[CANCER RESEARCH 36. 646-651, February 1976]
Experimental Oncornavirus Vaccines in the CaP
D. S . Y o h n , 2 R. G . O l s e n ,
J . P. S c h a l l e r ,
E. A . H o o v e r ,
L. E. M a t h e s ,
L. H e d i n g ,
and G. W. Davis
The Department of Veterinary Pathobiology, The Ohio State University, Columbus, Ohio 43210
Summary
An experimental approach to the immunoprophylatic control of feline oncornavirus-mediated diseases has included
induction of antivirus immunity and antibodies to the feline
oncornavirus-associated membrane (tumor) antigens. A
suitable model for exploring the effectiveness of killed oncornavirus vaccines in the cat has been provided by the use
of feline sarcoma virus. Immunization of seven pregnant
queens over a 6-week period with ultraviolet light-inactivated Gardner-Arnstein feline sarcoma virus resulted in significant protection among 12 kittens challenged with a tumor-forming Dose 90 at 7 days of age. This immunity was
not present in kittens challenged at 35 days of age. Among
12 kittens born of queens immunized during pregnancy with
ultraviolet light-inactivated Kawakami-Theilen feline leukemia virus and challenged with the same live virus at 4
days of age, significant protection was noted, ranging from
prolongation of survival time to complete protection in 3
kittens. In general, the higher the antibody titer in the
mother, the more effective the protection afforded the
kittens.
Immunization of 43 kittens during their first 5 weeks of life
with the same vaccines used in adult cats did not immunize
sufficiently to protect against feline sarcoma virus challenge at 5 weeks of age. Neutralizing antibody responses in
these kittens were significantly lower than in pregnant
queens. That kittens of this age are immunologically responsive was established, since complete protection of 9 kittens
to feline sarcoma virus was obtained by immunization with a
crude tumor extract inactivated with 5 to 7 megarads of Tirradiation. All these kittens developed feline oncornavirusassociated membrane antibodies while 3 developed demonstrable levels of virus-neutralizing antibodies.
The results of these studies are believed indicative that
killed virus vaccines and tumor vaccines can be effective
immunoprophylatic measures in the control of RNA tumor
virus oncogenesis in the cat. Developments in this model
system should be relevant to any consideration given similar vaccines in humans.
Introduction
The feline leukemia-sarcoma virus-induced diseases provide excellent models in which the role of immunity to
mammalian oncornaviruses can be evaluated, since these
' Presented at the symposium "Immunological Control of Virus-associated
Tumors in Man: Prospects and Problems," April 7 to 9, 1975, Bethesda, Md.
Supported in part by NIH Contract CP-43217 and American Cancer Society
Grant IM 27.
2 Presenter.
646
diseases occur naturally (17), can be produced experimentally in outbred animals, and are transmitted horizontally in
nature. Accordingly, experimental studies w i t h postnatal
viral inoculation simulate the natural pathogenesis of these
diseases. Thus it is reasonable to develop and evaluate
RNA tumor virus vaccines in the cat as a means of preventing experimental and naturally occurring oncornavirusinduced disease.
There is evidence that both active (12, 15) and passive (5)
immunity can be induced to feline oncornaviruses and that
each protects against oncornaviral disease. Precedent for
the feasibility of using immunoprophylaxis to prevent leukemia has been shown in the murine oncornavirus systems (1,
7, 13).
The objectives of the studies reported here were to characterize the immune response of the cat to feline oncornavirus and to determine whether oncogenesis by feline oncornaviruses could be prevented by immunoprophylaxis. The
eventual objective of this work is to provide a nucleus of
data that may be applied to future development of vaccines
against human oncornaviruses.
Our ultimate goal will be to define the various viral vaccine and host factors that lead to optimum production of
antiviral neutralizing antibody, anti-tumor antigen antibody,
and protection from oncogenesis in vivo following viral
challenge.
Materials
and Methods
C a t s . All cats used in this study were from a closed
breeding colony maintained at The Ohio State University.
These animals are of hysterectomy-derived ancestry and are
maintained in isolation (Shope units) from conventional
cats and other laboratory animals. The cats are similar in
most respects to conventional cats but are uniformly free of
antibody to the feline oncornaviruses and other feline viruses.
