Chimeric vaccine flaviviruses

 

The invention relates to medicine and relates to chimeric live attenuated infectious virus, which is used to create chimeric flavivirus vaccines. The invention includes a chimeric live attenuated infectious virus containing the yellow fever virus, which has the nucleotide sequence encoding a protein DDM-E, demeterova, shortened or metirovan so that the functional protein DDM-E is not expressed, but in the genome of yellow fever virus integrated nucleotide sequence encoding a protein DDM-Th of a second, different, flavivirus so that the protein DDM-E second flavivirus is expressed, as well as the nucleic acid molecule encoding the virus. The advantage of the invention is to create chimeric viruses, which can be used as live attenuated vaccines. 3 C. and 15 C.p. f-crystals, 7 ill., 6 table.

The present invention relates to attenuated viruses used as vaccines against diseases caused by flaviviruses.

Some members of the family flaviviruses represent a real or potential threat to the health of the population of the planet in the tea millions of residents of the Far East, at risk of this disease. The dengue viruses at a frequency of up to 100 million cases of primary dengue fever and more than 450 thousand cases of dengue haemorrhagic fever in the world is one of the most serious human diseases transmitted by arthropod vectors. Other flavivirus also cause a number of endemic diseases of different nature and able to conquer new regions due to changes in climatic conditions, the population dynamics of vectors and violations of the natural environment caused by human activities. These flavivirus include, for example, a virus encephalitis St. Louis, which causes sporadic but serious acute illness in the middle East, South East and West of the USA; West Nile virus, which causes fever, accompanied by acute encephalitis, which is widely distributed in Africa, the Middle East, the former USSR and in parts of the districts of Europe; the virus encephalitis Murray valley, which leads to local neuropathology, distributed in Australia; and tick-borne encephalitis virus, which is widespread on the territory of the former Soviet Union and in Eastern Europe, where widespread his case who is another member of the family flaviviruses: it is characterized by genome organization and duplication parameters similar, but not identical, in comparison with the above described flaviviruses. The HCV virus is mostly transmitted by parenteral type, it causes chronic hepatitis, which can develop into cirrhosis and hepatocellular carcinoma, and in the U.S. is the leading cause of liver lesions, which require orthotopic liver transplantation.

The Flaviviridae family of viruses isolated from alpha viruses (such as WEE, VEE, EEE, SFV, etc) and currently has three types - the actual flavivirus, pestivirus and hepatitis C. a Fully formed Mature virions flaviviruses consist of three structural proteins - shell (E), capsid (C) and membrane (M), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The immature flavivirus detected in infected cells revealed premembrane protein (DDM), which is a precursor protein M

After binding of virions to the cell receptors of the host protein E undergoes an irreversible conformational transformation under the influence of the acidic environment of the endosome, resulting due to the binding of the bilayer membrane of the virion and endocytotic vesicles, in the viral genome Perm in such a bind: it determines the that proteolytic cleavage of the precursor DDM is necessary for the emergence of competent binding of virions and with full infektsionistu viruses (Guirakhoo et al., 1991, J. General Virol, 72, Pt. 2:333-338). The influence of ammonium chloride in the late stages of viral replication were obtained virus encephalitis Murray valley (MVE) containing DDM: it has been shown that they are incompetent to merge. When using a specific sequence of peptides and monoclonal antibodies, it was shown that propeptide DDM interacts with amino acids 200-327 in the protein that is Such interaction is necessary to protect the protein E from irreversible conformational rearrangement determined by the maturation process in acidic vesicles in associtation metabolic mechanism (Guirakhoo et al., 1992, Virology, 191:921-931).

Cleavage of the precursor DDM with the formation of protein M occurs shortly before the release of virions with the participation of furino-like cell protease (Stadler et al., 1997, J. Virol, 71:8475-8481) that is required to enable hemagglutination activity, fusogenic activity and infectivity of virions. Protein M cleaved from the polypeptide his predecessor (DDM) for consensus serial the shell together with the protein that is

Sequences that result in the processing, are conservative not only flaviviruses, but also to other proteins that are not closely related viruses, such as proteins ARE of coronaviruses mice, RE of alpha viruses, influenza viruses and R160 retroviruses. Cleavage of the polypeptide predecessor is essential for the infectivity of the virus, but not for the education of its virions. It was shown that in the case of viral Chimera TBE/dengue-4 change site processing DDM reduce neurovirulence this Chimera (Pletnev et al., 1993, J. Virol. 67:4956-4963), which is consistent with earlier data that efficient processing of precursor DDM is necessary for the full manifestation of infectivity (Guirakhoo et al., 1991, CIT. above; 1992, CIT. above; Heinz et al., 1994, Virology, 198:109-117). Antibodies to the polypeptide DDM can mediate the expression of immunity, apparently due to neutralization facing virions, including reprezentirovanii DDM. The site of proteolytic cleavage of the protein RE of VEE virus (includes 4 amino acids) has been delegated with the use of site-directed mutagenesis infectious clone (Smith et al., 1997, ASTMH Meet., Dec. 7-11). Deletion mutants replicated with high efficiency, and proteins REO manifestation of 100% seroconversion and manifestation protection of all immunogenic monkeys from a lethal outcome.

