Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-07-26
2004-03-02
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S204100, C424S211100, C435S235100, C435S236000, C435S440000
Reexamination Certificate
active
06699479
ABSTRACT:
The present invention is concerned with a Newcastle disease virus (NDV) mutant that expresses its V protein at a reduced level, a vaccine comprising the NDV mutant and the use of the NDV mutant for the manufacture of a vaccine to protect birds against ND
Newcastle disease (ND) is one of the devastating diseases of poultry and has substantial economic impact on the poultry industry. NDV is the etiologic agent of this disease and belongs to the family Paramyxoviridae. Newcastle disease is complicated in that different isolates and strains of the virus may induce substantial variation in the severity of the disease. In general, the younger the chicken the more acute and severe the disease. The infection may take place by either inhalation or ingestion of the virus. The infectious form of the virus spreads from one bird to another.
In order to reduce the economic losses due to ND in the poultry industry, vaccination of chickens, particularly those raised for commercial consumption, is carried out throughout the world on a routine basis. Examples of live (lentogenic) NDV vaccine strains commonly used are the V4, Hitchner B 1, F and La Sota strain. However, these vaccine strains still cause light to moderate vaccination reactions, in particular in the respiratory tract upon primary vaccination of young birds.
Mild NDV vaccine strains have been developed which do not cause (respiratory) vaccination reactions upon administration to young birds: U.S. Pat. No. 5,250,298 (University of Delaware) discloses a live, cold-adapted temperature-sensitive mutant of the Hitchner B1 strain, designated as CaTs. U.S. Pat. No. 5,149,530 (Duphar Int. Res. B.V.) describes a NDV strain, designated as NDW, which is a mutant derived from the Ulster 2C strain. U.S. Pat. No. 5,750,111 (Akzo Nobel N.V.) discloses a mild vaccine strain, designated as the C2 strain, which does not induce adverse reactions in one-day-old chicks.
Currently available NDV vaccines can only be administered to hatched chickens through drinking water, aerosol, eye drops or by parenteral routes. These methods of applications have some disadvantages, most importantly expensive because of the labour needed for their application. Recently, the use of vaccines, such as herpesvirus of turkey and infectious bursal disease virus vaccines as embryo vaccines (Sharma and Burmester, Avian Diseases 26, 134-149, 1982 and Sharma, Avian Diseases 29, 1155-1169, 1985) has proved to be effective and economical. Moreover, embryo vaccination was found to be advantageous due to early age of resistance to the specific disease and administration of a uniform dose of vaccine into each egg using semiautomatic machines with multiple injection heads.
It should be noted that many vaccines used conventionally for post-hatch vaccination of birds cannot be used for in ovo vaccination. Late stage embryos are highly susceptible to infection with most vaccine viruses examined, including those vaccine viruses which can safely be used in one-day-old hatched chicks. Consequently, conventional vaccines must be modified for in ovo use.
Currently, there is no suitable commercially available ND vaccine that can be applied in ovo, mainly due to high level of embryo mortality associated even with two of the mildest commercially available NDV vaccine strains: NDW and C2. U.S. Pat. No. 5,427,791 (Regents of the University of Minnesota) discloses the use of chemical mutagenic agents to produce NDV mutants of the Hitchner BI strain that are non-pathogenic for late stage embryos. Chemical treatment of the BI strain with ethyl methanine sulfonate (EMS) resulted in the mutant virus NDV-B1-EMS which could be safely administered to chicken eggs at embryonation day 18. However, such mutagenic process leads to the introduction of random mutations in the genome of the virus in an uncontrolled, non-reproducible way. Such random mutations may influence properties of the virus other than those associated with the safety in ovo, such as properties of the virus related to the immunogenicity. Moreover, disadvantageously, each egg passage step of this strain must be carried out in the presence of the mutagenic agent EMS because of the property of the mutant to revert back to the parent B1 strain which is not safe for embryos.
Recently, genetic modification of non-segmented negative stranded RNA viruses has become possible by the development of a process referred to as “reverse genetics” (reviewed in Conzelmann, J. Gen. Virology 77, 381-389, 1996; Conzelmann, Annu. Rev. Genet. 32, 123-162, 1998 and Palese et al., Proc. Natl. Acad. Sci. 93, 11354-11358, 1996). The established reverse genetics system that enable controlled genetic manipulation of negative strand RNA viruses has potential applications for the development of novel vaccine strains.
NDV is a member of the family Paramyxoviridae and its negative-strand RNA virus genome contains six genes encoding six major structural proteins (3′NP-P-M-F-HN-L 5′). A general feature of paramyxoviruses, however, is the presence of additional structural or non-structural viral proteins resulting from the use of alternative reading frames and RNA editing of their P gene (reviewed by Kolakofsky et al., J. Virology 72, 891-899, 1998). Like other paramyxoviruses, NDV is also found to edit its P gene by inserting one or two G residues at the editing locus (UUUUUCCC). The three mRNAs encode the P protein (unedited), the V ORF (with +1 frame-shift) and the W ORF (with +2 frame-shift) (Steward et al., J. Gen. Virology 74, 2539-2547, 1993). Translation of the P, V and W specific mRNAs result in the expression of three proteins which have the same N-terminal halve but which differ in their C-terminal halves as a result of the use of different reading frames down-stream of the editing locus.
Peeters et al. (J. Virology 73, 5001-5009, 1999) and Römer-Oberdörfer et al. (J. Gen. Virol. 80, 2987-2995, 1999) described the generation of infectious NDV entirely from cloned cDNA by the reverse genetics system. It is shown in Peeters et al (1999) that the virulence of a NDV vaccine strain can be increased dramatically by modifying the amino acid sequence at the cleavage site of the F
0
protein. It is also suggested that that elimination of expression of the V protein of NDV may result in an attenuated phenotype in birds (Peeters et al., 1999, supra).
It is an object of the present invention to identify a NDV mutant which can be used for the manufacture of a vaccine for the protection of birds against ND which can be administered not only to young birds after hatch, but which can also be administered safely in ovo.
A new NDV mutant has been identified herein that displays not only mild, attenuated properties for young hatched chickens similar to those displayed by the commercially available mild NDW and C2 vaccine strains, but which in contrast to the NDW and C2 vaccine strains, can also safely be used for embryo vaccination.
The invention provides a NDV mutant that expresses its V protein at a reduced level (NDV V
−
), characterised in that the mutant is phenotypically V protein positive and wherein ≦6% of its P-gene derived mRNAs in infected cells encode V ORF.
It has been found that a NDV mutant as defined above causes significantly less embryo mortality, even if administered to embryos at 11 days of age. This is in contrast to the parent lentogenic vaccine strain from which the mutant is derived. This vaccine strain kills all the embryos before they hatch. Additionally, it was found that a NDV V
−
mutant does not affect hatchability of the eggs, particularly of embryonated commercial chicken eggs and that chickens hatched from embryo vaccinated eggs were protected against virulent NDV challenge. These unexpected combined properties of a NDV V
−
mutant make such a mutant especially suitable for the manufacture of a vaccine for in ovo administration.
Surprisingly, it has been found that NDV mutants that are not able to express V protein generated by reverse genetics techniques can not be rescued after passage of transfection sup
Mebatsion Teshome
Schrier Christina Carla
Akzo Nobal N.V.
Foley Shanon
Milstead Mark W.
Mosher Mary E.
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