V i r u s e s . The GA-FeSV 3 (10) used in vaccination studies
was obtained from Electronucleonics Inc. (Bethesda, Md.)
as tissue ~:ulture-grown and sucrose gradient-purified virus.
Virus for in vitro serum neutralization studies was the STFeSV (19) prepared as cell-free 10% tumor homogenates.
Homogenized tumor suspensions were clarified at 2,300 •
3 The abbreviations used are: GA-FeSV, Gardner-Arnstein feline sarcoma
virus; ST-FeSV; Snyder-Theilen feline sarcoma virus; R-FeLV, Rickard feline
leukemia virus; KT-FeLV, Kawakami-Theilen feline leukemia virus; FeSV,
feline sarcoma virus; FeLV, feline leukemia virus; FOCMA, feline oncornavirus membrane antigen; SN, serum-neutralizing antibody; IrA, immunofluorescence.
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Experimental Oncornavirus Vaccines in the Cat
g for 20 min, followed by an 18,000 x g clarification for 1
min. Supernatants were aliquoted and stored at - 7 0 ~. The
R-FeLV (18) was prepared from 20% t u m o r homogenates of
thymus, spleen, and bone marrow in our 2rid and 4th in vivo
passages. These homogenates were clarified at 2,300 x g
for 20 min, aliquoted, and stored at - 7 0 ~ and - 1 7 0 ~. The
KT-FeLV (16) was prepared from tissue culture supernatants
of feline lymphoblastoid line FL-74 (20); these preparations
were concentrated by ultracentrifugation and twice banded
in sucrose gradients.
Vaccine Preparation. UV inactivation of purified GAFeSV was accomplished at a surface dose rate of 150
ergs/sq mm/sec for an accumulated total close of 35,000
ergs/sq mm. The Dio for the ST-FeSV was approximately
3,500 ergs and was close to that obtained for the murine
sarcoma viruses (21). Formalin inactivation of GA-FeSV was
performed by the method of Fink and Rauscher (6). Immediately prior to use, inactivated virus vaccines were emulsified
in an equal volume of Adjuvant 65 or Freund's complete
adjuvant.
Inactivation of virus in t u m o r antigen vaccines was by 7irradiation. Aliquots of a 10% homogenate of ST-FeSV-induced tumor cells were treated with various doses of 7radiation by Dr. Darwood B. Rowley, at the United States
Army Nadic Laboratories. Vaccination experiments used
tumor cell vaccines irradiated with 5, 6, and 7 megarads,
respectively.
In Vitro Virus Assay Methods. FeSV's were assayed for
focus-forming activity in early passages (3 to 15) of feline
embryo cells. Actively growing cells were seeded in plastic
Petri dishes (2 x 105/dish) in 4.5 ml of Medium L-15 and 15%
fetal calf serum. After 24 hr, the growth medium was removed and the cells were treated with 1 ml DEAE-dextran
(40 ~g/ml) in Medium L-15 without fetal calf serum. After 20
min, this medium was removed and the plates were rinsed
with 5 ml of growth medium. Monolayers were then overlaid
with 0.2 ml of appropriate virus dilutions for 2 hr. Plates
were rocked at 10- to 15-min intervals to maintain an even
distribution of the inoculum. The latter was removed and
replaced with 5 ml of growth medium. Monolayers were fed
with fresh m e d i u m on the 7th day after infection and fixed
with buffered formalin and stained with Giemsa, 3 to 4 clays
later. Foci were counted at x25 or •
magnification with a
dissecting microscope.
R-FeLV was assayed for helper focus-inducing activity on
a sarcoma-positive, leukemia-negative feline cell line kindly
supplied by Fischinger et a/. (8). This cell line is designated
subclone 81 of a Moloney murine sarcoma virus transformed CCC cat cell line 8c (9). The focus-induction assay
procedure was similar to that described above for ST-FeSV
on feline embryo cells except that McCoy Medium 5A with
15% fetal calf serum was used as growth m e d i u m (8).
