Dna construct viable chimeric recombinant flavivirus, the vaccine against tick-borne encephalitis virus, the method of preventing infection of a mammal with a virus tick-borne encephalitis

 

The invention relates to biotechnology. Proposed DNA construct viable chimeric recombinant flavivirus containing the structural genes encoding proteins preM and E Langat virus, a protein With Langat virus or mosquito virus non-structural genes of the mosquito flavivirus. The DNA construct used in the composition of the vaccine against tick-borne encephalitis virus. A vaccine against tick-borne encephalitis virus used in the method of preventing human infection with the tick-borne encephalitis virus. The vaccine provides long-lasting protection against subsequent infection with tick-borne encephalitis virus. 3 S. and 12 C.p. f-crystals, 7 tab., 7 Il.

Field of invention the Present invention relates to a chimerical viral vaccine against tick-borne encephalitis virus (VIK) and other virulent relatives. More specifically, the invention relates to a chimerical virus containing the genes for structural proteins gem and E Langat virus (LGT), associated with the genes of non-structural protein mosquito flavivirus.

The level of technology in the Family Flaviviridae comprises, within the kind of portable arthropods of flaviviruses, more than sixty antigenically-related viruses containing positively to close the Press, New York, 961-1035, 1996). Among them, the virus mosquito yellow fever, Japanese encephalitis virus, dengue viruses (DEN) viruses of tick-borne encephalitis (tick-borne encephalitis), the latter are endemic in most European countries, Russia, India and Northern China. Tick-borne encephalitis is transmitted exclusively by ticks and can be subdivided into two serologically distinct subtype: Eastern subtype (prototypical strain Sofjin), predominant in Siberia and far East regions of Russia, and the Western subtype of (prototypical strain Neudoerfl), common in Eastern and Central Europe. Tick-borne encephalitis causes serious encefalite disease with a mortality rate ranging from 1% to 30%.

All flaviviruses the same genome organization: 5'-C-preM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3', where the first three genes encode the proteins of the capsid (C), premembrane (REM) and envelope (E), and the remaining genes encode non-structural proteins. Gomologichnosti between mosquito and tick flaviviruses is relatively low. (Chambers et al., Annu. Rev. Environ. 44:649-688, 1990; Pletnev et al. . Virology 174:250-263, 1990). However, gomologichnosti among mosquito flaviviruses and tick flaviviruses relatively high (lacoco-Connors et al. Virology 188:875-880, 1992).

There are four serotypes of dengue virus (type 1 - type 4), distinguished by neutralizaci tests using polyclonal sera (Bancroft et al. Pan Am. Hith. Org. Sci. Publ. 375:175-178, 1979; Henchal et al., Am. J. Trop. Med. Hyg. 31:548-555, 1982). These four serotypes of dengue virus have the same genomic organization. The complete nucleotide sequence determined for dengue virus types 3 and 4 and several strains of the virus type 2, including neurovirulence for mice new Guinea mutant (Mackow et al. Virology 159:217-228, 1987; Zhao et al., Virology 155:77-88, 1986; Osatomi et al., Virology 176:643-647, 1990; Irie et al., Gene 75:197-211, 1989; Mason et al., Virology 161:262-267, 1987; Hahn et al., 162:167-180, 1988).

Despite the considerable evolutionary distance between DEN and tick-borne encephalitis, was created viable chimeric flavivirus containing genes With REM-E or REM-E structural protein of the virulent far Eastern Russian tick-borne encephalitis with other genes nonstructural proteins and 5'- and 3'-non-coding sequences derived from DEN [tick-borne encephalitis(CME)/DEN and tick-borne encephalitis(IU)/DEN respectively] (Pletnev et al., Proc. Natl. Acad. Sci. U. S. A. 89:10532-10536, 1992).

Tick-borne encephalitis(IU)/DEN saves neurovirulency for mice its tick-borne encephalitis ancestor, from which derived its genes gem and E, but he has no peripheral neurovirulence VIK, i.e., the ability to extend from a peripheral position in the Central nervous system (CNS) and cause fatal encephalitis. However, mice previously inoculated with chimeric Vij doses of highly virulent tick-borne encephalitis. Neurovirulency this Chimera was significantly reduced by the introduction of its RHEM, E or non-structural viral protein 1 (NS1) of a single mutation (Pletnev et al., J. Virol. 67:4956-4963, 1993). These amino acid substitution caused the restriction of viral replication in tissue culture cells as monkeys and mosquitoes. However, parenteral inoculation of these additionally attenuated chimeric mutants were generated in mice full resistance to fatal encephalitis caused intracerebral inoculation neurovirulence chimeras tick-borne encephalitis(IU)/DEN.

The Langat virus (LGT) is the least virulent of all flavivirus tick-borne encephalitis complex, but closely antigenic associated with highly virulent far Eastern tick-borne encephalitis (Calisher et al., J. Gen. Virol. 70:37-43, 1989; DeMadrid et al., J. Gen. Virol. 23:91-96, 1974; lacoco-Connors et al. Virus Res. 43:125-136, 1996) and has a high level of gomologichnosti sequence (lacoco-Connors et al., Virology 188:875-880, 1992; Mandl et al. Virology 185:891-895, 1991; Shamanin et al., J. Gen. Virol. 71:1505-1515, 1990). The LGT virus was tested in experimental live vaccine against tick-borne encephalitis in the early 70's (Ilenko et al., Bull. Wid. Hith. Org. 39:425-431, 1968; Mayer et al. Acta. Virol. 19:229-236, 1975; Price et al., Bull. Wid. Hith. Org. 42: 89-94, 1970). Several strains LGT, which were attenuated for mice and monkeys, were selected and tested for 800000 adults who mandates virus Elancia, was associated with a very low incidence of encephalitis, i.e. one in 20,000 vaccinations (Mandl et al., above).

Currently available experimental vaccine produced by inactivation of tick-borne encephalitis formalin; however, this vaccine has several limitations. For example, this vaccine is highly immunogenic, therefore, to generate a protective immune response aboutsa re-vaccination. Even when the reaction of antibodies to the vaccine are present, the vaccine is not able to provide a protective response to the virus in up to 20% of the population. Therefore, there remains a need for reliable, more effective vaccine against tick-borne encephalitis. The present invention provides such a vaccine.

Disclosure of the invention One implementation of the present invention is a viable chimeric recombinant flavivirus containing the first section of a nucleic acid operatively encoding structural proteins gem and E virus, Langat, operatively associated with the second plot nucleic acid operatively encoding non-structural proteins mosquito flavivirus. Preferably Langat virus is a strain TR nemotivirovannoe virus Langat or more attenuated mutant strain E5 Langat. Predpochtitelno dengue is type 4. Alternative mosquito flaviviruses is the yellow fever virus. In accordance with another aspect of this preferred implementation of the first section of nucleic acid operatively encodes a protein capsid mosquito flavivirus or virus Langat. In yet another aspect of this preferred implementation recombinant flavivirus further comprises at least one mutation. Preferably recombinant flavivirus inserted into the expression vector. Mainly the expression vector is a plasmid.

