Vaccines against feline leukemia

Vaccines affording protection against the diseases resulting from infection of cats with feline leukemia virus are produced from cells infected with that virus or from the virus alone.

The present invention relates to a vaccine and particularly to a vaccine 
offering protection against the diseases associated with the infection of 
cats with feline leukemia virus (FeLV). 
It is known that feline leukemia virus may be transmitted between 
individual cats, thereby causing infection by the virus of a relatively 
high proportion of cats in a colony or in a population. It is thought that 
infection of cats with feline leukemia virus is responsible for diseases 
other than leukemia, for example immunosuppression and non-responsive 
anemias. The present invention provides vaccines and immunizing procedures 
which produce, in cats, high titres of anti-body against FeLV associated 
antigens and protection of the animals against infection with this virus. 
Vaccines will be described which provide high titres of antibody against 
FeLV associated antigens in the blood of cats in the absence of any 
lasting virus infections. Methods will also be described of providing 
sufficient primary immunization against FeLV to enable a cat which is 
subsequently infected by the virus to mount a secondary response and 
thereby overcome the infection. In all cases, it is shown that the 
vaccines of the present invention provide sufficient protection for an 
animal to enable it to overcome a subsequent infection with FeLV, without 
inducing a lasting infection in the animal. In many cases, protection is 
achieved without the injection of infective materials. 
The vaccines of the present invention may comprise preparations of cells, 
infected with FeLV, or preparations of the virus alone which has been 
separated from the cells in which it is grown. In both cases, the virus 
may be grown in a variety of different cells and cultured by conventional 
means. Thus, feline lymphoblasts may be cultured in suspension and these 
cells may transmit the virus particles from generation to generation. 
Similar cell cultures may be derived from cells of a non-feline species 
such as a dog or human being and these too may carry the virus infection 
from generation to generation. Alternatively, it is possible to culture 
feline embryo cells in monolayers. These cells may be infected and 
collected after a suitable period of infection. Cells from non-feline 
species may also be cultured in monolayers and infected. It is further 
possible to maintain tumor cells from an infected cat in continuous 
culture. It is found that the virus particles from such cells may display 
only low infectivity for cats while remaining capable of immunizing cats 
against infection with a fully-infective virus. In all cases, the culture 
of these cells is achieved using conventional conditions which provide for 
the replication of the virus. 
It has now been found that cells infected with FeLV produce large 
quantities of virus-associated antigen on the surface of the cell membrane 
and that this membrane antigen is immunogenic. Antibodies raised in a cat 
by injection of infected cells are therefore active against cells in the 
animal which are infected with the virus. Such antibodies are also active 
against cells which have become infected and transformed into malignant 
tumor cells. However, it is also recognized that the virus particles 
themselves are immunogenic and can form the basis of an effective vaccine. 
Such a vaccine will be active against infecting virus particles rather 
than infected cells but both vaccines are considered to be within the 
scope of the present invention. 
The vaccines of the present invention may therefore comprise infected cells 
or virus separated from infected cells and each may be presented to the 
animal in a variety of forms. 
Thus, whole live cells which have been infected with FeLV and which have on 
their surfaces large amounts of virus-associated antigen may be 
administered to the animals. By selection of a suitable dose of cells, it 
is possible to obtain high titres of antibody against the virus or 
virus-infected cells without the retention of any live virus in the 
animal. The administration of infected cells to an animal naturally 
carries the risk of infecting the animal itself. However, by selecting the 
dose of the cells to be administered, it is possible to obtain 
immunization without lasting infections. 
It is naturally preferred that non-infective material should be used as a 
vaccine providing it is sufficiently immunogenic. Whole cells may be 
either rendered inactive by suppression of their growth or killed by a 
large number of treatments already known in the art. For example, live 
cells may be rendered inactive by treatment with RNA/DNA transcription and 
translation inhibitors such as mitomycin D. Alternatively, infected cells 
may be killed by, for example, irradiation, hydroxylamine or thermal 
inavtivation. Two preferred agents for killing infected cells are 
paraformaldehyde and acetylethyleneimine, used either separately or in 
conjunction with one another. In all cases, the conditions used for 
killing the infected cells should be sufficient to substantially destroy 
their infective activity without removing their immunogenic properties. 