In V/vo Virus Assay Methods. Vaccinated and nonvaccinated kittens received injections in the flank area with infectious FeSV at 5 to 6 weeks of age after active immunization,
or during the 1st week of life in passive immunization studies (pregnant queen immunization). Inoculation sites were
palpated at frequent intervals, and tumor sizes were measured with calipers to determine mean t u m o r induction times
and to estimate t u m o r growth rates. Serum samples were
collected at weekly intervals during the 1st 6 to 8 weeks, and
biweekly thereafter. Cats inoculated with FeSV were observed for 35 to 40 weeks, while those given FeLV were
observed 12 to 18 months. All cats that expired or were
euthanized at terminus were necropsied, and the extent of
metastases was determined.
Serological Tests. Indirect i m m u n o f l u o r e s c e n t antibody
titers to FOCMA were determined according to procedures
described by Essex et al. (4).
SN's were determined by a modified microtest procedure
originally described by Jarrett et al. (14), using Falcon Microtest II plastic plates. Twofold serum dilutions (inactivated at
56 ~ for 30 min) were prepared in Leibowitz Medium L-15
with 10% 7-globulin-free fetal calf serum in plastic Falcon
microtest plates (0.1 ml/well). Five to 10 focus-forming units
of FeSV (ST t u m o r homogenate) in a volume of 0.025 ml
were added to all but the serum and the cell control wells.
Plates were shaken briefly and incubated for 60 min at room
temperature or at 4 ~. Ten thousand feline embryo indicator
cells were added in a volume of 0.025 ml. After shaking, the
plates were incubated for 18 hr at 37 ~, and the supernatant
medium was then replaced. Medium was again replaced
after 3 to 4 days before being fixed on the 11th or 12th day in
10% buffered formalin and stained with Giemsa. The total
number of foci in 3 wells/dilution was counted. Virus survival was plotted as a function of serum dilution, and survival curves were constructed for 3 or more points. Serumneutralizing titers represented that serum dilution that neutralized 80% of the viral infectivity (2). Hyperimmune goat
anti-KT-FeLV served as positive antibody control for all SN
assays.
Results
Owing primarily to the greater rapidity of detectable clinical responses, most studies were performed with FeSV
rather than FeLV.
Serum Neutralization Responses in Cats Immunized
with Killed Feline Oncornavirus Vaccines. In order to evaluate the i m m u n o g e n i c i t y of inactivated Ga-FeSV, 43 kittens
and 7 pregnant queens were i m m u n i z e d (Table 1 ). No significant levels of serum-neutralizing antibodies were observed
in kittens during the 1st 4 weeks (4 injections of 3 x 10 l~
virus particles). In contrast, 6 of 7 pregnant queens given 4
injections of the same vaccines responded with a mean SN
titer of approximately 1:8. By 7 weeks, the mean SN titer of
the queens was 1:14, whereas only 1 group of kittens, those
given FeSV inactivated with UV and emulsified in Adjuvant
65, indicated some response. By 2 to 5 weeks postchallenge
(Table 1, Columns 7 to 10), this group showed a mean SN
titer of approximately 1:5. No other significant SN antibody
titers developed in the kittens. None of the queens or kittens
produced antibodies to FOCMA.
Effects of Immunization of Kittens with UV or Formalininactivated GA-FeSV on Tumor-growth Patterns and Survival Times. Two groups of 14 kittens given killed GA-FeSV
vaccines and challenged with a 90% t u m o r dose of FeSV at
5 to 6 weeks of age (Table 2) showed no significant differences in t u m o r latent periods and in mean survival times
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D. S. Yohn et al.
Table 1
SN antibody responses of kittens and queens to inactivated GA-FeSV
Geometric highest SN titers achieved/time interval
Vaccine"
GA-FeSV(UV-65)
GA-FeSV(For-65)
GA-FeSV(UV-FCA)
None
GA-FeSV(UV-65)
Recipients
20 kittens b
13 kittens
10 kittens
17 kittens
7 queens"
0-2 wk
<2.0
<2.0
<2.0
<2.0
2.4
_+ 0.0"
+ 0.0
- 0.0
_+ 0.0
+ 0.2
2-5 wk
(0/3)"
(0/9)
(0/6)
(0/5)
(1/7)
2.6
3.0
2.7
2.5
7.4
_ 0.2
+-- 0.3
_+ 0.2
_+ 0.3
___0.2
5-7 wk
(5/20)
(4/13)
(4/10)
(4/17)
(6/7)
3.2
2.7
2.4
2.4
14.0
_ 0.2
+-- 0.2
_+ 0.1
-+ 0.2
- 0.3
7-10 wk
(7/16)
(4/13)
(4/12)
(2/14)
(7/7)
4.6
2.1
3.7
3.7
_ 0.4
+- 0.1
_+ 0.3
-_+ 2.4
(7/11)
(1/14)
(3/9)
(4/14)
Abbreviations and numbers in parentheses in Column 1: UV, UV inactivation; For, formalin inactivation; 65,
Adjuvant 65; FCA, Freund's complete adjuvant.
