GB2451428A - Process for preparing an in ovo combination viral vaccine - Google Patents

Abstract

A process for the preparation of a viral vaccine, the process comprising culturing at least two different viruses in an embryonated avian egg. The process preferably consists of inoculating the egg with a first virus and subsequently inoculating the egg with additional viruses, the inoculation further preferably takes place 8 to 14 days after fertilization of the egg. The different viruses are preferably Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), and are further preferably the La Sota, B1 or lentogenic strains of NDV and the VMG 91 strain of IBDV. Also claimed is a stabilising solution comprising 0.5 to 10% w/w polyvinylpyrrolidone, 1 to 30% w/w bactopeptone and 0.5 to 10% w/w sodium glutamate.

Description

PROCESS FOR PREPARING VIRAL VACCINE

The present invention relates to a process for the preparation of viral vaccines, in particular vaccines containing a combination of two or more viruses. The process is particularly suitable for the preparation of vaccines against common avian diseases, especially Newcastle Disease and infectious bursal disease (Gumboro Disease).

Newcastle disease presents an exceptional risk for the poultry industry in almost all countries of the world, irrespective of whether it concerns intensive or extensive, poultry production. The global economic damage caused by this disease is enormous.

The causal agent is Newcastle disease virus (NDV), which contains a non-segmented, single-chain RNA and belongs to the genus Paramyxovirus of the family Paramyxoviridae. In the group of avian paramyxoviruses there are 9 serotypes, marked from PMV-1 to PMV-9; NDV is PMV-l or serotype 1. Pathogenic NDV endangers all domestic fowl, although chickens and turkeys are most susceptible.

Significant losses (mortality, drop in egg yield) can also occur in wild fowl farming management (Alexander, D. J. (2003): Newcastle Disease, Other Avian Paramyxoviruses, and Pneumovirus Infections. Pages 63-99. in: Diseases of Poultry.

11th ed., Editor-in-Chief Saif Y. M. Blackwell Publishing, Ames, Iowa, USA).

Control of Newcastle Disease is regulated by law in many countries and, worldwide, and Newcastle disease is seen as the most important of all diseases that put poultry health at risk. Specific immunoprophylaxis plays the most important role in the prevention of disease and vaccines may be either live attenuated strains administered as aqueous suspensions of previously freeze-dried preparations (with the addition of adequate protective substances), or inactivated preparations, usually in the form of oil emulsions.

Infectious bursal disease (IBD), also knows as Gumboro disease, is an acute and highly contagious viral disease of young chickens, characterised by significant damages of the bursa of Fabricius and immunosuppression. A two-segment, two-chain RNA virus of the family Birnaviridae causes the disease. Control and prevention of IBD is possible only by timely vaccination and implementation of bio-protection measures. The use of mild, live vaccines prevailed till the end of I 980s when unsuccessful results of vaccination were reported worldwide. Vaccines containing vaccinal strains of intermediary virulence were subsequently introduced (Lukert D. P., Y. M. Saif (2003): Infectious Bursal Disease. Pages 16 1-179. In: Diseases of Poultry. 11th ed., Editor-in-Chief Saif Y. M. Blackwell Publishing, Ames, Iowa, USA).

It is common practice to use freeze-dried vaccines against Newcastle and Gumboro disease for the protection of the health of poultry, with tens of billions of doses being administered every year. in general, separate administration of each vaccine within 4 or more days is recommended, although there are examples of concomitant use of both live vaccines (Terziá, K., S. ajavec, F. Kraun, V. Savié (2005): Simultaneous use of live vaccines against infectious bursal disease, Newcastle disease and coccidiosis in broilers. Praxis veterinaria 53, p. 91-102). A dogma prevailing in the everyday practice is that infectious bursal disease virus (IBDV) is immunosuppressive and that it inhibits the development of immunity against Newcastle disease.

In the manufacture of "live" vaccines against viral poultry diseases, it is usual to produce the virus either in embryonated specific pathogen free (SPF) chicken eggs or in cell cultures. However, the use of SPF embryonated eggs is more common, particularly in the culture of NDV, as higher yields of virus are obtained.