The present invention also provides a host cell stably transformed with recombinant flaviviruses, as described above, providing the mentioned expression construct DNA. Preferably the host cell is prokaryotic. In another aspect of this preferred execution of the tick-borne encephalitis virus is selected from the group consisting of viruses Eastern subtype, Western subtype, Omsk hemorrhagic licorace, louping ill, kazanowski forest disease, Negishi or Powassan.

Another implementation of the present invention is a vaccine against tick-borne encephalitis virus containing chimerical recombinant flavivirus, who spent mammals against infection by tick-borne encephalitis virus. Another implementation of the present invention encompasses immunogenic composition containing the above-described chimerical recombinant flavivirus in a pharmaceutically acceptable carrier.

The present invention also provides a method of preventing tick-borne encephalitis infection in a mammal, containing the step of introducing to a mammal effective to prevent tick-borne encephalitis number of chimerical recombinant flavivirus, and this chimerical flavivirus the first section contains a nucleic acid operatively encoding structural proteins, REM and E Langat virus or protein With mosquito flavivirus, and structural proteins gem and E virus, Langat, operatively associated with the second plot nucleic acid operatively encoding non-structural proteins mosquito flavivirus, in a pharmaceutically acceptable carrier. Preferably the mammal is man. Mainly the step of introducing is intranasal, transdermal, subcutaneous, intramuscular or intravenous. In one aspect of this preferred implementation is effective to prevent tick-borne encephalitis ranges from about 1 g to about 1000, the Method may further comprise the introduction of one or the persons stimulate the immune response, directed against discussed above chimerical recombinant flaviviruses, mlekopitayushchikh containing the step of introducing to a mammal effective to prevent tick-borne encephalitis number of chimerical recombinant flavivirus, and the chimerical flavivirus the first section contains a nucleic acid operatively encoding structural proteins, REM and E Langat virus or protein With mosquito flavivirus, and structural proteins gem and E virus, Langat, operatively associated with the second plot nucleic acid operatively encoding non-structural proteins mosquito flavivirus, in a pharmaceutically acceptable carrier.

A brief description of the drawings Fig.1 shows a comparison of tick-borne encephalitis viruses/DEN and LGT/DEN.

Fig. 1A shows the location of the 5'-terminal sequences of non-coding area (NCA) attenuated tick-borne flavivirus LGT, strain TR (PEFC. 1), its more attenuated derivative LGT strain E5 (PEFC. 2) and virulent tick-borne encephalitis-strains Sofjin (VCAS; th. 3) and Neudoerfl (VCAN; th. 4) and virulent North American strain of Powassan (PHS; th. 5). Nucleotides identical to the sequence of strain LGT E5, indicated by dots, and the input to alignment gaps are shown by dashes. Rooms follower is zevah GCC-sequences weakened tick flavivirus LGT, strain TR (PEFC. 1), its more attenuated derivative LGT strain E5 (PEFC. 2) and virulent tick-borne encephalitis-strains Sofjin (VCAS; th. 3) and Neudoerfl (VCAN; th. 4) and virulent North American strain of Powassan (PHS; th. 5). Nucleotides identical to the sequence of strain LGT E5, indicated by dots, and the input to alignment gaps are shown by dashes. Sequence numbers are right. The initiation codons and stop underlined.

Fig. 2A-2C illustrate the growth of the parent and chimerical of flaviviruses in LLCMK2 cells apes (Fig.2A), Vero cells apes (Fig.2B) and the cells mosquitoes (Fig.2C). The inoculum chimerical virus was grown in cages of mosquitoes. The inoculum of the parent viruses LGT TP21 and E5 were grown in Vero cells apes. The inoculum of virus parent DEN for cells mosquitoes and cells of apes grew, respectively, in the cells of mosquitoes and Vero cells apes. LLCMK cells2or Vero apes were infected: (i) virus DEN, TP21 or E5 with the multiplicity of infection (CI) equal to 0.01, or (ii) chimerical virus TR/DEN or E5/DEN with KI equal to 0.5. Cells C6/36 mosquito was infected with: (i) virus DEN, TR/DEN or E5/DEN when KI is equal to the oral titer was determined by analysis of the plaques on the same cells, which were used to study virus replication. Plaques were counted on the seventh or eighth day after infection. Reducing the size of the plaque shows that viruses are limited in growth phenotype.

A detailed description of the preferred executions of the Present invention includes the observation of the fact that chimerical viruses LG/DEN containing structural proteins gem and E strains TP21 or E5 LGT, find a limitation in the growth and formation of plaques in inoculation in cell cultures of apes. E5 is more attenuated derivative TP21 (Thind et al., Amer. J. Epidemiol. 84:198-213, 1966), which differs from it in 24 nucleotides, producing 11 changes the encoding, four of which - protein E (table 2). These chimeras LG/DEN were at least 5000 times less neurovirulent than the parent viruses LGT in the newborn mouse (table 3). These chimeras were also no detectable visibility of neuroinvasion after intraperitoneal inoculation of 105plaque-forming units (PFU) Swiss mouse or 107The BATTLE SCID mouse. On the contrary, TR or E5 matter DM50the mouse SCID equal of 0.004 and 0.06 FIGHT accordingly. Despite the fact that the Chimera otsutstvoval the Bo from viruses excited LGT-neutralizing antibodies and resistance to fatal encephalitis, caused by the introduction LGT TP2L Thus, the construct LG/DEN useful as a vaccine against all strains of tick-borne encephalitis, including Eastern and Western subtypes, all of which are closely antigenically related. Although to obtain chimerical vaccine was used DAN as mosquito virus, refers to the use of other strains of dengue virus (i.e. types 1, 2 and 3) due to the high level of gomologichnosti sequence and close antigenic relationship of these other types and type 4. Additionally, the use of chimerical vaccine containing cDNA of non-structural protein of any mosquito flavivirus (including yellow fever virus), optionally containing protein With mosquito flavivirus or virus Langat, in combination with cDNA LGT, encoding proteins gem and E, also corresponds to the scope of the invention. These chimerical constructs containing mutations, insertions, deletions or substitutions in any of the viral genes, which does not detract from the ability of the construct to provide protective immunity to the introduction of the virulent parental virus tick-borne encephalitis.