Kittens received 4 weekly immunizations of 3 • 101~inactivated virus particles beginning at 4 to 6 days of age prior
to challenge with a 90% tumor dose of GA-FeSV at 35 to 40 days of age.
~' Mean _+ S.E.
" Numbers in parentheses in Columns 3 to 6: number of cats with SN titers ->1:4/total number of cats tested/time
period.
" Queens received 3 x 10 I~ inactivated virus particles within the 1st 4 days after copulation and again at 1,2, 3, 5, and
7 weeks during pregnancy (6 injections, total).
Table 2
The effect of immunization with inactivated GA-FeSV on tumor growth and survival times following
inoculation with live GA-FeSV
Tumor response b
Immunization category"
GA-FeSV(UV-65)
GA-FeSV(For-65)
Nonimmunized
No. of
cats
Rapid progressor
Slow progressor
and/or temporary regressor
14
14
15
7
6
2
2
7
6
Complete regressor
and/ornotu- Mean latent
mor
period"
5
1
7
3.5 +_ 0.8"
4.7 _+ 1.4
4.1 +__1.7
Survival
time (wk)
28.9 +- 9.9
29.0 _+ 6.7
34.4 +_ 5.5
a Regimen given in Table 1, Footnote a.
b The number of animals showing the designated tumor growth pattern.
' Time in weeks between challenge with live virus and appearance of tumor, excluding animals
that did not develop a detectable tumor.
" Mean _+ S.D.
than did a n o n v a c c i n a t e d group. Similarly, no differences
were observed in t u m o r - g r o w t h patterns a m o n g n o n i m m u nized and i m m u n i z e d kittens. A p p r o x i m a t e l y 50% of kittens
in both categories died from their t u m o r s or were sacrificed
d u r i n g terminus. These results indicate that the low levels of
a n t i b o d y induced in the kittens (Table 1) were not protective
against a 90% t u m o r dose of GA-FeSV.
Live Virus Challenge of Kittens Born of Queens Immunized with Inactivated FeSV or FeLV. Evidence of passively
transferred i m m u n i t y to FeSV or FeLV was s o u g h t by chall e n g i n g kittens born of queens i m m u n i z e d d u r i n g their pregnancy. In 1 part of this study, 12 kittens of Ga-FeSV(UV-65)vaccinated queens and 12 kittens from n o n v a c c i n a t e d
queens were c h a l l e n g e d in the 1st w e e k of life with GAFeSV (Table 3). All 12 kittens from n o n i m m u n i z e d queens
d e v e l o p e d t u m o r s , and 11 of 12 died from their t u m o r s
w i t h i n 21 weeks of age. In contrast, only 6 of the 12 kittens
from i m m u n i z e d q u e e n s developed p r o g r e s s i n g tumors,
w h i l e t u m o r s regressed in 3, and 3 failed to develop t u m o r
d u r i n g the 36-week o b s e r v a t i o n period. In general, kittens
born of queens with the highest n e u t r a l i z i n g a n t i b o d y titers
(->1:16) at time of delivery were a f f o r d e d the greatest protec-
648
tion. No serological tests were made on c o l o s t r u m ; however, it seems likely that the protection was provided by
maternal antibody.
