Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-07-06
2004-04-13
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S184100, C424S278100, C424S093200, C424S204100, C435S440000, C435S173300, C435S236000
Reexamination Certificate
active
06719981
ABSTRACT:
The present invention relates to attenuated rabies virus mutants and live attenuated anti-rabies vaccines comprising said mutants.
Rabies is a disease that can occur in all warm-blooded species and is caused by rabies virus (RV). Infection with RV followed by the outbreak of the clinical features in nearly all instances results in death of the infected species. In Europe, the USA and Canada wild life rabies still exists and is an important factor in the cause of most human rabies cases that occur. On the other hand, urban rabies constitutes the major cause of human rabies in developing countries.
Rabies virus (RV) is a non-segmented negative-stranded RNA virus of the Rhabdoviridae family. RV virions are composed of two major structural components: a nucleocapsid or ribonucleoprotein (RNP), and an envelope in the form of a bilayer membrane surrounding the RNP core. The infectious component of all Rhabdoviruses is the RNP core which consists of the RNA genome encapsidated by the nucleocapsid (N) protein in combination with two minor proteins, i.e. RNA-dependent RNA-polymerase (L) and phosphoprotein (P). The membrane surrounding the RNP core consists of two proteins: a trans-membrane glycoprotein (G) and a matrix (M) protein located at the inner site of the membrane.
The G protein, also referred to as spike protein, is responsible for cell attachment and membrane fusion in RV and additionally is the main target for the host immune system. The amino acid region at position 330 to 340 (referred to as antigenic site III) of the G protein has been identified to be responsible for the virulence of the virus, in particular the Arg residue at position 333. All RV strains have this virulence determining antigenic site III in common.
An effective way to control rabies is vaccination with inactivated RV or with attenuated vaccine strains of RV. In general, attenuated live anti-rabies vaccines are preferred because they often evoke a long lasting immune response usually based on both humoral and cellular reactions. Currently available attenuated live anti-rabies vaccines are based on attenuated RV vaccine strains including the SAD Bern strain or the SAD B19 strain, however these vaccines still have undesired residual pathogenicity.
Several attempts have been made to obtain non-pathogenic RV strains for use in a live vaccine. European Patent 350398 describes an avirulent RV mutant SAG1 derived from the Bern SAD strain of RV in which the glycoprotein possesses Ser instead of Arg at position 333. The avirulent mutant SAG1 was obtained under selection pressure of specific monoclonal antibodies on the SAD Bern strain. In adult mice SAG1 has been found to be non-pathogenic. However, pathogenic revertants of the attenuated virus occurred at a frequency of 1 in 10,000 (Lafay et al, Vaccine 12. pp. 317-320, 1994). The genetic instability of this mutant renders it unsuitable for safe vaccination.
European patent application 583998 describes another attenuated RV mutant, SAG2, in which Arg at position 333 has been substituted by Glu in the glycoprotein. SAG2 is non-pathogenic for adult mice when administered by various routes. SAG2 is currently used for oral vaccination of foxes particularly in France. Because this mutant also has the potential to revert to the pathogenic parental strain, the vaccine is produced in the presence of specific monoclonal antibodies to prevent reversion (Blancou and Meslin, 1996; In Laboratory techniques in rabies, pp. 324-337). Since these specific monoclonal antibodies are not present in inoculated animals, vaccination with such mutant still has the risk that the mutant reverts to virulence in the inoculated animal resulting in disease outbreaks in the inoculated animals and possible spread of the pathogen to other animals.
Hence there is an ongoing need for attenuated live anti-rabies vaccines which do not have residual pathogenicity or the potential to revert to the pathogenic variant. The present invention provides for such vaccines.
According to the present invention it was found that stable, attenuated RV mutants could be obtained by a mutation in the G-protein gene of the viral genome, said mutation comprising substitution of the Arg
333
codon with a codon that differs by all three nucleotides from the Arg
333
codon. For the purpose of this invention, the term “Arg
333
codon” is defined as the codon in the G-protein gene of the viral genome that encodes Arg
333
in the G protein. The term “Arg
333
” is defined as the Arg residue at position 333 of the RV G protein. In RV strain SAD and strains derived therefrom the Arg
333
codon is AGA and mutation of this codon into a codon that differs by all three nucleotide from said Arg
333
codon resulted in stable and attenuated RV mutants. Preferably the Arg
333
codon was mutated into GAC, CAG, TCC, GAG, CAC or CAT. Similar mutations can be carried out with other RV strains to obtain stable attenuated mutants. Mutations according to the invention were found to be stable and the resulting RV mutants were attenuated and did not revert to pathogenicity. These stable, attenuated RV mutants are very suitable for use in a vaccine. A great advantage of the invention is furthermore that vaccines comprising the RV mutants according to the invention can be produced without the need of specific monoclonal antibodies. Hence vaccine production becomes more simple and easier to carry out.
Thus in a first aspect the present invention provides for recombinant RV mutants comprising a mutation in the viral genome, whereby said mutation comprises at least a substitution of the Arg
333
codon with a codon that differs by three nucleotides from said Arg
333
codon. Preferably the mutants are mutants of an RV strain in which the Arg
333
codon is an AGA triplet. More preferably the mutants according to the invention are mutants of RV strain SAD and its derivatives, especially RV strain SAD B19.
Preferred RV mutants according to the invention are RV mutants in which the Arg
333
codon AGA has been substituted with a GAC triplet, CAG triplet, TCC triplet, GAG triplet, CAC triplet or CAT triplet. Much preferred RV mutants are RV mutants in which the Arg
333
codon AGA has been substituted with a GAC triplet or CAC triplet. Particularly preferred are recombinant RV mutant strains SAD D29 and SAD H31, in which the Arg
333
codon in the genome of RV strain SAD B19 has been substituted with a GAC triplet and CAC triplet, respectively.
Thus the present invention provides for stable, attenuated recombinant RV mutants in which the G protein of said mutant comprises an amino acid at position 333 which is encoded for by a codon which differs by all three nucleotides from the Arg
333
codon of the parental virus. It was found that the recombinant RV mutants according to the invention are non-pathogenic in immune competent animals and were found to be highly stable. Surprisingly, even after 25 passage experiments in cell culture no alterations were observed. All cell culture passages were carried out in the absence of monoclonal antibodies. Moreover the mutants remained non-pathogenic for adult mice even after a passage in suckling mice. The substitutions at position 333 of the G protein in no way affected the growth rate of the virus in BSR cells and the final titre was similar to the parental strain. This makes the recombinant RV mutants according to the invention very suitable for use in a live anti-rabies vaccine.
In addition to substitution of the Arg codon at amino acid position 333 in the G protein, the recombinant RV mutants according to the present invention may comprise other substitutions that affect the amino acids of Antigenic site III of the glycoprotein. Preferably these substitutions are made in the codons that encode the amino acids of Antigenic site III of the glycoprotein, more preferably in the codons that correspond to amino acid position 330 and/or 336 in the G protein. The recombinant RV mutants according to the present invention may furthermore comprise other mutations or modifications including heterologous genes e.g. a gene enc
Conzelmann Karl Klaus
Mebatsion Teshome T
Akzo Nobel N.V.
Milstead Mark W.
Scheiner Laurie
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