MXPA01005272A - Stable, attenuated rabies virus mutants and live vaccines thereof - Google Patents

Abstract

The present invention relates to recombinant rabies virus mutants comprising a mutation in the viral genome, whereby said mutation comprises at least a substitution of theArg333 codon in the gene encoding the G protein with a codon that differs by three nucleotides from said Arg333 codon. These rabies virus mutants have a glycoprotein G that comprises an amino acid at position 333 which is encoded by a codon that differs by all three nucleotides from the Arg codon in amino acid position 333 in the glycoprotein of the parental virus. Said recombinant rabies virus mutants are stable and non-pathogenic in immune competent animals and are suitable for use in a live, attenuated anti-rabis vaccine.

Description

MUTANTS OF RABIES VIRUS ATTENUATED, STABLE AND LIVING VACCINES OF THEMSELVES The present invention relates to attenuated rabies virus mutants and live attenuated rabies vaccines comprising said mutants. Rabies is a disease that can occur in all warm-blooded species and is caused by the rabies virus (RV). Infection with RV followed by the onset of clinical features in almost all stages results in the death of the infected species. In Europe, the USA and Canada, wildlife rabies still exists and is an important factor in the origin of most cases of human rabies that occur. On the other hand, urban rabies is the main source of human rabies in developing countries.

Rabies virus (RV) is a non-segmented, negative filament RNA virus of the Rhabdoviridae family. RV viruses are composed of two main structural elements: a nucleocapsid or ribonucleoprotein (RNP), and an envelope in the form of a bilayer membrane that surrounds the nucleus of RNP. The infectious component of all Rhabdoviruses is the nucleus of RNP consisting of the RNA genome encapsulated by the nucleocapsid protein (N) in combination with two secondary proteins, ie RNA-dependent RNA (L) and phosphoprotein (P) . The membrane surrounding the RNP nucleus consists of two proteins: a trans-membrane glycoprotein (G) and a binding protein (M) located at the inner site of the membrane. Protein G, also referred to as protein tip, is responsible for cell binding and membrane fusion in the RV and is additionally the main target for the host's immune system. The region of the amino acid at position 330 to 340 (referred to as antigenic site III) of the G protein has been identified as being responsible for the virulence of the virus, in particular the Arg residue at position 333. All RV strains have this virulence determining the antigenic site III in common. An effective way to control rabies is vaccination with inactivated RV or with attenuated RV vaccine strains. In general, live attenuated rabies vaccines are preferred because they often evoke a long-lasting immune response usually based on both cellular and humoral reactions. The currently available attenuated rabies vaccines are based on attenuated RV vaccine strains including the SAD Bern strain or the SAD B19 strain, however, these vaccines still have undesirable residual pathogenicity. Various attempts have been made to obtain non-pathogenic RV strains for use in a living vaccine. European Patent 350398 describes a non-virulent RV mutant SAG 1 derived from the SAD Bern strain of RV in which the glycoprotein has Ser in place of Arg at position 333. The non-virulent mutant SAG1 was obtained under the selection pressure of specific monoclonal antibodies in the SAD Bern strain. In adult SAG 1 mice it has been found to be non-pathogenic. However, the 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 makes it unsuitable for safe vaccination. The application of European Patent 583998 describes another attenuated RV mutant, SAG2, in which, Arg at position 333 has been replaced by Glu in the glycoprotein. SAG2 is non-pathogenic for adult mice when administered by several routes. SAG2 is currently used for the oral vaccination of foxes particularly in France. Because this mutant also has the potential to revert to the pathogenic origin 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 do not occur in inoculated animals, vaccination with such a mutant still has the risk that the mutant will revert to virulence in the inoculated animal resulting in a disease that sprouts in the inoculated animals and possibly spreads the pathogen to other animals. Therefore, there is an ongoing need for attenuated live rabies vaccines, which have no residual pathogenicity or the potential to revert to the pathogenic variant. The present invention provides such vaccines.

