MXPA02004033A - Attenuated recombinant rabies virus mutants and live vaccines thereof. - Google Patents

Abstract

The present invention describes recombinant RV mutants comprising a combined mutation in two different parts of the viral genome, involving the P and the G genes. The mutations in the P gene preferably encompass residues 139 to 170, more preferably residues 139 to 149, most preferably residues 143-149. The mutation can be a substitution or deletion of one or more amino acids in the above region, as well as combinations of deletion and substitution. Preferred mutants according to the invention may be obtained by deleting residues 143 to 149 or 139 to 149 of the phosphoprotein (P) of rabies virus and simultaneously replacing the Arg at position 333 of the glycoprotein into another residue, preferably Asp instead of Arg. Surprisingly, when these mutations were introduced into rabies viruses lacking Arg at position 333 of their G protein, a dramatic reduction in pathogenicity for suckling mice was observed. This unexpected finding has a profound advantage in developing more safe live attenuated rabies vaccines. The mutation in the G gene may comprise a mutation of the Arg333 codon into a codon that differs by one, two or three nucleotides from said Arg333 codon. Preferably the mutants are mutants of a RV strain in which all three nucleotides of the Arg333 codon are substituted.

Description

| F MOTANTES DB ATTACKED ECOMBINANT RABIES VIRUSES AND VACCINES LIVE OF THEMSELVES The present invention relates to attenuated rabies virus mutants comprising attenuation mutations combined in their glycoprotein and phosphoprotein genes. Rabies continues to be one of the most terrible infectious diseases that affect humans and animals, 10 despite the significant scientific advances in its prevention and control. Rage presents itself as a different problem in different parts of the world. In the most industrialized nations, the risk to human beings has been minimized significantly, mainly due to the 15 compulsory vaccination programs for dogs and other domestic animals. Although there is still rabies in wildlife in developed countries, the greatest remarkable progress has been made in the control and elimination of rabies from wildlife using oral immunization 20 of the wild carnivores. In contrast, rabies continues to be a major threat to public health, and it continues to cause numerous deaths of humans in less industrialized nations. Dog rabies is still epizootic in most countries 25 of Africa, Asia and South America, and in these countries the * Dogs are responsible for the majority of human deaths from the disease. Therefore, the introduction of new control strategies in addition to existing parenteral vaccination programs is a necessity. Because of the success in controlling rabies in wildlife, many developing countries are now volunteering to use oral dog vaccination. For the purpose of oral immunization of dogs, 10 the vaccine should be highly safe due to the very close contact between dogs and humans, than between wild animals and humans. Therefore, there is a progressive need for safe attenuated live rabies vaccines, which do not have residual pathogenicity or the potential to revert to 15 pathogenic variant. Rabies virus (RV) is a non-segmented negative-strand RNA virus of the Rhabdoviridae family. The rabies virus is composed of two main structural components: a nücleocapsid or ribonucleoprotein (RNP), and 20 an envelope in the form of a two-layer membrane surrounding the core of the ribonucleoprotein. The infectious component of all Rhabdoviruses is the nucleus of the ribonucleoprotein consisting of the RNA genome encapsulated by the nucleoprotein (N) in combination with two other 25 proteins, ie, RNA polymerase dependent on RNA (L) and phosphoprotein (P). The membrane surrounding the nucleus of the ribonucleoprotein consists of two proteins: a transmembrane glycoprotein (G) and a matrix protein () located at the inner site of the membrane. Protein G, which is also referred to as a spike protein, is responsible for cell attachment and membrane fusion in the rabies virus and is additionally the main target for the host immune system. It has been identified that the region of amino acids at positions 330 to 340 (referred to as the antigenic site III) of protein G is responsible for the virulence of the virus, in particular the Arg residue at position 333. - The currently available live attenuated rabies vaccines are based on attenuated rabies virus vaccine strains including the SAD Bern strain or the SAD B19 strain, however, these vaccines still have unwanted residual pathogenicity. Many attempts have been made to obtain non-pathogenic rabies virus strains for use in a live vaccine. European Patent 350398 describes a SAG1 mutant of avirulent rabies virus of the SAD Bern strain of rabies virus, in which the glycoprotein has Ser in place of Arg at position 333. The avirulent SAG1 mutant was obtained under selection pressure of specific monoclonal antibodies in the SAD Bern strain. In adult mice it has been found that SAG is non-pathogenic. Nevertheless, pathogenic reversals of the attenuated virus occurred at a frequency of 1 in 10,000 (Lafay et al., Vaccine 12, pages 317-320, 1994). The genetic instability of this mutant makes it unsuitable for safe vaccination. European Patent Application 583998 describes another attenuated rabies virus mutant, SAG2, in which Arg at position 333 has been replaced by Glu in the glycoprotein. SAG 2 is non-pathogenic for adult mice when administered by different routes. Because this mutant also has the potential to revert to the pathogenic parent strain, the vaccine is produced in the presence of specific monoclonal antibodies to prevent reversion (Blancou and Meslin, 1996; In Laboratory Techniques in Rabies, pages 324-337). Since these specific monoclonal antibodies are not present in inoculated animals, vaccination with that mutant still carries the risk that the mutant will revert to virulence in the inoculated animal, resulting in outbreaks of disease in the inoculated animals, and possible spread. from the pathogen to other animals. In addition, WO00 / 32755, the content of which is incorporated herein by reference, described stable attenuated rabies virus mutants, which possess Arg replacements at position 333 of the G protein with other amino acids that differ in all three nucleotides of the codon Arg333 in the mother virus. All of the live rabies vaccine viruses described above possess an amino acid that differs from Arg at position 333 of the glycoprotein are non-pathogenic for immunocompetent adult mice. However, they are still pathogenic when inoculated to suckling mice of 1-2 days of age, demonstrating the existence of residual pathogenicity, and the risk associated with it for immuno-deficient animals or humans. The present invention has conveniently solved this problem by generating rabies virus mutants, which have a reduced pathogenicity for lactating mice. The present invention describes recombinant rabies virus mutants comprising a combined mutation in two different parts of the viral genome, which involves * the P and G genes. Mutations in the P gene preferably encompasses residues 139 to 170, more preferably residues 139 to 149. The mutation may be a substitution or deletion of one or more amino acids in the anterior region, as well as combinations of deletion and substitution. Residues 138 to 172 of the rabies P virus have been mapped as the domains responsible for binding to the LC8 dinein light chain (Raux et al., 2000, J. Virol.

