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1. (WO1990006131) VACCIN VIVANT CONTRE LA GRIPPE AVIAIRE
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A LIVE VACCINE AGAINST NEWCASTLE DISEASE

The subject of the invention is a live virus vaccine against Newcastle disease and a process for preparing same.
For the prevention of Newcastle disease (ND) two avirulent Newcastle disease virus (NDV) strains are used as vaccine all over the world: strain LaSota of American origin and strains B-1 (lentogenic strains). These strains became widely used in the nineteen-fifties. Their use for vaccine production is simple and inexpensive: the strains can be grown in embryonated hen's eggs. The vaccine is easy to administer: in the drinking water or in virus aerosols large masses of chickens of almost any age can be immunized. Both virus strains induce immunity in two weeks. The birds remain protected against infection for about 1 to 3 months (depending on their age) after the first vaccination. Under intensive, large-scale conditions a drawback of strain B-1 is that it produces weak immunity. In the first few days or weeks after vaccination with strain LaSota the chickens often exhibit respiratory symptoms (vaccination reaction). The high prevalence of infectious bronchitis virus further increases the risk of "vaccination reactions". Vaccination may activate other latent infections (e.g. mycoplasma infection) which may aggravate the disease. The vaccine virus itself keeps circulating in the chichken flock for one or two weeks (Erdei et al., Magyar Allatorvosok Lapja, 1988) which is also an undesirable phenomenon. A further disadvantage of vaccination with the above strains is that immunization at 2 or 3 weeks of age depresses the chickens' growth rate for days: under large-scale farming conditions this can give rise to substantial losses.
The purpose of this invention is, besides eliminating the above-mentioned disadvantages, to develop a new type of live vaccine against ND.
This task has been solved by developing a vaccine using a lentogenic NDV strain, namely strain NDV-6/10.
Accordingly, the subject of the invention is a live virus vaccine against Newcastle disease containing strain NDV-6/10.
Another subject of the invention is a process for producing a live virus vaccine against ND by inoculating embryonated hen's eggs with strain NDV-6/10, incubating the eggs, then, after reaching the required virus titre, killing the embryos and collecting the allantoic fluid.
The best mode of the procedure is to incubate
after
embryonated hen's eggs for about 9 days and ,/ inoculating with strain NDV-6/10, to incubate at 35 to 40 °C (preferably at 37 °C) for 60 to 110 hours (preferably for 72 to 96 hours) until the required virus titre is reached. The required virus titre is at least 108 determined by titra tion in embryonated eggs and by the haemagglutination-inhibiton (HI) test. After the required virus titre has been reached, the embryos in the incubated eggs are killed (preferably by cooling), the allantoic fluid is collected and, after testing it for bacteriological sterility, formulated as a vaccine.
Strain NDV-6/10, which is the starting material of the live vaccine according to the invention, was deposited in the National Collection of Microorganisms (Budapest) under the accession number 001075 on December 1. 1988, furthermore, in Collection of Animal Pathogenic Microorganisms (Brno, Czechoslovakia) under the accession number on 1989.
Strain NDV-6/10 has been found suitable for producing a live vaccine on the basis of the following properties:
1. It has been selected from numerous avirulent field isolates on the basis of its much milder cytopathogenic effects in chick embryo kidney cell cultures (in vitro) as compared with the common vaccine strains (LaSote and B-1). While the common vaccine strains induce more pronounced syncytium formation, strain NDV-6/10 mainly causes only rounding off the cells. This is recognized as a mild cytopathogenic effect in case of NDV strains.
2. The strain maintains its CPE, i.e. induction of rounding-off, as a stable property. Such a special virus was selected from the population by plaque isolation.
3. Strain NDV-6/10 meets the requirements specified for avirulent (lentogenic) NDV strains (Exa-minations of poultry biologies, 1985).
a) a dose of about 100 EID50 (50 % of embryo infective dose), when inoculated into the allantoic cavity, does not kill the
10 day old chicken embryos within 5 days; b) one day old chicks (at least 10 birds per experiment) inoculated intracerebrally with a virus dose of 10 EID50 remain completely healthy and develop normally.

4. Chickens of any age, inclusing newly hatched ones, tolerate even a 100-fold immunizing dose administered by any route (virus aerosol, eye-drop or nasal drop) without developing clinical signs.
5. The immunizing dose of NDV-6/10 administered in the form of virus aerosol is 107.3 EID50/m3.

