NO742557L - - Google Patents

Description

Vaccine against Marek's disease.

The invention relates to vaccines for immunizing poultry against Marek's disease (leukosis type II), and in particular vaccines that are immunogenic and non-pathogenic for Marek's disease, methods for producing such vaccines, and methods for immunizing poultry with the vaccines.

Neoplasia is one of the most common diseases in poultry.

Most poultry tumor types have their histogenesis in the "blood system" and are classified as poultry leukosis complex. At least two different virus types, namely an RNA virus (also called RIF virus) and a DNA herpes virus ( among which JM virus was first isolated from nons as a prototype), can cause leukotic tumors. The etiological separation and identification of these diseases has led to the term lymphoid leukocyte or type I lymphoid leukocyte for RNA virus tumors and Mrek's disease or type II lymphoid leukocyte for DNA virus diseases.

Most natural tumors in poultry are of the Marek's disease type. Currently, it is estimated that the losses due to poultry tumors are approx. 200 million dollars per to ride

U.S.A.

Marek's disease infections in poultry flocks are both common and widespread. Almost all outdoor flocks are infected to some extent. Most children are infected at a young age and are chronically infected. Although the infection rate is up to 100% in most cases, neoplastitis as a result of such infection can only be found to a very small extent in some flocks and up to over 50% in other flocks.

Much research has been done to find treatments and preventive measures for Marek's disease, but until recently no satisfactory vaccine was available.

U.S. patent no. 3,642,574 describes a vaccine made from turkey herpes virus (HVT) which is non-pathogenic to chickens and which, via mechanisms not yet known, protects vaccinated chickens against the development of tumors from Marek's disease.

The vaccine described in the U.S. patent No. 3,642,574 immediately gained wide application as soon as it became available to poultry farmers, because of the severity of the disease and because until then no alternative treatment existed.

However, the HVT vaccine has recently been shown to be questionable as a satisfactory solution to the problem of Marek's disease since its tumor-preventing effect in many cases appears to be less than originally indicated. This may be because the HVT vaccine does not produce any significant level of protective or neutralizing antibodies against infections. Inoculation of hbns with HVT vaccine has been found to result in chronic viremia, as opposed to immunity that suppresses or eliminates the infection. Furthermore, hbns vaccinated with HVT vaccine are not immune to a very strong virus infection of Marek's disease and the protection provided by the HVT vaccine is more "cosmetic" than real. Furthermore, original statements that HVT did not grow in mammalian cells have now been shown to be uncertain.

U.S. patent no. 3,590,128 describes a vaccine for lymphoid leukocyte type I, as distinct from a vaccine against Marek's disease.

U.S. patent no. 3,674,861 describes a diluted vaccine against Marek's disease produced by serial transfers of virus in poultry.

It will be clear from this that there is an enormous need for an effective vaccine against Marek's disease and especially for such vaccines that will prevent or eliminate infection in vaccinated nons.

According to the invention, there is thus provided a vaccine which, when properly administered, is immunogenic and non-pathogenic for Marek's disease in poultry, and particularly hbns.

A virulent strain of the virus that causes Marek's disease, identified as the JM-V strain, was developed by rapid transfer of JM virus in hons over several hundred transfers. This JM-V strain is extremely pathogenic to hbns and the LD^Q dose for most genetic species hbner is 10^ per ml when the virus is given parenterally as a blood inoculum, as injected birds undergo a lymph tumor growth in the lbpet of 3 - 6 days after the inoculation.

It was found that day-old chicks given a large dose of JM-V virus usually succumbed. However, when given a smaller dose of JM-V virus, some chickens survived and developed from 1,000 - 10,000 neutralizing antibodies. These surviving chickens with high levels of antibodies were relatively immune to subsequent exposure to JM virus, but use of the JM-V strain is not believed to be a viable immunization method due to the extremely pathogenic nature of the JM-V strain.

To be satisfactory, a vaccine must first of all be safe for the vaccinated bird. It should also induce high levels of neutralizing antibodies so that the viremia resulting from vaccination is temporary so that the vaccinated bird can be virus-free when it is slaughtered and used for food.

The vaccine according to the present invention fulfills these criteria.

A sample of said virulent strain of virus for Marek's disease, identified as the JM-V strain, has been submitted

as a culture of this living organism with accession number VR-736 to the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. The organism will hereinafter be called strain or preparation no. VR-736. As previously mentioned, this organism is immunogenic but extremely pathogenic to poultry.