Volume 74, pages 10212-10216; Jacob et al., 20C0, Volume 74, pages 10217-10222). It has been suggested that the interaction between P and LC8 could be important for the pathogenesis of the rabies virus. However, a deletion of as many as 11 amino acids (residues 139-149) of this domain has no detectable effect on the pathogenicity of a conventional rabies vaccine strain. Surprisingly, when these mutations were introduced into rabies viruses lacking Arg at position 333 of their G protein, a dramatic reduction in pathogenicity was observed for suckling mice. This unexpected discovery had a profound advantage in developing safer living attenuated rabies vaccines. Preferred mutants according to the invention can be obtained by deletion of residues 143 to 149, or 139 to 149 of the phosphoprotein (?) Of the rabies virus, and simultaneously replacing Arg at position 333 of the glycoprotein in another residue. , preferably Asp instead of Arg. The mutation in the G gene may comprise a mutation of the Arg333 codon within a codon differing by one, two c three nucleotides from the Arg333 codon. Preferably the mutants are mutants of a strain of rabies virus in which the three nucleotides of the Arg333 codon are replaced. The mutants according to the invention are. preferably mutants of the SAD rabies virus strain, especially the SAD Bern and 5AD BIS rabies virus strains. The preferred rabies virus mutant according to the invention is a mutant of recombinant rabies virus in which the codon Arg333 in the genome of the rabies virus strain SAD B19 has been replaced with a GAC triplet, which codes for the Aspartic acid (Asp, D). We found that recombinant rabies virus mutants that possess the combined mutation in the genes? and G are not pathogenic or are much less pathogenic for lactating mice of 1-2 days of age, as opposed to individual mutants containing only the mutations described in the P gene or gene G. The introduction of the combined mutation within the The rabies virus genome rc affected the rate of virus growth in BSR cells, and the final titer was similar to the mother strain. In addition, the combined mutations introduced do not affect the immunogenicity of recombinant rabies viruses after oral administration in dogs. We measured similar r.ivele of rabies-specific antibody titers in dogs vaccinated orally with recombinant viruses that possessed either only the G protein mutation or the mutations combined in the proteins? and G. This makes the rabies virus mutants recombine in accordance with the invention, the safest live anti-virus vaccines. These highly alive live rabies vaccine viruses can be used not only to immunize against rabies, but also as vaccine vectors to protect humar, and / or animals from other infectious agents. In addition to 1 other five viral proteins, it has been shown that the rabies virus expresses a foreign gene in a stable manner for rr of 25 passages in series (Mebatsion et al., 1S9 PNAS, Volume 93, pages 7310-7314). Recently, the potential of vectors based on rabies virus as vaccines against other viral diseases such as HIV-1 was also demonstrated (Schnell et al., 2000, PNAS, Volume 9 pages 3544-3549). The introduction of the combined G and P protein mutations described above into rabies virus vectors will undoubtedly increase the safety of any vaccine vector based on rabies virus. The recombinant rabies virus mutants according to the invention can be obtained using recombinant DNA technology and site specific mutagenesis, to introduce the desired mutations, targeted genetic manipulation of the rabies virus can be performed using the reverse genetics system described by Schnell and collaborators, 1994; EMBO J. Volume 13, Number 1S pages 4195-4203, and European Patent Application 0 ~ 0 085, both of which are incorporated herein by reference. Site-specific mutagenesis can be performed using commercially available kits. A cDNA clone of infectious complet length (pSAD-L16) of the SAD B19 vaccine strain described in Schnell et al., 1994, was used as a basis for introducing single or combined mutations into the gene? and / G. The rabies virus mutants according to 1 invention can be obtained by a) the introduction of the desired mutation into the full-length cDNA clone of the rabies virus, b) the simultaneous expression of an RNA virus. of full-length antigenomic rabies of the modified and N, P, and L proteins of the rabies virus of the transfected plasmids within the cells of T7-RNA polymerase expression, and 3) isolation of the virus mutants of the rabies produced mediated-those cells. Recombinant rabies virus mutants according to the invention can be grown in cell culture derived from, for example, BHK cells or human diploid cells. The cultured viruses can be harvested by collecting the fluids and / or cells from the cell culture. The vaccine according to the invention can be prepared using standard techniques available in the art. In general, the vaccine is prepared by combining the attenuated recombinant rabies virus mutant, according to the invention, with a pharmaceutically acceptable diluent carrier. Carrier-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 pH regulators such as alkali metal phosphates (e.g., PBS) ), alcohols, polyole, and the like. In addition, the vaccine according to the invention may comprise other additives such as adjuvants, stabilizers, antioxidants, preservatives, and the like. Suitable auxiliaries include, but are not limited to, aluminum salts or gels, carbomers, nonionic block copolymers, tocopherols, monophosferase lipido A, muramyl dipeptide, oil emulsions (water / oil or oil / water), cytokines, and saponins such as Quil A. the amount of auxiliary added depends on the nature of the auxiliary itself. The stabilizers suitable 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. of pH as alkaline phosphates Suitable preservatives include, among others, thimerosai, merthiolate, and gentamicin The live attenuated anti-rabies vaccine according to the invention can be administered to warm-blooded mammals, including humans, dogs, foxes, raccoons , and skunks, by injection (intramuscularly, intradermally, or subcutaneously), spray or aerosol (intranasally), or orally.Preferably the vaccine is administered to the subjects orally, especially in the case of wildlife animals or dogs For oral administration, the vaccine is mixed with a suitable carrier such as, for example, proteins or oils of vegetable or animal origin. Oral ion, the vaccine formulation can also be encapsulated with baits prepared from substances that can be metabolized from animal and vegetable sources. The useful dose to be administered will vary, depending on the type of warm-blooded mammals to be vaccinated, the age, the passage and the mode of administration. In general, a suitable dose will vary between 102 to 108 TCID50 / mammal. The following examples will illustrate the invention, without limiting the invention thereto.