According to the present invention it was found that stable, attenuated RV mutants could be obtained by means of a mutation in the G protein gene of the viral genome, said mutation comprising the substitution of the codon Arg333 with a codon that differs by all the three nucleotides of codon Arg333- For the purpose of this invention, the term "codon Arg333" is defined as the codon in the G protein gene of the viral genome encoding Arg333 in protein G. The term uArg333"is defined as the Arg residue at position 333 of the RV G protein.In the strain of RV SAD and the strains derived from it, the codon Arg333 is AGA and the mutation of this codon in a codon differs by all three nucleotides of said codon Arg333 resulted in attenuated and stable RV mutants.Preferably the Arg333 codon was mutated in GAC, CAG, TCC, GAG, CAC or CAT.Similar mutations can be carried out with other strains of RV to obtain mutants attenuated, stable. Mutations according to the invention were found to be stable and the resulting RV mutants were attenuated and not reversed in pathogenicity. These stable, attenuated RV mutants are well suited for use in a vaccine. A further advantage of the invention is that vaccines comprising the RV mutants according to the invention can be produced without the need for specific monoclonal antibodies. Therefore, the production of the vaccine becomes simpler and easier to carry out. Thus, in a first aspect, the present invention provides recombinant RV mutants comprising a mutation in the viral genome, by means of which said mutation comprises at least one substitution of the Arg333 codon with a codon differing by three nucleotides of said codon Arg333. Preferably, the mutants are mutants of an RV strain in which, the codon Arg333 is a triplet AGA. More preferably, the mutants according to the invention are mutants of the RV SAD strain and its derivatives, especially the strain of RV SAD B19. Preferred RV mutants according to the invention are RV mutants in which the codon Arg333 AGA has been substituted with a triplet GAC, triplet CAG, triplet TCC, triplet GAG, triplet CAC or triplet CAT. The most preferred RV mutants are the RV mutants in which the codon Arg333 AGA has been replaced with a triplet GAC or triplet CAC. Particularly preferred are the mutant strains of RV SAD D29 and SAD H31 recombinants, in which, the Arg333 codon in the genome of RV strain SAD B19 has been replaced with a triplet GAC and triplet CAC, respectively. In this manner, the present invention provides the stable, attenuated recombinant RV mutants, in which, the G protein of said mutant comprises an amino acid at position 333 which is encoded by means of a codon that differs by all three nucleotides of the codon Arg333 of the origin virus. It was found that 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 pass experiments in the cell culture no alteration was observed. All steps of cell culture were carried out in the absence of monoclonal antibodies. Still further, the mutants remained non-pathogenic for adult mice even after passage in the absorbent mice. The substitutions at position 333 of the G protein did not affect in any way the growth rate of the virus in the BSR cells and the final concentration was similar to the origin strain. This makes the recombinant RV mutants according to the invention very suitable for use in a living rabies vaccine. In addition to the substitution of the Arg codon at position 333 of the amino acid in the G protein, the recombinant RV mutants according to the present invention can comprise other substitutions that affect the amino acids of the glycoprotein III site. Preferably, these substitutions are carried out in the codons encoding the amino acids of the antigenic site of the glycoprotein, more preferably in the codons corresponding to position 330 of the amino acid and / or 336 in the protein G. The recombinant RV mutants according to The present invention may further comprise other mutations or modifications that include heterologous genes, for example, a gene encoding a G protein of a different RV strain. Recombinant RV mutants according to the invention can be obtained using recombinant DNA technology and site-specific mutagenesis to introduce the desired mutation in contrast to the prior art alteration by chance using the monoclonal antibodies. Direct genetic manipulation of RV can be carried out using the reverse genetics system described in Schnell et al., 1994; EMBO J. Vol. 13, No. 18, pp. 4195-4203 and the application of European Patent 0 702 085, of which both are incorporated herein by reference. Site-specific mutagenesis can be carried out according to the method described by Kunkel, T.A., Roberts, J.D. and Zakour, R.A. (1987): Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymology Vol. 154, pp. 376-392. A full-length cDNA clone of the SAD B19 vaccine strain described in Schnell et al. , supra, was used as a basis for introducing codons that differed from the triplet Arg of the strain of RV origin in all three nucleotides for the generation of the recombinant RV mutants according to the invention. VR mutants according to the invention can be obtained by means of a) the introduction of the desired mutation in the RV full-length cDNA clone, b) the simultaneous expression of a full-length anigenomic RV RNA of the modified cDNA and the RV N, P, and L proteins of the transfected plasmids in the T7-RNA polymerase expression cells and 3) the isolation of RV mutant viruses produced by said cells. Recombinant RV mutants according to the invention can be grown in a cell culture derived from, for example, BHK cells or human diploid cells. In this way, the grown viruses can be harvested by collecting the fluids and / or cells from the cell culture of the tissue. In a further aspect, the present invention provides the attenuated living rabies vaccines comprising one or more recombinant RV mutants according to the invention. Preferably, an attenuated live rabies vaccine according to the invention comprises a recombinant RV mutant derived from the RV strain SAD B19. The live attenuated rabies vaccine according to the invention, which is especially preferred, comprises strains of recombinant RV mutant in which the codon Arg333 in the viral genome has been replaced with a triplet GAC and triplet CAC, respectively. More specifically, the vaccine according to the invention comprises a strain of recombinant RV mutant in which, the Arg333 codon in the viral genome has been replaced with the triplet GAC, resulting in a replacement of Arg with Asp at position 333 of the G protein. Particularly preferred are the vaccines comprising the recombinant RV mutant strain SAD D29. Vaccines according to the invention have the great advantage that they can be produced in the absence of specific monoclonal antibodies. The vaccines according to the invention can be prepared using standard techniques available in the art. In general, the vaccine is prepared by mixing the attenuated recombinant RV mutant according to the invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carriers or diluents that can be used to formulate a vaccine according to the invention are sterile and physiologically compatible such as for example sterile water, saline, aqueous regulators such as alkali metal phosphates (eg, PBS), alcohols, polyols, and the like. In addition, the vaccines according to the invention may comprise other additives such as adjuvants, stabilizers, anti-oxidants, preservatives and the like.