Preparation VR-736 grows well in chicken embryos. A method for changing the preparation or the strain according to the present invention so that it becomes non-pathogenic for hens and chickens consists in serial transfer through the yolk sac in the chicken embryo. You start with blood from a brooding chicken that has been previously inoculated with VR-736, approx. 0.1 ml of this blood is introduced into the yolk sac of a poultry embryo. 4-6 days old. About. 7 days after inoculation, 0.1 ml of the yolk from the infected fetus is taken out and re-introduced serially into a healthy 4-6 day old fetus. The procedure is repeated over approximately 50 transfers. The material that is coughed up at a relatively advanced infected stage is strongly antigenic and produces protective antibodies in chickens against the virus that causes Marek's disease (the JM strain), and will protect chickens against infections in the field.

Coughed material from later transfer stages can be stabilized with SPGA or other suitable stabilizer media and frozen in liquid nitrogen until shortly before use, and then diluted with a pharmaceutical carrier medium to the desired strength. The coughed-up material can optionally be frozen and used in a known manner.

Embedded material containing modified virus

from VR-736, frozen or lyophilized as described above, may be diluted with a pharmaceutically suitable carrier, e.g. phosphate buffered saline or tryptose phosphate medium which may contain "Arlacel A", "Bayal F", Freund's medium etc, for it is given to poultry. The diluted preparation can be injected parenterally or otherwise administered to chickens from one day old up to adulthood, depending on the results desired and the flock in question. As will be described in more detail later, adult broilers can be actively immunized with up-

the vaccine of the invention and these will congenitally transfer protective antibodies to the offspring and in this way one avoids vaccinating the hatched chickens individually when these are to be raised into broilers which are slaughtered at approximately 7 weeks of age. The congenital antibodies are only effective for 3 - 5 weeks, but the short time between the loss of congenital immunity and the time of slaughter is not long enough for significant tumors to form.

The idea of the invention consists in changing the highly pathogenic strain No. VR-736 into a non-pathogenic one. but still powerfully immunogenic strain or preparation by serial transfer through e.g. the yolk sac of the poultry embryo.

It is an object of the invention to provide

an effective vaccine for Marek's disease in poultry.

It is also a purpose of the invention to provide such a vaccine which is safe (non-pathogenic) and which provides virus-free vaccinated animals.

That these and other purposes and advantages of the invention are achieved according to the invention will be apparent from the following detailed description and preferred embodiments, in connection with the attached drawings where: Fig. 1 - 4 show curves of the antibody response at

use of the vaccine according to the invention.

JM-V preparation No. VR-736 was prepared by rapid transfer of virus causing JM Marek's disease via day-old chicks. The organic tissues selected for the first of these serial transfers were early tumors from the liver, spleen, kidneys, gonads and blood. These organs were chosen because it is in these organs in hers that tumor antibodies are produced. Blood was used after a number of initial transfusions.

The resulting JM-V strain No. VR-736 is highly pathogenic to hens and chickens as can be seen from the following Table I where one can see the steadily decreasing period that passes for inoculated chickens with increasing transmission numbers, for which the chickens die. It is seen that after 400 transfers all inoculated chickens are dead after 5 days, which shows the extremely lethal nature of preparation No. VR-736.

However, it was found that hons who were given a non-lethal but infectious dose of Preparation No. VR-736 and survived produced neutralizing antibodies against the Marek's disease virus. Although it was thus shown that the virulent preparation VR-736 elicited a high level of neutralizing antibodies, it was also clear that a modification of the virulent preparation was needed to produce a safe vaccine.

It was also found that a cell-free product prepared by sonicating blood infected with JM-V, followed by centrifugation and filtration through a "Millipore" filter 0.45/U would produce neutralizing antibodies in poultry. Cell-free material produced in this way provided protection in surviving birds against subsequent JM attacks, but a significant percentage of birds inoculated with this cell-free preparation died after inoculation so that cell-free JM-V was not considered useful as a vaccine against bMarek's disease. It was thus again clear that in order to produce a satisfactory vaccine one had to find some method to reduce the pathogenic nature of the JM-V preparation.

Preparation No. VR-736 (0.1 ml whole blood from moribund chicken) was injected into the yolk sacs of 4-7 day old poultry fetuses and then coughed 7 days after inoculation and transferred

again in the bird embryo. After a sufficient number of transfers, it was found that the preparation had been modified to the point where it was no longer pathogenic to chickens. It was now surprisingly found that this modified, non-pathogenic preparation had retained its strong immunogenic character, so that this modified agent is very useful as a vaccine against Marek's disease. Similar results can be obtained if a fibroblast culture is created from a

embryo transfer and the material from the fibroblast culture is transferred again to a culture medium in vitro.

The immediate number of transfers required

to prepare a non-pathogenic reagent derived from VR-736

is not definite as the change from pathogenic to non-pathogenic is both gradual and imprecise. After 10 transfers, however, mortality is considerably reduced and after 40 transfers the preparation appears to be sufficiently modified.