EXAMPLES: EXAMPLE 1: Generation and characterization of recombinant rabies virus with modifications in the binding site of the dinein light chain MATERIALS AND METHODS Construction of cDNA clones: With the purpose of introducing the desired mutations within the rabies virus genome, a 2.2 kb BstBl fragment comprising nucleotides 1497-3738 of the rabies virus strain SAD B19 was first cloned. (nucleotide numbering is in accordance with Conzelmann et al 1990, Viroiogy, Volume 175, pages 485-499; EMBL / GenBank accession number M31046) inside the vector pSK. Site-directed mutagenesis was performed using the "QuikChange Site Directed Mutagenesis Kit" in accordance with ccr. the manufacturer's instructions. The primer pairs # 142 and # 143 (Table 1) were used to suppress the nuclei at position 1940 to 1960, corresponding to amino acids 143 to 149 of the P protein of the rabies virus. A larger deletion covering nucleotides 1928 to 1960, corresponding to amino acids 139 to 149 of the P protein of the rabies virus, was also introduced, using oligonucleotides # 144 and 145 (Table.) The precision of the suppression introduced was confirmed. by sequencing the modified region Clones containing the desired deletions (7 or 11 amino acids) were digested with Ncol and SnaBl, and the respective fragments of -0.8 kb were used to replace the corresponding fragments of the virus cDNA clones. of full-length rabies The manipulation in pSAD-L16 (Schneii et al., 1994 EMBO J, Volume 13, pages 4195-4203), which represents the sequence pSAD-B19 gave rise to pL16-A? 7 or pL16-APll ( Figure 1), which possess deletions of 7 or 11 residues, respectively.After a similar exchange of modified DNA fragments of -0.8 kb in pSAD-D29, a full-length clone possessing Asp (D) instead of e Arg (R) at position 333 of the glycoprotein, full-length clones were obtained-pD29-AP7 and pD29-APll (Figure 1). These clones have the respective deletions in protein P and the substitution of R to D in protein G.

