Active and Passive Immunization of Cats With Inactivated Feline Oncornaviruses
1. 2
J. P. Schaller,3 E. A. Hoover,3 and R. G. Olsen 3. 4. 5 ABSTRACT-The humoral Immune responses of young «1 mo) and older (>6 mo) cats to killed and live feline leukemia virus (Fe LV), feline sarcoma virus (FeSV), and the feline oncornavlrus· associated cell membrane antigen (FOCMA) were evaluated. In addition, the biologic responses to oncogenic feline oncornavlrus challenge were analyzed In cats actively Immunized with Inac· tlvated Gardner-Arnstein FeSV (GA·FeSV) or passively Immu· nlzed with either the Rickard FeLV (R·FeLV) or GA·FeSV. Kittens were Immunized during the first month of life with UV· or formalin· Inactivated GA·FeSV and challenged at 5-6 weeks of age with a known oncogenic dose of GA·FeSV. Passive Immunization was evaluated In kittens that suckled mothers Immunized during pregnancy with purified GA·FeSV or R·FeLV. Kittens during the first month of life were capable of producing humoral antibody against FOCMA and keyhole·llmpet hemocyanin. Apparently linear disease responses to live R·FeLV from susceptibility (viremia with no FOCMA antibody) to resistance (no viremia and high FOCMA antibody level) were observed In cats from birth to 4 months of age. Newborn kittens failed to develop FOCMA and vlrus·neutralizlng (VN) antibody «1 :2) following challenge, whereas adult cats responded with significant levels of FOCMA and VN antibody 2 months after Infection. Thlrty·three young kit· tens Immunized with UV· or formalln·lnactlvated GA·FeSV failed to develop significant levels of VN antibody (~2) In contrast to 7 Immunized adult female cats that responded with significant levels (mean titer, 1:14). No evidence of protection from on· cogenlc challenge was observed In actively Immunized kittens. Moreover, the results suggested that vaccinated kittens were more susceptible to oncogenic virus challenge than were nonvac· clnated cats. Actively Immunized cats had a shorter survival time, greater primary tumor size, faster rate of tumor growth, and a greater extent of tumor metastases. In addition, significantly fewer Immunized cats responded to FOCMA and viral envelope antigens than did nonlmmunlzed cats. With the use o.f the same criteria, definitive evidence of protection from oncogenic virus challenge was observed In kittens that had been nursed by dams vaccinated during pregnancy.-J Natl Cancer. Inst 59: 14411450,1977.
has been shown to playa role in the prevention of FeL V infection in cats (5) and also protection against neoplastic disease in mice vaccinated with whole murine oncornavirus (12-14) or specific, highly purified, viral envelope glycoprotein (gp 71) (15). Successful immunoprophylaxis against FeLV- or FeSV-induced disease must, therefore, demonstrate the ability to induce either levels of anti-FOCMA antibody protecting against tumor cell growth or VN antibody protecting against virus infection. To this end, vaccination against feline oncornaviruses has been attempted with the use of live FeL V (16) and inactivated FeL V and FeSV (17, 18) as well as live (19) and inactivated tumor cells (18,20). Although vaccination with whole live tumor cells induced protective levels of anti-FOCMA and VN antibody (19), the potential dangers of introducing oncogenic FeL V into the environment and of inducing immune complex disease due to introduction of unpurified tumor cell membrane material make the use of live tumor cells in field studies undesirable. The use of live attenuated FeLV as a vaccine would also be unlikely, since it possibly could revert to a fully oncogenic form. Circumventing the hazards of using live tumor cells, we have recently demonstrated effective levels of protective immunity in cats with the use of heat-killed tumor cells (20) or irradiation-inactivated tumor homogenates (18). This procedure, however, did not prevent viremia and, therefore, emphasizes the need for a potent VN antibody-inducing vaccine to prevent virus infection. One factor found to influence susceptibility of cats to the induction of malignant feline oncornavirus disease is age (5, 21). In light of the established age-related susceptibility pattern observed for FeSV (subgroups A and B) (21) and two FeLV isolates (subgroups A and A, B, and C) (5), attempts at the determination of the efficacy of active immunization
The capacity to resist feline oncornavirus-induced neoplastic disease in the cat was shown to be associated with an efficient immune response to a tumor cell membrane antigen (1-5). High levels of antibody against FOCMA were correlated with tumor regression or failure to develop tumors in cats inoculated with FeSV (1-4) or FeLV (5). By contrast, low levels of antibody to FOCMA were found in cats with progressive neoplastic disease responses. In addition, a similar immune relationship was found in cats horizontally exposed to FeL V or FeSV as cage mates in the laboratory (6) or the natural environment (7-10). Protective levels of FOCMA antibody were also found in kittens born of queens that had been exposed horizontally to FeSV (1, 3). In one study, VN antibody in cats receiving oncogenic doses of FeSV did not always correlate with the presence of high FOCMA antibody titer or with the absence of tumor (4). In that study, however, the failure to correlate VN antibody with FOCMA antibody and tumor status could be due to a persisting viremia (11) effectively removing newly synthesized antibody from circulation. By contrast, VN antibody
ABBREVIATIONS USED: FOCMA = feline oncomavirus-associated cell membrane antigen; FeSV = feline sarcoma virus; FeLV = feline leukemia virus; VN = virus-neutralizing; LSA = lymphosarcoma; SPF = specific-pathogenfree; GA-FeSV=Gardner-Amstein FeSV; R-FeLV=Rickard FeLV; STFeSV = Snyder-Theilen FeSV; 0 10 = dose of UV measured at the sample surface that reduces viral infectivity by 1 loglo; FCA = Freund's complete adjuvant; KLH=keyhole-limpet hemocyanin; G-MuLV=Gross strain of murine leukemia virus; F -MuL V = Friend strain of murine leukemia virus.
VOL. 59. NO.5, NOVEMBER 1977
Received December 7, 1976; accepted May23, 1977. Supported by Public Health Service contract NOI-CP53571 from the Division of Cancer Cause and Prevention within the Virus Cancer Program of the National Cancer Institute. 5 Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210. 4 The Ohio State University Comprehensive Cancer Center, 357 McCampbell Hall, 1580 Cannon Drive, Columbus, Ohio 43210. 5 We thank Mr. Larry Mathes, Ms. Dagmar Imel, Mr. Steve Torvik, and Mr. Ken Milliser for their valued technical assistance and Dr. Jean Powers and Mr. Ed Herderick for I;j.elpful assistance with statistical treatment of data. I
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SCHALLER, HOOVER, AND OLSEN
are hampered. because of the short period of greater susceptibility in cats to feline oncornavirus challenge. Although epidemiologic studies have demonstrated that some adult cats are susceptible to the induction of LSA by FeL V (22), the relatively low incidence and long latent period for disease induced by contact with infected cats render this method unsuitable as a challenge in vaccine studies. This age factor may be responsible for the failure to induce protective levels of VN antibody with killed FeSV. Also, the failure of kittens to respond to killed FeSV in contrast to the positive response of adult cats (18) may in some way be related to age-related susceptibility. The present report expands on those observations. It presents evidence that suggests a selective nonresponsiveness of young cats to oncornavirus envelope antigen. This report also attempts to characterize the differences in immune competence to live and killed feline oncornaviruses in both young and older cats. Results may yield pertinent useful information not only in regard to the basis for age-related susceptibility of cats to their oncornaviruses but also in regard to the development of an efficient feline oncornavirus vaccine. Prior observations documented the unsuccessful active vaccination of kittens (17, 18, 23) and apparently successful immunization of adult pregnant dams (17, 18). These studies have been extended and we now report the apparent increased susceptibility of cats to FeSV disease induction following active immunization of young cats with inactivated FeSV. In addition, evidence for effective passive immunization of kittens that were nursed by vaccinated queens as an effective means of immunoprophylaxis against FeL V is further substantiated.