About. 50 transfers are recommended for extra security. The necessary number of transfers is a number that provides a non-pathogenic vaccine as opposed to the pathogenic starting material (VR-736).

There have been negligible losses from Marek's disease among

about. 2,000 chickens vaccinated with modified reagent produced after 50 transfers via hbn embryo and later exposed to virus that caused Marek's disease in untreated animals, while the losses were considerable among hbns vaccinated with HVT vaccine and exposed to virus in the same way.

This is illustrated using the following table II,

where the action of JM and JM-V (Preparation No. VR-736) is shown for separate groups, of day-old chicks, one group was vaccinated by injection of 2,000 plaque-forming units of HVT vaccine and the other group was injected with 10 CEID^q of vaccine according to the present invention which was prepared by 50 passes of preparation No. VR-736 through bird embryos and gave the modified non-pathogenic but highly immunogenic material. The recommended dose for marketed HVT vaccine is 1,000 platelet-forming units.

In the above table, a trial chicken was considered "attacked" if it either died or showed lymphedema-like lesions of the Marek's disease type. CLD, CID and CEID are abbreviations for respectively chick lethal dose, chick infectious dose and chick embryo infectious dose.

The vaccine according to the invention can also be used to vaccinate hatchlings and the birds will transfer congenital immunity to the offspring, which lasts for 3 - 5 weeks on average, and after this period the chickens are immunologically better able to fight field infections with Marek's disease. Since broilers are usually slaughtered approx. At 7 weeks of age, such broilers for slaughter will not have time to develop significant tumor formations if they become infected with Marek's disease after the congenital immunity is gone.

A preferred plan for immunization of poultry flocks where the birds are to be reared beyond a few weeks of age is to vaccinate parenterally with e.g. 100 CEID^q of the vaccine according to the invention and to vaccinate the offspring at about 1 day old to give the animal immunity after the innate immunity is gone. A further vaccination of the offspring approx. 3 weeks old or something later strengthens the immunity.

The following Table III shows the effect of passive (female) antibodies on virus attack of cell-free JM virus on chicken offspring from dams vaccinated with 100 CEID^q of cell-free JM-V material, compared with chickens from nons

vaccinated with 5,000 platelet-forming units of known HVT vaccine,

and compared to chickens from unvaccinated queens. The attack consisted in all. cases of 0.1 ml JM virus (10^'<8>CID^q per ml)

in specified dilution. As can be seen from the table below, cell-free JM-V material was much more effective in providing passive immunity to progeny from vaccinated dams than known HVT vaccine. Furthermore, attempts at reisolation of JM virus showed that the virus level

in chickens from nones vaccinated with cell-free JM-V material var. very little compared to the HVT-vaccinated group. This dramatically illustrates the antibody response that can be achieved with JM-V material. As previously mentioned, however, cell-free JM-V is not completely safe, and even if surviving animals are protected, a certain percentage will die due to Marek's disease by vaccination with unmodified JM-V.

The relationship regarding the present vaccine's safety in relation to unmodified cell-free JM-V material is clearly illustrated by the following Table IV which shows the number of surviving chickens vaccination one day old, followed by a booster vaccination after three weeks with unmodified cell-free JM-V, compared with the number of surviving chickens upon vaccination according to the invention. In each case, 100 CID^Q vaccine was used per chicken for the first vaccination and the second vaccination. The birds that did die did so as a result of the vaccination, which shows the dangers involved in using unmodified material compared to the safety achieved by using embryo-overfort material according to the invention.

In the above table, the numbers indicate the number of survivors/number of vaccinated for each group.

Some of the survivors from both groups of vaccinated animals© (vaccinated with cell-free JM-V and with fetal series-transferred material) as well as a group of animals vaccinated with HVT., were then exposed to 2,000 IA-q JM-V virus 6 weeks old. As can be seen from Table V, both cell-free and embryo-overferted material gave total protection, while HVT gave a clear but not complete protection compared to unvaccinated control animals.

In all cases, the numbers denote survivors/experimental animals.

The immune response (serum neutralizing strength) of susceptible P-line chicks vaccinated one day old and three

..weeks old with 2,000 PFH known HVT vaccine or 100 CEID^q of vaccine according to the invention or naturally field-infected with Marek's disease is shown in fig. 1. As can be seen from fig. 1

the immune response to vaccination according to the invention is much greater than for unvaccinated birds or birds treated with HVT vaccine.