Recovery and propagation of recombinant viruses: transfected approximately 1.5 x 10c 3SR cells (Buchholz et al., 1999, J. Virol, Volume pages 251-259) with a mixture of plasmids containing: g of pT7T-N, 2.5 μg of pT7T -P, 2.5 μg of pT7T-L and with 1 of one of the full-length plasmids, using Strata mammalian transfection kit (CaP04 protocol). The supernatants of the transfected cells were removed, and infection of the cells was monitored by direct immunofluorescence with a nucleoprotein conjugate anti-rabies virus (Centocor). The recombinant viruses were further passed 2 or 3 times and the resulting virus stock was titrated by endpoint dilutions.

Replication of recombinant viruses in vitro: To compare the efficiency of virus production under multiple cycles of infection, T7 BSR cells were infected with a multiplicity of infection (m.o.i.) of 0.02. Supernatants were harvested two days after infection and were titrated by endpoint dilutions. For the one-step growth curve analysis, it is grown. 106 cells BSR-T7 / 5 in dishes of 3.2 centimeters of diérr.etrc during the night, and were infected in duplicates with ur.a multiplicity of infection (MOI) of 10 with the different recombinant viruses. After 2 hours of incubation at 37 ° C, the inoculum was removed and the cells were washed three times with PBS. The cells were supplied with 2.5 milliliters of fresh medium, and were further incubated at 37 ° C. At 4, 18, 24 and 48 hours after infection, IZZ μ was removed. of culture supernatants, and were titered in duplicate in BSR-T7 / 5 cells.

RT-PCR and Sequence analysis; To determine whether recombinant viruses maintain the introduced deletions, total RNA was isolated from BSR cells infected with level four deposition of the respective in-house viruses. RT-PCR was performed on 1 total RNA isolated from infected cells. The PCR products were analyzed on 1 percent agarose gels, and used directly for sequencing.

Protein composition of mutant viruses. To analyze the protein composition of the recombinant viruses, ~107 BSR-T7 / 5 cells were infected at an MOI of 0.02, and incubated for 2 days. The virions were then purified in the supernatant, and concentrated by centrifugation through a 20 percent sucrose cushion in a Beckman SW28 rotor at 25,000 revolutions per minute for 9C minutes. The pills were resuspended and mixed with pH buffer of protein sample to break the virions. The viral proteins were then decomposed from the purified virions by gel electrophoresis, sodium dodecylsulfate polyacrylamide (SDS-PAGE) and transferred to the PVDF membranes (illipore). After incubation with a blocking solution, the membranes were incubated with rabbit serum raised against the rabies virus ribonucieoprotein, S50 (Mebatsion, et al., Cell 84, pages 941-951, 1996) or an anti-human antibody. -LC3 pe-liclonal rabbit, R4058 (ing, SM, and RS Patel-King, J. Biol. Chem. 270, pages 11445-11452, 1995). The membranes were then incubated with goat anti-rabbit immunoglobulin G, conjugated with peroxidase. Proteins were visualized after incubation with peroxidase substrate (Vector).