MATERIALS AND METHODS Cats. - The adult cats were from an SPF colony derived originally from gnotobiotic cats obtained by hysterectomy (24). The kittens were from SPF cats placed in isolation rooms far from contact with other animals. Each litter was housed separately until weaned (2-3 mo), after which queens were placed in separate cages. Before being vaccinated, all adult female cats were negative for FOCMA and VN antibody. Viruses. -GA-FeSV (25) used in vaccines or as challenge inocula was obtained as commercially prepared stocks of gradient-purified, tissue-culture-grown virus (Electro-Nucleonics, Inc., Bethesda, Md.). R-FeLV (26) used for vaccine was obtained as commercially prepared stocks, whereas challenge virus was prepared from feline LSA tumor homogenates. Tumor homogenates were prepared as 20% homogenized tumor tissues in Hanks' balanced salt solution. ST-FeSV and subgroup-specific FeLV preparations used invirus neutralization assays were processed as described previously (27). Virus immunogen preparation. - UV inactivation of GAFeSV and R-FeLV was done by the method described by Yoshikura (28). Stock virus preparations were placed into plastic petri dishes and irradiated at a distance of 13 cm with the use of a germacidal low-pressure mercury lamp. The dose rate, measured at the sample surface with a BlakRay Ultraviolet Intensity Meter (Ultra-Violet Products, Inc., San Gabriel, Calif.), was 150 ergs/mmllsecond.
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Samples were irradiated for an accumulated dose of 35,000 ergs/mml . ST-FeSV preparations of similar absorbance yielded a 0 10 of 3,500 ergs/mml, close to that obtained for the murine sarcoma viruses (28). Formalin inactivation of GA-FeSV was performed by the method of Fink and Rauscher (12). Immunizing virus doses were emulsified in equal volumes of either adjuvant 65 or FCA. A ctive immunization regimen. - Beginning at 4 days of age, kittens were given 4 weekly im immunizations. The inoculum (0.2 ml of purified virus and 0.2 ml of adjuvant) for each immunization was given in alternating flank and shoulder muscle areas. Twelve kittens from 5 litters were immunized with UV-inactivated GA-FeSV, whereas, in a second regimen, 14 kittens from 4 litters were immunized with formalin-treated FeSV. Kittens were challenged 7-10 days after the last injection with 0.2 ml of infectious GA-FeSV. One cat from each litter served as a nonimmunized but virus-challenged control. Passive immunization regimen.-Adult pregnant dams were immunized six times during pregnancy with either UVinactivated GA-FeSV or R-FeL V. Each immunization consisted of 0.2 ml of inactivated virus emulsified in 0.2 ml adjuvant 65 for the GA-FeSV and with 0.2 ml FCA for the R-FeL V vaccine. Female cats were removed from the SPF colony 3-5 days post coitus. Immunizations were begun on the day the cats were transferred and at I, 2, 3, 5, and 7 weeks thereafter. Cats that became pregnant delivered within 1-2 weeks following the last immunization. Twelve kittens from 6 dams immunized with UV-inactivated GAFeSV and 12 kittens from 4 nonimmunized dams were challenged with GA-FeSV within 5-8 days after birth. Thirteen kittens from 5 dams immunized with UV-inactivated RFeLV and 7 kittens from 3 nonimmunized dams were challenged with R-FeLV within 1-4 days after birth. Evaluation of disease response after challenge. -During vaccination and following virus challenge, weekly serum ' samples were collected for 8 weeks after challenge and biweekly thereafter. Inoculation sites were examined, and subcutaneous tumors were measured twice weekly with the use of calipers. FeSV-challenged cats were observed until they either died from neoplastic disease or were 36-40 weeks old. R-FeLV-challenged cats that survived beyond 36 weeks were held for 12-18 months. Cats that survived beyond 40 weeks were assigned a survival time of 40 weeks in all calculations. Severely debilitated cats not expected to survive more than 1 or 2 days were killed. as were some healthy cats between 36 and 40 weeks of age. All cats were necropsied and examined for the extent of tumor metastases. Serology. -FOCMA antibody titers were determined as described by Essex et al. (1) using the FL-74 target cells. VN antibody was determined as described by Schaller et al. (4) and Schaller and Olsen (27). Complement-fixing antibody to KLH antigen was determined as described by Olsen and Yohn(29). Virus infectivity. -Sera were assayed for the presence of infectious FeLV by a method described earlier (27) with the use of sarcoma-positive, leukemia-negative murine sarcoma virus-transformed BIC cells (30). FeL V gs antigen in leukocytes. - The presence of FeL V gs antigen in circulating leukocytes was determined as described by Hardy et al. (31). VOL. 59, NO.5, NOVEMBER 1977
1443
IMMUNIZATION OF CATS WITH INACTIVATED FELINE ONCORNAVIRUS
Immunization with KLH. - Two litters of newborn kittens were immunized at various times after birth with 2 mg KLH in 0.5 ml. Cats in each litter were given 1, 2, or 3 immunizations at various times within the first month of life. All cats were bled weekly.
A-A
(FeLV+ FOCMA-)
---.
(FeLV- FOCMA+)
RESULTS Age·Related Differences in Susceptibility and 1m· munologlc Responses in the Cat to Live FeLV The marked immunologic response differences observed between young kittens and adult cats in their ability to form VN and FOCMA antibodies to infecting R-FeLV are shown in text-figure 1. Adult cats within 5 weeks after virus exposure developed significant immune responses to both virus and tumor cell antigens (FOCMA). Mean titers reached over 1:32 and between 1:8 and 1:16 for VN and FOCMA antibody, respectively. Antibody levels peaked after 8 weeks. By contrast, newborn kittens were apparently immunologically unresponsive to infectious R-FeLV, since they had no significant levels of either VN or FOCMA antibody. Cats tested at intermediate ages had intermediate antibody responses. As reported earlier (5) and summarized here, a drastic transition from susceptibility to resistance was observed between young and old cats (text-fig. 2). Almost 100% of newborn kittens and a large proportion of 2-weekold to 2-month-old kittens became viremic, failed to develop significant levels of VN or FOCMA antibody, and developed thymic LSA. By contrast, over 90% of 4- and 12-month-old cats developed significant VN and FOCMA antibody responses and failed to develop detectable viremia. Over 90% of these cats never developed LSA-related disease within the 12-month period of study. Transition from susceptibility to resistance was apparently half complete at 2 months of age and complete by 4 months of age. 6
. - . VN Ab (ADULT) 0 - 0 VN Ab (NEWBORN) A-A FOCMA Ab (ADULT) l l - l l FOCMA Ab(NEWBORN)
5
_ _ _•
................
•
~-.-----.----.----.----,-------~r-,
NB
234 AGE ( months)
12
TEXT-FIGURE 2.-The disease response pattern in cats of various ages to inoculation with R-FeLV. Represented is the proportion of cats per age group that responded with either FeLV-positive lymphocytes and no FOCMA antibody (FeLV+ FOCMA --poor prognosis) or FeLV-negative lymphocytes and FOCMA antibody (FeLV- FOCMA+-good prognosis). Percentages were based on the use of 28. 9. 6. 4. 14. and 211 cats inoculated as newborns (NB) and at 2 wk. 1 mo. 2 mo. 4 mo. and 12 mo. respectively.
Age·Related Immune Response Differences to Killed Feline Oncornaviruses The ability of adult cats and newborn kittens to respond to killed (UV- or formalin-inactivated) GA-FeSV is illustrated in text-figure 3. A significantly greater VN antibody response was observed in vaccinated adult female cats after the fourth immunization as compared with that response seen in kittens. By 2 weeks later, the mean VN antibody level in kittens was approximately 1:2, while it had reached nearly 1:16 in adult female cats. All cats in this study received the same virus dose at each vaccination. The levels of antiviral reactivity did not differ significantly between the 2 groups of vaccinated kittens and their littermate controls.