When using paraformaldehyde as an agent to kill infected cells, the 
concentration and period of incubation should be such that substantially 
all of the cells are killed without an unacceptable reduction in their 
immunogenic activity. Similar agents to those described above may also be 
used in order to kill the live virus separated from the cell cultures, 
although the exact conditions used to kill the virus may differ from those 
used to kill cell-virus mixtures. 
Conventional adjuvants may be added to the immunogenic material comprising 
the vaccine, in order to stabilize the material and enhance the immune 
reaction against it. Among those adjuvants suitable for this purpose, are 
Freund's complete or incomplete adjuvant, killed hemophilus pertussis or 
polynucleotide compositions. The preferred proportion of adjuvant may be 
established by experimentation. 
The immunogenic material used in the present vaccines may be presented to 
the animal in any conventional formulation. For example, it may be 
suspended in a buffer solution in which its activity is preserved, 
freeze-dried, or maintained as a frozen suspension. The vaccine, in a 
suitable form for administration, may be injected into the animal by any 
suitable route. For practical reasons, the subcutaneous or intra-muscular 
routes are preferred. 
It is possible by means of the present invention to provide immunity in an 
animal with a single administration of a suitable vaccine. Alternatively, 
it is possible to produce a primary response by an initial injection and a 
subsequent strong secondary response by a further injection at a later 
date. The former procedure is preferred as the animal need only be 
vaccinated on one occasion. It is further possible to provide a primary 
response with a single injection of vaccine, such that a subsequent 
natural infection of the animal will then result in a strong secondary 
reaction to the infection which will be sufficient to reverse the 
infection causing the reaction and thus to prevent the animal from 
contracting any of the diseases associated with infection by FeLV. It is 
shown below that the use of the present vaccines allow all of these 
procedures to be used successfully.

EXAMPLE 1 
Vaccination of cats with live cells infected with FeLV provide high titres, 
in the blood of these animals, of antibodies against FeLV associated 
antigens. When high doses of cells are administered, the virus persists in 
the blood although no symptoms of disease are manifest. However, with 
lower doses of cells, infected with virus of lower infectivity, all trace 
of virus are removed from the blood within one month and high antibody 
titres are maintained, thus protecting the animal against subsequent 
infection by the virus. 
Monolayer cultures of feline embryo fibroblasts are grown according to 
conventional procedures. The cells are chronically infected with FeLV of 
known sub groups. The infected cells were grown for sufficient time to 
allow extensive replication of the virus. They are then harvested, 
suspended in buffer at a known concentration of cells, packed by 
centrifugation and injected subcutaneously. 
The animals are monitored each month for antibody to feline leukemia virus 
cell membrane antigen. At the end of the period of study, the animals were 
sacrificed and their tissues analyzed, by the procedures below, for the 
presence of live virus. 
(a) Samples from many different tissues are viewed with an electron 
microscope for the presence of visible virus particles. 
(b) Tissues from the animals are cultured in the presence of uninfected 
monolayer cells for 28 days. They are subsequently examined with an 
electron microscope and by immunofluorescence for the presence of virus 
particles and virus antigen. The results of immunization with live, 
infected cells are shown in the Table below. 
TABLE I 
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Average Virus 
Peak Duration 
Isolation 
Cells Antibody Study at Virus 
Type Number Titre (months) 
necropsy 
Subgroup 
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FEA 2 .times. 10.sup.9 
120 6 - 13 + A + B 
FEA 7 .times. 10.sup.8 
96 6 + A 
FEA 4 .times. 10.sup.7 
202 1 - 3 - A + B + C 
FL 4 .times. 10.sup.7 
248 1 - 3 - A + B + C 
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FEA Monolayer feline embryo fibroblasts. 
FL Suspension feline lymphoblasts. 
All cats immunized with live infected cells subsequently develop high 
titres of antibodies against FeLV. When high doses of cells are injected, 
the presence of virus in the tissues persists for 6 to 12 months although 
no symptoms of disease were apparent. In contrast, immunization with lower 
doses of cells also result in the development of high antibody titres but 
no virus is detectable in these animals after a period of 1 to 3 months. 