In the 2nd part of this study, all 7 F e L V - i n o c u l a t e d kittens
f r o m n o n i m m u n i z e d queens died within 19 w e e k s after RFeLV challenge, whereas a significant p r o p o r t i o n (25%) of
kittens from R-FeLV(UV-65)-immunized q u e e n s failed to develop disease after m o r e than 70 weeks of age. Of the 12
c h a l l e n g e d kittens f r o m FeLV-immunized queens, 7 died
f r o m t h y m i c l y m p h o m a and 2 died from FeLV-related diseases (17). Their mean survival was 32 weeks, a significantly
p r o l o n g e d survival time, c o m p a r e d with kittens f r o m nonimm u n i z e d queens.
FeSV Challenge of Kittens Immunized with Irradiated
Tumor Cell Vaccine. Table 4 summarizes t h e effects of
various doses of ~/-radiation on infectivity of a ST-FeSV tumor cell h o m o g e n a t e . The untreated h o m o g e n a t e contained nearly 8 x 107 and 1.4 x 106 f o c u s - f o r m i n g units per
ml of FeLV and FeSV, respectively. Irradiation w i t h 3 megarads reduced titers of both viruses by nearly 103 while 4
m e g a r a d s eliminated all infectivity d e t e c t a b l e in vitro. In
vivo s a r c o m o g e n i c activity for n e w b o r n kittens was still
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Experimental
Oncornavirus
V a c c i n e s in t h e C a t
Table 3
Influence of i m m u n i z a t i o n of p r e g n a n t queens with inactivated FeSV and FeLV vaccines on
susceptibility of kittens to FeSV or FeLV
Tumor growth response
Immunization of
queens a
GA-FeSV(UV-65)
None
R-FeLV(UV-65)
None
Challenge of kittens
Disease response b
GA-FeSV'
GA-FeSV'~
R-FeLV"
R-FeLV"
9/12
12/12
9/12
7/7
Progressors
6
11
9
7
Regressors
No tumor or
leukemia
3
1
0
0
3
0
3
0
(20)d
(21)
(32y
(19)
Regimen given in Table 1, Footnote a.
b Number of kittens responding/number receiving injections.
' Kittens were challenged between 5 and 8 days of age.
d Numbers in parentheses, mean survival time in weeks.
'~ Kittens were challenged between 2 and 4 days of age.
i p = 0.05 (x2).
Table 4
Effect of T-radiation on inactivation of FeLV and FeSV in a 10%
tumor cell homogenate of a ST-FeSV-induced tumor
In vitro assay (foci-forming
Radiation
dose (megarads)
units/ml)
FeLV"
FeSVb
in vivo
None
3
4
5
6
7
7.9 • 107
2.2 • 103
0
0
0
0
1.4 • 106
5.7 • 102
0
0
0
0
+
+
NT
-
Sarcomagenesis
a Titrated on CCC cells.
b Titrated on feline embryo cells.
" NT, Not tested.
present after 3 megarads; however, doses of 5 megarads or
more abolished in vivo o n c o g e n i c i t y .
The i m m u n e response of 9 cats i m m u n i z e d with ST-FeSV
t u m o r cell vaccines irradiated with 5, 6, or 7 megarads,
respectively, is s u m m a r i z e d in Table 5. V i r u s - n e u t r a l i z i n g
antibodies were induced in only 3 animals.
Target cells used in the indirect IFA assay for F O C M A
were FL-74 cells (feline h e m o p o i e t i c cell line s h e d d i n g KTFeLV) and a KT-FeLV-infected dog t h y m u s cell. All kittens
were negative for antibodies to F O C M A prior to i m m u n i z a tion. S i g n i f i c a n t IFA titers to F O C M A developed after 3
weeks of i m m u n i z a t i o n . The specificity of the FOCMA response was s u p p o r t e d by the e v i d e n c e that (a) IFA to
F O C M A in FL-74 cells was not removed by a b s o r p t i o n with
cat tissues, and (b) similar IFA patterns to F O C M A were
obtained w i t h the KT-FeLV-infected d o g t h y m u s cells. Moreover, noninfected t h y m u s dog cells were not reactive with
FOCMA-positive cat sera.
An i m p o r t a n t aspect of this study (Table 5) was that i m m u nized cats were resistant to ST-FeSV challenge. Only cats
immunized with the 7-megarad-treated t u m o r cells developed tumors. However, these t u m o r s regressed or failed to
progress. It seems likely that the 7-megarad dose of Tirradiation destroyed some a n t i g e n i c i t y of the h o m o g e n a t e .