The invention is a chimeric live attenuated infectious viruses, which include: (a) the first yellow fever virus (e.g., strain 17D), representing the live attenuated vaccine virus, the genome of which the nucleotide sequence encoding a protein DDM-E or demeterova, or shortened, or metirovan so that the functional protein DDM-E first flavivirus is not expressed; and (b) integrated into the genome of the first flavivirus nucleotide sequence encoding a viral envelope components (proteins DDM (E) a second, different flavivirus, thus, what protein DDM-E second flavivirus is expressed from the composition of the modified genome of the first flavivirus.

Thus, the chimeric virus comprises genes and encoded products required for intracellular multiplication relating to the first flavivirus genes and encoded products of the shell of the second flavivirus. Therefore, the virus containing all antigenic determinants responsible for the induction of neutralizing antibodies in infected chimeric virus, in this case will generate antibodies only in relation to the second flavivirus.

Preferred living briteney, is yellow fever virus. At least one of the known vaccine uses such a live attenuated virus: this vaccine is known under the brand name YF17D and used for vaccination of humans for over 50 years. The YF17D vaccine is described in a large number of publications, including materials of Smithburn et al. and Friston (Smithburn et al., 1956, "Yellow Fever Vaccination, World Health Org., p. 238; Freestone, 1995, In "Vaccines", 2d ed., eds Plotkin et al. , W. B. Saunders, PA). In addition, the yellow fever virus has been studied at the genetic level (Rice et al. , 1985, Science 229:726-733), and also provided information about the correlation of genetic and phenotypic parameters (Marchevsky et al., 1995, Amer. J. Trop. Med. Hyg., 52:75-80).

The preferred flaviviruses for use as the second flavivirus in the chimeras of the present invention, respectively, which are sources immunitysec antigen, are the Japanese encephalitis virus (JE), dengue virus (DEN, for example, any of the types of dengue 1-4), the virus encephalitis Murray valley (MVE) virus encephalitis St. Louis (SLE), West Nile virus (WN), tick-borne encephalitis virus (TBE) and hepatitis C virus (HCV). In addition, flaviviruses suitable for use as the second flavivirus are virus Cugnini, virus Central is asamushi forest disease virus Omsk hemorrhagic fever. The preferred chimeric virus of the present invention, the sequence encoding a protein DDM-E second flavivirus, is used to replace a sequence that encodes a protein DDM-E, in the genome of the first live yellow fever virus. The preferred chimeric virus sequence encoding a protein DDM-E, is derived from an attenuated virus strain, such as a vaccine strain. Also, as this will be the description hereinafter, the region of DDM in the composition of such a protein may carry the mutation, which prevents the processing with the formation of the Mature membrane protein.

Also the present invention is for the prevention or treatment flaviviruses infection in mammals, such as man, by introducing a chimeric flavivirus of the present invention that mammals, the use of chimeric flavivirus of the present invention in the preparation of medicinal products intended for the prevention and treatment flavivirus infections, molecules of nucleic acids encoding the chimeric flavivirus of the present invention, and methods of producing chimeric flavivirus of the present invention.

The invention has several advantages is smojiniu, they can be used for the formation of long-term immunity. Because these viruses include in genome replication genes attenuated virus (e.g., yellow fever virus strain 17D), the chimeric virus is attenuated to such an extent, which makes it safe for use in respect of the person.

Other options and advantages of the present invention will be clear from the following detailed description, drawings and claims.

Fig. 1 schematically represents the steps of the genetic manipulations that have been implemented with the aim of constructing a chimeric virus of the present invention, including yellow fever virus and Japanese encephalitis virus (YF/JE).

Fig. 2 shows growth curves of chimeric viruses YF/JE of the present invention in cell culture, suitable for the production of human vaccines.

Fig. 3 is a graph showing a comparison of the growth of viruses RMS (Research Master Seed, YF/JE SA14-14-2) and YF-Vax in the cell line MRC-5.

Fig.4 is a graph and a table showing the results of the analysis of neurovirulence chimeric virus YF/JE of the present invention, carried out on mice.

Fig.5 is a schematic diagram dvuhmestnoj system unit described in connection with the creation of chimeric virus YF/JE.

Fig. 6 is a schematic representation of the structure of modified clones YF, designed by deleterevision part of the NS1 protein and (or) the expression of foreign proteins under the control of its own customers log in to the ribosome (IRES). The figure shows only part of the E/NS1 genome of the virus. The stop codon is introduced in the section that encodes the C-end of the envelope protein that is a Translation down is initiated within intergenic open reading frame (ORF) by the action of factor IRES-1, thereby determining the expression of foreign proteins (such as proteins E1 and (or) E2 virus HCV). The second factor IRES (IRES-2) controls the translation initiation of unstructured segment of the yellow fever virus, which is expressed densely Packed truncated NS1 proteins (for example, NSldel-1, NSldel-2 or NSldel-3). The size of the deletions in the structure of NS1 is inversely proportional to the size of the ORF, which is connected with the IRES-1.