From economical view-point this is not inferior to the common vaccine strains.
6. A favourable property of NDV-6/10 is its pronounced heat stability. Thus, the virus dose intended for administering to chickens does not decrease during vaccination (via the drinking water or virus aerosol). As compared with the common vaccines, less virus is lost during virus propagation, lyophilization and storage.
7. An advantage of strain NDV-6/10 is that lower serum HI titres are accompanied by at least as strong protection (immunity) as that produced by the common vaccines. The advantage of lower serum HI titres is that it is easier to distinguish birds (flocks) which were only vaccinated from those which were vaccinated and subsequently infected by wild virus but did not fall ill: the basis of differentiation is that the latter have rapidly rising HI titres.
8. Strain NDV-6/10 induces moderate respiratory response. The risk of eliciting respiratory reaction has been reduced by partly adapting the strain to intestinal epithelial cells. SPF chickens were infected with 0.2 ml virus suspension (titre: 106) orally. Four days later, from the cloaca the virus was reisolated and grown in embryonated eggs, and the obtained virus suspension was again inoculated into chicks. This passage was repeated ten times.
9. Using highly specific immunαlogical reagents (monoclonal antibodies) and by a highly sensitive immunological test (ELISA), strain NDV-6/10 can be differentiated from all other avirulent (lentogenic) NDV strains known from the literature, including the common vaccine strains (LaSota and B-l). Moreover, strain NDV-6/10 can be identified individually. By this means it can be easy determined whether an avirulent virus strain isolated from any respiratory condition is identical or not with the vaccine strain NDV-6/10.
The possibility of individual identification can be considered as a basic requirement of modern vaccine. Strain NDV-6/10 is the first Newcastle disease vaccine strain which meets this requirement.
Owing to its above-listed properties, strain NDV-6/10 can be used for immunization against ND in several different forms.
The vaccine according to the invention can be administered in virus aerosol, in the drinking water, or dropped on the conjunctiva (eye-drop) or intranasally (nose-drop).
First immunization can be carried out on chicken flocks at day to few weeks of age. Lasting protection of growing and adult birds can be ensured by repeated immunization. Protection can be substantially prolonged by repeated immunization. Thus,
the chicken flock can be protected against ND through its whole life-time, by immunizations with strain. NDV-6/10 alone.
The live vaccine of the invention is suitable for use in combinations, too. Young and growing flocks can be immunized with the NDV-6/10 vaccine even if they are intended to be immunized with an inactivated or, possibly, combined vaccine before season.
the leaying Namely, the NDV-6/10 vaccine ensures good booester effect of the inactivated vaccine.
The vaccine of the invention ensures a suit able protection even if the flocks are immunized also with other vaccines (e.g. against Marek's disease, infectious bronchitis, infectious bursal disease) either simultaneously or within a short period of time.
Newcastle disease virus is a good inducer of interferon. Strain NDV-6/10 can also be used for induction of interferon production or for eliciting a similar effect, the more so since this strains is completely harmless.

Immunization trials
A. Immunization of broilers
First immunization of birds at day to few weeks of age with strain NDV-6/10 is especially recommended as it does not cause any vaccination reaction. Growth of the live vaccine virus strain in the organism is a precondition of the development of immunity. However, this growth is inhibited by yolk-derived maternal antibodies, especially if their level is high at the time of vaccination. This inhibitory effect can be diminished in the following ways:
a) If the first immunization is carried out at 2-3 weeks of age, when the level of maternal antibodies is already lower, the above growth-inhibiting effect will be less pronounced. However, the susceptibility of chickens to natural infection tends to in crease with the age and, if natural infection does not occur, a severe outbreak may emerge.
b) If the virus is administered by dropping it onto the conjunctiva or nasal mucosa, at the point of entry the maternal antibodies will not markedly inhibit the growth of virus. This method of immunization is. however, rather labdursome and time consuming and, thjs, is difficult to use in large hatcheries.
c) Most of the veccine virus contained in the spray of large particle size (droplet) (30 to