The complete nucleotide sequence was determined for genome nemotivirovannoe LGT virus (strain TR) and its more attenuated derivative E5 received potamic constructs of the expression vector LG/DEN by replacing the genes of structural or non-structural proteins LGT TP21 or LGT E5 corresponding genes DEN using well-known from the prior art in molecular biology methods, described in such sources as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1988; Ausubel, Current Protocols in Molecular Biology, 1989). Only two of the 16 tested chimerical constructs (shown in Table 1A) was found to be viable, which was determined by their ability to grow and develop in culture viral particles (table 1B). These two chimerical virus contains genes gem and E strain TR or strain E5 virus LGT and all other sequences of the virus DEN. These chimeras differed in their derivatives from LGT sequences of only four amino acid positions in the protein (table 2). Chimerical viruses TR/DEN and E5/DEN was analyzed from the point of view of efficiency of their growth in cell culture, neurovirulence and neuroinvasiveness (peripheral neuroinvasiveness) for mice, immunogenicity and effectiveness of protection. These properties were compared with the same properties of their parent LGT, as well as the properties previously described for highly virulent closely related tick-borne encephalitis.

Chimerization LGT TP21 or LGT E5 mosquito DEN significantly reduces the replicative ability of the resulting virus in cells of apes compared with any of the ro is by using DEN, with the assumption that this may be a General phenomenon for viruses group of tick flaviviruses. Thus, chimerical viruses showed a very low neurovirulence its parent mosquito DEN and not higher neurovirulency for mice its parent tick LGT-virus.

The present invention relates to recombinant chimerical DNA construct containing a DNA fragment encoding the proteins gem and E LGT-virus nonstructural proteins of dengue virus and vector. This fragment can encode rekultivirovanie proteins or mutant proteins containing point mutations, insertions, deletions or the like, which do not reduce the reliability and effectiveness of vaccines derived from this chimerical construct. The reliability and effectiveness of any such chimerical virus can be determined using methods described here without undue experimentation.

In yet another implementation of the present invention relates to a chimerical vaccine against tick-borne encephalitis to humans, containing DNA encoding the proteins gem and E rekultivirovannyh TR and LGT E5 and nonstructural proteins of dengue virus. These chimerical vaccines evaluated in primates is STI, measured neurological symptoms after direct intranasal or intracerebral inoculation or peripheral inoculation; (3) immunogenicity, denoted by the type and amount or antibody-based test response following viral infection that meets the immunogenicity and protective efficacy; and (4) protective efficacy, as measured by resistance to infection with virulent virus tick-borne encephalitis following immunization. Chimerical vaccine, showing markedly reduced virulence, but retaining sufficient immunogenicity in monkeys was assessed during clinical tests on humans.

For use as a vaccine, the chimerical flavivirus according to the invention comprise the formula with a pharmaceutically acceptable excipient and parenteral introduced into a mammal, preferably human. "Pharmaceutically acceptable" means that the agent must be acceptable in the sense of compatibility with other ingredients of the formula, and also in the sense of security for the patient. Such carriers are phosphate-buffered saline (PBS) solution and lactate ringer's solution. Implied modes of administration include intradermal, intravenous, subcutaneous and any other mi etc. The number of vaccine drug, administered to a mammal, typically range from about 1 to 1000 g, preferably from about 50 g to 500, the Method may further comprise the introduction of one or more booster injections of chimerical flavivirus in the amount of from about 1 g to about 1000, Although the exact number of recombinant virus can vary depending on the individual, this quantity can be optimized using conventional experiments of the type dose-response, well known to experts.

Flavivirus LGT and DAN were obtained, chemerisov and evaluated on neurovirulency and protective efficacy against infection parent LGT-virus, as described in the following examples.

Example 1 Sources of virus and virus isolation Strain TR nemotivirovannoe LGT was originally isolated from ticks in Malaya in 1956 (Gordon-Smith, Nature 178:581-582, 1956). Then he was moved 11 times in the mouse brain and twice in Vero cells apes. LGT TP 21 used here was obtained from Dr. R. Shope (Yale University, New Haven, CT) from the collection of the Rockefeller center. Attenuated strain LGT E5 was obtained from strain TR by 42 transfers within seven e is, 966; Thind et al., Amer. J. Epidemiol. 84:214-224, 1966). This virus was obtained from Dr. J. Huggins (USAMRIID, Frederick, MD). The Langat virus was purified from platelets three times on Vero cells under the soft agar to prepare viral raw material, which was titrated109plaque-forming units PFU/ml of virus TR and 1.2109PFU/ml of virus E5. Vero cells were grown at 37oWith in minimum mandatory environment (BMU) Needle plus 10% fetal calf serum (FCS) and were incubated in an atmosphere of 5% CO2. Virus DEN (clone 2A), extracted from the DNA construct of full-length genome of strain 814669 dengue virus type 4, was used as the parental virus DEN (Lai et al., Proc. Natl. Acad. Sci. U. S. A/ 88:5139-5143, 1991). All these viruses are publicly available in the research community.

Example 2 Genomic sequence of the virus Langat Cleaning LGT and the allocation of its RNA was performed as previously described for tick-borne encephalitis (Pletnev et al. Virology 174:250-263, 1990). First strand cDNA was synthesized using the system SuperscriptTMpurification of reverse transcriptase (GibcoBRL, Life Technologies) and synthetic nucleotide primer, complementary to the saved 22 3'-terminal nucleotides strains of tick-borne encephalitis (Mandl et al. , J. Virol. 65:4070-4077, 1991; Wallner et al. Virology 213:169-178, 1995) Imerese (PCR) was used to amplify three overlapping cDNA fragments of the genome LGT TP21 or LGT E5. Sequences of PCR primers were derived from the published site coding LGT TP21 (lacoco-Connors et al. Virology 188: 875-880, 1992; Mandl et al. Virology 185:891-895, 1991). The PCR fragment corresponding to the 5'-noncoding the plot was produced using a primer containing the first 21 saved 5'-terminal nucleotides of the genome of the tick-borne encephalitis (Dobrikova et al. , Bioorg. Chem. 21:528-534, 1995; Mandl et al., Virology 166:197-205, 1988; Mandl et al., J. Virol. 65:4070-4077, 1991). All ascending primers contained the Pvul site crushing. Each PCR product was cloned in E. coli BD1528 using p5'-2(NotI, Xhol, HindIII) vector (Cahour et al. , Virology 207:68-76, 1995). The complete nucleotide sequence of the genomes of TP21 and E5 were determined by building sequences of three overlapping cDNA clones on both strands of DNA using the method of termination dideoxynucleotide chain (Sanger et al., Proc. Natl. Acad. Sci. U. S. A. 74: 5463-5467, 1977). Were constructed sequences for several independent clones of each third of the genome.

