Cross-protection between JMV Marek's disease-derived tumour transplant, Marek's disease and turkey herpesviruses

Cross-protection tests were conducted using attenuated JMV (JMV-A) Marek's disease-derived lymphoblasts, glutaraldehyde-treated JMV tumour cells, attenuated Marek's disease virus (MDV, strain HPRS-16/att) and turkey herpesvirus (HVT, strain FC126) as vaccines, and virulent JMV and MDV (HPRS-16) for challenge. The JMV and JMV-A preparations were free of MDV, leukosis and reticuloendotheliosis viruses. Vaccination of chickens with attenuated MDV or with HVT provided good protection against both JMV lymphoblastosis and Marek's disease (MD). In one experiment HVT (cell-free) caused a better resistance to JMV than to MD. Inoculation of JMV-A always resulted in a 100% resistance to virulent JMV. However, JMV-A did not induce any appreciable resistance to MD, even when the birds were challenged with MDV by contact exposure. Control experiments revealed that high doses of normal lymphocytes from uninfected chickens also had a protective effect against JMV. The 50% protective dose varied from 10(7) to 10(8) lymphocytes. JMV tumour cells inactivated by glutaraldehyde were used in different experiments but rarely caused a clear-cut protection against virulent JMV. The results of this study suggested that a one-way relationship exists in vivo between HVT or MDV and JMV lymphoblastic leukaemia. However, resistance induced against JMV tumour cells appeared to be related to histocompatibility antigens at least as much as to tumour-specific cell surface antigens. The results obtained failed to provide clear evidence for or against vaccinal resistance to MDV being dependent on the action of a common Marek's disease tumour-associated surface antigen (MATSA) additional to the immune response to viral antigens.     

Propagation and assay of virulent and attenuated JMV Marek's disease-associated tumour cells

JMV tumour cells were shown to cause a lethal lymphoblastic leukaemia in young chickens as well as in chicken embryos. The incubation period was very short but dose-dependent. Chickens died in 4 to 12 days, embryos in 7 to 14 days, after inoculation. Embryo-passaged attenuated JMV (JMV-A) caused the same lesions in embryos as virulent JMV. The dose-response relationship depended on the route of inoculation and on the quality of the tumour cell preparation. Intramuscular (i.m.) inoculation of leukaemic blood or embryo lymphoblasts provided the most satisfactory response. Intraperitoneal (i.p.) inoculation and lymphoblastic chicken spleens as a source of JMV were definitely less suitable. The dose-response curves obtained in yolk sac-inoculated embryos were similar to the curves obtained by i.m. inoculation of chickens. Only 4 to 10 lymphoblasts were needed per lethal dose (50%) in chickens and 50 to 80 in embryos. The pathogenicity and antigenicity of JMV and JMV-A were strictly cell-associated. No Marek's disease (MD) virus or any other avian virus could be detected, either by various virus isolation procedures, or by serological methods. Contact transmission of JMV to other chickens did not occur. Antibodies against surface antigens on JMV lymphoblasts were detected in JMV and JMV-A chicken hyperimmune sera. These sera reacted against MD lymphoblastoid cell lines (HPRS-1 & 2, MSB-1) as well as MSB-1 anti-serum, but all sera reacted also against thymus lymphocytes from normal chickens. The results of absorption tests suggested that the surface antigens of JMV lymphoblasts and of the tested cell lines were not identical. The majority of tumour cell surface antigens appeared to represent genetically specific histocompatibility or lymphocyte antigens. A common MD tumour-associated surface antigen (MATSA) could not be identified serologically (FA test) on the tumour cells studied.     

