Br. J. Cancer (1975) 31,

Suppl. II, 147

THE RELATION OF IMMUNE RESPONSE TO PATHOGENESIS, VACCINATION AND EPIDEMIOLOGY IN VIRUS INDUCED LEUKAEMIA W. F. H. JARRETT From the University of Glasgow Veterinary School, Department of Veterinary Pathology, Bearsden Road, Bearsden, Glasgow, G61 1QH

Summary.-The antigenic systems of oncornaviruses and particularly feline leukaemia virus (FeLV) are reviewed briefly. The use of immunological methods in studying the epidemiology of -the disease is described. The incidence of FeLV infection as judged by a serological survey is at least 100 times greater than that of leukaemia in the cat population. Horizontal transmission, due to virus replication in respiratory and alimentary epithelial cells, is common. A method of producing high titres of antibody against membrane antigens of virus infected cells is described; the use of such vaccination is discussed in relation to several epidemiological facets of feline leukaemia virus infection. Leukaemia viruses are well known to cause immunodepression to heterologous antigens. The hypothesis is advanced that depression of the humoral antibody response to leukaemia virus antigens and cell membrane antigens may be an early event allowing establishment and replication of virus in haemic and the lymphatic tissues. Subsequent depression of cell mediated immunity through direct action of thymic cells is known to take place in the cat system. This may allow further spread of the virus with replication in epithelial cells which are not susceptible to cytotoxic action. Thus the primary events leading to leukaemogenesis may be an interplay between immunostimulation and immunodepression. THE VIRUSES which are known to cause leukaemia, lymphosarcoma and fibrosarcoma in the cat are oncornaviruses. They have a similar structure to avian, murine and other leukaemogenic viruses. Recent work has shown that there is a striking general similarity between the proteins of the virions of the fowl, mouse and cat. This is reflected in the function of these proteins as antigens (Table I). In very general terms, oncornaviruses have 2 main antigenic systems, the group specific antigens (gsa) and the sub-group specific antigens (sgsa). The group specific antigens are common to most of the leukaemogenic viruses of any single species (the case of endogenous viruses will be described later), i.e., all of the mouse viruses have cross reacting gsa's, as have all of the cat. In addition, one polypeptide (p30, Table I) carries a determinant shared by several virus groups

from different mammalian species, the interspecies antigen. Thus, antisera against mouse p30 give a line of identity with cat p30 on double diffusion. These group specific polypeptides are onlyweakly antigenic in their host of origin; they do not appear to be involved in any protective immunological mechanism. Heterologous antisera can be made against them and these can be used to detect the presence of virus genome in cells. The sub-group specific antigens are glycoproteins located on the surface of the virion and on the membranes of cells which are producing virus by budding. At present, feline leukaemia viruses (FeLV) are known to have at least 3 sub-groups, A, B and C (Sarma and Log, 1971). The viruses of one sub-group have in common (1) surface antigens, (2) receptors which determine host-cell infectivity range and (3) the ability to interfere on superinfection

W. F. H. JARRETT

148

TABLE I.-The Structural Proteins of Feline Leukaemia Viruses Symbol plO

p12 p'5 p30 gp35 gp75 p

=

Approx. M.W. 10000 12000 15000 30000 35000 75000 polypeptide; gp

=

Site Core RNP Virion surfaace Extra core Extra core Surface of membrane glycoprotein.

Antigenicity Species gsa Species gsa Species gsa Sp. & interspec. gsa Sub-group specific

by homologous but not heterologous virus. These 3 virus-host functional relations are almost certainly associated with the subgroup specific glycoproteins. It is likely that the same receptors are involved in virus neutralization and at least in part in membrane antigen immunofluorescence. Cats can, under certain circumstances, produce fairly high levels of antibody against these antigens in contradistinction to gsa. These antibodies are almost certainly involved in immunity to the virus and to the infected cell respectively. The method of demonstrating antibodies which we have found most useful in immunological and epidemiological studies is the FOCMA (feline oncornavirusassociate cell membrane antigen) test (Essex et al., 1971; Jarrett, 1972). In the feline fibrosarcoma system it has been shown that regression of solid tumours is associated with the development of FOCMA antibody titres and that this antibody can be transmitted maternally, giving rise to refractoriness to tumour induction in kittens (Essex et al., 1971). In field cases of leukaemia we have found that 75% have no FOCMA antibody titre and the others have very low levels (Fig. 1). Also, in surveys of normal cats in rural and urban areas (see later) antibody levels are usually low. In studying methods of vaccination against feline leukaemia we inoculated cats with a range of doses of live cells which were bearing virus associated and virus antigens on their surfaces. This was originally in an attempt to mimic the sarcoma system as we thought that it might be necessary to present to the immune system cell walls bearing antigen arrays; it was also