Age·Related Immune Response to Cell·Associated FOCMA
'"
01 4
o
....J
A_A",
o
4
.~~:==:::::=----==,~
R-FeLV
2
4 6 8 10 12 WEEKS AFTER R- FeLV INJECTION
14
TEXT-FIGURE I.-Mean FOCMA and VN antibody (Ab) responses in newborn kittens and adult cats (~4 mo old) given injections of infectious R-FeLV (tumor homogenate). VN titers were determined against FeLV subgroup A on sarcoma· positive. leukemia-negative murine sarcoma virus-transformed 8lC cells. Each point represents the mean titers obtained with 6 adult cats and from 10 to 18 newborn kittens from 6 litters. VOL. 59. NO.5. NOVEMBER 1977
In contrast to the apparent lack of immune reactivity in kittens to killed virus, significant responsiveness was demonstrated against FOCMA in kittens vaccinated with crude cell-derived FOCMA preparations (table 1). While no significant differences in FOCMA antibody response were detected in catS' given 2 or 3 immunizations with heattreated FL-74 cells, kittens receiving 3 immunizations of irradiated ST-FeSV-induced tumor homogenate at 1, 2, and 3 weeks of age responded with a slightly lower antibody response.
Humoral Immune Response of Young Cats to KLH The immune competence of young kittens to foreign antigen was evaluated by immunization of kittens within the first month after birth with KLH. Complement-fixing antibody to KLH was evaluated after the immunization of 6
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~
Q
Q
Q
+ K+ K+
~4K
,
SCHALLER, HOOVER, AND OLSEN
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Q
K
+
o
TABLE 2.-Complement-fixing antibody response of kittens immunized with KLH
+
/4)
0::
W
lI-
E:;3
Cat No.
1 2 3 4 5 6
oCO
I-
~2 z > z
tJ1
8
WEEKS 3
2
9
3 4
6
7
7 10 5 20 16 11 13 13 14 10 12 9
7
TABLE l.-Anti-FOCMA response in cats vaccinated with crude cell-derived FOCMA preparations
ST-FeSYinduced tumor homogenate FL-74 (heattreated) FL-74 (heattreated) FL-74 (heattreated)
Age at immunization wk
response/ total No. of cats tested
Geometric mean highest FOCMA antibody titer±mean deviation
No. of cats w~th positive
3
1,2,3
10/10
3.0±1.4b
2
4,5
3/3
4.3±1.1 c
2
9,10
3/3
6.0±0.7c
3
6,7,8
10/11
4.7±1.4b
° ST-FeSY-induced tumor homogenates (10%) were inactivated by y-irradiation as described by Olsen et al. (20), whereas FL-74 vaccines consisting of 1 X 10 8 heat-killed cells per immunization were prepared as described by Yohn et al. (18). b FOCMA antibody titers were achieved within 1 wk following last immunization±mean deviation. Titer values were significantly different as determined by the Mann-Whitney U test for nonparametric ranking. c FOCMA antibody titers were achieved after 3d wk following last immunization± mean deviation.
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40.0 Positive 31.7 >40.0 Negative Negative >40.0 25.2 Positive 24.0 Positive 27.2 Positive 21.4 b Negative 29.1 Positive 36.0 Positive Negative >40.0 35.9 Positive 24.7 Positive 28.8±4.7
Viremia prior to necropsy Negative Positive Negative Negative Positive Positive Negative Negative Positive Positive Negative Positive Positive
>35.5±7.0 61
100 100
54 10
"Dams were immunized beginning 3-5 days after copulation and at I, 2, 3, 5, and 7 wk thereafter with 2X10 UV-inactivated densitygradient-purified R-FeLV. All cats were challenged within the first 4 days of age with 1 ml of 20% R-FeLV tumor homogenate. b Died from intussusception of intestine and had no evidence of disease when necropsied. Mean survival time±sD. d Significantly different (P>O.025) from the survival time of kittens from vaccinated queens. " Progressive response-cats with tumors that grew and eventually led to debilitation of the animals. C
nificant antibody responses to both FOCMA and viral andevelopment of viremia. Therefore, absolute levels of protigens were observed in adult cats that received R-FeLV; tective antibody are likely below these levels, possibly at or however, newborn kittens failed to develop significant levels below the limits of detectability by current assay methods, of VN or FOCMA antibody. Cats that were given live since significant VN antibody titers could not be demonR-FeLV at intermediate ages, between birth and 6 months, strated in passively immunized kittens in the current study. responded with FOCMA and VN antibody levels ranging Attempts to induce active immunity in kittens during the between the responses of the young and old cat groups. first month of life were unsuccessful, and evidence existed that vaccinated cats were more susceptible to FeSV chalThe age dependency for immune response and disease inlenge than were nonvaccinated littermate controls. Vacciduction to live FeL V in cats was similar to that found for Gnated cats had shorter survival times, larger tumors, tumors MuLV in rats (12), in which a total shift from susceptibility that grew faster, more severe diseases before being killed, to resistance occurred at 2 weeks of age. Age-related susceptibility has also been observed for FeSV (21). The inability of and more extensive tumor metastases than did nonvaccinated cats. That the vaccinated cats were more severely afyoung cats to respond immunologically to infecting virus at fected than nonvaccinated cats was further supported by an early stage of development may either be due to a lack of the fact that significantly fewer vaccinated cats developed immune competence, the overwhelming of a competent imFOCMA and VN antibody by 4-5 months of age. The detecmune response by challenge virus, or to a greater susception of VN antibody in several progressor cats was as tibility of target tissues in the young. A lack of immune comreported earlier (4, 18) and probably represents ineffective petence selective for viral proteins is suggested, since the levels found in viremic cats during transient periods of ciryoung cats responded as well as did older cats to FOCMA culating antibody excess. These studies suggest the possibiliand KLH antigen. However, if young cats were able to rety of either an enhancement or immunosuppressive mecha- _ spond to viral envelope antigens, possibly newly synthesized nism for killed FeSV in kittens. antibody is removed from circulation, in the face of antiThe age of the cat did not appear to be a factor in the imgen excess, and deposited in the form of immune complexes mune response to a foreign antigen (KLH) or the FOCMA. (34,35). Kittens during the first month of life responded as well as The induction of malignant melanomas, associated with did older cats to FOCMA-containing preparations and fibrosarcomas in cats inoculated with GA-FeSV as reported KLH. Young and old cats responded poorly to inactivated earlier (36), was also observed in these studies in 18-42% of GA-FeSV, a finding consistent with previously reported obcats with fibrosarcomas. The low incidence of melanomas as servations (23, 33). Adult cats (>6 mo old), however, showed compared with the incidence presented earlier may be due significantly greater VN antibody responses than did young either to sc rather than intradermal inoculation of the virus (>1 mo old) cats to inactivated GA-FeSV. Likewise, imor the failure to detect small focal melanotic areas within mune responses to live R-FeLV were age-dependent. Sigthe primary tumor.
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VOL. 59, NO.5, NOVEMBER 1977
IMMUNIZATION OF CATS WITH INACTIVATED FELINE ONCORNAVIRUS
Protection against oncomavirus challenge has been observed in mice following active and passive immunization with inactivated F-MuLV (12) and following passive immunization with G-MuLV (13, 14). F-MuLV can induce leukemia in adult mice and. in addition. is strongly immunogenic in either the live or formalin-inactivated states (12). These characteristics are not apparently shared with the feline oncomaviruses and may likely explain the lack of correlation with F-MuLV regarding the effectiveness of active immunization. Unlike F-MuLV and the Rauscher strain of murine leukemia virus, susceptibility of rats to G-MuLV is restricted to the early postnatal period and, in this respect, may be similar to the experimentally determined susceptibility of cats to their oncomaviruses (5, 21). The basis for the enhanced susceptibility in vaccinated cats may be due either to immunologic enhancement or immunosuppression induced or stimulated by killed FeSV. The possibility for an enhancement mechanism is suggested by the detection of sporadic low levels of VN antibody just prior to and after challenge of vaccinated cats (18). In this regard, the fact that young cats of similar ages were immunocompetent for virus-specified tumor antigen (FOCMA) as well as KLH implies an adequate immune response capability and young cats should, therefore, respond to viral antigen. Moreover, adult cats may possess natural antibodies to feline oncomaviruses that are passively transmitted postnatally to kittens. Present in low titers, these antibodies may also be directed toward determinants that do not participate in virus neutralization, but which bind to virus and to virusspecific proteins or tumor cell membranes (37). These antibodies may hypothetically participate in a classical enhancement-type phenomenon. Immunosuppressive effects of oncomaviruses have been well documented (38, 39) and could altematively be responsible for the observed enhanced susceptibility in vaccinated cats. That viral antigen may be implicated in immunosuppression is at least suggested by the observation that immunosuppressive effects induced by the Moloney strain of murine leukemia virus did not depend on the degree of viral replication (40) and, furthermore, by the observation that the degree of immunodepression in animals infected with F-MuLV is dose-dependent (41). The ubiquitous presence of FeLV, as well as its possible immunosuppressive effects, is illustrated by the observation that the FeL V gs antigen-positive state was detected in 1 of 2 to 2 of 3 of all cats with non-neoplastic disease (42, 43). Further, Essex et al. (44) found that FeL V -infected cats are fivefold more likely to develop non-neoplastic diseases than are noninfected FeL V cats housed together. In addition, depressed cellular immune function has been observed in Fe LV-infected cats (45, 46). The failure to stimulate active immune responses in kittens could also have been due to an ineffective method of virus inactivation. The methods used could have altered sensitive virus structural antigens, thereby resulting in the presentation of inadequate viral antigen required for effective immunization. In spite of this possibility, however, adult cats did respond with significant levels of VN antibody in contrast to the low levels of VN antibody responses observed in kittens. These data indicate that active vaccination of kittens with UV - or formalin-inactivated FeSV is unsuccessful in inducVOL. 59, NO.5, NOVEMBER 1977
1449
ing protective immunity and, furthermore, may result in increased susceptibility. This result, together with the demonstration of effective protection induced by passive immunization with FeSV or FeLV, suggests a possible immunization protocol for ultimate immunoprophylaxis against FeL V or FeSV. This regimen would include immunization of adult cats with whole or purified viral protein followed by booster immunizations of pregnant mothers. Immunity would then be provided to offspring; the offspring would be protected from virus infection during the period of maximum susceptibility. Immunization of young cats could then be initiated with inactivated virus or purified viral protein and/or FOCMA following decay of matemal antiviral antibody at approximately 3-4 months after birth.