Animal and avian viruses are highly sensitive and therefore the virus is generally combined with various substances that provide a protection of the virus during freeze drying; this provides a final product that is in the form of a cake containing 1-3% water. In order to take account of the possible loss of activity of the virus (i.e. a drop in the virus titre per dose of product) during the manufacturing and storage of the vaccine, the manufacturing process may start with a surplus of virus particles.

Alternatively, the product can be stabilised with appropriate protective substances.

Problems related to the stabilisation of live vaccines for veterinary medicine have been comprehensively described in WO 2007/919128.

The most common type of vaccine in use today is the single component freeze-dried vaccine. However, there are certain specific two-component avian vaccines currently available on the market, for example a combination vaccine against NDV and avian infectious bronchitis virus and a combination vaccine against fowl pox virus and avian encephalomyelitis.

The manufacturing process for such two-component vaccines consists essentially of the production of two separate viral suspensions which are mixed and then freeze dried.

The possibility of providing a combined vaccine comprising the La Sota strain of NDV and IBDV was explored by Terzié, K., S. ajavec, F. Kraéun, V. Savié (2005): "Simultaneous use of live vaccines against infectious bursal disease, Newcastle disease and coccidiosis in broilers"; Praxis veterinaria 53, p. 91-102. In addition, UA I 6452U describes the preparation of a two-component vaccine against infectious bursal disease and Newcastle disease in which the vaccine viruses (IBDV strain HG- 05; NDV strain La Sota) were separately propagated in chick embryo fibroblast cell culture and freeze-dried with the addition of skim milk and peptone.

EP 0332241 relates to combined poultry vaccines of which one component is strain Ma5 of the avian infectious bronchitis virus. There are no details in the document about how the vaccines are prepared but it appears to indicate that the viruses are cultivated separately and mixed before administration to the birds.

Combined vaccines are advantageous because it is more cost effective to give a single dose of a combined vaccine instead of separate doses of two or more vaccines.

In addition, the stress associated with vaccination is reduced by having a single dose rather than separate doses.

It would be an advantage to provide a combined vaccine in which the viral strains are produced and freeze dried simultaneously as this would significantly reduce manufacturing costs.

Therefore, in a first aspect of the present invention, there is provided a process for the preparation of a viral vaccine, the process comprising culturing at least two different viruses in an embryonated avian egg.

In general, the egg in which the viruses are cultured will be a chicken egg, especially an SPF chicken egg.

In the process of the present invention, the egg may be inoculated with a mixture of the at least two different viruses. Alternatively, the process may comprise inoculating the egg with a first virus and subsequently with additional viruses. Inoculation may take place about 8 to 14 days after fertilization, typically about 10 to 11 days after fertilization.

Preferably, one of the at least two different viruses is Newcastle disease virus (NDV) or infectious bursal disease virus (IBDV) and in a particularly preferred embodiment of the invention, both NDV and IBDV are cultured in the embryonated egg.

Generally, suitable strains of NDV for use in vaccines are lentogenic strains, particularly the La Sota and Bi strains. Strain VMG 91 is a particularly suitable IBDV strain.

Preferably the eggs are inoculated with the virus in the form of suspensions of suitable dilution. For example, the NDV suspension may have a titre range of from 10° to 106 E1D50 / 0.lml, preferably from 10 to l0 EID50 / 0.lml. The IBDV suspension may contain virus with TCID50 in the range of 10° to 106 per 0.lml, preferably from I 35 to I 0 TCID50 / 0.1 mIN After inoculation, the egg is preferably incubated for at least 72 hours. During this time, it is preferred that candling of the egg is carried out at 24 hour intervals to determine viability. After incubation, the viral products are harvested to obtain a viral suspension comprising the at least two different viruses.

It is preferred that only eggs which remain viable for at least 48 hours after inoculation are harvested.

The different viruses may replicate in different parts of the egg. For example, when NDV is one of the viruses, replication occurs mainly in allantoic fluid and, to a lesser extent, in embryos. On the other hand, replication of IBDV occurs mainly in embryos and to a lesser extent in the allantoic fluid.

