

DECH group that was negative for p27 from the analysis resulted in a loss of significance

when the remaining five cats in the DECH group were compared with any five cats in the FeLV-control group at 10 weeks p.i. These results suggest that there were no DEC induced

changes in p27 antigenemia. To be certain that the recorded measurements were the linear part of the OD curve and hence giving accurate determination of p27 levels between the

groups, we titrated (1:2-1:64) the 17 week bleeding samples from all treatment groups. In

all cases the samples yielded a similar and linear p27 OD titration (data not shown). The AZT treatment group demonstrated a significantly different antigenemia profile when

compared with that of the other groups (Fig. 5). The mean OD units for the AZT treatment

group reflects transient antigenemia which was significantly lower than the placebo group during Weeks 2 and 4 p.i. (P < 0.0164). During Weeks 6-10 p.i. p27 was undetectable in

all AZT-treated cats (Fig. 5).

3.4. Serum antibody to gp70

The FeLV control and DECL groups produced weak and transient Ab titers (Fig. 6) that peaked at 4-6 weeks p.i. and, in most cases, were below baseline measurement at 10 weeks p.i. Antibody in five of six cats of the DECH treatment group was detectable only at 10 weeks p.i. and only at low levels. One cat in the DECH group produced a strong Ab response comparable with the cats in the AZT treatment group. The DECH cat with high Ab titer was also the cat with low p27 levels in the circulation (individual data not shown). As with

190 P. Nelson ef al. / Velerinary Immunology and lmmunopathology 46 (1995) 181-194

the p27 data, the AZT-treated cats distinguished themselves from the other groups in serum antibody titers to gp70. All cats in the AZT-treated group developed strong antibody titers by 6 weeks p.i. that remained high at 10 weeks p.i. (Fig. 6).

3.5. Disease development in FeLV, infected cats

Cats infected with the FeLV, isolate of FeLV commonly develop lymphosarcomas

(LSA) within a year following infection (Essex et al., 1975). All of the placebo treated FeLV infected cats in this study became moribund and were euthanized within a year of

infection, with a median survival time of 35.5 weeks (Table 1). All of these cats were subsequently shown histologically to have lymphosarcomas, primarily in the thymus. Cats

in the DECH and DECL treatment groups also developed LSA. However, the frequency of LSA development (4/6 for DECH and 5/6 for DECL) was lower than the FeLV control

group (6/6) as of this writing. Moreover, the mean survival time for both the DECH (69 weeks p.i.) and DECL (72 weeks p.i.) were significantly greater than the control group

(35.5 weeks p.i.) (Table 1) . The AZT treatment group also showed a significantly greater

mean survival time ( 2 102 weeks pi.), and at this time only 2/6 cats have developed LSA

(Table 1) .

Table 1 Disease development and mortality in FeLV infected cats

Group

AZT

Mean survival

time (weeks p.i.)”

102”

No. of deaths/

total

216

Cat Time to death Disease/necropsy

ID No. (weeks pi.) findings

4 90 LSA’

6 98 LSA

DECH 69” 4/6 3 33

6 4-l

4 50

2 70

LSA

LSA

Fibromas

LSA

DECL 72” 5/6 4 33 LSA

I 49 LSA

6 58 LSA

2 94 LSA

3 92 LSA

FeLV CTL 35.5 616 6 20 LSA

3 26 LSA

2 29 LSA

5 35 LSA

4 51 LSA

I 52 LSA

“The standard error of the means is 8.728 weeks. Analysis of variance indicates significant differences in survival

times among all treatment groups (P < 0.0008 ) bThe AZT mean survival time was significantly longer than DECH. DECL, and FeLV CTL (rejection level 0.05).

‘LSA. lymphosarcoma. dDECH and DECL mean survival times were significantly longer than FeLV CTL (rejection level 0.05).

P. Nelson et al. /Veterinary Immunology and lmmunopathology 46 (1995) 181-194 191

4. Discussion

The need for non-toxic, effective anti-HIV-l therapies prompted us to evaluate in the

feline/FeLV, model the therapeutic activity of diethylcarbamazine citrate (DEC), a pur-

ported immunomodulator with anti-retroviral activity (Kitchen, 1987, 1988; Kitchen and

Cotter, 1988; Kitchen et al., 1988; Kitchen and Matber, 1989). As a positive therapeutic

regimen, we used AZT, which has been shown to be effective prophylactically in several

animal models for AIDS (Tavares et al., 1987; Zeidner et al., 1990a,b), including the