Inoculation of mice. Intramuscularly (i.m.) or intracranially (i.c.) groups of 6 lactating Balb / c mice of 1-2 days of age each were inoculated with a volume of 0.03 milliliters of virus suspensions at different concentrations. The mice were observed to see if they had rabies symptoms for a total of 21 days. A brain suspension was prepared to 20 percent of the killed mice, and virus isolation was done in the cell culture. The lethal dose that killed 50 percent of the animals (LD50) was calculated using the method of Reed and Muenc.

Table 1. Sequence of primer pairs that will be used to introduce deletions within the gene? of rabies virus (strain SAD-B19) Sequence pairs in 5 '-3' orientation Position of P.-JClee-ices primers or amino acids suDrimidos # 142 GGA AAG TCT TCA GAG GGC CGA GAG CTC AAG 1940 A 1960 (amino acids 143 = # 143 CTT GAG CTC TCG GCC CTC TGA AGA CTT TCC 149 protein? # 144 CCC AAC CCT CCA GGA GGC CGA GAG CTC AAG 1928 A 1960 (amino acids 133 to # 145 CTT GAG CTC TCG GCC TCC TGG AGG GTT GGG 149 gold-eir.a?)! RESULTS The safety of live oral rabies vaccines is a major public health concern. The rabies virus murants that have a modification in the Arg 333 position of the glycoprotein are the most attenuated strains currently available. However, these strains are still sor-pathogenic for lactating mice of 1-2 days of age, demonstrating the potential danger of those live vaccines for immunocompromised animals or humans. Therefore, a safer strain for immunization against rabies using live vaccines is greatly needed. It was shown that the phosphoprotein (P) of the rabies virus interacts with the cytoplasmic dinoin LC8 light chain. The LC8 fixation domain was mapped to the central part of? (Residues 138 to 172). It is suggested that the interaction between? and LC8 could be involved in the pathogenesis of the rabies virus. Rabies virus mutants were constructed which contained deletions of 7 (amino acids 143 to 149) or 11 residues (amino acids 139 to 149) in the LC8 binding domain of the P protein. All mutants were successfully recovered in cell cultures. Identical strands were obtained from those of the mother viruses for all mutant viruses (Figure 2) and all the mutants were propagated in the cell culture as efficiently as the er parent strains. different time points (Figure 3).