100 nm gets also onto the conjunctive or nasal mucosa, and this is very favourable. A certain part of the viral carticles is inhalatec by the birds and, this, depending on the virulence of the vaccine strain, may give rise to vaccination reactions. This needs not be reckoned with after vaccination with strain NDV-6/13.
d) 0.1 to 0.2 ml of inactivated vaccine injected simultaneously wiτh NDV-6/10 administered by eye-drop or intranasally can prolong immunity; however, the procedure is very laboursome.
e) Vaporization of the fluid containing the vaccine virus to fine croplets ( < 10 nm) is the most favourable in terms of immunity induced, as with the aerosol the virus gets not only onto the conjunctiva and nasal mucosa but also into the trachea and lungs. The common vaccine strains, when given in this form to young birds, may give rise to a severe vaccination reaction. One of the greatest advantages of strain NDV-6/10 is that it can be administered either as spray or as aerosol to birds younger than 2-3 weeks (even in the hatchhery).
The above statements have been concluded by evaluating the results of extensive experiments. In the first experiments we found that day-old chicks having high maternal antibody level immunized with strain NDV-6/10 in the drinking water or as virus aerosol at 21, then at 35 or 49 days of age, showed a similar immunity against challenge performed 2 weeks after the second immunization as chicks immunized with strain LaSota by a similar way.
In later experiments strain NDV-6/10 was used as a spray already at day of age. Chicks immunized in this way did not show any vaccination reactions but showed a steadier body mass gain than chicks immunized with Phylavac or Clon 30 (that is train LaSota) at 21 days of age.
In repeated comparative experiments we studied whether the immunity induced by strain NDV-6/10 alone administered as eye-drop at day of age was different from that induced by NDV-6/10 eye-drop plus 0.1 to 0.2 ml inactivated vaccine administered simultaneously. The same experiment was performed with groups of chicks vaccinated with NDV-6/10 in spray formulation. Chickens of all groups survived challenge at 4 weeks of age. After challenge at 6-7 weeks of age, only 1 of 60 chickens immunized with NDV-6/10 as eye-drop or spray plus inactivated vaccine died (98 % protection). In the spray-vaccinated and challenged group (n = 18) 77 % protection, while in the group (n = 17) challanged after vaccination with NDV-6/10 as eye-drop 88 % protection was observed.
After having ascertained the innocuity of strain NDV-6/10 administered in the drinking water, as eye-drop or spray, we started to test the vaccine in aerosol form, using the MASTERDROP (MD-2500) generator patented by Geza Tamasi and his co-workers and an also patended aerosol stabilizing agent (DROY'S DROPS). This apparatus generates particles smaller than 7 nm which float in the air for 25 - 30 minutes. A virus dose of 107.3/m3 is inhaled by chickens present in the given air for 30 minutes.
In the first experiment 45 chickens aged 47 days and free from NDV antibodies were immunized with an aerosol containing only NDV-6/10 virus. Another group of 45 chickens was exposed to an aerosol containing both NDV-6/10 and Bronchovac I virus. Three weeks after vaccination the serum of chickens exposed only to NDV-6/10 virus contained ND antibodies in a geometric mean titre (GMT) of 1:27.8, whereas the geometric mean titre of ND antibodies was 1:33.8 in the group exposed to both viruses. After challange with virulent NDV on day 40 after vaccination only 1 of the 90 birds died (99 % protection); even that one belonged to the group vaccinated against NDV alone.
Encouraged by these favourable results, we started vaccination trials at large-scale farms in chickens between 1 and 10 days of age. So far these trials have included almost 80,000 broilers. Around day 30, the serum of immunized chickens having maternal antibodies contained ND antibodies in a GMT of 3.4 to 9.4. These chickens showed 98 to 100 % protection against a dose of 106 LD50 of virulent virus inoculated subcutaneously. In two groups immunized with an aerosol contaning NDV-6/10 virus plus infectious bronchitis virus (strain H120), there was observed 100 and 92 % protection, respectively. Fifty days after the immunization, chickens of the latter group showed 75 % protection to experimental challenge. In the other groups, 90 to 100 % of the chickens survived very strong challenge on day 50 to 54.
These experiments clearly show that a single immunization of chicks with strain NDV-6/10 at day to 10-day of age ensures a good protection on broiler chickens for the entire rearing period (until 7 to 8 weeks of age). This protection is satisfactory also if the broilers are simultaneously immunized with an infectious bronchitis virus strain harmless to day-old chicks. The comparative studies have shown that aerosol immunization gives the best result, but good protection can be achieved also by administering NDV-6/10 virus in spray form or onto the conjunctiva. Inactivated vaccine (0.1-0.2 ml) given simultaneously enhances the protection; however, it does not seem to be necessary in broilers. It is of outsta ding importance that birds immunized between 1 and 10 days of age did not show any vaccination reaction and their body mass increased steadily. Comparing their daily body mass gain to, that seen on the day before, the sudden stop, which is often seen in birds vaccinated conventionally at 2 or 3 weeks of age. did not occur in the 3rd or 4th week of life. Body mass at slaughter varied depending on the circumstances; however, it was always satisfactory, and the losses always remained below 5 %.