Suitable adjuvants include but are not limited to aluminum salts or gels, carbomers, nonionic blocopolymers, tocopherols, monophosferase lipid A, muramyl dipeptide, oil emulsions (w / ouo / w), cytokines, and saponins such as Quil A. The amount of adjuvant added depends on the nature of the adjuvant itself. Suitable stabilizers for use in a vaccine according to the invention are for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and regulators such as alkaline phosphates. Suitable preservatives include, among others, thimerosal, mertiolate, and gentamicin. The live attenuated rabies vaccines according to the invention can be administered to warm-blooded mammals, including humans, dogs, foxes, raccoons and skunks by injection (intramuscular, intradermal, or subcutaneous), spray or aerosol (intranasally), or by orally. Preferably, the vaccine is administered to subjects orally, especially in the case of wild animals or stray dogs. For oral administration, the vaccine is mixed with a suitable vehicle such as, for example, proteins or oils of animal or vegetable origin. For oral delivery, the formulation of the vaccine can also be encapsulated with baits prepared from metabolizable substances of vegetable or animal origin. The useful dose to be administered will vary, depending on the type of warm-blooded mammals to be vaccinated, the age, weight and manner of administration. In general, a suitable dose will vary between 102 to 108 TCID5o / mammal. The following examples will illustrate the invention without limiting the invention thereof.

METHOD AND MATERIAL Construction of cDNA clones Site mutagenesis directed by the method of Kunkel et al., 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods Enzymology, Vol. 154, pp 376-382 was performed with 21 mer oligonucleotides to change three nucleotides of pT7T-G (Conzelmann and Schnell, 1994; J. Virology, Vol. 68 , No. 2, pp. 713-719). The resulting plasmids encoded the modified RV glycoprotein (G protein) in which, Arg at position 333 (SAD B19 position 4370-4372) of the mature RV G protein was replaced with different amino acids (see Table I). In order to incorporate the introduced mutations into a full-length RV cDNA clone (pSAD L16), a fragment of StullPpuMI cDNA comprising SAD B19 4015-4470 nucleotides was changed.