The sequence analysis showed that the genome TR or E5 was long 10940 or 10941 nucleotide, respectively, and contained a single open reading frame that encodes polyprotein from 3414 amino acids. Sequence data were ispolzovany for communication of the virus LGT TP21 or E5 with other members of the genus of flaviviruses. About 84.2 per cent. On the contrary, the total gomologichnosti sequences LGT and DEN, mosquito flavivirus, was only 39.4 percent. Among tick flaviviruses LGT and tick-borne encephalitis structural proteins C, REM and E are the least similar (74, 75 and 88% of gomologichnosti respectively), while non-structural proteins are more similar sequences (90-95%).

Fig. 1A-1B show the sequence 5'- and 3'-non-coding area (NCA) viruses LGT E5 and TP21 compared with previously published sequences of Powassan virus, tick-borne encephalitis viruses of the European subtype of (prototypical strain Neudoerfl) and the far Eastern subtype (strain Sofjin). 5 NKU LGT E5 is the length 130 nucleotides and differs from 5 NKU virus TP21 with the exception of nucleotide G at position 61 and the presence of nucleotides instead of G at position 35. The combination of 5 NKU genomes of complex TBE virus shows that the two domains between nucleotides 1-30 and 82-129 stored in the respective areas of tick-borne encephalitis virus, LGT and Powassan virus (PHS). The sequence between these domains is hypervarieties, and the genomes of SURFACE and LGT undergo in this area is an exception in comparison with strains of tick-borne encephalitis. Among tick flavivirus 3'-non-coding sequence varies considerably in length: LGT E5 or TR contain the IG.1B) showed that last 95 3'-terminal nucleotides are very similar in sequence all flaviviruses. Only four nucleotide at the site is distinguished by a virus LGT E5 or TR from far Eastern strain of tick-borne encephalitis. The difference in length 3 NCU tick flaviviruses was observed primarily between the stop codon and the last 325 3'-terminal nucleotide, the latter is a plot that shows a high degree of sequence similarity. Genome LGT contains: (i) the insertion of the 172 or 80 nucleotides in this area compared with the far Eastern strain of tick-borne encephalitis or SURFACE and (ii) exception 182 nucleotides compared to the European strain of the subtype of tick-borne encephalitis. 3 NCU strain LGT E5 differs from its parent LGT TP21 inserting dinucleotide (AU) between the positions 10515 and 10516 and with the exception of s and U in the positions and 10599 10633, respectively.

The full sequence of the parent LGT TP21 was compared with the sequence of its weaker derived LGT E5 with an attempt to identify potential genetic determinants of neuroinvasiveness and neurovirulence. This analysis revealed 24 nucleotide differences, eleven of which caused amino acid substitution in the corresponding polyprotein (table 2). Amino acid event of changes protein E, showed that C1436>U was represented in three of the four clones. Interestingly, mutation of Asn668>Asp protein E strain E5 corresponds to the substitution, discussed earlier in protein E partially attenuated mutant tick-borne encephalitis (Holzmann et al., J. Virol. 64:5156-5159, 1990; Mandl et al., J. Virol. 63:564-571, 1989). To identify the relative importance of the 11 amino acid differences for virulence strains TR and LGT E5 genome cDNA DAN introduced different combinations of viral genes LGT by replacing the corresponding genes DEN, as described in Tables 1A, 1B.

Example 3
Construction of chimeras LGT/DEN and determining the viability of the
Chimerical viruses LG/DEN was built to analyze the genetic basis of low neurovirulence, tissue tropism and peripheral invasionist virus Langat and to develop reliable and effective vaccine is a live attenuated virus against antigenically related tick-borne encephalitis. cDNA DEN full length was used to build the chimerical constructs containing the genes for C-preM-E preM-E, NS1-NS2A, NS1-NS2A-NS2B-dNS3 or NS2B-NS3 LGT, with other sequences, derived from DEN (table 1A). In each example, the limit of the sequence of the corresponding cDNA fragments LGT TP21 and LGT E5 used to konstruirovanie. Natl. Acad. Sci. U. S. A. 89:10532-10536, 1992) was used to replace two or more genes LGT corresponding genes DEN. Oligonucleotide-directed mutagenesis was performed to introduce customers instead of Clal unique Asp718 site at the 3' end of the sequence of dengue p2A(XhoI). To simplify the construction of chimerical cDNA LGT TR(CME)/DEN or LGT E5(CME)/DEN (table 1A) plot cDNA encoding genes, REM and E DIN coming from BglII (nucleotide 88) to the Xhol site (nucleotide 2342), was replaced by the corresponding sequence of the virus TR or E5. To build chimerical cDNA LG(IU)/DEN containing genes gem and E strain TR nemotivirovannoe virus Langat or more weakened derived E5, were created four different connections between the genome WITH DEN and genome REM LGT in chimerical DNA plasmids (table 1A). For example, the construct number 3 LG(IU)/DEN were prepared as follows. The PCR fragment containing the genes REM-E between the introduced site > PST TP21 (nucleotide 422) or E5 (nucleotide 423) and the Xhol site (TP21 nucleotide 3279 or E5 nucleotide 2380) near the 3' end of the gene, was inserted into the vector DEN substituting the appropriate sequence DEN, producing a Chimera that contains genes gem and E VIK described previously (Pletnev et al., Proc. Natl.verified by constructing sequences by site. The functional integrity chimerical viruses was demonstrated by their ability to direct the synthesis of protein using lysate of reticulocyte rabbit or temporal expression of the virus T7-cowpox (EIroy-Stein et al., Proc. Natl. Acad, Sci. U. S. A. 86:6126-6130, 1989).

Example 4
The growth of chimeras in cell culture
The RNA transcripts of the full length, made with templates chimerical cDNA, described above, were tested for infectivity by transfection of cells LLCMK2apes, Vero cells of apes and cells C6/36 mosquito in the presence of DOTAP (Pletnev et al., above). Nine days after transfection the cells in the plate with 24 holes were transferred to a plate with 6 holes and a glass slide. On day 12, and then again on 16, 20, 24, 28, 32, 36 and 40 days, cells were split and moved. In addition, the cells in each of these days were tested by immunofluorescence assay (ELISA) for the presence of antigens DEN and LGT using a solution of 1: 300 DEN or LGT-specific hyperimmune mouse ascitic fluid (GMAG). Infectious virus was played only two of the 16 constructs (8 LGT TR/DEN and 8 LGT E5/DEN), shown in Table 1B, and only in the cells of mosquitoes. These chimerical viruses IN for chimeras attenuated strain E5 LG/DEN, containing genes gem and E LGT E5. When ELISA showed that 70-100% of the cells were infected cells in the plate with 6 grooves mixed with twice as many uninfected cells, and the resulting mixture was inoculates in the bottle 75 cm2held incubation for 7 days. Infected cells were collected together with the environment, was mixed with an equal volume of fetal bovine serum (FBS), frozen at -70oWith and used later as an inoculum for the preparation of suspensions of virus-offspring. The titer of such viral suspensions were determined by analysis of plaques in cells C6/36 mosquito.