In view of this, both the embryos and the allantoic fluid are harvested. Generally, the allantoic fluid and the embryos will be collected separately.

Embryos may be collected and homogenised with the addition of a buffer solution, especially a phosphate buffer to form a homogenised embryo suspension (one part of embryos and two parts of buffer solution). The homogenised embryo suspension may be stored at a temperature of 2-8°C until needed.

When the virus in the homogenised embryo suspension is infectious bursal disease virus, it is preferred that the suspension has a virus titre of at least 0 TCID50/ml but more preferably at least i0" TCID50IrnI.

Allantoic fluid may be collected according to known methods. When the virus cultivated in the allantoic fluid is Newcastle Disease virus, it is preferred that the allantoic fluid has a virus titre of at least i0 E1D50/mI but more preferably at least 10983 E1D50/mI.

and may be mixed with a stabiliser solution after collection to form a stabilised allantoic fluid solution. A particularly preferred stabiliser solution comprises an aqueous solution of polyvinylpyrrolidone, sodium glutamate and Bacto peptone.

Optionally, the stabiliser solution also comprises potassium dihydrogen phosphate and potassium hydroxide.

This stabilising solution forms a further aspect of the invention.

More particularly, the stabiliser solution may comprise from 0.5 to 10% w/v polyvinylpyrrolidone, I to 30% w/v bactopeptone and 0.5 to 10% w/v sodium glutamate in water, preferably purified water More preferably, the stabiliser solution comprises an aqueous solution containing about 2% w/v polyvinylpyrrolidone, about 10% w/v bactopeptone and about 2% w/v sodium glutamate.

The stabiliser solution may also contain additional ingredients, for example potassium dihydrogen phosphate, which may be present in an amount of from about 0.5 to 10% w/v, more usually about 0.5 to 3% w/v and typically about 1.4% w/v; and potassium hydroxide, which may be present in an amount of from about 0.1 to 5% w/v, more preferably about 0.1 to 1% w/v and typically about 0.4% w/v.

The stabilised allantoic fluid solution may comprise allantoic fluid and stabiliser in a volume ratio of from 4:1 to 1:4 allantoic fluid to stabiliser, preferably 4:1 to 1:1 allantoic fluid to stabiliser and usually about 2.5:1 allantoic fluid to stabiliser. The mixture of harvested allantoic fluid and stabiliser solution may be frozen and stored until needed.

The stabilised allantoic fluid solution may be mixed with homogenised embryo suspension to form a freeze drying mixture and therefore, in a further aspect of the invention, there is provided a freeze drying mixture comprising 15-45% v/v allantoic fluid, 15-45% v/v homogenised embryo suspension and 5-30% v/v stabilisér solution, with the balance being water.

The homogenised embryo suspension is as described above and comprises a suspension of homogenised embryos in a phosphate buffer such as phosphate buffered saline. The stabiliser solution is also as defined above.

A preferred freeze drying mixture comprises about 30% v/v allantoic fluid, 30% v/v homogenised embryo suspension, 20% v/v stabiliser solution and 20% v/v water.

The freeze drying mixture may be prepared by mixing stabilised allantoic fluid solution as described above with homogenised embryo suspension; and adding purified sterile water. Optionally, further stabilising solution may also be added to the mixture.

A typical freeze drying mixture may comprise 42% v/v stabilised allantoic fluid solution (containing 12% v/v stabiliser solution), 30% v/v homogenised embryo suspension; 8% stabiliser solution; and 20% water.

Each litre of freeze drying mixture is sufficient to provide approximately 106 doses of vaccine.

After freeze drying, the freeze dned vaccine must be reconstituted with water before use. The amount of water added to the freeze dried vaccine will depend upon the intended method of administration and the age of the birds to be treated. The water is preferably sterile.

When the vaccine is intended for use in drinking water, each thousand doses will typically be added to 5-25 litres of water. Typically, when the vaccine is intended for I week old birds, each 1000 doses is added to 10 1 of water and when the vaccine is intended for 2-3 week old birds, each 1000 doses is added to 20 1 of water.