A g e - r e l a t e d Factors in the R e s p o n s e to FeLV. With the
Table 5
Evaluation of killed ST-FeSV tumor cell vaccine as determined by in
vivo protection and FOCMA antibody p r o d u c t i o n
Tumor or
vaccine
(megarads)
Cats tested ~
Responsiveness
to ST-FeSV challenge
5
5
5
5
6
6
6
7
7
7
0
509-3
509-4
509-5
509-6
620-2
620-3
620-4
295-2
295-3
295-4
3d
No tumor
No tumor
No tumor
No tumor
No tumor
No tumor
No tumor
Regressor
Nonprogressor
Nonprogressor
All died
FOCMA antibody b titers
FL-74
C thy C
1:4
1:32
1:8
<1:2
1:8
1:2
1:2
1:2
1:16
1:2
1:32
1:16
NT
1:16
NT
NT
NT
1:8
NT
NT'
Immunized 3 times prior to challenge at 35 days of age.
b FOCMA antibody after the 3rd immunization as tested on FL-74
cells and canine thymus cells (C thy C) infected with KT-FeLV virus.
" NT, not tested.
" Three nonimmunized cats challenged at 35 days of age.
e x c e p t i o n of 1 e x p e r i m e n t wherein kittens b o r n of queens
i m m u n i z e d with UV-inactivated R-FeLV were c h a l l e n g e d at
4 days of age with R-FeLV (Table 3), all o t h e r studies were
p e r f o r m e d with FeSV. In o r d e r to assess the efficacy of
FeLV vaccines by means o t h e r t h a n disease i n d u c t i o n
a n d / o r survival, a w o r k i n g k n o w l e d g e of s u s c e p t i b i l i t y to
infection and i m m u n e responses t o i n f e c t i o n at various
ages is necessary.
Table 6 s u m m a r i z e s the s e r o l o g i c a l , v i r o l o g i c a l , and biological response of n e w b o r n , w e a n l i n g , and y o u n g adult
cats to R-FeLV and KT-FeLV infection.
Cats i n o c u l a t e d w h e n n e w b o r n d e v e l o p e d persistent
FeLV viremia but little or no n e u t r a l i z i n g or F O C M A antibody. The converse generally was true of cats i n o c u l a t e d in
the p o s t w e a n i n g or y o u n g adult period of life. L y m p h 0 s a r coma, aplastic anemia, or l y m p h o i d d e p l e t i o n disease has
o c c u r r e d in many of the cats in the g r o u p i n o c u l a t e d as
n e w b o r n s , whereas o n l y 1 of the 20 i n o c u l a t e d older cats
has d e v e l o p e d aplastic a n e m i a after 20 weeks. This cat was
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Table 6
Immunological and disease responses of cats inoculated at different ages with R-FeL V or
KT-FeLV
Virus
R-FeLVb
KT-FeLVe
Age at inoculation
FOCMA
antibodies
Virus neutraliz'ing antibodies
FeLV gs
antigen a
FeLV-related disease
Observation period (wk)
Newborn
Newborn
3/21
0/4
0/7
0/4
20/21
4/4
14/21
4/4
8-30
8
3/25
0/11
24/25
18/25
8/10
5/6
4/4
7/10
6/6
NT~
1/10
1/6
1/4
0/10
0/6
1/4
17/20
13/16
3/20
1/20
Totals
R-FeLVb
R-FeLVb
KT-FeLV"
Totals
3-4 mo.
1 yr
3 mo.
30
30
20
a Presence of feline group-specific antigen in peripheral leukocytes as determined by
the Hardy test.
One ml of a 20% tissue homogenate containing 105 infectious units of virus (by focus
induction on CCC cells).
" One ml containing 10~ infectious units.
NT, not tested.
only 1 of the 3 in this group that developed persistent
viremia.
Discussion
These studies were initiated to evaluate the feasibility of
i m m u n i z i n g an outbred mammal, the cat, against horizontally transmissible oncogenic RNA t u m o r viruses with killed
virus vaccines. The initial vaccine studies were performed
with FeSV, owing primarily to the relative rapidity of evaluation of protective efficacy, compared with similar studies
with FeLV.
