Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-25
2002-11-26
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C424S184100, C424S204100, C536S023720
Reexamination Certificate
active
06485940
ABSTRACT:
The present invention is concerned with an IBDV mutant comprising the nucleotide sequences encoding a variant E VP2 protein in segment A of the viral genome and that is capable to infect and replicate in CEF tissue culture, a vaccine comprising this mutant and the use of this mutant for the manufacture of a vaccine.
Infectious bursal disease virus (IBDV) is a member of the Birnaviridae family. Viruses in this family have a very similar genomic organisation and a similar replication cycle. The genomes of these viruses consist of 2 segments (A and B) of double-stranded (ds) RNA. The larger segment A encodes a polyprotein, which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4. VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of birnaviruses, and contains the antigenic regions responsible for the induction of neutralising antibodies. The VP4 protein is the virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. The larger segment A possesses also a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene. This second open reading frame encodes a protein VP5 of unknown function that is present in IBDV infected cells. The smaller segment B encodes VP1, a 90 kDa multifunctional protein with polymerase and capping enzyme activities.
For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes may be differentiated by virus neutralisation (VN) tests. Furthermore, subtypes of serotype 1 have been isolated. These so-called “variant” viruses of serotype 1 can be identified by cross-neutralisation tests, a panel of monoclonal antibodies or RT-PCR. These subtypes of serotype 1 of IBDV have also been described in literature, for example: classical, variant-E, GLS, RS593 and DS326 strains (Van Loon, et al. Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 179-187, 1994).
Infectious Bursal disease (IBD), also called Gumboro disease, is an acute, highly-contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate mortality rates. Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius, which is essential to the defence mechanism of the chicken. The IBD-virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
For many years the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine, to chickens that had been primed with attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevent early infection with IBDV and diminish problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
Recently, very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe. The current vaccination programs failed to protect chicks sufficiently. Vaccination failures were mainly due to the inability of live vaccines to infect the birds before challenge with virulent field virus.
A further cause of acute disease in vaccinated flocks is the emerging of antigenic variants in the mid-1980s, in particular in the USA. The most important new antigenic subtypes of serotype 1 IBDVs were the Delaware variant E and GLS strains. Until then, vaccines used were based on live attenuated or killed IBDV strains of the classical type only. However, despite the fact that classical IBDV vaccine strains induce a certain degree of cross-protection against infection of chickens by other subtype strains (and vice-versa), substantial economic losses have been sustained as a result of vaccination with vaccines based on only one of the subtype viruses.
As a result of these developments in the field it became necessary to incorporate both classical and variant IBDV subtype vaccine strains into the IBDV vaccines to obtain a broad spectrum protection.
As described above, both live and killed vaccines based on classical strains are generally used in the field. The killed classical vaccines are commonly administered per injection, whereas the live classical vaccines are administered per injection or by one of the inexpensive, mass application techniques, such as spray-, aerosol and drinking water vaccination.
Delaware variant E viruses identified so far are highly virulent and can be administered as an inactivated vaccine only. Until now, there has been only one successful attempt to adapt and attenuate a variant E strain in cell culture. This vaccine strain designated as 89/03 is able to induce protection against variant E infection, but has the disadvantage that it does not take if administered by one of the mass application routes. This vaccine virus needs to be administered by injection, subcutaneously or intramuscularly (Hein et al., Proceedings of the 43th Western poultry disease conference, pages 102-103, 1994, Sacramento, USA).
For protecting birds against infection by IBDV of the GLS subtype, only inactivated vaccines are available.
Mundt (J. Gen. Virology 80, 2067-2076, 1999) identified the structural requirements at the molecular level which allows IBDV strains that were only able to infect and replicate in vivo in the bursa of Fabricius, to infect and replicate in chicken embryo fibroblast (CEF) tissue culture as well. Two amino acid exchanges in the VP2 protein at positions 253 (Gln to His) and 284 (Ala to Thr) were necessary and sufficient for the ability of IBDV bursa strains (BU) to infect CEF and other tissue cultures. In particular, two chimeric D78/variant E IBDV mutants with a tissue culture (TC) phenotype are disclosed in this document.
In view of the above, a need existed for a live attenuated Delaware variant E IBDV vaccine that is efficacious if administered via a mass application route. Furthermore, a need existed to obtain a broad spectrum IBDV vaccine strain that affords adequate protection against IBDV strains of two or more subtypes. Such a broad spectrum vaccine prevents that separate vaccine viruses for each IBDV subtypes have to be manufactured and formulated into a combination vaccine.
It is an object of the present invention to provide an IBDV mutant that is able to induce a protective immunity in chickens against virulent strains of at least two IBDV subtypes. It is a further object to provide an IBDV mutant which affords protection against virulent IBDV variant E strains and that at the same time can be administered via a mass application technique commonly used for live IBDV vaccination.
It has been found now that these objects have been met by providing an IBDV mutant comprising the nucleotide sequences encoding a variant E VP2 protein in segment A of the viral genome and that is capable to infect and replicate in CEF tissue culture, characterised in that the mutant comprises one or more mutations in the variant E VP2 coding region such that,
(i) the codon for the amino acid at position 254 encodes Glycine, and/or
(ii) the codon for the amino acid at position 270 encodes Threonine.
It has been shown by the present inventors that the chimeric IBDV D78/variant E bursa (BU) mutant disclosed in Mundt et al. (1999, supra), displaying the culture (BU) and immunogenicity characteristics of a variant E strain, is virulent and induces complete lymphocyte depletion after administration to chickens. Furthermore, it is shown in Example 2 that the corresponding tissue culture (TC) mutant of this strain is less virulent and is able to induce a partial protective immune response if administered per injection. However, at the same time the T
Loon Van Adriaan Antonius Wilhelmus Maria
Mundt Egbert
Akzo Nobel N.V.
Park Hankyel T.
Ramey III William P.
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