To determine the level of expression of the P mutant proteins, and to elucidate the association between 1 = protein P and LC8, the proteins of the purified viruses were analyzed by Western immunostaining. The spots were incubated with a rabbit serum RNP anti-rabies virus (S50) or a polyclonal anti-LC8 antibody (R4053). the amounts of protein P or the indexes between proteins? and N of the mutant viruses were indistinguishable from those of the mother viruses, indicating that deletions of 7 or 11 amino acids from the LC8 binding site have no influence on the expression of mutated P proteins. In contrast to the respective mother viruses, no protein corresponding to LC8 could be detected in the mutant viruses with suppression at the LC8 binding site (Figure 4). This result demonstrates that the incorporation of LC8 within the membrane of the virions is triggered by a specific interaction between P and LC8, and the introduced deletions were sufficient to completely block the association between protein P and LC8. To compare the pathogenicity of the mutants derived from SAD-L16, intravenous mice of 1-2 days of age were inoculated intramuscularly: the mother virus, or with one of the mutants to be suppressed. (pL16-AP7 or pL16-APll) at a dose of 100 or 100,000 focus / mouse forming units. As shown in Figure 5, all lactating mice inoculated with 100,000 focus / mouse forming units died of rabies within 7 days after inoculation. Mice in all groups inoculated with 100 focus / mouse forming units died of rabies within 10 days after inoculation (Figure 5), indicating that the deletion of 7 or 11 residues of the LC8 binding site of the protein P does not significantly decrease the pathogenicity of the rabies virus for lactating mice. Mutants were then generated that contained the identical deletion of 7 or 11 amino acids in their P protein, as well as a substitution of Arg (R) for Asp (D) at position 333 of their G protein (Figure 1). These mutants (D29-AP7 and D29-AP11) grew in the cell culture at a titer similar to that of SAD-D29, which contained only the R substitution by D (Figures 2 and 3). To compare the pathogenicity of the combined mutants with that individual mutant rail, 1-day-old suckling mice were inoculated intramuscularly at a dose of 100 or 100,000 focus / mouse forming units. Within the following days after the inoculation, all mice inoculated with both doses of SAD-D29 died of rabies. In contrast, only 83 percent or 50 percent of the racor.es that had received a dose of 100,000 focus / mouse forming units of D29-AP7 or D29-AP11, respectively, speak. died of rabies 10 days after infection. Fifteen days after infection, mortality reached 100 percent and 83 percent, respectively, and remained the same until the end of the 21-day observation period (Figure 6). Surprisingly, no mortality occurred in the groups of mice inoculated with D29-AP7 or D29-APIL at a dose of 100 focus-forming units / mouse by the end of the 10 days after infection. Until the end of the 21-day observation period, no mortality had occurred in the group of mice inoculated with D29-AP11, whereas in the group of mice that were inoculated with D29-AP7 there was less than 17 percent (one of each six) of mortality (Figure 6). These unexpected results demonstrate that the combination of the mutation of the LC8 binding site and the amino acid change in Arg333 of the G protein considerably attenuates the virulence of the rabies virus for lactating mice. The degree of attenuation of 3-?? ?? 11 mediante was then analyzed by administration of graded doses within 1-day-old lactating mice by intramuscular or intracranial runes. As shown in Figure 7, the dose of D29-AP11 that was required to kill 50 percent (LD50 per 30 μ?) Of the 2-day-old lactating mice inoculated intramuscularly was 556 focus-forming units, while that the LD50 of the SAD-D29 virus was only 18 focus-forming units. This shows that D29-AP11 is attenuated as much as 30 times when administered by the intramuscular route. Interestingly, a very similar LD50 of 10 and 14 focus-forming units was obtained for SAD-D19 and 029-120, respectively, when the strains were administered by intracranial routes (Figure 7). This extraordinary attenuation after intramuscular administration, but not after intracranial administration, shows that D29-AP11 is inefficiently spread from a peripheral site of infection to the CNS, in comparison with the mother virus. These results demonstrate that the elimination of the LC8 ligand and the simultaneous substitution of R333 considerably attenuates 1 = pathogenicity of the rabies virus after inoculation. peripheral, and can be helpful in designing and developing vaccines based on highly safe live rabies virus.

Example 2: Safety and efficacy of recombinant rabies viruses in dogs, after oral administration MATERIALS AND METHODS To determine the safety and efficacy of recombinant viruses after oral administration, rabies seronegative dogs, three to four months of age, were divided into three groups of 4 dogs each. Groups 1 and 2 received a dose of 108 focus-forming units (ffu) in a volume of 2 milliliters of SAD-D29 or D29-? 11, respectively, by oral instillation. Group 3 served as a control. Saliva swabs were taken on days 1, 3, and 7 after vaccination to test the excretion of the vaccine virus by inoculation into cell cultures. Blood samples were taken from all the animals before vaccination, 2 and 8 weeks before the first immunization. Groups 1 and 2 were boosted with similar doses of the respective vaccines, 8 weeks after the first immunization. Again, blood samples were collected 2 weeks after the booster immunization. Heat samples were inactivated by heat for 30 minutes at + 56 ° C, and stored at -20 ° C until use. The absence / presence of antibodies against rabies was determined by the fast fluorescence fccc inhibition test (RFFIT), as described in "HC Laboratory Techniques in Rabies" (1996). Throughout the experiment the dogs were observed daily to see the general health conditions.