Fig.7 is a graph showing the response of neutralizing antibodies in mice, immunogenic chimeric vaccine YF/JE SA14-14-2.

The present invention is a chimeric flavivirus, which can be used in vaccination against flavivirus infections. Design and analysis of chimeric flavivirus of the present invention, such as chimeras,and encephalitis Murray valley (MVE), virus encephalitis St. Louis (SLE), West Nile virus (WN) virus, tick-borne encephalitis (TBE) and hepatitis C virus (HCV) was carried out as follows.

Flavivirus proteins was obtained by broadcasting a single long open frame (coding, for example, structural proteins: capsid (C), the precursor membrane (WG-M) and envelope (E) and nonstructural proteins) and then by a series of posttranslational processing stages proteolytic cleavage. The chimeric flavivirus of the present invention, as discussed above, are characterized by the substitution of sequences encoding proteins WG-M & E one flavivirus, sequences of protein-coding WG-M & E other flavivirus. Thus, the creation of such chimeric flaviviruses includes the formation of new combinations of bands and premembrane proteins, on the one hand, and enveloped and non-structural proteins (NS1), on the other hand, coming from two different flaviviruses. The splitting between the sections of each of these two groups of proteins (and the WG-M, on the one hand, and E and NS1) occurs in the natural proteolytic processing flavivirus proteins and requires the presence of signal sequences, f is s according to the present invention the virus signal sequence provided Chimera significant stability at this level, to accurately place the splitting of proteins between segments/WG and E/NS1 was effective. These signal sequences, supporting the chimeras described below. On the other hand, any of the many known signal sequences may be used in the design so that the composition of chimeras to join sequences encoding proteins With and WG-M or E and NS1 (see, for example, von Heijne, 1983, Eur. J. Biochem., 133:17-21; von Heijne, 1985, J. Mol. Biol., 184: 99-105), or, for example, using the known regulatory sequence, a specialist in the art may construct additional signal sequences that can be used in the chimeras of the present invention. Typically, for example, the signal sequence must include as its last amino acid residue with a small uncharged side chain, such as alanine, glycine, serine, cysteine, threonine or glutamine. Other requirements for the signal sequences are well known in the art (see, for example, von Heijne, 1983, CIT. above; von Heijne, 1985, CIT. above). Also the signal sequence of any virus, part of the chimeras can be stored completely or sobrannogo virus YF/JE Obtaining a full-sized matrices cDNA for chimeras YF/JE of the present invention, described below is based on a strategy similar to the one previously used by the developers of the regeneration process of YF17D material cDNA for the molecular-genetic study of the process of the propagation of the virus of yellow fever. This strategy is described, for example, Nestorovich et al. (Nestorowicz et al., 1994, Virology, 199:114-123).

In short, getting chimeras YF/JE of the present invention includes the following. The genomic sequence of yellow fever virus embed the two plasmids (YF5'3'IV and YFM5.2) that encode a sequence YF from nucleotides 1-2276 and 8279-10861 (YF5'3'IV) and from 1373-8704 (YFM5.2) (Rice et al., 1989, New Biologist, 1:285-296). Full-matrix cDNA obtained by ligating the appropriate restriction fragments obtained on the material of these plasmids. This method is most suitable from the viewpoint of ensuring stable expression sequences YF and obtain RNA transcripts with high levels of specific infectivity.

Strategy applicants used them in the construction of chimeras, includes replacement of YF virus sequences in the plasmid YF5'3'IV and YFM5.2 the corresponding sequences from the genome JE from the beginning of the protein DDM (478-th nucleotide; 128-I amino acid) carneceria stages, ensuring the integration or elimination of restriction sites in the composition of the sequences and YF and JE necessary to perform the ligation in vitro. The matrix structure intended for regenerating chimeric virus YF(C)/JE (DDM), shown in Fig.4. Using a similar strategy was designed second Chimera encoding a full structural component (DDM) of Japanese encephalitis virus.

Molecular cloning of the structural component of the JE virus Clones autotentichno genes encoding structural proteins of Japanese encephalitis virus, were obtained on material strain JE SA14-14-2 (live attenuated vaccine strain of JE virus), because of the biological characteristics and molecular parameters of this strain is well studied (see, for example, Eckels et al. , 1988, Vaccine, 6:513-518; viral strain JE SA14-14-2 is available from the Center for disease control in Fort Collins, Colorado and Arboviral research center, Yale University, new haven, Connecticut, USA, which are official in the United States reference centres for studying arboviruses world health organization). Was obtained viral strain JE SA14-14-2, located on cultivation level PDK-5: it pink. Used by applicants strategy involved the cloning of the structural segment in two parts that overlap by restriction site Nhel (1125 nucleotide genome JE), which can then be used to make ligating in vitro.