Chimeras TR/DEN and E5/DEN were reinforced once in cells C6/36 in bottles of 75 cm2. Viral RNA was then isolated and back transcribed using oligo 2634, which is complementary to the sequence DEN between nucleotides 5090-5110 (PEFC. 11). Single-stranded cDNA was used as template, PCR is used with pairs of primers oligo 239 (PEFC. 12) oligo 444 (PEFC. 13). The PCR products were cleaved with HindIII and BamHI and cloned in the vector pGEM3. The sequence of sites connections LG/DEN was confirmed by direct analysis of the cloned DNA inserts.

During the initial characters DAN and preM LGT genes or E LGT and NS1 DEN. It is important that chimeras differ in their descending from LGT sequences in only four amino acid positions in the protein (table 2). In addition, immunoprecipitate viral proteins from infected cell cultures mosquito showed that both of these chimeras produced the expected proteins. LGT-protein preM TR/DEN or E5/DEN was sensitive to cleavage by endoglycosidase F or H, while the protein E resisted cleavage by these enzymes. This shows that LGT-protein E, expressed in cells of the mosquito any of these chimeras was not glycosylated, despite the presence of a potential glycosylation site in the E-sequence.

Chimeras LGT TP21 and E5/DEN were compared with each other and with their parent viruses with regard to replication model and the maximum reproduction in cells of apes and mosquitoes (Fig.2). Inoculation with multiplicity of infection (CI) equal to 0.01, chimeras were grown to a moderate titer in cells mosquitoes, namely from 104,8up to 106,0PFU/ml on the Contrary, the growth of their parent virus LGT TP21 or E5 in the cells of mosquitoes was completely restricted fees when cell cultures were analyzed using analysis of plaques in the cell mosquito who was naujalis, if for inoculation of cells mosquitoes less parental virus LGT (within CI 0.01 to 100). In addition, using ELISA failed to detect the observed viral replication in either of these examples. The titer achieved chimeras in the cells of mosquitoes on the fifth day, was compared with DEN 10-100 times. Unlike DAN these two chimeras were initiated chronic recitations infection of cells mosquitoes. Also compared to DEN these two chimeras were produced smaller plaques. The size of the plaques was equal to average 5.0 mm for TR/DEN, 2.0 mm for E5/DEN and 11.5 mm for DIN.

When analyzing cells of apes as cell substrates had different hierarchy of viral replication. Chimerization LGT TP21 or E5 with DAN significantly reduced the efficiency of viral replication in cells of apes compared with the parent virus LGT TP21 or E5, and compared with DEN. Therefore, chimerical viruses LG/DEN growing up in cages of mosquitoes were incapable belascoaran in the cells of apes. In addition, these chimeras are not replicated as efficiently in cells of apes, as was shown by the analysis of plaques in cells of mosquitoes.

Her efficiency and grown to high titer, that is approximately 108up to 109PFU/ml, a level greater than the level achieved DEN.

Cells of apes were not completely resistant to chimerical viruses due to the fact that the virus multiplied in permissive insect cell culture, initiated a slow and partially limited viral replication in LLCMK cells2or Vero apes, inoculated with KI equal to 0.5. In addition, the spread of the virus in these cell cultures was different from the distribution in the parent virus LGT THR or E5, which was cytopathic and reached high titers of the fifth day of inoculation with the CI of 0.01. In contrast, infection of cells of apes any of these chimeras with KI of 0.5 did not produce cytopathic effects and progressed very slowly, as monitored using ELISA. For infection 80-100% of the cells of apes was required incubation period of 24-48 days. At this time established chronic infection without cytopathic effects, and these chronically infected cells could be maintained in the incubation and subculture within 10 months without obvious visible effect. The reproduction of the virus from these cleocib, and it was found that it reduced by 90% compared to the level achieved during the growth of any of the chimerical viruses in cells of mosquitoes. Reproduction of any of the chimerical viruses from infected cells LLCMK2apes do not form plaques on these cells. The same situation was observed for chimeras LGT E5/DEN growing up in Vero cells apes. On the contrary, reduced the number of very small weak plaques produced by Chimera LGT TR/DEN in these cells, which correlated with reduced replication of the chimeras in the cells of apes. While chimerical virus TR/DEN or E5/DEN collected from cells of apes, chronically infected for 8 months, was moved in mosquito cell culture and reached a high titer, replication of the virus in the cells of apes supported their limited growth and belascoaran.

These results show that chimerization LGT TP21 or LGT E5 using DAN significantly reduces replicative ability of these chimeras in the cells of apes than any of the parental viruses. This represents a contrast to the previous is edah apes (Pletnev et al., above).

Neurovirulency and neuroinvasiveness chimerical virus TP21, E5 or LG/DEN were studied on live models-mice, as described in Examples 5 and 6 below.

Example 5
Research neurovirulence
Neurovirulency viruses LGT TP21, LGT E5, DEN and chimerical TR/DEN or E5/DEN was estimated at outbred Swiss mice three days of age. A group of 10-14 mice were inoculators vnutriarterialno (CC) decimal virus solutions in the range of 102up to 107The BATTLE in 20 l modified eagle medium (ISI), containing 0.25% human serum albumin. Mice were followed for 28 days on the subject of fatal or non-fatal encephalitis.

Rekultivirovannyh LGT TP21 was highly virulent, as shown by its SD50is equal to 0.4 FIGHT a newborn mouse, that is one BATTLE was fatal to newborn mice (table 3). Neurovirulency more attenuated strain LGT E5 was in the same situation 50 times lower. DEN was still less neurovirulent as illness or death was observed only when the newborn mouse was inoculates large dose. Vnutricerepnae SD50DEN was estimated 8000 FIGHT. Chimerical TR/DEN or E5/DEN prooi inoculation into the brain of newborn mice. Thus, vnutricerepnae SD50TR/DEN was $ 2500 FIGHT that approximately 6250 less than that of its parent LGT TP21 (0,4 FIGHT). E5/DEN was further weakened with CC SD50>105The COMBAT. In addition, E5/DEN was 44 times less neurovirulent than TR/DEN. Thus, chimerical virus TR/DEN or E5/DEN kept for mice low neurovirulency its parent DEN, not high neurovirulency its parent virus Langat. Similarly, chimeras LG/DEN were at least 6250 times less neurovirulent for mice than their parent LGT TP21 or E5. This is a significant reduction in neurovirulence seems corallium with limited growth of these chimeras in the cells of apes.