Serological characterisation of Marek's disease tumour-associated surface antigens on Marek's disease lymphoma cells and on cell lines derived from Marek's disease lymphomas

Antisera produced in rabbits against Marek's disease lymphoma cells and against four lymphoma-derived cell lines (HPRS lines 1 and 2, MSB-1 and RPL-1) appeared, after extensive absorption with normal chicken cells, to react specifically by indirect immunofluorescent staining with antigens unique to Marek's disease tumour cells. The antigens present on the four cell lines were shown by antiserum titration and absorption tests to be related but not identical. The possibility is discussed that cellular transformation by Marek's disease virus involves the modification of a normal lymphocyte antigen and the modification of different allotypes of this antigen results in related but not identical neoantigens.     

Independence of chicken major histocompatibility antigens and tumor-associated antigen on the surface of herpesvirus-induced lymphoma cells.

Capping of chicken major histocompatibility (MHC) antigens on normal thymus, spleen, and peripheral blood leukocytes was demonstrated, although MHC antigens appeared to be present on only 15 to 18% of normal thymus cells. MHC antigen capping also occurred on cells from a Marek's disease herpesvirus-induced transplantable lymphoma (MDCT-NYM1). Capping of a Marek's disease tumor-associated surface antigen (MATSA) could be induced on MDCT-NYM1 lymphoma cells as well as on cells of two Marek's disease in vitro lymphoblastoid cell lines (MDCC-MSB1 and MDCC-LS1). Cocapping of MHC antigens and MATSA did not occur on MDCT-NYM1 lymphoma cells. The results suggest that MHC antigens and MATSA are not structurally associated on the cell membrane.     

The role of histocompatibility antigens in cell-mediated cytotoxicity against Marek's disease tumour-derived lymphoblastoid cell lines

The cytotoxic activity of spleen cells from Marek's disease (MD) virus-infected chickens against syngeneic and allogeneic tumour cell lines was compared, using MD lymphoma-derived lymphoblastoid cell lines obtained from two inbred and two outbred chicken strains. Activity was significantly greater against allogeneic than against syngeneic target cells, although some activity against syngeneic cells was detected. Cold target cell inhibition tests confirmed that unlabelled normal spleen cells could block the cytolysis of tumour cell targets bearing the same histocompatibility antigens as the spleen cells. It was concluded that the tumour-specific antigens against which the effector cells were reacting may be modified histocompatibility antigens and that the enhanced cytotoxicity seen with allogeneic target and effector cells may be an artefact analogous to an adjuvant effect.     

Potency tests with HVT-, MHV- and JMV vaccines against Marek's disease

Turkey herpesvirus (HVT)(FC 126) vaccine made in W. Germany, Marek's herpesvirus (MHV) (CVI 988) vaccine used in the Netherlands and experimental JMV vaccine were tested in laboratory and field trials for protection against Marek's disease. The tests were carried out with 780 SPF chicks and 3200 commercial white Leghorn chicks (with maternally derived antibodies to HVT and MHV). There were no differences in potency of HVT- and MHV vaccines. Both vaccines showed increased protection with an increased interval between vaccination and challenge. A significant protection (> 80%) resulted with both vaccine viruses after the 8th day following vaccination. JMV vaccine (embryo-adapted strain JMV-A-164) provided a 40 to 60% protection against Marek's disease depending on the time of challenge. However, there was a complete protection against the highly pathogenic JMV tumour cell inoculum. Hyperimmunisation of adult birds with JMV vaccine did not reduce the mortality in their progeny from Marek's disease.     

Differentiation between strains of Marek's disease virus and turkey herpesvirus by immunofluorescence assays