Pooling RNA was extracted from monolayer cultures of infected cells, LLC-MK2and synthesis of the first chain antisense cDNA was performed using reverse transcriptase with antimuslim primer (nucleotides 2456-2471 nucleotide sequence of the genome JE) carrying grouped restriction sites XbaI and NarI necessary, respectively, for cloning first part of the vector pBluescript II KS(+) and then the composition of plasmids YFM5.2 (NarI). After synthesis of the first chain cDNA was used to amplify by PCR sequences JE in nucleotides 1108-2471 using the same antisense primer and a sense primer (nucleotides 1108-1130 nucleotide sequence of the genome JE), including grouped restriction sites XbaI and NsiI required for cloning in the composition, respectively, pBluescript and YFM5.2 (NarI). Sequence JE were verified by restriction digestion and direct nucleotide sequencing. Nucleotide sequentially who eat antisense primer, corresponding to nucleotides 1116-1130 genome JE, and sense primer corresponding to nucleotides 1-18 genome JE: they both contain the restriction sites EcoRI. The obtained PCR fragments were cloned into the composition of pBluescript, and the sequence JE verify by direct nucleotide sequencing. All of the above together is the cloning of the genome sequences of JE in nucleotides 1-2471 (amino acids 1-792).

The construction of derived variants YF5'3'IV/JE and YFM5.2/JE To enable the C-terminal part of the protein shell JE in the site of cleavage YF E/NS1 unique restriction site Narl was made with the composition of plasmids YFM5.2, using oligonucleotide-directed mutagenesis of the signal sequence within the site of cleavage of the E/NS1 (nucleotides YF 2447-2452; amino acids 816-817) obtaining YFM5.2 (NarI). The transcripts, read with such matrices, supporting such a change, were tested for their infectivity: they showed specific infectivity, similar to the parent matrices (approximately 100 plaque-forming units on 250 ng of transcript). The sequence of JE in nucleotides 1108-2471 was subcloned from several independently obtained PCR clone pBluescript/JE part plasmids YFM5.2 (NarI) using the gorodki (nucleotides 1-118), adjacent to the site encoding the prM-E JE, were obtained by PCR amplification.

For obtaining sequences containing the connection capsid code YF and protein prM JE, antisense primer covering this segment, was used along with the sense primer corresponding to nucleotides 6625-6639 part YF5'3'IV, with the aim of forming PCR fragments, which are then used as antisense primers for PCR in combination with the semantic primers complementary sequences of the pBluescript vector, upstream of the restriction site EcoRI, for the purpose of amplification sequence JE (encoded in the reverse orientation in the vector pBluescript) from 477-th nucleotide (N-end protein prM) through available within the restriction site NheI to 1125-th nucleotide. The resulting panorama-fragments embedded in the plasmid YF5'3'IV using the restriction sites NotI and EcoRI. This design includes the SP6 promoter preceding the 5'-noncoding segment YF, and next in the following sequence: codes YF (C) JE (DDM), also including the restriction site NheI (1125 nucleotide JE) required for ligation in vitro.

Design YFM5.2 and YF5'3'IV, including restriction sites necessary for Legerova the th protein of JE as the site of ligation in vitro, excessive NheI site in the plasmid YFM5.2 (5459-th nucleotide) was removed. This was accomplished Nesmelova mutation sequence YF the nucleotide 5461 (T-->P: amino acid meaning - alanine, 1820-I amino acid). This site was included in the YFM5.2 by ligating the appropriate restriction fragments, and the YFM5.3(NarI)/JE contributed by replacing NsiI fragment/NarI encoding chimeric sequence YF/JE.

To create a unique 3'-restriction site for ligation in vitro, the website BspEl was formed so as to be located below the AatII site, normally used to create a full-sized matrices on the material plasmids YF5'3'IV and YFM5.2. (In the sequence that encodes the structural components of the JE virus, there are multiple restriction sites AatII, which does not allow to use them for the purposes of ligating in vitro). The restriction site BspEl created by Nesmelova mutation engine 8581-th nucleotide YF (A-->P: amino acid meaning - serine, 2860-I amino acid) and made part of YFM5.2 by replacing the appropriate restriction fragments. Unique restriction site was introduced in the composition YFM5.2/JE by replacing the fragment XbaI/SphI, and the plasmids YF5'3'IV/JE(DDM) by 3-is derived plasmids YFM5.2 (BspI) deletional sequence YF, located between the EcoRI sites at nucleotides 1 and 6912.

Currency cDNA JE-Nakayama part chimeric plasmids YF/JE because Of the non-obviousness of whether to function properly obtained by PCR structural segment JE SA14-14-2 in the context of sequences of chimeric virus, the applicants used a cDNA clone representing the Nakayama strain of Japanese encephalitis virus, which was studied in experiments on expression and its ability to induce the production of immunity to it (see, for example, McIda et al., 1987, Virology, 158:348-360; strain JE-Nakayama available from the Center for disease control in Fort Collins, Colorado and Arboviral research center, Yale University, new haven, Connecticut, USA). cDNA JE-Nakayama was built in the composition of the chimeric plasmids YF/JE using available restriction sites (HindIII to PvuII and BpmI to MunI) to replace a full plot of coding DDM-E in the composition dvuhmestnoj system, with the exception of a single amino acid in the 49th position (serine), which was left intact for the use of restriction site NheI for ligating in vitro. Full segment JE in clone Nakayama was sequenced to verify the authenticity replacement cDNA (table. 2).