The surprise was a huge reduction in neurovirulence chimeras LGT TR(IU)/DEN and LGT E5(IU)/DEN compared to tick-borne encephalitis(IU)/DEN (table 3). Chimeras TR/DEN and E5/DEN were respectively 125 and 5500 times less neurovirulent than tick-borne encephalitis is a Chimera.

Example 6
Research neuroinvasiveness
Neuroinvasiveness (peripheral neurovirulency) the parent or chimerical viruses was evaluated in female Swiss mice three weeks of age, which were, 106or 107The BATTLE of the virus LGT TP21 or LGT E5; or (ii) 105The BATTLE of the virus TR/DEN, E5/DEN or DEN. Mice were observed for 21 days, and the surviving mice were taken blood for evaluation or antibody-based test reactions. Survivors mouse on the following day received the WB 100 or 1000 WB SD50the parent virus TP21 and was observed up to an additional four weeks.

LGT TP21 was also moderately virulent for mice three weeks of age at inoculation peripheral route. WB SD50approximately 5103The COMBAT. On the contrary, chimerical TR/DEN showed lower peripheral neuroinvasiveness adult mice when WB SD50>105The COMBAT. Attenuated strain LGT E5 showed a much lower peripheral virulence than its parent LGT TP21. Only 10-20% of adult mice inoculated WB using the 107The BATTLE LGT E5, manifested symptoms of encephalitis, making it difficult to detect the impact of chimerization on neuroinvasiveness. However, data related to LGT TR/DEN show that chimerization LGT TP21 using DEN reduces or eliminates neuroinvasiveness LGT for mice.

More sensitive analysis of neuroinvasiveness contained inoculation immunomedics the functions, including functional macrophages, normal or increased function of NK-cells and increased hemolytic activity of complement. In this analysis of female mice SV-17 ICR/scid/scid three weeks of age (Bosma et al., Nature 301:527-530, 1983) groups of 5 animals were inoculators WB: (i) 105or 107The BATTLE of the virus DEN, TR/DEN or E5/DEN or (ii) the decimal solutions LGT TP21 or LGT E5 from 104up to 107The COMBAT. Then these mice were observed for mortality within 6 weeks.

The ability immunoregulatory SCID mice to detect neuroinvasiveness TP21 or E5 was approximately 106.3-108.8times greater than was observed for immunocompetent Swiss mice (table 3). Markedly increased susceptibility to fatal disease was noted when SCID mice were inoculators WB any of the parent LGT viruses. SD50LGT TP21 for immunoregulatory mice, WB-the route was equal 0,004 FIGHT (table 3). In addition, LGT E5 was also highly virulent for SCID-mice when WB route, with SD50equal to 0.06 FIGHT. The incubation period before the onset of encephalitis was 12 days, and death occurred within the following five days. As the parent virus DEN, both chimerical LGT-virus did not show obnaruzhenie 6-week postinoculation observation period. These discoveries show that chimerical TR/DEN or E5/DEN completely lost neuroinvasiveness his LGT-parent for SCID-mice.

Itself the parent virus TP21 or weakened derived strain E5 may not be suitable for people as a live attenuated vaccine against tick-borne encephalitis complex viruses. The appearance associated with encephalitis vaccine, although with a very low frequency (10-4.3), which was observed during large-scale clinical trials with the most attenuated vaccine candidate (virus Elancia) against tick-borne encephalitis, support this view (lacoco-Connors et al., Virology 188:875-880, 1992; Smorodincev et al., in Tick-borne Encephalitis and its Vaccine Prophylaxis, Leningrad, 1986). Live attenuated encephalitis flavivirus possibly not must have a very low level of virulence. In contrast to the parent LGT viruses assessment chimerical viruses TR/DEN and E5/DEN on the model of SCID-mice showed that these two chimeras were completely avirulent and showed no neuroinvasiveness in this very sensitive analysis system, since a large dose (107FIGHT any of these chimerical viruses could not affect the Central nervous system and cause encephalitis or death after inoculation in perifericheskaya encephalitis necessary proteins, encoded by genomic sites LGT other than genes gem and E.

Neuroinvasiveness should be distinguished from neurovirulence. So, LGT E5 showed moderate neurovirulency inoculation of the virus directly into the brain of newborn mice (DM50equal to 20 COMBAT), while its SD50when inoculation WB was >107The COMBAT. It is clear that neurovirulency required LGT to cause encephalitis by peripheral inoculation route, as if the WB inoculation. However, to achieve a virus of the brain require other properties of these flaviviruses.

Example 7
Immunogenicity and protective efficacy chimerical viruses LGT/AEH4 against virus Langat
To determine the immunogenicity and protective efficacy of chimeras LG/DEN used the mouse. Mouse inoculators WB using the 102The BATTLE TR or E5 or 105The BATTLE TR/DEN or E5/DEN, or antibody-based test developed strong reaction to the virion TP21, as measured by fermentativnogo immunosorbent assay (ELISA; table 4). In addition, these mice developed high levels of neutralizing antibodies to LGT TP21, as measured by the reduction of plaque. In contrast, mice inoculated with the WB using the 105The BATTLE DIN, failed the inoculation TR/DEN, E5/DEN, TP21 or E5 mouse infected by WB using 100 or 1000 WB SD50TP21 (table 5). All mice that were previously immunized with TR/DEN or E5/DEN, has developed high titers of neutralizing antibodies and were completely protected from WB infection TP21. In contrast, mice immunized with WB using DEN, were protected only partially: three of ten mice died at infection 100 WB SD50LGT TP21. All 30 unimmunized control animals fell ill with encephalitis and 25 of them then died. As discussed previously, these results indicate that the immune response to non-structural proteins DAN was unable to provide complete protection from LGT TP21 for mice.

In a subsequent study (table 6) each of the 5 mice inoculated with 105The BATTLE DIN, couldn't resist infection with 1000 WB SD50LGT TP21, while each of the five mice immunized with WB using the 105The BATTLE chimerical TR/DEN or E5/DEN experienced the same infection LGT TP21. Thus, it appears that proteins gem and E LGT are the major protective antigens of chimeras responsible for full resistance to lethal infection LGT.

Perhaps the immunogenicity and protective efficacy of radicals outstudy in viral drugs inoculated WB, instead of using antigens produced replicated in vivo virus. This assumption arose by assessment or antibody-based test responses and protective efficacy of viral preparations containing only 10 BATTLE. In addition, chimeras was assessed at two levels of infectivity, namely 103or 105The BATTLE (table 6). When each of the three evaluated doses of virus were tested without modification or after complete decontamination using UV irradiation. Before performing this study, the time required for complete decontamination of infectivity using UV was determined 60 minutes using a kinetic analysis.