B. Immunization of breeding and laying flocks Our experience gained on about 90 thousand birds can be summarized as follows.
The first immunization was performed as described for the broilers. As breeders and commercial laying hens producing for the market are immunized also against Marek's disease at day of age (in the hatchery), our experimental groups also received this vaccine in the hatchery. The chickens of two large flocks included in the trial were immunized in the hatchery also against infectious bronchitis: vaccine H120 was administered as a spray of large particle size. Chickens transported to the farm were vaccinated with NDV-6/10 aerosol between 2 and 10 days of age. No vaccination reaction was observed and in the first

2 months of rearing the mortality rate remained below

3 %.
At 30 days of age the antibody titres were higher than that in broilers of the same age (probably because of the more favourable management technology and the absence of forced rearing). There was 100 % protection against challenge. In one flock (the chickens of which had not been vaccinated against infectious bronchitis in the hatchery) the chickens were immunized with vaccine strain H120 administered in the drinking water at 34 to 37 days of age. Challenge at 60 days of age showed 84 h protection against ND.
In another flock, where the chickens had been vaccinated subcutaneously or in spray against Marek's disease and infectious bronchitis at day of age, they received a vaccine against infectious bursal disease in around the age of 1 month, then they were vaccinated with strain H120 also via the drinking water at 5 to 6 weeks of age. Chickens challenged with a dose of 106 LD50 of NDV at 45 to 53 days of age showed 96 % ppotection.

One flock, intended for further breeding, w /;as repeatedly vaccinated with NDV-6/10 aerosol about at 7 weeks of age. Three weeks later the level of

ND serum antibodies was substantially elevated (GMT: 1:24) and the value was 1:12 even at 4 months of age.
A total of 32,836 pullets were vaccinated only against Marek's disease and with strain NDV-6/10 at day of age. At 5 weeks of age they received first the less virulent (H120), then the more virulent (H52 ) infectious bronchitis vaccine in drinking water. At

10 weeks of age the HI-GMT was between 4.4 and 7.8 in the different groups. At that time the chickens received a second vaccination with strain NDV-6/10. As a result, the serum ND antibody titres markedly increased.
Before the laying season, at 4-5 months of age we tested two possibilities. The commercial laying flock was given a third vaccination by aerosol.

Depending on the antibody titres, aerosol immunization can be repeated here.
In the two other breeding flocks after two aerosol vaccinations performed at day of age and at around 7 weeks of age, the immunization before the laying season was carried out by administering a comtrivalent
bined inactivated/vaccine against ND-infectious bronchitis-infectious bursal disease.
It can be stated that the immunization with

NDV-6/10 aerosol can be carried out in laying flocks.

following the vaccination against Marek's disease and optionally against infectious bronchitis at day of age, without vaccination reactions on any day after transfer to the farm. Higher HI titres will be obtained, however, if aerosol immunization against ND is performed at 7-10 days of age. If there is no risk of epidemic, vaccination can be delayed until the birds are 2 to 3 weeks old.
Flocks immunized with the NDV-6/10 vaccine will have firm protection against ND even if, depending on the local immunization technology, the birds receive also other vaccines at the appropriate times. NDV-6/10 aerosol vaccine administered at week 7 provides satisfactory protection until the onset of the laying season. At that time protection can be prolonged by a further aerosol vaccination or the flock can be vaccinated with an inactivated (preferably trivalent) vaccine.
We observed also during aerosol immunization that under poor managemental and feeding conditions the HI titres are lower than those developing under optimum conditions. Therefore, the immunized flocks should be checked regularly by the HI test and, in case of need, immunization should be repeated.
The NDV-6/10 aerosol vaccine usually induces lower antibody titres than does the LaSota vaccine. At the same time, cell-mediated immunity is very pronounced. This may account for the finding that a challenge with virulent NDV in a dose of 106 LD50 billed only those birds which had possessed no, or very low titres of, antibodies before challenge (the only exception was a bird with a titre of 1:4). At the same time, the majority of aerosol-immunized birds (that had become Hl-negative during the long time that had elapsed since the immunization) survived the challenge. The serum of most survivors was positive even in dilutions around 1:2000.
The advantage of the vaccine of the invention prepared from strain NDV-6/10 is that it is completely harmless, does not cause any vaccination reaction, and can be used for immunizing poultry flocks αf any age. It does not have an adverse influence on body mass gain. It is easy to administer: large numbers of birds can be immunized via the drinking water or in spray, without having to take the birds in hand one by one. Immunization with other vaccines does not adversely interfere with the immunity developing to ND. Thus, vaccination with NDV-6/10 can be integrated into the immunization programme of any flock.
The invention is illustrated in the following example, without the intention of limitation.

Example 1
Production of live virus vaccine
Hen's eggs with 9 day old chicken embryos are inoculated with virus strain NDV-6/10 (MNG 001075) and incubated at 37 °C for 72 to 96 hours. The virus titre is determined by titration in embryonated eggs and by the HI test. After the virus has reached a minimum titre of 108, the embryos are killed by cooling, the allantoic fluid is collected and checked for bacteriological sterility and formulated as a vaccine.