< the full-size cDNA-matrices in principle correspond to as described by rice et al. (Rice et al., 1989, New Biologist, 1: 285-296) (see Fig.1). In the case of chimeric matrices plasmids YF5'3'IV/JE(DDM) and YFM5.2/JE split restrictase NheI/BspEI and ligation in vitro performed using 50 ng of purified fragments in the presence of DNA ligase of phage T4. The products of ligation linearized with XhoI to facilitate transcription. Transcript with SP6 promoter synthesized using 50 ng of purified matrix (quantitative control is carried out by incorporating labeled3H-UTP) and RNA integrity check using electrophoresis in sedentarism agarose gel. Output ranges from 5-10 g RNA per 1 reaction when using this procedure, most of the material presents a full-length transcripts. Transfection of RNA transcripts in the presence of cationic liposomes carried out as described by rice et al. (CIT. above) for YF17D. In initial experiments, we used cells, LLC-MK2for transfection and quantification of the virus on the material of which the applicants determine the suitability of these cells for virus and belascoaran testing of the parent strains of YF and JE. In table. 1 shows typical results of transfe the full line Vero were used to obtain infective populations of viruses, evaluation of labeled proteins and neutralization tests.

Sequencing the nucleotide sequence of the chimeric cDNA-matrices To identify the exact sequences of the envelope protein of strain SA14-14-2 and Nakayama conducted analysis of the nucleotide sequence of the plasmid comprising a chimeric cDNA YF/JE in respect of clones JE-segment. The differences between the nucleotide sequences of these structures in comparison with the published sequences (McAda et al., CIT. above) shown in the table. 2.

Structural and biological characterization of chimeric viruses YF/JE
Genomic structure of chimeric viruses YF/JE obtained in experiments for transfection was tested using the method of polymerase chain reaction with repertorium for viral RNA isolated from monolayers of infected cells. These experiments are done to prevent the likelihood that populations of viruses have been contaminated during the transfection procedure. For these experiments, the virus first passage were used to initiate the cycle of infection to prevent any artifacts that may be caused by the presence of residual transtitional viral RNA. Total RNA pools, e is with repertorium, using specific YF and JE primers, which allow you to select the entire structural segment in the form of two PCR products with a length of approximately 1000 nucleotides. These products are then analyzed by restricting splitting on prospective sites found within the sequences JE strains SA14-4-14-2 and Nakayama and providing differentiation of these viruses. Using this approach has been demonstrated chimeric nature of viral RNA, and for selected viruses was confirmed by the presence of the appropriate restriction sites. Then was verified by real-border-DDM, which was intact at the level of this sequence was applied sequencing section of the connection part chimeras YF/JE-DDM.

The presence of the envelope protein of JE in these two chimeras were tested using thus anticorodal, specific JE, and neutralization test for manifestation of plaques using antisera specific against YF and JE. Immunoprecipitate35S-labeled extracts of cells, LLC-MK2infected with these chimeras, using a monoclonal antibody to protein F of JE virus, showed that the envelope protein E Eoba to immunoprecipitate any protein from the cells, infected with yellow fever virus. Both hyperimmune serum to YF and JE showed cross-reactivity to the two envelope proteins, however, the number of replications is reproduced dimensional differentiation of two proteins: the virus YF - 53 kDa, deglycosylation; JE virus 55 kDa glycosylated. The use of monoclonal antibodies to YF in conditions thus proved ineffective: therefore, the specificity was determined in this analysis from monoclonal antibodies to JE. Neutralization test to reduce the number of plaques (PRNT) was performed in respect of chimeric viruses and clean viruses YF and JE SA14-14-2 using hyperimmune ascitic fluid (ATSS), specific YF and JE, and purified immunoglobulin G specific YF (monoclonal antibody 2 E10). These antisera were significant differences in their title, required for a 50% reduction in the number of plaques when testing chimeras compared with controls (clean) viruses in this experiment (table. 3). Thus, it is necessary for neutralization epitopes expressed infectious chimeric viruses YF/JE.

Growth parameters in cell culture
Sposobnostyam and from mosquitoes. In Fig.2 shows the cumulative growth curves of chimeras in cell line LLC-MK2after multiple infections with low density (0.5 to plaque-forming units per cell). In this experiment, were used for comparisons viruses YF5.2IV (cloned derivative) and JE SA14-14-2 (non-clone). Both chimeric virus has reached its maximum output level virus, approximately 1 log higher than the output of any of the parent viruses. In the case of chimeric YF/JE SA14-14-2 peak output of the virus was noted in 12 hours later than the same indicator chimeras YF/JE Nakayama (respectively, 50 and 38 hours). The chimeric YF/JE Nakayama showed significantly greater cytopathic effect in comparison with the chimeric YF/JE SA14-14-2 in the same cell line. A similar experiment was conducted with cells From 6/36 in multiple infection with a low density (0.5 to plaque-forming units per 1 cell). In Fig.2 also shows the dynamics of the growth of the virus in this cell line bespozvonochnykh. Similar levels of virus were observed in all aspects of testing in this experiment, which further confirms the conclusion that the chimeric virus breeding efficiency does not infringed.