To date, these studies have established that kittens can
be effectively protected against FeSV by active immunization with crude irradiated t u m o r cell vaccines. This observation is important since it argues against the possibility that
kittens are not responsive to i m m u n o g e n s responsible for
inducing i m m u n i t y to Oncornaviruses. Such a possibility
was raised by the observation that 4 injections of UV-irradiated or formalin-inactivated FeSV failed to protect kittens
and to induce detectable levels of serum-neutralizing antibodies by the the time kittens were 5 to 6 weeks of age, the
time of challenge with a 90% t u m o r - i n d u c i n g dose of virus,
whereas the same n u m b e r of injections induced detectable
levels of neutralizing antibodies in 6 of 7 pregnant queens.
Further immunization of queens with 2 additional doses of
vaccine resulted in serum-neutralizing antibody titers that
ranged from 1:8 to 1:32 (mean, 1:14). A significant number
of kittens, 6 of 12, born of these queens survived a normally
lethal dose of FeSV.
These data suggest that our apparent inability to effectively protect 5- to 6-week-old kittens by direct immunization with killed virus vaccines may be attributable to an
insufficient number of immunizations, to an insufficient
a m o u n t of i m m u n o g e n in the vaccine, or to an inappropriate vaccine regimen.
Another factor may be that the virus challenge was too
great. However the fact that a significant proportion of 1-
650
week-old kittens (6 of 12) born of immunized queens survived a normally lethal dose of FeSV argues against this
possibility. Furthermore, the solid protection afforded 5week-old kittens to FeSv by immunization with irradiated
crude tumor cell vaccines indicates that sufficient protection against the viral dose used can be achieved in 5- to 6week-old kittens.
The tentative conclusion is that the vaccine regimen used
in the kittens was insufficient and a regimen that will induce
significant levels of viral neutralizing antibodies must be
sought. It is not implied that induction of neutralizing antibodies will necessarily be protective; however, the results of
the queen immunization indicated a strong correlation between induction of virus-neutralizing antibodies and protection to virus challenge. In order to establish this correlation
conclusively, the FeSV system should provide this information more rapidly than the FeLV system.
Ultimately, it is desired that the efficacy of a viral vaccine
be evaluated in the FeLV system under simulated natural
conditions, i.e., an evaluation of protection against horizontally transmitted virus by virus shedders to contacts. For
these studies, markers of infection (other than disease) in
the contacts may be required. These may include detection
of viremia (11), of antiviral antibodies by neutralization or
complement-fixation inhibition, development of FOCMA antibodies, and alterations in immunological responses attributable to FeLV infection or leukemogenesis.
We are currently examining kittens from newborn to 4
months of age for an age group of cats that responds immunologically to FeLV and FeLV-associated antigen (FOCMA)
but are still moderately to highly susceptible to FeLV infection. Among this age group, evaluation of an FeLV vaccine
for production of active immunity to FeLV should be experimentally feasible. The results to date (Table 6) indicate that
infection of 3-month-old kittens with R-FeLV or KT-FeLV
results in humoral antibody responses to viral envelope
antigens and to FOCMA. The development of FOCMA antibodies may prove to be a valuable marker, since immunization with killed virus vaccines has not induced FOCMA
CANCER RESEARCH VOL. 36
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Research.
Experimental
a n t i b o d i e s . A c c o r d i n g l y , it s h o u l d be p o s s i b l e to i m m u n i z e
w i t h killed v i r u s and i n d u c e n e u t r a l i z i n g a n t i b o d i e s , challenge at 3 m o n t h s or y o u n g e r w i t h R - F e L V or K T - F e L V , and
m o n i t o r p r o t e c t i o n by the a b s e n c e of F O C M A a n t i b o d y form a t i o n . I n f e c t i o n of 3 - m o n t h - o l d k i t t e n s w i t h R - F e L V o r KTFeLV, as d e m o n s t r a t e d by n e u t r a l i z i n g and F O C M A antib o d y f o r m a t i o n , did not result in c l i n i c a l d i s e a s e or c o n s i s t ent v i r e m i a ( H a r d y test), t h e r e b y n e g a t i n g t h e s e as reliable
indices of F e L V i n f e c t i o n in this age g r o u p . Yet to be d e t e r m i n e d is w h e t h e r y o u n g e r kittens, 6 to 8 w e e k s of age,
develop persistent viremia following infection with R-FeLV
or KT-FeLV.