When the vaccine is intended for use directly in the crop of the birds, each dose may be added to ImI of water (2500 doses in 2.5 I of water).

in a further aspect of the present invention there is provided a vaccine composition obtainable by a process according to the invention.

In addition there is provided a vaccine composition comprising IBDV in a titre of at least I o20 TCID50 per dose and NDV in a titre of at least 1050 E1D50 per dose.

Preferably, the vaccine composition comprises IBDV in a titre of at least 1 0 0 TCID50 per dose and NDV in a titre of at least 1060 E1D50 per dose.

A typical dose comprises the following constituents IBDV �=i�° NDV �=i060 Polyvinyipyrrolidone 0.02 mg Bactopeptone 0.10 mg Sodium glutamate 0.02 mg Potassium dihydrogen phosphate 0.0136 mg Potassium hydroxide 0.004 mg together with water (up to 3% v/v).

1 5 As set out above, Jentogenic strains of NDV are preferred, particularly the La Sota and BI strains. Strain VMG 91 is a particularly suitable IBDV strain.

The invention will now be described in greater detail with reference to the following examples and the drawings in which: Figure 1 is a plot showing the HI-titre of specific anti-NDV antibodies in the blood serum of chickens after vaccination with either two-component vaccine against IBD and ND or with ND vaccine.

Figure 2 is a plot of ELISA-titre of specific anti-IBDV antibodies in the blood serum of chickens after vaccination with either two-component vaccine against IBD and NV or with vaccine against IBD.

Example 1

Embryonated SPF eggs (Valo Lohmann) were incubated for 11 days at 37.5°C and 55% humidity with turning of eggs 5 times a day.

Newcastle disease virus (NDV) and Gumboro disease virus (GDV) were propagated in embryonated chicken eggs for 72 hours. Candling of eggs was done every 24 hours.

Preparation of antigen for inoculation Three groups of antigen were prepared for inoculation of the embryonated chicken eggs as follows: 1. ND virus -inoculum litre of ND virus was 10 E1D50/0,1 ml 2. IBD virus -inoculum litre of IBD virus was i039 1C1D5010,1 ml 3. A mixture of NDV and IBDV was prepared -5 nil of each inoculum of virus I and 2 were mixed.

Inoculation of embryonated eggs Groups of embryonated SPF eggs were inoculated as follows: Group 1 -Each of 40 embryonated SPF eggs was inoculated with 0.1 ml of mixture of NDV and IBDV.

Group 2-Each of 10 embryonated SPF eggs was inoculated with 0.1 ml of IBD virus Group 3 -Each of 10 embryonated SPF eggs was inoculated with 0.1 ml of ND virus Groups 2 and 3 were inoculated with individual viruses for the control of virus growth.

During incubation, the replication of NDV occurred mostly in allantoic fluid, and to lesser extent in embryos, whilst the replication of IBDV occurred mostly in embryos and to lesser extent in allantoic fluid.

Embryos that had died within 24 hours were discarded as non-specific deaths.

Harvest was performed after 72 hours of incubation.

Allantoic fluid was taken separately from each egg containing a live embryo or an embryo that had died between 24 and 72 hours after inoculation, and a haemagglutination (HA) titre of NDV was determined in each.

Embryos were sorted according to group and according to whether they were alive or dead after 72 hours. Embryos from groups 1, 2, and 3 which were live at 72 hours were designated as groups 1/1, 2/1 and 3/1 while embryos which were dead after 72 hours were designated as groups 1/2, 2/2 and 3/2. After the harvest, the embryos were homogenised with the addition of phosphate buffer solution.

For the determination of titre of IBDV the embryos from group I (both live or dead within 72 hours) and group 2 (only live) were collected and homogenised with the addition of a double quantity of PBS.

Analytical Methods Haemagglutination test For the determination of the haemagglutination titre of NDV, double serial dilutions of the harvested virus suspension were prepared.