Comparison of dynamics of growth RMS (YF/JE Oh vaccines produced in cell lines, acceptable for vaccines intended for human use. A commercial vaccine against yellow fever YF17D (YF-Vax) was obtained from Connaught Laboratories, Swiftwater, PA. Cells MRC-5 (diplodia embryonic cells of the human lung) were acquired in ATSC (171-CCL, Batch F-14308: 18th passage): they were grown in culture medium of EMEM with the addition of 2 mm L-glutamine, salt solution Earl made to bring up to 1.5 g/l for sodium bicarbonate, 0.1 mm nonessential amino acids and 10% fetal serum of calves.

To compare the kinetics of growth RMS (Research Master Seed, YF/JE SA14-14-2) and YF-Vaxcells were grown to 90% confluently and have them infecting RMS or YF-Vaxdensity (MOI) of 0.1 B. about.E. Considering the fact that cells MRC-5 usually grow slowly, these cells maintained after infection for 10 days. The samples were frozen at 7-10 days and their infectivity was determined in the test for the formation of plaques using Vero cells.

Vaccine YF-Vaxand the chimeric YF/JE was increased in cells MRC-5 to moderate titers (Fig.3). The peak titer was approximately 4.7 log10B. about.E. for YF-Va 10 B. about.E. on the 6th day.

Testing neurovirulence in normal adult mice
The virulence properties of the chimeras YF/JE SA14-14-2 were analyzed using young adult mice: applied intracerebrally inoculation. Groups of 10 mice (males and females line ICR aged 4 weeks to 5 individuals of each sex in each group) were inoculated with 10 thousands plaque-forming units chimeras YF/JE SA14-14-2, YF5.2IV or JE SA14-14-2 and observed daily for 3 weeks. The results of these experiments are illustrated in Fig.4. Mice infected with the parent strain YF5.2IV, died after approximately one week after infection. Mortality or signs of disease were not detected in mice infected with a strain of Japanese encephalitis JE SA14-14-2 or Chimera. The inoculum used in these experiments were titrated in the moment of infection, and subgroups of surviving mice was tested for the presence of neutralizing antibodies, which was necessary to confirm that the infection took place. When comparing the tested specimens noted that the titers of antibodies against JE SA14-14-2 were similar to those identified in animals that were infected or that staminate chimeras YF/JE SA14-14-2 against mice, illustrated in PL. 4. In these experiments, all mice infected with yellow fever virus YF5.2.IV, died within 7-8 days. In contrast, none of the mice infected with the chimeric virus YF/JE SA14-14-2, did not die within two weeks after infection.

The results of experiments devoted to the study of neurovirulence and pathogenicity of chimeras YF/JE, illustrated in PL. 5. In these experiments, chimeric viruses were used to infect 3-week-old mice at doses varying from 10 thousand to 1 million plaque-forming units when they intraperitoneal administration. None of the mice infected chimeras YF/JE Nakayama or YF/JE SA14-14-2, did not die within 3 weeks after infection: this indicates that these viruses are not able to cause disease after peripheral inoculation. In mice infected with a chimeric YF/JE SA14-14-2, was formed neutralizing antibodies against the virus Japanese encephalitis JE (Fig. 7).

The design matrices cDNA necessary to create chimeric viruses yellow fever/dengue fever (YF/DEN)
Construction of chimeric viruses yellow fever/dengue fever (YF/DEN), described below, was carried out in principle in the same way, that is using a similar strategy using natural or artificially built-in restriction sites and, for example, oligonucleotide primers are shown in table. 6.

Construction of the chimeric virus YF/DEN
Although some molecular clones of viruses of dengue fever have been formed previously, there is a problem of stability of fragments of viral cDNA present in the composition plasmid systems, as well as the efficiency of propagation of the received virus. For the experiment, the applicants have chosen the clone DEN-2, formed by Dr. Peter Wright (Dept. Environ, Monash Univ., Clayton, Australia), because this system is relatively efficient from the point of view of the regenerate virus, and it is important that it is based on dvuhmestnoj system similar to the one used in the methodology of the applicants. The full sequence of this clone DEN-2 is available and, accordingly, facilitates the construction of chimeric matrices YF/DEN, because it requires only minor modifications to the clone YF. The necessary steps are as follows.