REFERENCES (1) EsSEX M, KLEIN G, SNYDER SP, et al: Antibody to feline oncornavirusassociated cell membrane antigen in neonatal cats. Int J Cancer 8:384-390,1971 (2) - - : Feline sarcoma virus (FSV) induced tumours: Correlation between humoral antibody and tumour regression. Nature 223:195-196,1971 (3) ESSEX M. SNYDER SP, KLEIN G: Relationship between humoral antibodies and the failure to develop progressive tumors in cats injected with feline sarcoma virus. In Unifying Concepts of Leukemia (Dutcher RM, Chieco-Bianchi L, eds). Basel: Karger, 1973, pp 771-777 (4) SCHALLER JP, ESSEX M, YOHN DS, et al: Feline oncornavirusassociated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats inoculated with Gardner-Arnstein feline sarcoma virus. J Natl Cancer Inst 55:13731378, 1975 (5) HOOVER EA, OLSEN RG, HARDY WD JR, et al: Feline leukemia virus infection: Age-related variation in response of cats to experimental infection.J Nat! Cancer Inst 57:365-369,1976 (6) ESSEX M, HARDY WD JR, COTTER SM, et al: Immune response of healthy and leukemic cats to the feline oncornavirus associated cell membrane antigen (FOCMA). In Comparative Leukemia Research 1973 (Ito Y, Dutcher RM, eds). Tokyo: Univ Tokyo Press, 1975, pp 431-436 (7) COTTER SM, HARDY WD JR, ESSEX M: The association of the feline leukemia virus with lymphosarcoma and other disorders. J Am Vet Med Assoc 166 :449-454, 1975 (8) ESSEX M: Horizontally and vertically transmitted oncornaviruses of cats. Adv Cancer Res 21:175-248, 1975 (9) - - - : Tumors induced by oncornaviruses in cats. Pathobiol Annu 5:169-196,1975 (10) ESSEX M, COTTER SM, HARDY WD JR, et al: Feline oncornavirusassociated cell membrane antigen. IV. Antibody titers in cats with induced and naturally occurring leukemia and other diseases. J Nat! Cancer Inst 55:463-467, 1975 (11) ALDRICH CD, PEDERSEN NC: Persistent viremia after regression of primary virus-induced feline fibrosarcoma. AmJ Vet Res 35:13831388, 1974 (12) FINK MA, RAUSCHER FJ: Immune reactions to a murine leukemia virus. I. Induction of immunity to infection with virus in the natural host.J Nat! Cancer Inst 32:1075-1082,1964 (13) IOACHIM HL: Prevention of Gross virus-induced leukemia in progeny of immunized female rats. Cancer Res 30:2661-2664,1970 (14) IOACHIM HL, GIMOVSKY ML, KELLER SE: Maternal vaccination with formalin-inactivated Gross lymphoma virus in rats and transfer of immunity to offspring. Proc Soc Exp Bioi Med 144:376-379,1973 (15) HUNS MANN G, MOENNIG V, SCHAFER W: Properties of mouse leukemia viruses. IX. Active and passive immunization of mice against Friend leukemia with isolated viral GP 71 glycoprotein and its corresponding antiserum. Virology 66:327-329, 1975 (16) HARDY WD JR, ZUCKERMAN EE, MCCLELLAND AJ, et al: Feline leukemia virus control and vaccination. In Comparative Leukemia Research (Clemmenson J, Yohn DS, eds). Basel: Karger, 1976, pp 511-514
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(17) OLSEN RG. SCHALLER JP. HOOVER EA. et al: Experimental on· comavirus vaccines in the cat. In Comparative Leukemia Research (ClemmensonJ. Yohn DS. eels). Basel: Karger. 1976. pp 515-517 (18) YOHN DS. OLSEN RG. SCHALLERJP. et al: Experimentaloncomavirus vaccines in the cat. Cancer Res 36:646-651. 1976 (19) JARRETI WF. MACKEY L. JARRETI O. et al: Antibody response and virus survival in cats vaccinated against feline leukaemia. Nature 248:230-232. 1974 (20) OLSEN RG. HOOVER EA. MATHES LE. et al: Immunization against feline oncomavirus disease using a killed tumor cell vaccine. Cancer Res 36:3642-3649. 1976 (21) SNYDER SP. DUNGWORTH DL: Pathogenesis of feline viral fibrosar· comas. Dose and age effects. J Nat! Cancer Inst 51 :793-798. 1973 (22) ESSEX M. COTIER SM. CARPENTER JL. et al: Feline oncomavirus· associated cell membrane antigen. II. Antibody titers in healthy cats from pet household and laboratory colony environments. J Nat! Cancer Inst 54:631-635.1975 (23) HARDY WD JR: Immunology of oncomaviruses. Vet Clin North Am 4:133-146.1974 (24) ROHOVSKY MW. GRIESEMER RA. WOLFF LG: The germfree cat, Lab Anim Care 16:52-59.1966 (2.5) GARDNER MB. RONGEY RW. ARNSTEIN p. et al: Experimental trans· mission of feline fibrosarcoma to cats and dogs. Nature 226: 807-809. 1970 (26) RICKARD CG. POST JE. NORANHA F. et al: A transmissible virus· induced lymphocyte leukemia of the cat. J Nat! Cancer Inst 42:987-996. 1969 (27) SCHALLER JP. OLSEN RG: Determination of subgroup feline on· comavirus neutralizing antibody. Infect Immun 12:1405-1410. 1975 (28) YOSHIKUKA H: Ultraviolet inactivation of murine leukemia and sarcoma viruses. Int J Cancer 7: 131-140. 1971 (29) OLSEN RG. YOHN DS: Demonstration of antibody in cat sera to feline oncomavirus by complement fixation inhibition. J Nat! Cancer Inst 49:395-403. 1972 (30) FISCHINGER PJ. BLEVINS CS. NOMURA S: Simple quantitative assay for both xenotropic murine leukemia and ecotropic feline leukemia viruses.JVirol14:177-179.1974 (31) HARDY WD JR. HIRSHAUT Y. HESS P: Detection of the feline leuke· mia virus and other mammalian oncomaviruses by immuno· fluorescence. In Unifying Concepts of Leukemia (Dutcher RM. Chieco·Bianchi L. eds). Basel and New York: Karger. 197!1. pp 778-799 (32) HOOVER EA. SCHALLER JP. MATHES LE. et al: Passive immunity to feline leukemia virus: Evaluation of immunity from dams naturally
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infected and experimentally vaccinated. Infect Immun 16:54-59. 1977 (33) JARRETI WF: The relation of immune response to pathogenesis. vaccination. and epidemiology in virus· induced leukaemia. Br J Cancer 31:147.1975 (34) YOSHIKI T. MELLORS RC. STRAND M. et al: The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J Exp Med 140:1011-1027.1974 (3.5) HOLLIS WV JR. AOKI T. BARRERA O. et al: Detection of naturally occurring antibodies to RNA·dependent DNA polymerase of murine leukemia virus in kidney eluates of AKR mice. J Virol 13:448-454. 1974 (36) MCCULLOUGH B. SCHALLER J. SHADDUCK JA. et al: Induction of malignant melanomas associated with fibrosarcomas in gnotobiotic cats inoculated with Gardner·feline fibrosarcoma virus. J Nat! CancerInst 48: 1893-1896. 1972 (37) OLD LJ. BOYSE EA: Antigens of tumors and leukemias induced by viruses. Fed Proc 24: 1009-1017. 1965 (38) NOTKINS AL. MERGENHAGEN SE. HOWARD RJ: Effect of virus in· fections on the function of the immune system. Annu Rev Microbiol 24:525-538.1970 (39) DENT P: Immunodepression by oncogenic viruses. Prog Med Virol 14:1-35.1972 (40) CERNY J. PROFFITT MR. EsSEX M: Immunosuppression by Moloney leukemia virus: Lack of correlation between virus replication and the immunosuppressive effect. J Nat! Cancer Inst 56:819-822. 1976 (41) CEGLOWSKI WS. FRIEDMAN H: Immunosuppression by leukemia viruses. I. Effect of Friend disease virus on cellular and humoral hemolysin responses of mice to a primary immunization with sheep erythrocytes.J Immunoll01:594-604. 1968 (42) COTIER SM. HARDY WD JR. ESSEX M: The association of the feline leukemia virus with lymphosarcoma and other disorders. J Am Vet MedAssoc 166:449-454.1975 (43) ESSEX M: Horizontally and vertically transmitted oncomaviruses of cats. Adv Cancer Res 21:175-248. 1975 (44) ESSEX M. HARDY WD JR. COTTER SM. et al: Naturally occurring persistent feline oncomavirus infections in the absence of disease. Infect Immun 11:470-476. 1975 (4.5) PERRYMAN LE. HOOVER EA. YOHN DS: Immunologic reactivity in the cat: Immunosuppression in experimental feline leukemia. J Nat! CancerInst 49: 1357 -1 365. 1972 (46) COCKERELL GL. KRAKOWKA SG. HOOVER EA. et al: Lymphocyte mitogen reactivity and enumeration of circulating B· and T ·cells during feline leukemia virus infection in the cat. J Nat! Cancer Inst 57:1095-1099. 1976