Jarret e t al. (15) have o b t a i n e d p e r s i s t e n t v i r e m i a in cats
infected at 6 m o n t h s of age w i t h 109.3 F e L V - i n f e c t e d feline
e m b r y o f i b r o b l a s t s . T h i s i n f e c t i o n led to high a n t i b o d y titers
to virus s u b g r o u p g l y c o p r o t e i n a n t i g e n s c o m m o n to the
infected cells and v i r i o n e n v e l o p e s . T h e s e a n t i b o d i e s are
r e p o r t e d to h a v e the s a m e s p e c i f i c i t y as t h o s e r e s p o n s i b l e
for virus n e u t r a l i z a t i o n and cell c y t o t o x i c i t y but are measured by an i n d i r e c t i m m u n o f l u o r e s c e n c e test on the infected cells. T h e i r r e l a t i o n s h i p to F O C M A a n t i b o d i e s w a s
not clarified. In this s t u d y , w e w e r e able to i n d u c e virusn e u t r a l i z i n g a n t i b o d i e s in the a b s e n c e of F O C M A and cytot o x i c a n t i b o d i e s , and v i c e v e r s a .
The studies by Essex e t al. (3) have e s t a b l i s h e d a c o r r e l a t i o n b e t w e e n p r e s e n c e of a n t i b o d y to the F O C M A and the
c a p a b i l i t y of t h e cat to resist feline o n c o r n a v i r u s - i n d u c e d
neoplasia. On the basis of these s t u d i e s , it is c o n c e i v a b l e
that a v a c c i n a t i o n r e g i m e n t h a t i n c l u d e d i m m u n i z a t i o n w i t h
F O C M A will b e n e c e s s a r y for e f f i c i e n t i m m u n o l o g i c a l prot e c t i o n f r o m F e L V - and F e S V - i n d u c e d d i s e a s e . Results described h e r e i n revealed t h a t i m m u n i z a t i o n of k i t t e n s w i t h
irradiated S T - F e S V t u m o r h o m o g e n a t e s (free of r e p l i c a t i n g
virus) (a) i n d u c e d s p e c i f i c a n t i - F O C M A r e s p o n s e s and (b)
p r o t e c t e d the k i t t e n s f r o m s u b s e q u e n t S T - F e S V c h a l l e n g e ,
even t h o u g h v i r u s - n e u t r a l i z i n g a n t i b o d y titers w e r e very l o w
or u n d e t e c t a b l e . F u r t h e r s t u d i e s are n e e d e d to d e f i n e a
n u c l e i c a c i d - f r e e t u m o r i m m u n o g e n t h a t i n d u c e s antiF O C M A r e s p o n s e and p r o v i d e s active i m m u n i t y to o n c o g e n i c virus c h a l l e n g e . The e x p e r i m e n t a l s t u d i e s r e p o r t e d
here w i t h S P F cats may be i n t e r p r e t e d to s u p p o r t the c o n c e p t (11) t h a t b o t h a n t i - F e L V and a n t i - F O C M A may be req u i r e d for s o l i d i m m u n i t y to virus i n f e c t i o n and t u m o r f o r m a tion. The u l t i m a t e c o m b i n i n g of v a c c i n a t i o n w i t h b o t h viral
e n v e l o p e and F O C M A i m m u n o g e n s m a y p r o v i d e u l t i m a t e
p r o p h y l a c t i c c o n t r o l of feline o n c o r n a v i r u s - i n d u c e d diseases and h o p e f u l l y serve as a useful m o d e l for c o n t r o l of
s i m i l a r d i s e a s e s in o t h e r o u t b r e d a n i m a l s y s t e m s .
Oncornavirus
Vaccines
in the Cat
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FEBRUARY 1976
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Research.
651
Experimental Oncornavirus Vaccines in the Cat
D. S. Yohn, R. G. Olsen, J. P. Schaller, et al.
Cancer Res 1976;36:646-651.
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