FeLV, model (Tavares et al., 1987; Haschek et al., 1990) used in this study. Both AZT and DEC prolonged survival of FeLV infected cats while only AZT affected concentrations

of plasma p27 and antibodies to gp70. Infection with FeLV, resulted in a sustained decrease in total leukocyte numbers char-

acterized primarily by a reduction of neutrophils. Compared with untreated controls, treat- ment of FeLV-infected cats with DECL, DECH, or AZT appeared to delay the neutropenia

but did not inhibit it. In these three treatment groups, the initial decrease in neutrophil counts was delayed by approximately 2 weeks, and then the neutrophil profile essentially paralleled

that of the FeLV-infected untreated cats (Fig. 1). FeLV infection also caused a mild

reduction in B cell and CD4+ lymphocyte numbers. Interestingly, DEC treatment at both

high and low doses appeared to promote a greater expansion of CD4+ T lymphocytes and

B lymphocytes following the initial lymphopenia as compared with the FeLV control group (Figs. 2 and 3). These results support previous reports that DEC has positive immunomo-

dulating qualities (Orange and Austen, 197 1; Piessens et al., 1981; Kitchen, 1987; Kitchen and Cotter, 1988; Kitchen and Mather, 1989).

Despite the ability of DEC to modulate the phenotypic profiles of circulating WBC, DEC was minimally effective in reducing FeLV p27 plasma levels and preventing functional

impairment of immune cells. ELISA assays for circulating FeLV, p27 showed that, with the exception of one animal in the DECH group, the DEC treated cats and the FeLV,

infected placebo-treated cats developed high and sustained levels of ~27. However, com-

pared with FeLV-control, there was a significant (P < 0.05) reduction in p27 levels by 10

weeks p.i. in the DECH group. At 17 weeks and 29 weeks p.i. ( 19 weeks after termination

of treatment) there was no significant difference in p27 levels between the DECH group and the FeLV control group, whereas the cats in the AZT group had undetectable ~27. It is

possible that the immunomodulatory effects of DEC are transient and that continued admin- istration would have caused additional reduction in p27 levels. It is more likely however,

that the perceived reduction in p27 levels by 10 weeks in the DECH group was an artifact introduced by the single cat in this group that was negative for p27 at that time. Omission

of this animal from the statistical analysis eliminated the difference between the DECH group and the FeLV control group by 10 weeks p.i.

Cats in the FeLV control and DEC groups with high levels of circulating p27 produced weak antibody responses to FeLVR gp70. In most of these cats the antibody response was transient, and many were negative by 8-10 weeks post infection (Fig. 6), suggesting a

profound immunosuppression. The DECH cats showed their greatest anti-gp70 response by 10 weeks p.i. However, these titers were still low and not significantly different from the FeLV control group. The weak anti-gp70 antibody results reported here are consistent

192 P. Nelson et al. /Veterinary Immunology and Immunopatho1og.v 46 (1995) 181-194

with previous reports of weak immune responses to FeLV antigens with progressive FeLV infections (Hardy et al., 1973; Hoover et al., 1977).

In contrast to the results described herein, Kitchen and Cotter ( 1988) reported that DEC treatment increased the serum antibody titers to the FOCMA in cats exposed to feline

leukemia virus. It is not clear why the results of these two studies should differ so markedly. Cats treated by Kitchen and Cotter ( 1988) had long-term FeLV, infections, whereas we tested cats with early acute stage FeLV, infections. Additional studies will be required to

address this. In the same study, Kitchen and Cotter ( 1988) reported that infectious virus in

the serum was undetectable after 1 month of DEC treatment in 12 of 14 FeLV cats initially showing high titers of infectious virus. One of 12 DEC treated cats in our study showed

this pattern of high antibody titer and low circulating viral antigen.

Despite the fact that DEC treatment failed to measurably alter the immunosuppression associated with FeLV, infection, as indicated by low to absent antibody response to the

virus and plasma p27 levels, the positive effect on FeLV-induced lymphocyte changes suggest that there was some subtle immunomodulatory effect of DEC. This is supported by

the observation that the long-term survival of cats in both the DECH and DECL groups was significantly greater than the FeLV, control group (P <0.05). It is possible that DEC

positively influenced other immunological parameters, such as cell mediated immune

responses, that are important in resistance to FeLV,. It is well documented that there is an

age dependent resistance to FeLV induced disease that is associated with maturity of macrophage resistance to infection (Hoover et al., 1976). It will be of interest to determine if DEC has any effect on macrophage function.