RESULTS All dogs remained clinically healthy throughout the 3-month observation period. All swabs of saliva collected at different time points were tested negative for the presence of vaccine viruses, indicating the absence of virus excretion at the indicated times. To determine the effect of deletion of 11 amino acids on the P protein on the immunogenicity of the virus, the dogs were immunized with SAD-D29 or D29-AP11 by oral instillation. Two weeks after the first immunization the rabies-specific titers in both groups of animals did not differ significantly (Figure 8). Since the administration of rabies vaccines by the oral route is less effective than parenteral administration, a booster dose was given 8 weeks after the first immunization. As shown in Figure 8, there was a comparable dramatic increase in antibody titers specific for rabies in both groups of aninaids immunized with either SAD-D29 or D29-AP11. These results demonstrate that the deletion of as many as 11 amino acids from the LC8 binding site of the P protein of rabies virus did not affect the immunogenicity of the virus when administered orally in dogs.

BRIEF DESCRIPTION OF THE FIGURES: Figure 1. A schematic representation of the order. of the rabies virus ger in the negative chain genomic RNA. (A) Order of the SAD-L16 gene, a recombinant rabies virus possessing the authentic sequence of SAD-319 (Conzelmann et al., 1990, Virology, Volume 175, pages 485-499). (B) Order of the SAD-D29 gene, a recombinant rabies virus that possesses a substitution of Arg (R) for Asp (D) at position 333 of the G protein of SAD-B19. The amino acid sequence (positions 138 to 150) is shown around the LC8 dinein light chain binding site of the phosphoprotein (protein P). 7 or 11 are mutants with the indicated deletions of 7 or 11 amino acids at the LC8 binding site of the P protein. Figure 2. Infectious titers of mutants of recombinant rabies virus. BSR cells were infected at one m.o.i. of 0.02 and incubated for 2 days. Supernatants were harvested and titrated by final dot dilution. The titles were expressed in focus formation units (ffu) logio / milliliter. -d7 and -dll represents -7 and -11, respectively. Figure 3. One-step growth curves of recombinant rabies viruses. BSR-77/5 cells were infected with the recombinant rabies viruses SAD-L16 (L16),? · 1? -7 (L7), L16-AP11 (Lll), SAD-D29 (D29), D29-AP7 ( D7), and D29-AP11 (Dll) at an MOI of 10. The aliquots of the cell culture supernatants were collected at the indicated time points, and viral titers were determined er. duplicates by serial dilutions. The titles were expressed in focus formation units (ffu) logio / milliliter. Figure 4. Recombinant rabies virus protein composition. Approximately 1C BSR-T7 / 5 cells were infected with the recombinant rabies viruses SAD-LI6 (L16), L16-AP7 (LA7), 1.16- ???? (A11), SAD-D29 (D29), 029 - ?? 7 (DA7), and D29-AP11 (DAll) at an MOI of 0.02. Two days after infection, the virions of the supernatants were purified on a 20 percent sucrose cushion, and the viral pills were analyzed by Western blotting. Using a protein marker as an indicator, the same spot was cut into two parts at approximately the ~ 20 kD position. Was the upper part of the stain incubated with Ry? anti-rabies virus (A), and the lower part of the spot was incubated with a rabbit polyclonal LC8 antibody (3). The incorporation of LC8 could only be detected in the SAD-L16 and SAD-D29 mother viruses, but not in the recombinant viruses that had deletions at the LC8 binding site. Figure 5. Pathogenicity of the rabies virus mutants, strain SAD-L16, L16-AP7 (L16-d7), and L16-AP11 (Li6-dll) in 1-day-old lactating mice. The lactating mice were inoculated with doses of 100 focuser / mouse forming units or 100, 000 focuser / rator forming units, intramuscularly, and were observed daily for a total of 21 days. The results recorded at the end of days 7 and 10 were presented. All animals in the three groups died of rabies within 10 days after inoculation. Figure 6. Pathogenicity of the rabies virus mutants, strain SAD-D29, D29-AP7 (D29-d7), and D29-AP11 (D29-dll) in 1-day-old lactating mice. Lactating mice were inoculated with doses of 100 focus / mouse forming units or 100,000 units. focus / mouse formers, intramuscularly, and were observed daily for a total of 21 days. The results recorded at the end of days 7, 10, 15, and 21 were presented. No mortality occurred in the group of mice that were inoculated with 10C focus / mouse forming units of D29-AP11 (D29-dll). Figure 7. Comparison of the degree of pathogenicity of recombinant rabies virus after intracranial (i.c.) or intramuscular (i.m.) inoculations. Two-day-old suckling mice were inoculated with SAD-D29 (D29), or D29-AP11 (Dll) by intracranial or intramuscular route at the indicated doses. After intracranial inoculation, the LD50 of SAD-D29 and D29-AP11 were 10 and 14 focus forming units / 30 μ ?, respectively. Whereas after intramuscular inoculation the LD50 of 029 - ?? 11 was 556 focus forming units, as opposed to only 18 focus forming units for SAD-D29, it is indicated that D29-AP11 is attenuated as much as 30 times. Figure 8. Immunogenicity of recombinant rabies virus vaccines after oral administration. Rabies seronegative dogs, three to four months old, were divided into three groups of 4 dogs each. Groups 1 (D29) and 2 (Dll) received a dose of 103 focus forming units (ffu) in a volume of 2 milliliters of SAD-D29 or D29-AP11, respectively, by oral instillation. Group 3 served as a control. Groups 1 and 2 were boosted with similar doses of the respective vaccines 8 weeks after the first immunization. Antibody titers specific for rabies were determined by the rapid fluorescent focus inhibition test (RFFIT) at the indicated time points.