VOL. 59. NO.5. NOVEMBER 1977
1. 2
J. P. Schaller,3 E. A. Hoover,3 and R. G. Olsen 3. 4. 5 ABSTRACT-The humoral Immune responses of young «1 mo) and older (>6 mo) cats to killed and live feline leukemia virus (Fe LV), feline sarcoma virus (FeSV), and the feline oncornavlrus· associated cell membrane antigen (FOCMA) were evaluated. In addition, the biologic responses to oncogenic feline oncornavlrus challenge were analyzed In cats actively Immunized with Inac· tlvated Gardner-Arnstein FeSV (GA·FeSV) or passively Immu· nlzed with either the Rickard FeLV (R·FeLV) or GA·FeSV. Kittens were Immunized during the first month of life with UV· or formalin· Inactivated GA·FeSV and challenged at 5-6 weeks of age with a known oncogenic dose of GA·FeSV. Passive Immunization was evaluated In kittens that suckled mothers Immunized during pregnancy with purified GA·FeSV or R·FeLV. Kittens during the first month of life were capable of producing humoral antibody against FOCMA and keyhole·llmpet hemocyanin. Apparently linear disease responses to live R·FeLV from susceptibility (viremia with no FOCMA antibody) to resistance (no viremia and high FOCMA antibody level) were observed In cats from birth to 4 months of age. Newborn kittens failed to develop FOCMA and vlrus·neutralizlng (VN) antibody «1 :2) following challenge, whereas adult cats responded with significant levels of FOCMA and VN antibody 2 months after Infection. Thlrty·three young kit· tens Immunized with UV· or formalln·lnactlvated GA·FeSV failed to develop significant levels of VN antibody (~2) In contrast to 7 Immunized adult female cats that responded with significant levels (mean titer, 1:14). No evidence of protection from on· cogenlc challenge was observed In actively Immunized kittens. Moreover, the results suggested that vaccinated kittens were more susceptible to oncogenic virus challenge than were nonvac· clnated cats. Actively Immunized cats had a shorter survival time, greater primary tumor size, faster rate of tumor growth, and a greater extent of tumor metastases. In addition, significantly fewer Immunized cats responded to FOCMA and viral envelope antigens than did nonlmmunlzed cats. With the use o.f the same criteria, definitive evidence of protection from oncogenic virus challenge was observed In kittens that had been nursed by dams vaccinated during pregnancy.-J Natl Cancer. Inst 59: 14411450,1977.
has been shown to playa role in the prevention of FeL V infection in cats (5) and also protection against neoplastic disease in mice vaccinated with whole murine oncornavirus (12-14) or specific, highly purified, viral envelope glycoprotein (gp 71) (15). Successful immunoprophylaxis against FeLV- or FeSV-induced disease must, therefore, demonstrate the ability to induce either levels of anti-FOCMA antibody protecting against tumor cell growth or VN antibody protecting against virus infection. To this end, vaccination against feline oncornaviruses has been attempted with the use of live FeL V (16) and inactivated FeL V and FeSV (17, 18) as well as live (19) and inactivated tumor cells (18,20). Although vaccination with whole live tumor cells induced protective levels of anti-FOCMA and VN antibody (19), the potential dangers of introducing oncogenic FeL V into the environment and of inducing immune complex disease due to introduction of unpurified tumor cell membrane material make the use of live tumor cells in field studies undesirable. The use of live attenuated FeLV as a vaccine would also be unlikely, since it possibly could revert to a fully oncogenic form. Circumventing the hazards of using live tumor cells, we have recently demonstrated effective levels of protective immunity in cats with the use of heat-killed tumor cells (20) or irradiation-inactivated tumor homogenates (18). This procedure, however, did not prevent viremia and, therefore, emphasizes the need for a potent VN antibody-inducing vaccine to prevent virus infection. One factor found to influence susceptibility of cats to the induction of malignant feline oncornavirus disease is age (5, 21). In light of the established age-related susceptibility pattern observed for FeSV (subgroups A and B) (21) and two FeLV isolates (subgroups A and A, B, and C) (5), attempts at the determination of the efficacy of active immunization
The capacity to resist feline oncornavirus-induced neoplastic disease in the cat was shown to be associated with an efficient immune response to a tumor cell membrane antigen (1-5). High levels of antibody against FOCMA were correlated with tumor regression or failure to develop tumors in cats inoculated with FeSV (1-4) or FeLV (5). By contrast, low levels of antibody to FOCMA were found in cats with progressive neoplastic disease responses. In addition, a similar immune relationship was found in cats horizontally exposed to FeL V or FeSV as cage mates in the laboratory (6) or the natural environment (7-10). Protective levels of FOCMA antibody were also found in kittens born of queens that had been exposed horizontally to FeSV (1, 3). In one study, VN antibody in cats receiving oncogenic doses of FeSV did not always correlate with the presence of high FOCMA antibody titer or with the absence of tumor (4). In that study, however, the failure to correlate VN antibody with FOCMA antibody and tumor status could be due to a persisting viremia (11) effectively removing newly synthesized antibody from circulation. By contrast, VN antibody
ABBREVIATIONS USED: FOCMA = feline oncomavirus-associated cell membrane antigen; FeSV = feline sarcoma virus; FeLV = feline leukemia virus; VN = virus-neutralizing; LSA = lymphosarcoma; SPF = specific-pathogenfree; GA-FeSV=Gardner-Amstein FeSV; R-FeLV=Rickard FeLV; STFeSV = Snyder-Theilen FeSV; 0 10 = dose of UV measured at the sample surface that reduces viral infectivity by 1 loglo; FCA = Freund's complete adjuvant; KLH=keyhole-limpet hemocyanin; G-MuLV=Gross strain of murine leukemia virus; F -MuL V = Friend strain of murine leukemia virus.