Mice were observed for 21 days, and the surviving 22 days after inoculation took blood for evaluation or antibody-based test response as measured by ELISA or test reduction neutralization of plaque. Surviving mice on day 24 WB were infected with 1000 WB SD50(5107The COMBAT) LGT TP21 and were examined in the next four weeks.

This low number, like 10 FIGHT any chimeras, regularly incited neutralizing and ELISA antibodies with a titer of 1:50 or higher (table 6), whereas only 1 of 10 mice that received UV-deactivated 105FIGHT him the appropriate antibodies. The response of these mice infected TP21 (1000 WB SD50), consistent with an immunological reaction. Only one of the 10 mice, WB inoculated with 10 the BATTLE of Himera, could not resist the infection, while 7 out of 10 mice that received UV-deactivated 105The BATTLE, died after infection. It seems that successful immunization these two chimeras primarily affects the action of viral replication, and not on the effect of a large mass of previously formed viral antigens.

Example 8
Immunogenicity and protective efficacy chimerical viruses LGT/DEN against infection with tick-borne encephalitis
To determine the immunogenicity and protective efficacy of chimeras LG/DEN used the mouse. Twenty-three days after inoculation TR/DEN, E5/DEN or DEN mice were infected with 103or 105The FIGHT tick-borne encephalitis or one of its solenoidality related tick-borne flaviviruses, such as a virus Omsk hemorrhagic fever virus forest disease Kyasanur, Negishi virus, Powassan virus or a virus, Central European tick-borne encephalitis. All previously immunized with TR/DEN or E5/DEN mice produce high titers of neutralizing antibodies and fully protected from the WB infection tick-borne encephalitis. Naprawianie control the mice developed encephalitis and died. These results indicate that the immune response to non-structural proteins DEN not able to provide complete protection to mice against tick-borne encephalitis or solenoidality relatives.

In an additional study, none of the groups of mice inoculated with 105The BATTLE DIN, could not resist the infection of tick-borne encephalitis, while each of the groups of mice immunized with the WB using the 105The BATTLE chimerical TR/DEN or E5/DEN experienced the same infection tick-borne encephalitis. Thus, it appears that proteins gem and E LGT represent major protective antigens of chimeras responsible for full resistance to lethal infection with tick-borne encephalitis.

Perhaps the immunogenicity and protective efficacy of the parent viruses LGT and their DEN-chimeras arises from immunization with the previously formed antigens present in viral drugs inoculated WB, and not of the antigens produced replicated in vivo virus. This assumption arose by assessment or antibody-based test responses and protective efficacy of viral preparations containing only a limited amount of virus. In addition, chimeras was assessed at two levels of infectivity, namely 103or 105The COMBAT. When each of the three on Before performing this study time required for full decontamination infectivity using UV was determined 60 minutes using a kinetic analysis.

Mice were observed for 21 days, and the surviving 22 days after inoculation took blood for evaluation or antibody-based test response as measured by ELISA or test reduction neutralization of plaque. Surviving mice on day 24 WB were infected with 1000 WB SD50Tick-borne encephalitis and were examined in the next four weeks.

This low number, like 10 FIGHT any chimeras, regularly incited neutralizing and ELISA antibodies with a titer of 1:50 or higher (table 6), while only a small percentage of mice that received UV-deactivated 105The BATTLE of Himera, has developed a low level of ELISA antibodies, and none of the mice had not developed a noticeable neutralizing antibodies. The response of these mice infected TR (1000 WB SD50), consistent with an immunological reaction. Only a small percentage of mice, WB inoculated with 10 the BATTLE of Himera, could not resist the infection, while the majority of mice that received UV-deactivated 105The BATTLE, died after infection. It seems that successful immunization these two chimeras primarily affects the action Viru the aims chimerical flavivirus vaccines in primates
Intranasal infection of monkeys was an experimental system infection in natural conditions, for testing and predicting the effectiveness of tick-borne encephalitis vaccines for use in humans. Hambleton, P., et al. Infect. Immun., 40:995-1003 (1983). This report showed that the responses of rhesus macaques to intranasal tick-borne encephalitis infection similar to the reactions of people, with the exception that there were no signs of fever. The objects of this study associated with vaccinated monkeys against infection with tick-borne encephalitis are: (1) evaluation of the immunogenicity of different chimerical recombinant flavivirus vaccine candidates; and (2) evaluation of the protective efficacy of the above vaccines against infection with virulent strains of tick-borne encephalitis or closely related viruses.

We used a group of adult rhesus monkeys (Masasa mulatta) of both sexes, weighing from 1.8 to 5.1 kg Animals were placed in cages and they were given power in accordance with standard protocols known and accepted in the prior art. For infection and all procedures sampling of animals was done anesthesia by intramuscular injection with the help of such well-known from the prior art of a suitable agent, such as ketamine hydrochloride (Vetalar; Parke, Davis &Yong complex tick-borne encephalitis. For example, the virus Russian spring-summer encephalitis virus (far Eastern), Central European encephalitis (Western), Omsk gemorragicheskoj fever, louping ill, forest disease Kyasanur, Negishi or Powassan can be used to test the effectiveness of the vaccines of the present invention.

A group of monkeys, selected for the study was divided randomly into experimental group and control group. The experimental group of monkeys was vacciniavirus effective dose chimerical flaviviruses vaccine of the present invention, while the control group of monkeys was left untreated. All monkeys were infected with 3108to 5108The BATTLE standard infecting drug selected tick-borne encephalitis. Animals were infected intranasally to give results similar to the results observed in humans infected with tick-borne encephalitis in vivo.

Blood (10 ml) were taken at the test monkeys for testing at intervals after infection with the help of well-known from the prior art standard methods. These samples were divided into component parts, and filtered samples sinnamonlove fluid (CSF) was taken in monkeys by cisternal puncture or other acceptable ways before infection and after 9-11 days after infection. These samples were filtered and stored at -20oWith in accordance with acceptable procedures known from the prior art. Part of unfiltered CSF were studied under the microscope for the presence of leukocytes.

Monkeys wordplays intervals from 4 days to 14 weeks after infection and at the last moment by intravenous injection of pentobarbital sodium. Necropsy was performed immediately. The brain and the spinal cord was removed, and tissue pieces from each region gathered with the aim of shedding virus. The remainder of the Central nervous system (CNS) were fixed in buffered 10% neutral formalin, as part of the liver, spleen, lung, kidney, and small intestine. After processing by standard methods and conclusions in paraffin wax of all these tissues were into fragments and stained with hematoxylin and eosin. Selected parts of the CNS was also painted by hematoxylin phospholipases acid, Luxology durable blue - crazylove purple and modification of Pisa and Marsland method Belkovskogo for neurons.