Claims (1)

1. CLAIMS 1. A mutant of recombinant rabies virus comprising a mutation in the protein G, as well as a mutation in the protein P. 2. A mutant according to claim 1, wherein the mutation in the protein? it envelops one or more of the amino acids at positions 139 to 170 of the rabies virus phosphoprotein. 3. A mutant according to claim 1 or 2, wherein the mutation in the P gene is a deletion of amino acids 143 to 149, or 139 to 149.. A mutant according to any of claims 1-3, wherein the mutation in protein G is a substitution of the amino acid of arginine at position 333 for another amino acid. 5. A mutant according to claim 4, wherein the codon encoding Argj33 is replaced by a codon that differs in all three nucleotides of the codon Arg333 of the parent virus. 6. A mutant according to any one of the preceding claims, wherein the Arg33j is replaced with Asp. 7. A mutant according to claim 6, wherein codon A g333 is replaced with GAC. 8. A mutant according to any of the preceding claims, wherein the codon rgj33 is replaced with GAC, and wherein the mutation in the gene? is a deletion of amino acids 139 to 149. 9. A mutant according to any of the preceding claims, wherein said mutant is a mutant of the SAD Bern or SAD 3i9 rabies virus strain. 10. Recombinant rabies virus according to any of claims 1 to 9, which additionally expresses a nucleic acid encoding a heterologous antigen. 11. A recombinant rabies virus according to claim 10, wherein the antigen is derived from a human or animal pathogen. 12. A recombinant rabies virus according to claim 10 or 11, which additionally expresses rgen that encodes an immunostimulatory protein. 13. Recombinant rabies virus according to any of the preceding claims, for use in a vaccine. 14. live attenuated rabies vaccine comprising a mutant of recombinant rabies virus, according to any of claims 1-8, and a pharmaceutically acceptable carrier. 15. The vaccine according to claim 14, characterized in that it is formulated for oral use. SUMMARY The present invention describes recombinant RV recombinants comprising a combined mutation in two different parts of the viral genome, which envelops the genes? and G. Mutations in gene P preferably encompass residues 139 to 170, more preferably residues 139 to 149, more preferably residues 143-149. The mutation may be the substitution or elimination of one or more amino acids in the anterior region, as well as combinations of elimination and substitution. Preferred mutants according to the invention can be obtained by means of residues 143 to 149 or 139 to 149 of the phosphoprotein (P) of rabies virus and simultaneously replenish the Arg at position 333 of the glycoprotein in another residue, pre Asp instead of Arg. Surprisingly, when these mutations were introduced into rabies viruses that suffered from Arg at position 333 of their G protein, a dramatic reduction in pathogenicity was observed for suckling mice. This unexpected finding has a profound advantage in the development of safer live attenuated rabies vaccines. The mutation in gene G may comprise a mutation of the codon Arg333 to a codon that differs by one, two or three nucleotides from that codon Arg333 - Preferably the mutants are mutants of an RV strain in which all three nucleotides of the codon Arg333 are replaced.