VOL. 59. NO.5, NOVEMBER 1977
Received December 7, 1976; accepted May23, 1977. Supported by Public Health Service contract NOI-CP53571 from the Division of Cancer Cause and Prevention within the Virus Cancer Program of the National Cancer Institute. 5 Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210. 4 The Ohio State University Comprehensive Cancer Center, 357 McCampbell Hall, 1580 Cannon Drive, Columbus, Ohio 43210. 5 We thank Mr. Larry Mathes, Ms. Dagmar Imel, Mr. Steve Torvik, and Mr. Ken Milliser for their valued technical assistance and Dr. Jean Powers and Mr. Ed Herderick for I;j.elpful assistance with statistical treatment of data. I
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SCHALLER, HOOVER, AND OLSEN
are hampered. because of the short period of greater susceptibility in cats to feline oncornavirus challenge. Although epidemiologic studies have demonstrated that some adult cats are susceptible to the induction of LSA by FeL V (22), the relatively low incidence and long latent period for disease induced by contact with infected cats render this method unsuitable as a challenge in vaccine studies. This age factor may be responsible for the failure to induce protective levels of VN antibody with killed FeSV. Also, the failure of kittens to respond to killed FeSV in contrast to the positive response of adult cats (18) may in some way be related to age-related susceptibility. The present report expands on those observations. It presents evidence that suggests a selective nonresponsiveness of young cats to oncornavirus envelope antigen. This report also attempts to characterize the differences in immune competence to live and killed feline oncornaviruses in both young and older cats. Results may yield pertinent useful information not only in regard to the basis for age-related susceptibility of cats to their oncornaviruses but also in regard to the development of an efficient feline oncornavirus vaccine. Prior observations documented the unsuccessful active vaccination of kittens (17, 18, 23) and apparently successful immunization of adult pregnant dams (17, 18). These studies have been extended and we now report the apparent increased susceptibility of cats to FeSV disease induction following active immunization of young cats with inactivated FeSV. In addition, evidence for effective passive immunization of kittens that were nursed by vaccinated queens as an effective means of immunoprophylaxis against FeL V is further substantiated.
MATERIALS AND METHODS Cats. - The adult cats were from an SPF colony derived originally from gnotobiotic cats obtained by hysterectomy (24). The kittens were from SPF cats placed in isolation rooms far from contact with other animals. Each litter was housed separately until weaned (2-3 mo), after which queens were placed in separate cages. Before being vaccinated, all adult female cats were negative for FOCMA and VN antibody. Viruses. -GA-FeSV (25) used in vaccines or as challenge inocula was obtained as commercially prepared stocks of gradient-purified, tissue-culture-grown virus (Electro-Nucleonics, Inc., Bethesda, Md.). R-FeLV (26) used for vaccine was obtained as commercially prepared stocks, whereas challenge virus was prepared from feline LSA tumor homogenates. Tumor homogenates were prepared as 20% homogenized tumor tissues in Hanks' balanced salt solution. ST-FeSV and subgroup-specific FeLV preparations used invirus neutralization assays were processed as described previously (27). Virus immunogen preparation. - UV inactivation of GAFeSV and R-FeLV was done by the method described by Yoshikura (28). Stock virus preparations were placed into plastic petri dishes and irradiated at a distance of 13 cm with the use of a germacidal low-pressure mercury lamp. The dose rate, measured at the sample surface with a BlakRay Ultraviolet Intensity Meter (Ultra-Violet Products, Inc., San Gabriel, Calif.), was 150 ergs/mmllsecond.
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Samples were irradiated for an accumulated dose of 35,000 ergs/mml . ST-FeSV preparations of similar absorbance yielded a 0 10 of 3,500 ergs/mml, close to that obtained for the murine sarcoma viruses (28). Formalin inactivation of GA-FeSV was performed by the method of Fink and Rauscher (12). Immunizing virus doses were emulsified in equal volumes of either adjuvant 65 or FCA. A ctive immunization regimen. - Beginning at 4 days of age, kittens were given 4 weekly im immunizations. The inoculum (0.2 ml of purified virus and 0.2 ml of adjuvant) for each immunization was given in alternating flank and shoulder muscle areas. Twelve kittens from 5 litters were immunized with UV-inactivated GA-FeSV, whereas, in a second regimen, 14 kittens from 4 litters were immunized with formalin-treated FeSV. Kittens were challenged 7-10 days after the last injection with 0.2 ml of infectious GA-FeSV. One cat from each litter served as a nonimmunized but virus-challenged control. Passive immunization regimen.-Adult pregnant dams were immunized six times during pregnancy with either UVinactivated GA-FeSV or R-FeL V. Each immunization consisted of 0.2 ml of inactivated virus emulsified in 0.2 ml adjuvant 65 for the GA-FeSV and with 0.2 ml FCA for the R-FeL V vaccine. Female cats were removed from the SPF colony 3-5 days post coitus. Immunizations were begun on the day the cats were transferred and at I, 2, 3, 5, and 7 weeks thereafter. Cats that became pregnant delivered within 1-2 weeks following the last immunization. Twelve kittens from 6 dams immunized with UV-inactivated GAFeSV and 12 kittens from 4 nonimmunized dams were challenged with GA-FeSV within 5-8 days after birth. Thirteen kittens from 5 dams immunized with UV-inactivated RFeLV and 7 kittens from 3 nonimmunized dams were challenged with R-FeLV within 1-4 days after birth. Evaluation of disease response after challenge. -During vaccination and following virus challenge, weekly serum ' samples were collected for 8 weeks after challenge and biweekly thereafter. Inoculation sites were examined, and subcutaneous tumors were measured twice weekly with the use of calipers. FeSV-challenged cats were observed until they either died from neoplastic disease or were 36-40 weeks old. R-FeLV-challenged cats that survived beyond 36 weeks were held for 12-18 months. Cats that survived beyond 40 weeks were assigned a survival time of 40 weeks in all calculations. Severely debilitated cats not expected to survive more than 1 or 2 days were killed. as were some healthy cats between 36 and 40 weeks of age. All cats were necropsied and examined for the extent of tumor metastases. Serology. -FOCMA antibody titers were determined as described by Essex et al. (1) using the FL-74 target cells. VN antibody was determined as described by Schaller et al. (4) and Schaller and Olsen (27). Complement-fixing antibody to KLH antigen was determined as described by Olsen and Yohn(29). Virus infectivity. -Sera were assayed for the presence of infectious FeLV by a method described earlier (27) with the use of sarcoma-positive, leukemia-negative murine sarcoma virus-transformed BIC cells (30). FeL V gs antigen in leukocytes. - The presence of FeL V gs antigen in circulating leukocytes was determined as described by Hardy et al. (31). VOL. 59, NO.5, NOVEMBER 1977
1443
IMMUNIZATION OF CATS WITH INACTIVATED FELINE ONCORNAVIRUS
Immunization with KLH. - Two litters of newborn kittens were immunized at various times after birth with 2 mg KLH in 0.5 ml. Cats in each litter were given 1, 2, or 3 immunizations at various times within the first month of life. All cats were bled weekly.
A-A
(FeLV+ FOCMA-)
---.