The immune response (serum neutralizing strength) of susceptible chicks of P-line progeny and resistant N-line progeny with high or low content of passive (inherited) antibodies, vaccinated at one day and three weeks old with 0.1 ml embryo material after 18 and 21 transfers, respectively, via chicken embryo starting from blood from a brooding chicken which was previously inoculated with VR-736, is shown in fig. 2. As can be seen, unvaccinated control animals, both N- and P-line chickens, quickly lost most of their initial immunity and after three weeks had very low antibody levels. Fig. 3 illustrates the antibody level for P-line and N-line chickens by indirect fluorescence, which chicken series contained hby concentration or low concentration of passive (working) antibodies, vaccinated one day old and after three weeks with embryo vaccine derived from VR-736 according to the invention. It appears that the antibody concentration is much higher for animals vaccinated with the vaccine of the invention than for untreated birds. Fig. 4 illustrates the degree and duration of active immune response, measured by serum neutralization against JM virus in vitro, on 18-month-old P- and N-line chickens inoculated intramuscularly with 100 CEID,-n vaccine prepared as cell-free

50

JM-V material. The degree of immune response is seen to be much higher

for both P- and N-line chickens after vaccination and the immune response remains as seen over a longer period,

which again illustrates the antibody response to inoculation with JM-V derived material.

The vaccine according to the invention can be processed

in a regular way. As previously mentioned, the coughed-up yolk sac material can be used directly after many transfers starting from VR-736, e.g. by taking out 0.1 ml of material and injecting this into an animal. This material has a strength of around Hr CEID^q per ml.

For use on a larger scale, hosted material is needed

from the egg yolk is preferably stabilized with one of the many known stabilizing compositions. e.g. with an equal volume of stabilizer containing 7% DMSO, 10% fetal bovine serum and the rest medium 199. The temperature of the stabilized extracted material

ale drills are lowered at a rate of 1°C per minute to -60°C and stored in liquid nitrogen. For use, frozen, stabilized

hardened material is quickly thawed in water at room temperature and

dilute with a pharmaceutical diluent such as 1/4 strength tryptose phosphate medium or phosphate buffered saline at pH 7.2. The amount of diluent is typically 100 - 1,000 times the volume of thawed material, depending on the vaccine's strength and method of use.

The vaccine can optionally be used frozen for easier storage

and treatment and the vaccine can be obtained in a cell-free form by crushing the cells through ultrasound treatment, freezing, etc.

The vaccine can be given in many ways, including subcutaneously, intramuscularly or intra-abdominally by injection with approx. 10 -

100 chicken fetus infection doses of 50% (CEID^q), or by other natural routes of infection such as per os, via the respiratory system or the city, in which cases 100 - 1,000 CEID^q may be necessary to achieve effective results. A particularly suitable method consists in making an aerosol spray containing the vaccine in a closed room containing the birds to be

be vaccinated.

A particularly important feature of the invention is that a reagent (VR-736) which is capable of inducing an exceptionally strong antibody response in the host animal is treated until the preparation has lost its pathogenic ability while retaining the ability to induce antibody- response in the host bird, which makes it possible to produce a vaccine that is both safe and effective.

The above detailed description of a preferred embodiment of the invention shall not limit this beyond the attached claims.

Claims (9)

1. Process for the production of an immunogenic and non-pathogenic vaccine for the immunization of poultry against Marek's disease, characterized in that a virulent reagent in the form of ATCC No. VR-736 is transferred to poultry embryos, this fetal material coughs up after several days, serially transfer the coughed material until the reagent proves non-pathogenic to live poultry and recover the material that is immunogenic and non-pathogenic to Marek's disease in poultry. 2. Method as stated in claim 1, characterized in that the coughed material is serially transmitted via poultry embryo. 3. Method as stated in claim 1, characterized in that the coughed material is serially transferred via cell cultures in vitro. 4. Method as specified in claim 2, characterized in that the fetal material is coughed up after approx. 1 week and transferred again to the yolk sac in the poultry embryo at intervals of about a week until a material has been obtained which is non-pathogenic for one-day-old chicks. 5. Method as stated in claim 4, characterized in that the fetal material is coughed up and transferred through at least 40 transfers. 6. Procedure for treating poultry, characterized by giving a vaccine derived from a virus in the form of ATCC No. VR-736, as well as a pharmaceutical carrier medium, to said poultry, in sufficient quantity to form high concentrations of antibodies in said poultry, which prevent the development of Marek's disease, and provide the vaccine to the breeding birds' offspring in an effective amount so that the offspring remain immune to Marek's disease after the expiration of the normal innate immunity. 8. Method as stated in claim 7, characterized in that the vaccine is given parenterally to the farmed birds and their offspring. 9. Method as stated in claim 7, characterized in that the vaccine is given to the offspring as an aerosol spray within a limited area.