Table I: RV cDNA clones and the codons that are encoded for the amino acid residues in the antigenic site III of the glycoprotein of the resulting recombinant RV mutant viruses.

*) Comparative examples: RV cDNA clones in which the codon differs by only two nucleotides of the Arg codon.

Recovery and propagation of antigenic site mutants III Transfection experiments were carried out as previously described (Conzelmann and Schnell, 1994, J. Virology, Vol. 68, No. 2, pp. 713-719). Approximately 10β BSR cells were infected with recombinant vaccinia virus vTF7-3 (Fuerst et al., 1986) and then transfected with a plasmid mixture containing 5 μg of pT7T-N, 2.5 μg of pT7T-P, 2.5 μg of pT7T-L and with 4 μg of a plasmid encoding the full-length antigenomic RNA when using the Stratgene mammalian transfection kit (CaPO4 protocol). The isolation of the transfectant virus and the elimination of the vaccine virus was carried out as described in Schnell et al. , 1994 supra. Infection of the cells was monitored by means of direct immunofluorescence with an anti-RV nucleoprotein conjugate (Centocor) and the recombinant RVs were also passed until infection of the entire monolayer was achieved. The strains of the resulting virus were concentrated by endpoint dilution. Twenty-five steps in series were carried out in BSR cell cultures at a multiplicity of infection (moi) of 0.01.

RT-PCR and sequence analysis To determine the stability of the recombinant viruses, 25 successive steps were carried out in the BSR cells. The RT-PCR was performed in 1 μg of the total RNA isolated from the infected cells using the "Giant Tube RT-PCR System" according to the instructions of the suppliers (Boehringer Mannheim) .The PCR products were analyzed in 1% of agarose gels and were used directly for the sequence.

Mice inoculation and virus neutralization Groups of 3-week-old NMRI mice were inoculated intracerebrally (ic) with 0.03 ml of a virus suspension (3,000 to 9,000,000 ffu / mouse) and observed for rabies symptoms. To determine if the pathogenic revertants appear after passing in absorbing mice, the recombinant viruses were inoculated ic in two-day-old mice. A 20% brain suspension was prepared from killed mice and inoculated into 3-week-old mice. Serum samples were collected from the surviving mice 21 days after infection. To determine the neutralizing activity of mouse serum, the 5-fold dilution of the serum was incubated with 40 ffu of CVS strain. After 1 hour the BHK cells were added in the virus-serum mixture, incubated for 24 hours and examined by means of direct influencescence. For data see the table Table II: Mutants of antigenic site III; mutants corresponding to the cDNA clones of Table I (rec = recombinant; RV = rabies virus; pfu = plaque formation units; ic = intracerebral; ffu = focus formation units; W = weaned mice 3 weeks after age; S = 2-day-old absorbent mice; Ab = antibody; ND = not done)

Claims (7)

1. CLAIMS 1. The mutant of recombinant rabies virus comprising a mutation in the viral genome, whereby said mutation comprises at least one substitution of the Arg333 codon with a codon that differs by three nucleotides from said Arg333 codon.
2. 2. A mutant according to claim 1, characterized in that said mutant is a mutant of the SAD strain.
3. 3. A mutant according to claim 1 or 2, characterized in that the codon Arg333 is replaced with a triplet GAC or a triplet CAC.
4. 4. A mutant according to claim 3, characterized in that the mutant is mutant strain of SAD D29 recombinant rabies virus.
5. 5. The recombinant rabies virus mutant according to any of claims 1-4, for use as a prophylactic or therapeutic agent.
6. 6. The recombinant rabies virus mutant according to any of claims 1-4, for use in a vaccine.
7. 7. The live attenuated rabies vaccine characterized in that said vaccine comprises a recombinant rabies virus mutant according to any of claims 1-4 and an acceptable pharmaceutical carrier.