(FeLV- FOCMA+)
RESULTS Age·Related Differences in Susceptibility and 1m· munologlc Responses in the Cat to Live FeLV The marked immunologic response differences observed between young kittens and adult cats in their ability to form VN and FOCMA antibodies to infecting R-FeLV are shown in text-figure 1. Adult cats within 5 weeks after virus exposure developed significant immune responses to both virus and tumor cell antigens (FOCMA). Mean titers reached over 1:32 and between 1:8 and 1:16 for VN and FOCMA antibody, respectively. Antibody levels peaked after 8 weeks. By contrast, newborn kittens were apparently immunologically unresponsive to infectious R-FeLV, since they had no significant levels of either VN or FOCMA antibody. Cats tested at intermediate ages had intermediate antibody responses. As reported earlier (5) and summarized here, a drastic transition from susceptibility to resistance was observed between young and old cats (text-fig. 2). Almost 100% of newborn kittens and a large proportion of 2-weekold to 2-month-old kittens became viremic, failed to develop significant levels of VN or FOCMA antibody, and developed thymic LSA. By contrast, over 90% of 4- and 12-month-old cats developed significant VN and FOCMA antibody responses and failed to develop detectable viremia. Over 90% of these cats never developed LSA-related disease within the 12-month period of study. Transition from susceptibility to resistance was apparently half complete at 2 months of age and complete by 4 months of age. 6
. - . VN Ab (ADULT) 0 - 0 VN Ab (NEWBORN) A-A FOCMA Ab (ADULT) l l - l l FOCMA Ab(NEWBORN)
5
_ _ _•
................
•
~-.-----.----.----.----,-------~r-,
NB
234 AGE ( months)
12
TEXT-FIGURE 2.-The disease response pattern in cats of various ages to inoculation with R-FeLV. Represented is the proportion of cats per age group that responded with either FeLV-positive lymphocytes and no FOCMA antibody (FeLV+ FOCMA --poor prognosis) or FeLV-negative lymphocytes and FOCMA antibody (FeLV- FOCMA+-good prognosis). Percentages were based on the use of 28. 9. 6. 4. 14. and 211 cats inoculated as newborns (NB) and at 2 wk. 1 mo. 2 mo. 4 mo. and 12 mo. respectively.
Age·Related Immune Response Differences to Killed Feline Oncornaviruses The ability of adult cats and newborn kittens to respond to killed (UV- or formalin-inactivated) GA-FeSV is illustrated in text-figure 3. A significantly greater VN antibody response was observed in vaccinated adult female cats after the fourth immunization as compared with that response seen in kittens. By 2 weeks later, the mean VN antibody level in kittens was approximately 1:2, while it had reached nearly 1:16 in adult female cats. All cats in this study received the same virus dose at each vaccination. The levels of antiviral reactivity did not differ significantly between the 2 groups of vaccinated kittens and their littermate controls.
Age·Related Immune Response to Cell·Associated FOCMA
'"
01 4
o
....J
A_A",
o
4
.~~:==:::::=----==,~
R-FeLV
2
4 6 8 10 12 WEEKS AFTER R- FeLV INJECTION
14
TEXT-FIGURE I.-Mean FOCMA and VN antibody (Ab) responses in newborn kittens and adult cats (~4 mo old) given injections of infectious R-FeLV (tumor homogenate). VN titers were determined against FeLV subgroup A on sarcoma· positive. leukemia-negative murine sarcoma virus-transformed 8lC cells. Each point represents the mean titers obtained with 6 adult cats and from 10 to 18 newborn kittens from 6 litters. VOL. 59. NO.5. NOVEMBER 1977
In contrast to the apparent lack of immune reactivity in kittens to killed virus, significant responsiveness was demonstrated against FOCMA in kittens vaccinated with crude cell-derived FOCMA preparations (table 1). While no significant differences in FOCMA antibody response were detected in catS' given 2 or 3 immunizations with heattreated FL-74 cells, kittens receiving 3 immunizations of irradiated ST-FeSV-induced tumor homogenate at 1, 2, and 3 weeks of age responded with a slightly lower antibody response.
Humoral Immune Response of Young Cats to KLH The immune competence of young kittens to foreign antigen was evaluated by immunization of kittens within the first month after birth with KLH. Complement-fixing antibody to KLH was evaluated after the immunization of 6
J NATL
CANCER INST
1444
~
Q
Q
Q
+ K+ K+
~4K
,
SCHALLER, HOOVER, AND OLSEN
Q
Q
K
+
o
TABLE 2.-Complement-fixing antibody response of kittens immunized with KLH
+
/4)
0::
W
lI-
E:;3
Cat No.
1 2 3 4 5 6
oCO
I-
~2 z > z
tJ1
8
WEEKS 3
2
9
3 4
6
7
7 10 5 20 16 11 13 13 14 10 12 9
7
TABLE l.-Anti-FOCMA response in cats vaccinated with crude cell-derived FOCMA preparations
ST-FeSYinduced tumor homogenate FL-74 (heattreated) FL-74 (heattreated) FL-74 (heattreated)
Age at immunization wk
response/ total No. of cats tested
Geometric mean highest FOCMA antibody titer±mean deviation
No. of cats w~th positive
3
1,2,3
10/10
3.0±1.4b
2
4,5
3/3
4.3±1.1 c
2
9,10
3/3
6.0±0.7c
3
6,7,8
10/11
4.7±1.4b
° ST-FeSY-induced tumor homogenates (10%) were inactivated by y-irradiation as described by Olsen et al. (20), whereas FL-74 vaccines consisting of 1 X 10 8 heat-killed cells per immunization were prepared as described by Yohn et al. (18). b FOCMA antibody titers were achieved within 1 wk following last immunization±mean deviation. Titer values were significantly different as determined by the Mann-Whitney U test for nonparametric ranking. c FOCMA antibody titers were achieved after 3d wk following last immunization± mean deviation.
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40.0 Positive 31.7 >40.0 Negative Negative >40.0 25.2 Positive 24.0 Positive 27.2 Positive 21.4 b Negative 29.1 Positive 36.0 Positive Negative >40.0 35.9 Positive 24.7 Positive 28.8±4.7
Viremia prior to necropsy Negative Positive Negative Negative Positive Positive Negative Negative Positive Positive Negative Positive Positive
>35.5±7.0 61
100 100
54 10
"Dams were immunized beginning 3-5 days after copulation and at I, 2, 3, 5, and 7 wk thereafter with 2X10 UV-inactivated densitygradient-purified R-FeLV. All cats were challenged within the first 4 days of age with 1 ml of 20% R-FeLV tumor homogenate. b Died from intussusception of intestine and had no evidence of disease when necropsied. Mean survival time±sD. d Significantly different (P>O.025) from the survival time of kittens from vaccinated queens. " Progressive response-cats with tumors that grew and eventually led to debilitation of the animals. C
nificant antibody responses to both FOCMA and viral andevelopment of viremia. Therefore, absolute levels of protigens were observed in adult cats that received R-FeLV; tective antibody are likely below these levels, possibly at or however, newborn kittens failed to develop significant levels below the limits of detectability by current assay methods, of VN or FOCMA antibody. Cats that were given live since significant VN antibody titers could not be demonR-FeLV at intermediate ages, between birth and 6 months, strated in passively immunized kittens in the current study. responded with FOCMA and VN antibody levels ranging Attempts to induce active immunity in kittens during the between the responses of the young and old cat groups. first month of life were unsuccessful, and evidence existed that vaccinated cats were more susceptible to FeSV chalThe age dependency for immune response and disease inlenge than were nonvaccinated littermate controls. Vacciduction to live FeL V in cats was similar to that found for Gnated cats had shorter survival times, larger tumors, tumors MuLV in rats (12), in which a total shift from susceptibility that grew faster, more severe diseases before being killed, to resistance occurred at 2 weeks of age. Age-related susceptibility has also been observed for FeSV (21). The inability of and more extensive tumor metastases than did nonvaccinated cats. That the vaccinated cats were more severely afyoung cats to respond immunologically to infecting virus at fected than nonvaccinated cats was further supported by an early stage of development may either be due to a lack of the fact that significantly fewer vaccinated cats developed immune competence, the overwhelming of a competent imFOCMA and VN antibody by 4-5 months of age. The detecmune response by challenge virus, or to a greater susception of VN antibody in several progressor cats was as tibility of target tissues in the young. A lack of immune comreported earlier (4, 18) and probably represents ineffective petence selective for viral proteins is suggested, since the levels found in viremic cats during transient periods of ciryoung cats responded as well as did older cats to FOCMA culating antibody excess. These studies suggest the possibiliand KLH antigen. However, if young cats were able to rety of either an enhancement or immunosuppressive mecha- _ spond to viral envelope antigens, possibly newly synthesized nism for killed FeSV in kittens. antibody is removed from circulation, in the face of antiThe age of the cat did not appear to be a factor in the imgen excess, and deposited in the form of immune complexes mune response to a foreign antigen (KLH) or the FOCMA. (34,35). Kittens during the first month of life responded as well as The induction of malignant melanomas, associated with did older cats to FOCMA-containing preparations and fibrosarcomas in cats inoculated with GA-FeSV as reported KLH. Young and old cats responded poorly to inactivated earlier (36), was also observed in these studies in 18-42% of GA-FeSV, a finding consistent with previously reported obcats with fibrosarcomas. The low incidence of melanomas as servations (23, 33). Adult cats (>6 mo old), however, showed compared with the incidence presented earlier may be due significantly greater VN antibody responses than did young either to sc rather than intradermal inoculation of the virus (>1 mo old) cats to inactivated GA-FeSV. Likewise, imor the failure to detect small focal melanotic areas within mune responses to live R-FeLV were age-dependent. Sigthe primary tumor.