Equimolar in virion with p30 Antibodies to these glycoproteins are neutralizing, cytotoxic and protective

466 s.,

%Cats

S. 4,

20 L

* 1

2 3 4 5 5

7

*

5 1 11

Log2 Ab Titre Fia. 1.-Antibody titres in spontaneous cases of lymphoma and leukaemia (hatched) compared with the titres in cats vaccinated with live cells infected with viruses of sub-groups A, B and 0.

considered likely that the effective immunogens might be preserved better in this way. High titres were achieved but some rather surprising results emerged. In one experiment giving approximately 109 cells infected with virus of sub-groups A and B, a mean titre of 128 was induced but the response did not reach a peak until 3 months. The titre persisted for 13 months and no abnormality of any kind was seen during that time; however, at necropsy the cats were still infected with FeLV. Cats inoculated with 107 cells which were infected with a different isolate containing virus of sub-groups A, B and C, reached their peak titre in one month and animals necropsied at both 1 and 3 months after infection had no detectable virus (Table II). In further investigating this phenomenon, we have inoculated groups of cats with 109, 108, 107 and 106

IMMUNE RESPONSE TO PATHOGENESIS, VACCINATION AND EPIDEMIOLOGY 149

TABLE II.-FOCMA Antibodies and Virus Survival in Cats Immunized with Infected Cells Cell no. 109

108 107

107

Type

Group mean

Monolayer fibroblasts Monolayer fibroblasts Monolayer fibroblasts Suspension culture, lymphoblasts

cells and similar groups with approximately 109, 108 and 107 tissue culture infectious units of FeLV purified virus of sub-groups A, B and C to compare the immunogenicity of cells and virus. All of the groups given cells responded with high antibody titres but none of the virus inoculated animals which had no preinoculation titre did so. Those which had a pre-inoculation titre showed a typical secondary response, indicating that the virus was immunogenic. It is a well known phenomenon that leukaemia viruses of the mouse and cat

be immunodepressant to other antisuch as sheep erythrocytes, bacteriophage or bacterial polysaccharides. One mechanism which has not been widely investigated is the effect of these viruses in depressing the immune system of the host to the virus or viral associated antigens in order to allow the establishment of a chronic viral infection and ultimately the survival of a clone of leukaemic cells which carry viral or virus associated antigens on their surfaces, i.e., directly allow the development of leukaemia. Our results raise interesting speculation both as to the protective immune reactions and to the mechanism of the pathogenesis of leukaemia. We are investigating these in detail but at the moment the possibility can be envisaged that (1) leukaemia virus infects an animal and multiplies first in cells of the bone marrow; (2) it produces new antigens on the infected cell surface; (3) the virus causes immunodepression of the humoral antibody response so that may gens

Peak titre

titre 128

(months)

Virus

3

+

128

3

+

128

1

-

128

1

-

neither membrane antigen directed nor virus neutralizing antibodies are produced; (4) this allows virus to grow in the haemopoietic cells and to spread via the blood infecting other, e.g., epithelial tissues possibly insusceptible to cytotoxic antibody and hence allowing virus to continue being replicated; (5) neonatal infection of kittens leads to viral thymectomy between 1 and 3 months after infection and this is associated with a deficiency in cell mediated immunity; (6) at some later point the definitive leukaemogenic effect or target cell strike takes place leading to to leukaemia. This concept satisfies the present evidence better than the earlier one of tolerance to viral associated or viral antigens by foetal exposure to vertically transmitted virus. We have never found any evidence of tolerance to these membrane antigens, every cat treated in the above fashion responding immunologically. We have carried out experiments using inactivation procedures on both virus and infected cells; antibody titres have been induced with these experimental vaccines and no detectable infection has resulted. We have also demonstrated that such regimens can give rise to a marked and sustained secondary response (Jarrett et al., 1974). The importance of this finding in relation to possible vaccination has been shown by epidemiological studies. In the Glasgow urban area, between 10% and 40°% of normal cats have antibody titres to FeLV whereas the leukaemia incidence is only 0.05% (Jarrett et al., 1973a).