Preparation of Double Serial Dilutions jtl of PBS was added to all wells of a microtiter plate in all wells. In the first row, tl of virus suspension was added and mixed then 25 iI of the mixture was transferred to the second row and mixed, following which 25 ti was added to the next row. The process was continued until the 12" row obtaining dilutions of 1:2; 1:4; 1:8; 1:16; 1:2048; 1:4096, i.e. ofbetween 1:2' and 1:212.

A 1% suspension of cockerel erythrocytes was added to each of the dilutions. The suspension was maintained for 45 minutes at room temperature, after which the reaction was read. The reciprocal value of the highest dilution of virus suspension that haemagglutinated was taken as the titre value. Test results are shown in Table I below and are expressed as mean values.

TCID

TCID50 is defined as the tissue culture infectious dose which will infect 50% of the challenged cells.

A primary culture of chicken fibroblasts was grown on 48-well microtitre plates.

To each well was applied 10 pi of a virus suspension at a dilution of between 102 and I -6 The plates were allowed to stand at room temperature for 30 minutes to allow adsorption to take place, after which 0.5ml of modified Eagles medium (MEM) was added to each well. The wells were then covered and allowed to stand at a temperature of 3 7°C. The results were read under a microscope after 24 hours and every subsequent 24 hours until a final reading was taken after 144 hours.

A result was considered to be positive if the cells in any particular well showed a cytopathogenic effect. The TCID50 was calculated using the Spearman-Karber method Results Table 1 -Mean values of titre of NDV (HA-titre) and IBDV (TCID5o Group Live! Number HA I TCID50/1 dead of titre ml ________ (his) embryos _____ ________ I/l live 23 210 108.1 1/2 dead (72) 12 2 _______ 2/1 live 6 2° 108.4 2/2 dead (72) 3 2° - 3/1 live 7 210 - 3/2 dead (72) 3 2 -The results in Table I show that the virus suspension obtained from Group I/I had a similar HA titre to the virus suspension obtained from Group 3/I and a similar TCID50 to the virus suspension obtained from Group 2/1.

Therefore, the simultaneous culturing of these two viruses in a single egg clearly does not affect the amount of each virus which can be obtained.

Example 2-Preparation of Vaccine Composition Preparation of stabiliser A stabiliser solution was prepared as follows: 1. 800 g of polyvinylpyrrolidone and 800 g of sodium glutamate were dissolved in 8 litres of purified water heated to 65°C.

2. 4 x 1 kg of Bacto peptone and 4 x 136 g of potassium dihydrogen-phosphate were dissolved in 4 x 2.5-3 L of purified water heated to 85°C.

3. 2 L of solution from the first stage were added to each bottle of the second stage (4 bottles), followed by 720 ml of I N solution of potassium hydroxide to pH 7.0 and then made to 10 1 volume with purified water.

4. sterile filtration of the solution Preparation of Mixture for Freeze-drying A mixture for freeze drying was prepared as follows Suspension of IBD virus -virus titre �= i0795 TCID5)/m1 of embryo homogenate Suspension of ND virus -virus titre �=i� E1D50/ml of suspension of allantoic fluid Mixture for freeze-drying contained (in one litre): 420 ml of suspension of NDV (containing 120 ml of stabiliser) 300 ml of suspension of IBDV ml of stabiliser ml of stenle purified water Filling volume for a vial of 2500 doses was 2.5 ml of freeze-drying mixture per vial.

Edwards freeze-drier was used.

Vaccine content: We add 250 ml of water, and then in each 0.1 ml (I dose)of the vaccine suspension titre is: IBDV -virus titre �=l 3 ° TCID50/dose NDV -virus titre �=1 060 E1D50/dose The vaccine was stored in a cooling chamber at 2-8°C.

Example 3 -Accelerated Stability Test Samples of the vaccine were stored in a thermostatic chamber at 37°C.

Samples of the vaccine were collected after 24, 72 and 168 hours and then kept in a cooling chamber at 2-8°C until analysis.

The results of accelerated stability test of two-component vaccine against infectious bursal disease, strain VMG 91 and Newcastle disease, strain La Sota (2500 doses/vial) are shown in Tables 2 and 3 below.