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VOL. 59, NO.5, NOVEMBER 1977
IMMUNIZATION OF CATS WITH INACTIVATED FELINE ONCORNAVIRUS
Protection against oncomavirus challenge has been observed in mice following active and passive immunization with inactivated F-MuLV (12) and following passive immunization with G-MuLV (13, 14). F-MuLV can induce leukemia in adult mice and. in addition. is strongly immunogenic in either the live or formalin-inactivated states (12). These characteristics are not apparently shared with the feline oncomaviruses and may likely explain the lack of correlation with F-MuLV regarding the effectiveness of active immunization. Unlike F-MuLV and the Rauscher strain of murine leukemia virus, susceptibility of rats to G-MuLV is restricted to the early postnatal period and, in this respect, may be similar to the experimentally determined susceptibility of cats to their oncomaviruses (5, 21). The basis for the enhanced susceptibility in vaccinated cats may be due either to immunologic enhancement or immunosuppression induced or stimulated by killed FeSV. The possibility for an enhancement mechanism is suggested by the detection of sporadic low levels of VN antibody just prior to and after challenge of vaccinated cats (18). In this regard, the fact that young cats of similar ages were immunocompetent for virus-specified tumor antigen (FOCMA) as well as KLH implies an adequate immune response capability and young cats should, therefore, respond to viral antigen. Moreover, adult cats may possess natural antibodies to feline oncomaviruses that are passively transmitted postnatally to kittens. Present in low titers, these antibodies may also be directed toward determinants that do not participate in virus neutralization, but which bind to virus and to virusspecific proteins or tumor cell membranes (37). These antibodies may hypothetically participate in a classical enhancement-type phenomenon. Immunosuppressive effects of oncomaviruses have been well documented (38, 39) and could altematively be responsible for the observed enhanced susceptibility in vaccinated cats. That viral antigen may be implicated in immunosuppression is at least suggested by the observation that immunosuppressive effects induced by the Moloney strain of murine leukemia virus did not depend on the degree of viral replication (40) and, furthermore, by the observation that the degree of immunodepression in animals infected with F-MuLV is dose-dependent (41). The ubiquitous presence of FeLV, as well as its possible immunosuppressive effects, is illustrated by the observation that the FeL V gs antigen-positive state was detected in 1 of 2 to 2 of 3 of all cats with non-neoplastic disease (42, 43). Further, Essex et al. (44) found that FeL V -infected cats are fivefold more likely to develop non-neoplastic diseases than are noninfected FeL V cats housed together. In addition, depressed cellular immune function has been observed in Fe LV-infected cats (45, 46). The failure to stimulate active immune responses in kittens could also have been due to an ineffective method of virus inactivation. The methods used could have altered sensitive virus structural antigens, thereby resulting in the presentation of inadequate viral antigen required for effective immunization. In spite of this possibility, however, adult cats did respond with significant levels of VN antibody in contrast to the low levels of VN antibody responses observed in kittens. These data indicate that active vaccination of kittens with UV - or formalin-inactivated FeSV is unsuccessful in inducVOL. 59, NO.5, NOVEMBER 1977
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ing protective immunity and, furthermore, may result in increased susceptibility. This result, together with the demonstration of effective protection induced by passive immunization with FeSV or FeLV, suggests a possible immunization protocol for ultimate immunoprophylaxis against FeL V or FeSV. This regimen would include immunization of adult cats with whole or purified viral protein followed by booster immunizations of pregnant mothers. Immunity would then be provided to offspring; the offspring would be protected from virus infection during the period of maximum susceptibility. Immunization of young cats could then be initiated with inactivated virus or purified viral protein and/or FOCMA following decay of matemal antiviral antibody at approximately 3-4 months after birth.
REFERENCES (1) EsSEX M, KLEIN G, SNYDER SP, et al: Antibody to feline oncornavirusassociated cell membrane antigen in neonatal cats. Int J Cancer 8:384-390,1971 (2) - - : Feline sarcoma virus (FSV) induced tumours: Correlation between humoral antibody and tumour regression. Nature 223:195-196,1971 (3) ESSEX M. SNYDER SP, KLEIN G: Relationship between humoral antibodies and the failure to develop progressive tumors in cats injected with feline sarcoma virus. In Unifying Concepts of Leukemia (Dutcher RM, Chieco-Bianchi L, eds). Basel: Karger, 1973, pp 771-777 (4) SCHALLER JP, ESSEX M, YOHN DS, et al: Feline oncornavirusassociated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats inoculated with Gardner-Arnstein feline sarcoma virus. J Natl Cancer Inst 55:13731378, 1975 (5) HOOVER EA, OLSEN RG, HARDY WD JR, et al: Feline leukemia virus infection: Age-related variation in response of cats to experimental infection.J Nat! Cancer Inst 57:365-369,1976 (6) ESSEX M, HARDY WD JR, COTTER SM, et al: Immune response of healthy and leukemic cats to the feline oncornavirus associated cell membrane antigen (FOCMA). In Comparative Leukemia Research 1973 (Ito Y, Dutcher RM, eds). Tokyo: Univ Tokyo Press, 1975, pp 431-436 (7) COTTER SM, HARDY WD JR, ESSEX M: The association of the feline leukemia virus with lymphosarcoma and other disorders. J Am Vet Med Assoc 166 :449-454, 1975 (8) ESSEX M: Horizontally and vertically transmitted oncornaviruses of cats. Adv Cancer Res 21:175-248, 1975 (9) - - - : Tumors induced by oncornaviruses in cats. Pathobiol Annu 5:169-196,1975 (10) ESSEX M, COTTER SM, HARDY WD JR, et al: Feline oncornavirusassociated cell membrane antigen. IV. Antibody titers in cats with induced and naturally occurring leukemia and other diseases. J Nat! Cancer Inst 55:463-467, 1975 (11) ALDRICH CD, PEDERSEN NC: Persistent viremia after regression of primary virus-induced feline fibrosarcoma. AmJ Vet Res 35:13831388, 1974 (12) FINK MA, RAUSCHER FJ: Immune reactions to a murine leukemia virus. I. Induction of immunity to infection with virus in the natural host.J Nat! Cancer Inst 32:1075-1082,1964 (13) IOACHIM HL: Prevention of Gross virus-induced leukemia in progeny of immunized female rats. Cancer Res 30:2661-2664,1970 (14) IOACHIM HL, GIMOVSKY ML, KELLER SE: Maternal vaccination with formalin-inactivated Gross lymphoma virus in rats and transfer of immunity to offspring. Proc Soc Exp Bioi Med 144:376-379,1973 (15) HUNS MANN G, MOENNIG V, SCHAFER W: Properties of mouse leukemia viruses. IX. Active and passive immunization of mice against Friend leukemia with isolated viral GP 71 glycoprotein and its corresponding antiserum. Virology 66:327-329, 1975 (16) HARDY WD JR, ZUCKERMAN EE, MCCLELLAND AJ, et al: Feline leukemia virus control and vaccination. In Comparative Leukemia Research (Clemmenson J, Yohn DS, eds). Basel: Karger, 1976, pp 511-514
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