Consistent with reports that FeLV resistant cats usually produce high titers of virus neutralizing and FOCMA antibody (Rojko et al., 1979; Hooveret al., 1980), all cats treated with AZT produced a strong antibody response to gp70 by 2-4 weeks post infection. Similar to our observations, Zeidner et al. ( 1990b) reported that most cats that were antigen negative

during AZT treatment remained negative for up to 38 days after treatment was discontinued.

However, these authors also showed that these cats were latently infected as evidenced by reactivating virus from bone marrow cultured in the presence of hydrocortisone. We also

observed that though FeLV LTR sequences could not be detected in PBL at 8 and 10 weeks p.i., FeLV LTR sequences were detected in bone marrow of negative AZT treated cats by

PCR/Southern analysis (P.D. Nelson, unpublished, 1993). The weak and transient antibody (anti-gp70) response in the FeLV-control and DEC

treatment groups is somewhat surprising. It is unlikely that the profound suppression in antibody response to virus gp70 in the FeLV-control group and in many cats of the DEC

treatment groups can be attributed simply to loss of T (CD4+) and B lymphocytes given the generally mild nature of the virus-induced lymphopenia. However, it is possible that

low antibody titers in the FeLV-control group are due to functional suppression of T and/ or B cells by FeLV,. In this regard, we have recently shown that peripheral CD4+ cells have a high FeLV provirus load as compared with CD8+ in the FeLV, control cats while provirus is undetectable in peripheral B cells (Nelson, 1993). Moreover, in AZT treated cats, FeLV provirus is undetectable in any circulating lymphocytes between 6 and 10 weeks p.i. (data not presented), suggesting that the immunosuppression observed in placebo and DEC treated cats may be associated with a preferential infection of CD4+ cells that is inhibited in the AZT treated cats. In support of this, we also reported that Con A stimulated

P. Nelson et al. /Veterinary Immunology and Immunopathology 46 (1995) 181-194 I93

lymphocytes from cats with symptomatic as well as asymptomatic FeLV infections were deficient in their ability to produce IL-2, a CD4+ T, derived cytokine (Tompkins et al.,

1989). Whatever the mechanism, it is clear that suppression of the antibody (anti-gp70) response to FeLV occurs early after infection and persists for at least 10 weeks (longest

time tested). Although DEC did not appear to alter the plasma p27 and anti-gp70 levels, both DECL

and DECH treatment groups had a significantly longer mean survival time than the placebo

treatment group. All placebo treated cats developed lymphosarcomas and survived a mean

of 35.5 weeks p.i., whereas 10 of 12 DEC treated cats developed lymphosarcomas and survived for approximately 70 weeks p.i. Factors contributing to the increased survival of

DEC treated cats are not evident from this study. Lymphoid tumors, and in particular virus induced tumors, are thought to be controlled by the cell mediated arm of the immune response. It would be of considerable interest to determine if DEC modulates the cell

mediated immune responses either directly or through alterations in cytokines (vis IL-2. IFN-y and TNF-a) that regulate effector cells (CD8’ T cells, LAK, macrophages).

As expected from previous studies (Zeidner et al., 1990b), AZT treatment inhibited

lymphoma development in FeLV, infected cats. We attribute this to the fact that AZT reduced the early viremia and allowed the development of a strong cell mediated immune response to FeLV, antigens that maintained the latent condition of infection and prevented

disease development. In summary, we have presented evidence that both AZT and DEC are capable of modu-

lating immune parameters and increasing the mean survival time when administered to cats during acute FeLV, infection. While AZT appears to prevent the early FeLV, viremia and

associated immunosuppression, the mechanism of action of DEC remains to be defined. These observations suggest that therapy with lower and/or less frequent doses of AZT combined with immunomodulators such as DEC may be effective in either maintaining the latent/asymptomatic infection or converting immunosuppressed, viremic cats to immuno- competent, latently infected cats. Zeidner et al. ( 1990a,b) showed that AZT plus IFN-(r therapy conferred more complete resistance to FeLV-FAIDS challenge when compared

with either treatment alone in addition to having some therapeutic efficacy against estab- lished infection.

Acknowledgments

This study was supported by NIAID NOI AI 722665 and NO1 AI 35155 through the Division of AIDS. The authors would also like to thank Dr. David Hutchinson of the Wellcome Research Laboratories, Beckenham, Kent, UK for generously providing the DEC used in this study. The authors acknowledge the excellent technical assistance of Tedd Childers, Chris Parrish, and Douglas Gebhard.

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