Table 2 -For NDV strain La Sota (EID50/vial) Sample 4°C 72 h/37°C A Jog 168 h/37°C A log 1 i09 -0.10 i09 -0.05 2 10942 1O° +0.08 i� +0.04 3 109Th 10968 +0.05 10998 +0.23 Table 3 -For IBDV (TCID50/vial) Sample 4°C 72 h/37°C A log 168 h137°C A log 1 107.2 10 -0.4 1069 -0.3 2 1 o i o +0.1 107.2 -0.1 3 l0° 10 +0.4 1069 -0.1 A log -Difference in results of samples kept at +4°C and 37°C The results of an accelerated stability test of two-component vaccine against infectious bursal disease, strain VMG 91 and Newcastle disease, strain B 1 (2500 doses/vial) are shown in Tables 4 and 5.

Table 4 -For NDV strain BI (EID50/vial) Sample 4°C 72 h/37°C A log 168 h/37°C A Jog 1 10980 10996 +0.16 10992 +0.12 2 10972 l0 +0.06 +0.01 Table 5 -For IBDV (TCID50/vial) Sample 4°C 72 h/37°C A log 168 h/37°C A log 1 10 10° -0.4 l0' -0.3 2 1 I O 0 1 2 -0.1 A log -Difference in results of samples kept at +4°C and 37°C The results in Tables 2 to 5 demonstrate that the vaccine is stable as there is no significant loss of activity even when it is kept at 37°C for 168 hours.

Example 4 -Use of Two-Component Vaccine A study was carried out for the purpose of proving the efficacy of vaccination of chickens with two-component vaccine against infectious bursal disease (Gumboro) and Newcastle disease. The vaccine used in the study was prepared from the strain VMG 91 (intermediary strain) of infectious bursal disease virus (IBDV) and the strain La Sota of Newcastle disease virus (NDV), both propagated in the same chicken egg.

Content of a vial of 2500 doses of two-component vaccine against IBD and ND was suspended so as to obtain in each vaccine dose (1 ml) the virus titre of I & 0 TCID50 for IBDV, 1060 E1D50 for NDV respectively.

In the study were included 63 SPF 10-day-old chickens. Nineteen chickens (Group 1) were vaccinated with Newcastle disease vaccine, 17 (Group 2) with two-component vaccine against infectious bursal disease and Newcastle disease, and 15 (Group 3) with vaccine against infectious bursal disease. In the control group were 12 unvaccinated chickens. Each bird was administered 1.0 ml of vaccine directly in the crop.

Blood samples for serological tests (HI-test for NDV and ELISA test for IBDV)) were collected on the day of vaccination and again in weekly intervals till the 28th day after the vaccination and the results are shown in Tables 6 and 7 and in Figures 1 and 2.

Hl-titre in the vaccinated chickens increased during the study period, and on day 21 following vaccination it amounted to in the group vaccinated with two-component vaccine, 2469 respectively, in the group vaccinated with Newcastle disease vaccine.

Chickens of the control group and Group 3 (vaccinated with IBD vaccine) were free from specific anti-NDV antibodies.

Table 6. HI-titre of specific anti-NDV antibodies in the blood serum of chickens after vaccination with either two-component vaccine against IBD and ND or ND vaccine DayO Day71Dayl4 Day2l Day28 Group 1 0 22.32 2 19 2469 Group 2 0 2294 2 2 50 2 81 Control 0 0 0 0 Table 7 -ELISA-titre of specific anti-IBDV antibodies in the blood serum of chickens after vaccination with either two-component vaccine against IBD and ND or vaccine against IBD DayO Day7 Day 14 Day 21 Day 28 Group 2 0 44 1034 2044 2785 Group 3 0 77 1194 2246 2995 Control 0 0 0 0 0 ELISA-titre in the vaccinated chickens increased during the study period and also in the group vaccinated with two-component vaccine and on day 28 post vaccination it 1 5 amounted to 2785, 2995 respectively, in the group vaccinated with infectious bursal disease vaccine.

Chickens of the control group and Group I (vaccinated with Newcastle disease vaccine) were free from specific anti-IBDV antibodies.