150

W. F. H. JARRETT

In the adjacent rural area, 6% of normal cats have antibody. Infection has been conclusively proved to be accomplished horizontally from cat to cat (Jarrett et al., 1973b), due to large amounts of virus being produced in oral and respiratory epithelial cells. This accords with previous epidemiological studies (Hardy et al., 1973). When cats carrying naturally induced antibodies are experimentally vaccinated or infected a secondary response occurs. Thus, there is now very encouraging evidence that the FeLV system is an excellent model for exploring prophylaxis and immunotherapy in leukaemia. Two other major factors must, however, be kept in mind: these are vertical transmission of virus and immunosuppression due to virus infection, both of which, at least theoretically, present difficulties in attempts to vaccinate and treat. It is known that oncornaviruses become integrated into the host cell genome. This is mediated by the transcription of viral RNA to DNA by the RNA dependent DNA polymerase system. The DNA is then integrated into the host cell genome. It may be derepressed, giving rise to productive virus infection, or it may remain repressed. When the latter occurs in germ cells, all cells of the foetus may be infected with provirus DNA. It has recently been suggested that many cat cells may contain integrated oncornavirus genome (series of papers in Nature, 11 July 1973). The surprising fact has emerged, however, that these oncornaviruses are different from the conventional leukaemogenic viruses already known in the cat, even to the extent of having different gsa's. Induced viruses of this type are not infective for cat cells but can infect human cells. They are thus a different group of viruses with different antigenic structure and host range. As a somewhat similar situation has been found in the fowl, it would appear possible that these vertically transmitted viruses are stable in an evolutionary sense. In the cat, they have not yet been shown to be young

leukaemogenic. There is therefore no evidence of vertical transmission of actual disease inducing virus in the cat and no evidence of immunological tolerance to the true leukaemogenic virus antigens. All normal cats which we have tested have been responsive to antibody induction by experimental vaccination procedures. We have also shown that cats infected neonatally with large dQses of FeLV become " virus thymectomized " and are These animals immunosuppressed. usually die from a variety of normally trivial respiratory and other infections. We have never seen destruction of the thymus in artificially immunized animals. It has not yet been proved in all cases whether the low titres or absence of antibody in cats with frank leukaemia is due to early immunosuppression of both humoral and cell mediated immunity allowing the disease to develop, or whether it is due to mopping up of antibody by tumour cells containing large amounts of antigen on their surface. It can be stated, however, that we have never seen leukaemia develop in animals which we have studied serially in time and which had a moderate antibody titre, and we have failed to demonstrate autologous antibody on the membranes of virus producing tumour cells when the serum of the animal did not contain demonstrable antibody. Cell mediated immunity and humoral antibody operate both individually and together in a variety of virus systems (Allison, 1973). It seems likely, from our evidence, that both systems are involved in the immunology and pathogenesis of leukaemia virus infection. It may be that virus neutralizing antibody is active against dissemination of the virus by blood and tissue fluids, FOCMA antibody against haemic or lymphoid cells bearing virus associated membrane antigens (cytotoxic effect) and CMI against the many other virus producing cells which we now know to be responsible for long-term virus replication and horizontal spread of the disease.

IMMUNE RESPONSE TO PATHOGENESIS, VACCINATION AND EPIDEMIOLOGY 151

This brief summary of current work emphasizes the value of the cat system as a model for the immunological manipulation of leukaemia. REFERENCE ALLISON, A. C. (1973) Cell Mediated Immunity. Proc. R. Soc. Med., 66, 1151. ESSEX, M., KLEIN, G., SNYDER, S. & HARROLD, J. (1971) Antibody to Feline Oncornavirus-associated Cell Membrane Antigen in Neonatal Cats. Int. J. Cancer, 8, 384. HARDY, W., OLD, L., HESS, P., ESSEX, M. & COTTER, S. (1973) Horizontal Transmission of Feline Leukaemia Virus. Nature, Lond., 244, 266.

JARRETT, W. F. H. (1972) Feline Leukaemia. J. clin. Path., 25, Suppl. (R. Coll. Path.), 6, 43. JARRETT, W. F. H., ESSEX, M., MACKEY, L. J., JARRETT, 0. & LAIRD, H. M. (1973a) Antibodies in Normal and Leukemic Cats to Feline Oncornavirus-associated Cell Membrane Antigens. J. natn. Cancer Inst., 51, 261. JARRETT, W. F. H., JARRETT, O., MACKEY, L. J., LAIRD, H. M., HARDY, W. & ESSEX, M. (1973b) Horizontal Transmission of Leukemia Virus and Leukemia in the Cat. J. natn. Cancer Inst., 51, 833. JARRETT, W. F. H., MACKEY, L. J., JARRETT, O.,

LAIRD, H. M. & HOOD, C. (1974) Antibody Response and Virus Survival in Cats Vaccinated against Feline Leukaemia. Nature, Lond., 248,

230. SARMA, P. S. & LOG, T. (1971) Viral Interference in Feline Leukaemia-Sarcoma Complex. Virology, 44, 352.