The rate of development of antibodies and their persistence in the blood is 
shown in FIG. 1. Animals are injected with 2.times. 10.sup.9 FEA cells and 
the level of antibodies in the blood monitored each month. It can be seen 
that a maximum antibody titre is reached within 3 months and that this 
persists at a high level beyond 12 months. It is therefore possible by 
selection of the dose of cells injected to immunize cats with live cells 
infected with FeLV and thereby to induce the development of high levels of 
anti-feline leukemia virus antibody in the blood stream. As will be shown 
below, such levels of antibody are capable of protecting a cat against 
subsequent infection by the virus. 
EXAMPLE 2 
Cats vaccinated with a vaccine comprising live cells containing FeLV are 
capable of withstanding infection when challenged with a high dose of 
virulent virus. 
Three cats, whose sera initially contain no detectable antibody, are each 
inoculated subcutaneously with 3.times. 10.sup.7 live feline 
lymphoblastoid cells chronically infected with FeLV, of low infectivity 
for cats and feline tissue culture. The cats are bled monthly for serum 
samples and the results of the immunofluoresence antibody test for 
anti-FeLV antibody are shown in FIG. 2. 
The antibody titre rises within the first month to a mean of 256 and all 
cats reach this antibody level. One month after inoculation the cats are 
challenged by injection with 10.sup.6 infectious units of strain FeLV5 
virus, known to be highly leukemogenic and pathogenic in cats. The cats 
are sacrificed and necropsied three months after challenge. The following 
investigation demonstrates that there is no remaining viral infection 
resulting from the challenge. 
1. No pathological or histological abnormalities are found. 
2. No virus is found by electron microscopic examination of bone marrow or 
tracheal tissue. 
3. Plasma from challenged cats is used to inoculate cultures of feline 
embryo monolayer cells and no virus is detected in the culture. 
4. Bone marrow cells are co-cultivated with feline embryo monolayer cells 
and no infection with virus can be detected. 
Similar, but unvaccinated, cats are also challenged with the same dose of 
infectious FeLV. They are killed and necropsied at the same time as the 
vaccinated cats and the same tests are applied for virus detection. 
However, virus particles are detected in all samples tested from these 
cats. The presence of live virus particles would lead in due course to 
symptoms of the particular diseases caused by FeLV infection. Vaccination 
of cats with live cells containing FeLV of low infectivity therefore 
produces in those cats an immunity against subsequent infection with 
virulent FeLV. 
EXAMPLE 3 
Cats may be treated with a vaccine in which the virus-containing cells are 
killed by treatment with paraformaldehyde. 
Feline lymphoblastoid cells (2.times. 10.sup.8), chronically infected with 
FeLV of low infectivity, are suspended in 1 ml. Hank's solution. 9 ml. of 
1% paraformaldehyde is added slowly while the mixture is stirred on an ice 
bath. The cells were kept in paraformaldehyde at 4.degree. C. for one hour 
and then washed in Hank's solution. Aliquots were prepared each containing 
6.times. 10.sup.7 cells in 1 ml. solution. 
Cats, having no detectable FeLV antibodies in their sera, are injected 
subcutaneously with an aliquot containing the killed cells prepared above. 
They are bled monthly for serum samples and the anti-FeLV antibody titres 
are shown in FIG. 3. 
High antibody titres are reached within three months of vaccination and 
persist for 3-4 months at which time the animals are killed. The same 
techniques described in Example 1 for the detection of virus particles are 
applied to all animals and no virus particles are detected. 
A high antibody titre, shown in Example 2 to protect cats against infection 
with live FeLV, may be stimulated in cats by vaccination with 
virus-containing cells killed by treatment with paraformaldehyde. 
EXAMPLE 4 
Animals with an initial anti-FeLV antibody titre of 4 may develop a high 
antibody titre upon immunization with inactivated infected cells. The 
initial antibody titre may have resulted from a natural infection or from 
prior immunization such as that described in Example 2. 
An animal with an initial antibody titre of 4 is injected with a vaccine 
prepared from infected feline embryo fibroblasts cells which have been 
inactivated with formaldehyde and acetylethyleneimine(AEI). The cells are 
cultured in monolayer and are infected and harvested as described before. 
3.times. 10.sup.8 cells are suspended in 100 ml of 0.05% formaldehyde for 
1 day at 22.degree. C. The cells are again washed twice in buffer and are 
resuspended in 200 ml of 0.05% AEI for one day at 22.degree.. The cells 
are again washed and resuspended in 20 ml of buffer to which has been 
added 2 ml of 20% sodium thiosulphate. The cells are resuspended in 4 ml 
of buffer and 0.5 ml of the suspension is injected subcutaneously into the 
animal. 