Most of the control monkeys infected intranasally tick-borne encephalitis, clinical neurological symptoms appeared between 10 and 15 days. These symptoms consisted in shaking hands, the rotation of the neck within 12-24 hours after the occurrence.

Clinical chemical analysis of blood and CSF shows that the changes in the test animals are the result of infection with tick-borne encephalitis, as well as provide a means of comparing animal and human data. In the blood: changes in the activity of certain blood components, such as certain enzymes serum, became apparent after approximately 10 days after infection. Could increase the activity of aspartate aminotransferase (AST). Could also increase the activity of dehydrogenase and creatine kinase. On the contrary, the activity exude alkaline phosphatase could be reduced progressively towards coinfections levels. In relation to the CSF: the activity of AST after infection could significantly increase, but the increase in the number of cells in the infection could not happen.

It was also observed other effects of a virus infection. Lesions were found in those infected animals showed clinical symptoms of the disease. These lesions were present principally in the cerebellum, the posterior part of the brain stem and vertikalno spinal cord, but weaker lesions could be present in the cerebral cortex and the midbrain. The virus can also temporarily move is necessary.

In contrast to the control group, the experimental group of monkeys were practically non-disease symptoms caused by infection with tick-borne encephalitis. The protection offered by a chimerical flaviviruses vaccine experimental group of monkeys, demonstrates the reliability and efficiency of chimerical flaviviruses of the present invention.

Example 10
A random experiment with chimerical viral vaccine LGT/AEH4
Chimerical viral vaccine LG/DEN produced as described in the above Examples, is used to test the reliability and effectiveness of these vaccines when used on people. This Example is based on research, about which wrote Harabacz, I., et al. Vaccine, 10(3): 145-150 (1992). One dose of the virus in the vaccine contains 1.0, 1.5 and 2.0 µg particles of recombinant virus. The test is designed in the form of expected multicenter controlled study "blind". A random sample of participants was carried out taking into account dose and vaccination schedule. Randomly ordered three dosage in the ratio of 1:1:1. Random assignment to the common or limited schedule is in the ratio 2:1. The research centres can be located in different European what W was seized by a group of healthy adults, regardless of gender. Age and gender balanced randomly distributed between the groups. All volunteers before the experiment is required to give its consent; the study was approved by the relevant ethical committees at the centers where the study was carried out.

In respect of the schedules of vaccination and surveillance: the "normal" schedule of immunization consists of vaccinations at 0, 28 and 300 days. Samples of blood or antibody-based test for analysis are taken in 0, 28, 42, 56, 300, 314 and 328 days. "Abbreviated" schedule of immunization consists of vaccinations at 0, 7 and 21 days. Blood samples collected at 0, 21, 28, 35, 49 and 321 days. Test the vaccine is administered by intramuscular injection in the deltoid muscle. For each subject within 28 days after immunization had been under surveillance to monitor side effects of the vaccine.

Or antibody-based test analyses were performed by using such standard immunological techniques, such as fermentatively immunosorbent assay (ELISA) test of inhibition of haemagglutination (TIG) and the test of neutralization (TN). These analyses are performed in accordance with well known from the prior art methods, for example, TIG can be performed in accordance with the method of Clark and Casals, Am. J. Trop. Med. Hyg., 7:561-573 (1958), ELISA, in accordance with the Heinz et al., Virolog is using doses of virus in 100 ID500.1 ml. of the Lower limits of seroconversion can be defined as 8 bit, 2 TON and 160 in ELIS.

The results show that chimerical flavivirus vaccine of the present invention are safe for use on humans. They also show that the immunized individuals produce seroconversion and produce antibodies to chimerical flavivirus vaccines. These antibodies produce results TIG and T, showing that the immune response achieved by the vaccine is to protect against infection with tick-borne encephalitis. Also this study showed that the side effects associated with vaccination, are small compared to tolerance reactions of the majority of immunized during the research subjects.

The above description of the invention given solely to support the understanding of the invention. It should be understood that variants of the invention, including all equivalents now known or later developed, shall be considered as included in the scope of the invention, limited only subsequent claims.


Claims

1. DNA construct viable chimeric recombinant flavivirus representing the organization the proteins RHEM, E Langat virus and protein With Langat virus or mosquito virus, operatively associated with the second plot nucleic acid that encodes a non-structural proteins mosquito flavivirus.

2. DNA construct viable chimeric recombinant flavivirus under item 1, in which the said Langat virus is a strain TR of Langat virus or strain E5 virus Langat.

3. DNA construct viable chimeric recombinant flavivirus under item 1, in which the aforementioned mosquito flavivirus is a dengue virus.

4. DNA construct viable chimeric recombinant flavivirus under item 3, in which the said dengue virus is a type of 4.

5. DNA construct viable chimeric recombinant flavivirus under item 1, in which the aforementioned mosquito flaviviruses is the yellow fever virus.

6. DNA construct viable chimeric recombinant flavivirus under item 1, which referred to the first section of the nucleic acid also encodes a protein capsid mentioned mosquito flavivirus.

7. DNA construct viable chimeric recombinant flavivirus under item 1, inserted into the expression vector.

8. DNA construct viable chimeric recombinant flavivirus is containing a series of DNA-construct viable chimeric recombinant flavivirus under item 1 in a pharmaceutically acceptable carrier.

10. The vaccine under item 9, which mentions the tick-borne encephalitis virus is a virus of the Eastern or Western subtype.

11. Method of preventing infection of a mammal with a virus tick-borne encephalitis, including the introduction of said mammal effective to prevent infection with tick-borne encephalitis virus number DNA construct viable chimeric recombinant flavivirus, these DNA construct viable chimeric flavivirus the first section contains a nucleic acid that encodes structural proteins RHEM, E Langat virus and protein With Langat virus or mosquito virus, operatively associated with the second plot nucleic acid that encodes a non-structural proteins mosquito flavivirus, in a pharmaceutically acceptable carrier.

12. The method according to p. 11, in which the aforementioned mammal is man.

13 the Method according to p. 11, in which the introduction mentioned effective for preventing infection with tick-borne encephalitis virus in the number of DNA-construct viable chimeric recombinant flavivirus is or intranasally, or intradermally, or subcutaneously, or intramuscularly, or intravenously.

14. FPIC is the number of DNA-construct viable chimeric recombinant flavivirus is within 1 - 1000

15. The method according to p. 11, characterized in that to impose an additional one or more booster injections mentioned DNA construct viable chimeric flavivirus.

 

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