Similarly dvuhmestnoj system used in relation to viruses YF5.2IV and YF/JE, system YF/DEN uses a unique restriction site within the sequence encoding the envelope protein E of DEN virus-2 as the point bit'3'IV/DEN (prM-E') and YFM5.2/DEN (E'-E) (see Fig. 5). Two restriction site, which is required for ligation in vitro chimeric matrix, marked by restrictase AatII and SphI. Recipient of the plasmid for the 3'segment of the gene sequence of the protein E DEN is a plasmid YFM5.2 (Narl[+]SphI[-]). This plasmid includes a restriction site NarI as part of the joint E/NS1 used to embed the C-terminal part of the protein E of JE virus. He was further modified by eliminating the additional restriction SphI site within the sequence that encodes a protein NS5, the applied method is directed Nesmelova mutagenesis. This allows you to embed a sequence of DEN-2 from the unique SphI site to site NarI using a simple directional cloning. Suitable fragment of DEN cDNA-2 was generated by PCR on the material derived from the DEN-2 clone MON310 provided by Dr. Wright. Primers for PCR consisted of 5'-primer flanking restriction site SphI, and the 3'primer homologous to the nucleotides comprising DEN-2, located immediately upstream from the signal site in connection E/NS1: substitute signal site due to substitutions that create a new kind of website, but also make a restriction site NarI. The resulting PCR-fragment condition is with a sequence which encode DDM and N-protein E, was constructed in the plasmid YF5'3'IV using chimeric primers for PCR. Chimeric primer comprising a 3'-end of the antisense sequence of a protein From the virus YF and 5'-end of the gene protein DDM DEN-2, was used along with the semantic primers flanking the promoter SP6 included plasmids YF5'3'IV, with the aim of creating a PCR product consisting of 771 nucleotides, characterized by a 20-pair extension representing the sequence of DDM virus DEN-2. This PCR product was then used for priming plasmids DEN-2 along with 3'-primer, representing nucleotides 1501-1522 sequence of DEN-2 and flanking restriction site SphI order to obtain the final PCR product consisting of 1800 nucleotides comprising a sequence of yellow fever virus YF from the restriction site NotI through the SP6 promoter, the 5'-noncoding segment YF and a sequence encoding a protein related to the sequence coding DDM-E DEN-2. The PCR product was Legerova part plasmids YF5'3'IV using the restriction sites NotI and SphI to obtain plasmid YF5'3'IV/DEN(prM-E).

Construction of chimeric matrices for other flaviviruses
The creation procedure described above for the poly system YF/DEN-2. Table. 6 shows features of a strategy for the creation of chimeric virus-based vaccine strain YF17D. It also shows a unique restriction site used for ligation in vitro, and chimeric primers for construction of hybrid proteins With/DDM and E/NS1. Sources cDNA for these heterologous viruses are easily accessible (MVE: Dalgarno et al., 1986, J. Mol. Biol, 187:309-323; SLE: Trent et al., 1987, Virology, 156:293-304; TBE: Mandl et al., 1988, Virology, 166: 197-205; dengue virus-1: Mason et al., 1987, Virology, 161:262-267; dengue virus-2: Deubel et al., 1986, Virology, 155:365-377; dengue-3: Hahn et al. , 1988, Virology, 162:167-180; dengue-4: Zhao et al., 1986, Virology, 155:77-88).

An alternative approach to the design of additional chimeric viruses is the creation of a hybrid protein With the/DDM by ligating "blunt ends" of the PCR-derived DNA restriction fragments, characterized by the ends that meet in the place of this hybridization, and 5'- and 3'-ends that flank the restriction sites suitable for installation in plasmid YF5'3'IV or in any intermediate plasmid, such as pBS-KS(+). Approaches to the use of chimeric oligonucleotide or legirovanie "blunt ends" should vary depending on the availability of unique restriction sites within the sequence, HCV antigens
Because of the structural proteins E1 and E2 of the HCV virus (HCV) non-homologous structural proteins described above flaviviruses, the strategy of expression of these proteins includes embedding within the region of the genome that is not "essential", so that all these proteins then expressibility together with proteins of yellow fever virus in the process of viral replication in infected cells. The plot, which is a target for embedding of sequences encoding these proteins is the sequence encoding the N-terminal part of the protein NS1, because the presence of complete protein NS1 is not strictly necessary for the propagation of viruses. Given the existing problems with the stability of the genome YF in the presence of heterologous sequences that exceed the normal size of the viral genome (approximately 10 thousand nucleotides) that can be used are described below detectiona strategy". In addition, DeleteMovie NS1 gene can give advantages chimeric flaviviruses systems "YF/flavivirus" described above, due to the fact that partial deletion of this protein can prevent the production of immunity to YF, based on antibodies to the NS1 protein, which thus relieves prli when previously introduced yellow fever vaccine or its introduction will need in the future.

This strategy involves the formation of a series vnutriramochnym deletions within the sequence that encodes a protein NS1 (found in plasmids YFM5.2, in conjunction with the construction of a stop codon at the end of the coding frame of the protein E, as well as a series of two IRES (the so-called "internal sites attach to the ribosome"). One of the sites IRES is located just down from the stop codon and provides expressii open frame in the area between coding sequences E and NS1. The second site IRES initiates the broadcast of the truncated NS1 protein, which provides the expression of the remaining polpredstavleniya nonstructural proteins. These derived variants are tested for the recovery of infectious virus, and the design with the largest deletion is used to embed alien sequences (e.g. genes, proteins HCV) in their first site IRES. A particular design may also serve as a basis for determining whether the deletion of the NS1 gene to suppress the vector-specific immunity in the context of the chimeric structures "YF/flavivirus" expressing DDM-E, in a way that was described above.