The animal has a pre-inoculation antibody titre of 4. Within one month, the 
antibody titre is 32 and this is maintained at 3 months. At the end of 
this period, no virus or virus particles can be detected in the animal by 
means of electron microscopy or immunofluorescence. It is therefore 
possible to initiate a strong secondary reaction in an animal having an 
antibody titre of 4 by injection of infected cells which have been 
inactivated by means of formaldehyde and AEI. The antibody titres achieved 
by such immunization are capable of protecting an animal against 
subsequent infection with FeLV and these antibody levels are maintained 
for a considerable period of time. 
EXAMPLE 5 
Immunization capable of inducing protective titres of anti FeLV antibody 
was achieved by the injection of infected feline embryo cells killed by 
treatment with formaldehyde. Feline embryo fibroblasts are grown in 
monolayer culture, infected and harvested as before. The cell suspension 
is added to 20 times its volume of 1/100 formaldehyde and stored at 
+4.degree. C. for one week. The cells are centrifuged at 5,000 r.p.m., 
washed and re-suspended in Alsever's Solution. In this form, the cells may 
be stored at minus 20.degree. C. for two months. 
The cells are diluted and 1.5.times. 10.sup.8 cells per animal are injected 
subcutaneously into mature adult cats. Peak antibody titres of 16 are 
achieved within one month of immunization. It is shown below that animals 
with this titre of antibody are able to successfully resist an infection 
with live virus by mounting a secondary reaction. In addition, no trace of 
virus or virus antigen could be detected by microscopy of the tissues of 
animals immunized by this procedure. 
EXAMPLE 6 
Animals in which there is a detectable anti-feline leukemia virus antibody 
titre are capable of developing a strong secondary response to infection 
with live virus which is capable of overcoming the virus and eradicating 
the infection. 
Animals with an initial antibody titre of 16, together with some animals 
with no detectable antibody titre, are injected with a high dose 
(10.sup.10 particles per animal) of live FeLV. 
The animals are monitored over a period of 7 months for the levels of anti 
feline leukemia antibodies in the blood, and these levels are shown in 
FIG. 4. Those animals which had no detectable antibodies when injected 
with virus became chronically infected and contracted many of the diseases 
associated with FeLV. In addition, many died. In contrast, those animals 
having an initial antibody titre of 16 developed a strong secondary 
reaction to the injected virus and after 6 months displayed an antibody 
titre of 256. There was no detectable disease or virus in these latter 
animals at the end of 7 months. It is clear therefore that animals with a 
relatively low initial antibody titre are capable of mounting a strong 
secondary reaction to infection with live virus. 
EXAMPLE 7 
A vaccine is prepared from virus separated from cells in which it is grown. 
Cats innoculated with such a virus raise titres of anti FeLV antibodies 
which allow them to subsequently resist infection with live virus by 
mounting a strong secondary immunological response. 
Infectious FeLV particles are grown in FEA monolayer cells and separated 
from the cells by conventional techniques of ammonium sulphate 
precipitation and density gradient centrifugation. The virus particles are 
added to the inactivating agent at the concentration given below and 
incubated for 6 hours at 4.degree. C. The inactivation by AEI was stopped 
by the addition of 20% sodium thiosulphate. One part of Freund's 
incomplete adjuvant was added to each part of the virus suspension. 
Cats are innoculated with approximately 10.sup.7 virus particles and the 
peak antibody titre reached within three months was recorded. It is shown 
in Table 2 that cats injected with inactivated virus subsequently produce 
moderate antibody titres against FeLV. These titres are such that a 
subsequent innoculation or injection would result in a strong secondary 
response with the production of high antibody titres. 
The animals were sacrificed after three months and samples of tissue and 
blood are examined by electron microscopy, immunofluorescence, and 
cocultivation with uninjected cells. No virus particles are detected. 
TABLE 2 
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Response - mean peak 
Inactivant antibody titre 
______________________________________ 
Formalin (0.05%) 11.3 
AEI (0.05%) 4 
Formalin (0.05%) 5.7 
+ 
AEI (0.05%) 
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NB. All injections included Freund's incompleted adjuvant (50%).