Two serological types of Marek's disease virus and a herpesvirus of turkeys have been differentiated by indirect immunofluorescence tests as (1) pathogenic strains of Marek's disease virus (MDV) and their attenuated variants: HPRS-16, HPRS-16/att, HPRS-B14, JM, JM/att, GA, VC and 'Oldenburg', a recent field isolate; (2) apathogenic strains HPRS-24 and HPRS-27 of MDV; (3) herpesvirus of turkeys strain FC126 and its HVT(A-) variant. Virus strains could not be distinguished on the basis of qualitative differences in immunofluorescent staining of intracellular virus-induced antigens. Results were similar whether chicken kidney, chicken embryo fibroblast or duck embryo fibroblast cell cultures were used. Fluorescence of virus-induced antigens was stronger with homologous than with heterologous antisera. Using the direct immunofluorescence technique Marek's disease virus and turkey herpesvirus infections could be distinguished. There were never any significant differences in the appearance and distribution of antigen in infected cells treated with homologous or heterologous antisera at dilutions of comparable activity using the indirect immunofluorescence technique. Antibody titres of antisera were 4 to 8-fold higher in the indirect immunofluorescence test against the homologous virus-induced antigens than against heterologous antigens. Cross-reactions between the 3 serological types could be prevented by absorption of antisera with the appropriate antigens. Cross-reactions could also be prevented by the appropriate dilution of antisera before use in the indirect immunofluorescence test.     

Protection against the JMV Marek's disease-derived transplantable tumour by Marek's disease virus-specific antigens

Chickens immunised with inactivated chicken kidney cells infected with Marek's disease (MD) virus were protected against the lethal effects of inoculation with the MD-derived tumour transplant JMV, which appears not to express viral antigens. It is concluded that the distinction between MD virus-specific and tumour-specific antigens is not as complete as has been thought.     

Marek's disease in turkeys: the induction of lesions and the establishment of lymphoid cell lines

The inoculation of turkeys with large doses of a virulent strain of Marek's disease virus (GA strain), but not of two other virulent strains (HPRS-16 and JM), was found to induce a disease resembling Marek's disease of the chicken. The most prominent lesions were lymphocytic leukaemia and lymphoid and reticular hyperplasia in the spleen and the liver. These developed after a prolonged latent period and the early histological changes (lymphoid cell destruction and reticuloendothelial cell hyperplasia) reported in chickens were not observed. Twelve cell lines were established from suspensions of spleen cells or of buffy coat cells from infected turkeys. These cells expressed both Marek's disease tumour-associated surface antigen and T-cell antigens. The cells carried the Marek's disease virus genome and when inoculated into chickens induced typical Marek's disease lymphomas. Nine of the cell lines were infected with an avian leukosis virus, but three lines were free of such infection. All cell lines had normal turkey karyotypes.     

Protection against Marek''s disease-derived tumor transplants by the nononcogenic SB-1 strain of Marek''s disease virus.

A series of experiments was conducted to study the in vivo protection against Marek's disease-derived tumor transplants by the nononcogenic SB-1 strain of Marek's disease virus. Intact, embryonally bursectomized (Bx), thymectomized (Tx), or cyclophosphamide (Cy)-treated chickens of four genetic lines were vaccinated with live or inactivated SB-1. JMV, a non-virus-producing transplant, and GA/Tr-1 and MDT-198, two virus-producing transplants were used for challenge. Optimal protection against JMV was present 7 days postvaccination, but there was significant protection even when SB-1 and JMV were administered together. Protection was abolished by an increase in the number of tumor cells used for challenge or by combined Tx and Cy treatment. Inactivated SB-1-infected cells were unable to induce protection against JMV challenge. Protection was also present against challenge with GA/Tr-1, but not against MDT-198, except in vaccinated, Bx chickens. It was concluded that protection against JMV was T-cell dependent and required the induction of neo-antigens not present in an inactivated SB-1 cellular preparation. The absence of protection in intact chickens against MDT-198 could not be explained.     

Local tumours induced in chickens by continuous cell lines of the JMV Marek's disease tumour transplant

Marek's disease (MD) tumour cell lines RPL-1 and JMV-1 were shown to express high oncogenic potential if inoculated intramuscularly. No more than 10(3) cells per dose were needed to cause primary tumours at the site of inoculation in 50% of 2-day-old chickens, but more than JO(6) to more than 10(7) cells were required to cause a 50% mortality due to lymphoblastic leukaemia as another parameter of oncogenicity. Herpesvirus of turkeys (HVT) was nonprotective against local tumour development of the transplants, and resistance to JMV tumours did not coincide with resistance to Marek's disease.     