HI-titre of anti-NDV antibodies was found to be higher in chickens vaccinated with two-component vaccine against infectious bursal disease and Newcastle disease in comparison with that recorded in chickens vaccinated only against Newcastle disease. However, there were no statistically significant differences between titre values.

ELISA-titre of anti-IBDV antibodies was insignificantly higher in the group of chickens vaccinated with a single component vaccine against 1130 in comparison with that recorded in chickens vaccinated with two-component vaccine against infectious bursal disease and Newcastle disease.

Claims (29)

1. A process for the preparation of a viral vaccine, the process comprising culturing at least two different viruses in an embryonated avian egg.

2. A process as claimed in claim 1 wherein the egg is a chicken egg.

3. A process as claimed in claim I or claim 2, comprising inoculating the egg with a mixture of the at least two different viruses.

4. A process as claimed in claim 1 or claim 2 comprising inoculating the egg with a first virus and subsequently inoculated with additional viruses.

5. A process as claimed in any one of claims I to 4, wherein inoculation takes place about 8 to 14 days after fertilization of the egg.

6. A process as claimed in any one of claims I to 5, wherein one of the at least two different viruses is Newcastle disease virus (NDV).

7. A process as claimed in claim 6, wherein the NDV is a lentogenic strain.

8. A process as claimed in claim 7, wherein the NDV is of the La Sota or the BI strain.

9. A process as claimed in any one of claims 6 to 8, wherein the egg is inoculated with a suspension of NDV having a titre range of from 100 to 106 E1D501 0.1 ml.

10. A process as claimed in any one of claims I to 5, wherein one of the at least two different viruses is infectious bursal disease virus (IBDV).

II. A process as claimed in claim 10, wherein the IBDV is of strain VMG 91.

12. A process as claimed in claim 10 or claim 11, wherein the egg is inoculated with a suspension of IBDV with TCID50 in the range of 100 to 106 per 0. ImI.

13. A process as claimed in any one of claims I to 11, the process comprising culturing both NDV and IBDV in the embryonated egg.

14. A process as claimed in any one of claims 1 to 13, wherein the culturing comprises incubating the egg for at least 72 hours and harvesting the viral products.

15. A process as claimed in claim 14, wherein the harvesting comprises collecting both the embryos and the allantoic fluid from the egg.

16. A process as claimed in claim 15, wherein the allantoic fluid and the embryos are collected separately.

17. A process as claimed in claim 16 comprising homogenising the embryos.

18. A process as claimed in claim 16 or claim 17 comprising stabilising the collected allantoic fluid with a stabiliser solution comprising polyvinylpyrrolidone, sodium glutamate and Bacto peptone.

19. A process as claimed in any one of claims 15 to 18 further comprising freeze drying a mixture of the homogenised embryos and allantoic fluid to obtain a freeze dried vaccine composition.

20. A process as claimed in claim 1 9, further comprising reconstituting the freeze dried vaccine composition with water.

21. A vaccine composition obtainable by a process as claimed in any one of claims 1 to 20.

22. A live vaccine composition comprising NDV and IBDV.

23. A live vaccine composition as claimed in claim 22, wherein the NDV is present in a titre of at least I o° E1D50 per dose.

24. A live vaccine composition as claimed in claim 22 or claim 23, wherein the NDV is of strain La Sota or Bi.

25. A live vaccine composition as claimed in any one of claims 22 to 24, wherein the IBDV is present in a titre of at least 1 0 ° TCJD5O per dose.

26. A live vaccine composition as claimed in any one of claims 22 to 25 wherein the IBDV is of strain VMG91.

27. A stabilising solution comprising from 0.5 to 10% w/w polyvinylpyrrolidone, I to 30% w/w bactopeptone and 0.5 to 10% w/w sodium glutamate.

28. A stabilising solution as claimed in claim 27 comprising about 2% w/w polyvinylpyrrolidone, about 10% w/w bactopeptone and about 2% w/w sodium glutamate.

29. A stabilising solution as claimed in claim 27 or claim 28 further comprising one or more additional components selected from potassium dihydrogen phosphate and potassium hydroxide.