Precipitating antigens associated with Marek's disease viruses and a herpesvirus of turkeys

Precipitating antigens associated with a number of Marek's disease virus strains and with a turkey herpesvirus have been analyzed. The 'A' antigen has been defined as the major soluble antigen in feather follicles of infected chickens, which is identical with the major antigen usually present in supernatants of chicken kidney cell cultures infected with strains of Marek's disease virus. 'BC' antigens are 2 or more antigens which are usually not noted in skin extracts but present in cultured cells infected with Marek's disease virus or turkey herpesvirus, in addition to the 'A' antigen. Some of the virus strains examined were positive and others negative for 'A' antigen, but all contained the 'BC' antigens. Results of agar-gel precipitin tests suggested a serological classification of the group of avian herpesviruses formed by Marek's disease viruses and turkey herpesvirus into 3 types. Pathogenic strains of Marek's disease virus and their attenuated A- variants, represented by the HPRS-16 strain (HPRS-16, JM, GA, VC, Oldenburg). Apathogenic Marek's disease virus, represented by the HPRS-24 strain. Turkey herpesvirus and its A- variants, represented by the FC126 strain. A serological subdivision corresponding to the different grades of pathogenicity of virus strains of the first type was not possible. Differences between antigens associated with the 3 types of virus were apparent from the antigen and antibody titres against homologous and heterologous reagents. Precipitin bands produced by homologous antigen and antibody were stronger than those produced by heterologous reagents. Differences between 'A' antigens of the 3 virus types were characterized by spur patterns of precipitin bands indicating a partial identity. At least 3 'BC' precipitin bands were noted; at least one was group-specific and one appeared to be type-specific.     

Spontaneous and induced herpesvirus genome expression in Marek''s disease tumor cell lines.

We incubated 31 newly established Marek's disease tumor cell lines at 41 degrees C for 48 h after subculturing and then examined them to determine the spontaneous rates of expression of viral internal antigen(s), viral membrane antigen(s), and virus isolation. All but two of the lines were isolated from tumors induced by clone-purified Marek's disease virus strain JM-10, GA-5, RB-1B, and BC-1A in nine different genetic strains of chickens with defined histocompatibility antigens. The line-to-line variations in the rates of spontaneous expression for the antigens or virus rescue were great, but the levels of expression were very low in most cases. The median rates of expression for viral internal antigen, viral membrane antigen, and virus isolation were 32, 8, and 2 positive cells per 10(5) cells, respectively (ranges, 0 to 20,280, 0 to 22,990, and 0 to 220 positive cells per 10(5) cells, respectively). The ratio of viral internal antigen expression to virus isolation was extremely variable and often high, whereas the ratio of viral internal antigen to viral membrane antigen expression was more consistent and generally low. The virus strain which induced the cell line influenced the level of virus genome expression, but the cell genotype did not. Cell lines transformed by JM-10 virus, which exhibited low oncogenicity, had significantly (p less than 0.01) higher rates of expression than cell lines transformed by CA-5 and RB-1B viruses, which exhibited high oncogenicity. Treatment with iododeoxyuridine or incubation at 37 degrees C induced increased rates of expression in most lines but not in all lines. The degree of enhanced expression was inversely proportional to the rate of spontaneous expression.     

Comparison of SV40 induced antigens on the surface of cultivated cells by a cytolytic microassay

By means of a direct cytolytic assay, SV40 specific antisera were shown to kill SV40 T antigen positive AL/N mouse cell lines, including lines which were repeatedly passed through the syngeneic mouse as tumors. The sera also killed a polyoma transformed SV40 T antigen negative AL/N cell line, but not untransformed or spontaneously transformed AL/N embryo cell lines. With the antisera tested there was also no lysis observed on SV40 T antigen positive Balb/c mouse, hamster or human cell lines, although by use of a competition assay SV40 specific common antigens were detectable on the surface of these cells.     

Stimulation of local solid tumour development of the nonproducer Marek's disease tumour transplant JMV by virus-induced immunosuppression

Chickens could be protected against lethal lymphoblastic leukaemia due to the nonproducer JMV Marek's disease (MD) tumour transplant by infection with the herpesvirus of turkeys (HVT) or various strains of MD virus. However, solid JMV tumours developed in MD virus-infected birds at the site of intramuscular or subcutaneous transplantation, but tumours never developed at the site of MD virus inoculation. The incidence and extent of local tumour growth, the development of metastases and the inhibition of tumour regression were related to the pathogenicity of the MD virus strains used for pre-treatment of the chickens. Infection of chickens with reticulo-endotheliosis virus (REV-C) or with chick syncytial virus (CSV), which are nonprotective against MD virus or JMV transplants, stimulated local tumour development of the attenuated JMV-A variant of the JMV transplant. Chickens which did not reject local tumours died of visceral JMV tumour metastases. A direct helper mechanism of viral infection on the oncogenicity of transplants was excluded. The results suggested that virus-induced immunosuppression stimulated the development of local JMV tumours which never occurred in normal chickens. Immunity to the JMV transplant, including resistance to lethal leukaemia and successful regression of local tumours, did not coincide with immunity to MD virus-induced visceral lymphomas or nerve lesions. Vaccinal induced tumour immunity evidently was defective. The significance of these results is discussed with reference to immunological functions of MD tumour-specific antigens.     

Expression of H-2 Antigens Durin Growth of Cultured Tumor Cells

H-2 antigens cell surface expression has been investigated in murine cultured tumor cells L1210 at different stages of their growth. H-2 antigens are maximally expressed during the mid-log phase of cell growth as judged by the sensitivity of cells to antibody mediated lysis in the complement dependent cytotoxic test and by the absorbing capacity for H-2 antisera. The yields of H-2 antigens solubilized from cells at different stages of growth correlated consistently with the cell surface expression of these antigens.

Mouse histocompatibility (H-2) antigens are genetically determined cell surface markers which are expressed on most murine cells and tissues. Since Medawar's (1) discovery that allograft survival could be prolonged by non-particulate murine transplantation antigens, it has been attempted to solubilize these antigens from the surface of cell membranes. A variety of methods have been applied and considerable efforts were made to characterize the biological as well as chemical natures of both human and murine histocompatibility antigens (2,3,4). Cultured lymphoid cells have been extensively utilized for the solubilization of these antigens, since such cells provide a genetically uniform source and are readily available in large amounts (5,6,7).

While determining optimal extraction conditions for H-2 antigens from cultured murine tumor cells L1210, we observed great variability in yields of antigen obtainable from batches of cells harvested during different stages of the cell population curve. We had previously found the greatest yields of soluble HL-A antigens in the log growth phase of cultured human lymphoid cells derived from donors free of malignancy (8). Moreover, antigenic expression on these cells was essentially unchanged throughout the cell growth (9) in contradistinction to the reported finding of variable expression of H-2 antigens during the growth of murine tumor cells induced by viruses (10,11,12). These observations prompted us to determine whether yields of soluble H-2 antigens obtained during different growth phases correlate with the degree of expression of these antigenic determinants on the surface of cultured murine tumor cells.     

Expression of H-2 Antigens Durin Growth of Cultured Tumor Cells

H-2 antigens cell surface expression has been investigated in murine cultured tumor cells L1210 at different stages of their growth. H-2 antigens are maximally expressed during the mid-log phase of cell growth as judged by the sensitivity of cells to antibody mediated lysis in the complement dependent cytotoxic test and by the absorbing capacity for H-2 antisera. The yields of H-2 antigens solubilized from cells at different stages of growth correlated consistently with the cell surface expression of these antigens.

Mouse histocompatibility (H-2) antigens are genetically determined cell surface markers which are expressed on most murine cells and tissues. Since Medawar's (1) discovery that allograft survival could be prolonged by non-particulate murine transplantation antigens, it has been attempted to solubilize these antigens from the surface of cell membranes. A variety of methods have been applied and considerable efforts were made to characterize the biological as well as chemical natures of both human and murine histocompatibility antigens (2,3,4). Cultured lymphoid cells have been extensively utilized for the solubilization of these antigens, since such cells provide a genetically uniform source and are readily available in large amounts (5,6,7).

While determining optimal extraction conditions for H-2 antigens from cultured murine tumor cells L1210, we observed great variability in yields of antigen obtainable from batches of cells harvested during different stages of the cell population curve. We had previously found the greatest yields of soluble HL-A antigens in the log growth phase of cultured human lymphoid cells derived from donors free of malignancy (8). Moreover, antigenic expression on these cells was essentially unchanged throughout the cell growth (9) in contradistinction to the reported finding of variable expression of H-2 antigens during the growth of murine tumor cells induced by viruses (10,11,12). These observations prompted us to determine whether yields of soluble H-2 antigens obtained during different growth phases correlate with the degree of expression of these antigenic determinants on the surface of cultured murine tumor cells.     

Preferential protection against marek's disease pathogenesis by immunisation with syngeneic virus-nonproducer lymphoblastoid cells

The effect of immunisation with virus non-producer Marek's disease (MD) lymphoma-derived lymphoblastoid cells on virus replication and lymphoid organ pathogenesis following MD virus challenge was studied with chickens from two related inbred lines and their F(1 )offspring. Protection against peripheral blood lymphocyte-associated viraemia as well as MD virus-caused weight changes of the spleen and bursa of Fabricius and degenerative bursal lesions was significantly greater among syngeneic and semi-syngeneic (F(1)) recipients than among allogeneic recipients of the lymphoblastoid cells. The results suggest that the protective immunity observed may be major histocompatibility complex (MHC) antigen-restricted and that non-infectious MD virus-transformed cells possess antigens that can induce anti-viral immunity.     

Antisera to human cytomegalovirus produced in hamsters: reactivity in radioimmunoassay and other antibody assay systems.

Hamsters immunized with human cytomegalovirus (CMV) concentrated and purified by polyethylene glycol precipitation and density gradient centrifugation produced antisera with high titers of specific viral antibody, and which showed no significant reactivity with human host cell components. The antisera had high titers of CMV antibody in complement fixation, indirect fluorescent-antibody (FA), and neutralization tests, but titers obtained by indirect radioimmunoassay (RIA) were markedly higher. The antisera were used to follow the development of CMV antigen in infected host cells by indirect RIA and indirect FA staining. Virus-specific antigen was first detectable by RIA at 8 h after infection, and by FA staining at 16 h; cells contained optimal amounts of antigen for RIA and FA assays at 72 to 100 h postinfection. Immune globulins from the antisera were labeled with 125I for use in direct RIA. The labeled globulins gave highly specific reactions with CMV-infected cells, including those infected with low-passage isolates, and showed no reactivity with cells infected with other human herpesviruses or certain other human viruses.     

Replication of Marek's Disease Virus in Cell Cultures Derived from Genetically Resistant Chickens

Various parameters of replication of Marek's disease virus (MDV) were studied in cells derived from genetically resistant (line 6) and susceptible (line 7) chickens. Cell-free and cell-associated preparations of three strains of pathogenic MDV (JM, GA, and Id-1) replicated equally well in cells of resistant and susceptible chickens. There were no demonstrable differences between the two genetic sources of cells for the chronological appearance and type of cytopathic effects and for the type of virus-induced antigens detectable by immunofluorescent and agar-gel precipitin tests. MDV propagated for about 4 weeks in cells of resistant line 6 chickens remained nonvirulent for line 6 and fully virulent for line 7 chickens. The lack of expression of genetic resistance to Marek's disease at cellular level contrasts the infection mechanisms of MDV from those of viruses associated with lymphoid leukosis in chickens.