The incorporation of nucleotides that encode proteins of the virus HCV E1, E2, and (or) E1+E2, is limited by the size of the deletions, Peremoga clone YF can be used shortened antigens of HCV virus. Proteins of HCV designed so that the N-terminal
the signal sequence is located immediately following the IRES site and a stop codon directly on the C-end. This design will be sent HCV proteins in the endoplasmic reticulum with subsequent secretion from the cell. Schematically, the strategy of receipt of such structures is shown in Fig.6. For this design can be used plasmids encoding proteins of HCV genotype I, for example, plasmids with HCV obtained from Dr. Charles rice of the University of Washington (Grakoui et al., 1993, J. Virol, 67: 1385-1395), which expressed this segment of the virus in processorbased systems and in the full-size clone of HCV is able to replicate.

Deletion mutants on splitting DDM, as alleged attenuated vaccine flavivirus
Additional chimeric viruses included in the present invention are mutations that prevent splitting processing of precursor DDM, such as mutations affecting the cleavage site of DDM. For example, the cleavage site of DDM in interest infectious flavivirus clones, such as dengue viruses, solid fuel, SLE and other, can be mutated using the method of directed (site-specificness deleterows or replaced. The fragment of the nucleic acid comprising the mutated genes DDM-E, can then be embedded in the vector of yellow fever virus by using the techniques described above. DeleteMovie in the gene DDM can be performed along with or without accompanying attenuating mutations, such as mutations in the gene of the protein E, before incorporation into the genome of the virus of yellow fever. These mutants possess advantages in comparison with monosubstituted mutants as possible vaccines, because they provide almost complete inability reversion deletirovannykh sequences and restore virulence.

Following the chimeric flavivirus of the present invention were deposited in the American type culture Collection (ATS), located in Rockville (Maryland, USA) in accordance with the Budapest Treaty with the date of incorporation January 6, 1998: chimeric virus of yellow fever 17D/dengue type 2" (YF/DEN-2; the Depository ATS VR-2593) and chimeric virus of yellow fever 17D/Japanese encephalitis" SA14-14-2 (YF/JE A1.3; the Depository ATS VR-2594).

Other options
Other options are also covered by the following claims. For example, genes with other medical value of the Oia vaccines against other important from a medical point of view flaviviruses (see, for example, Monath et al., 1995, "Flaviviruses", In "Virology", ed. by Fields, Raven-Lippincott, New York, Vol. I 961-1034).

Examples of other flaviviruses, genes from genomes which may be incorporated into the chimeric vectors of the present invention, are viruses fever Kunina, Central European encephalitis, spring-summer encephalitis, powstancow fever, kiesanowski forest disease and Omsk hemorrhagic fever. In addition, the genes is even more phylogenetically distant viruses can be embedded in vaccine yellow fever virus in the process of designing new vaccines.

Receipt and use of vaccines
Vaccines of the present invention is administered in amounts and using those methods, which can be easily identified by experts in the field of technology. Vaccines can be prepared and introduced, for example, in the same way as the yellow fever vaccine 17D, for example, in the form of clarified slurry infected tissue of chicken embryos or in the form of a liquid secreted from cell cultures infected with the chimeric virus of yellow fever. Thus, a live attenuated vaccine virus used in the preparation of a medicinal product in the form of sterile water nyh doses for tissue culture) in doses from 0.1 to 1.0 ml, intended for, for example, intramuscular, subcutaneous or intradermal injection. In addition, since flavivirus can be capable of human infection through mucous membranes, for example, through the mucous membranes of the mouth (Gresikova et al., 1988, "Tick-borne encephalitis". In "Arboviruses: Ecology & Epidemiology, ed. by Monath, CRC Press, Boca Raton, FL, Vol. IV, 177-203), the vaccine virus can be introduced along the way through the mucous membranes, in order to achieve a protective immune response. The vaccine can be given as a primary prophylactic agent in adults or children at risk of infection flavivirus infection. Also vaccines can be used as a secondary means for the treatment of infected flaviviruses patients by stimulating the immune response against flavivirus infection.

It may be desirable to use a vector system of the yellow fever vaccine designed to immunize the recipient against one of the viruses (e.g., Japanese encephalitis virus) and for subsequent reimmunization the same object against the second or third virus with the use of different chimeric constructs. A significant advantage of the chimeric system of the yellow fever virus is that the vector does not exp the barrier to the use of the chimeric vaccine as a vector for the expression of heterologous genes. These benefits are caused by deletion of the gene protein E of the yellow fever virus, which encodes a neutralizing protective antigens against yellow fever, and replace it with another heterologous gene which does not provide cross-protective effect against yellow fever. Although non-structural proteins of YF17D virus may play a role in the protection of, for example, due to the formation of antibodies to NS1, which is involved in mediated complement-dependent antibody lysis of infected cells (Schlesinger et al., 1985, J. Immunol, 135: 2805-2809), or by inducing T-cell response to NS3 protein or other viral proteins, it is practically impossible that these immune responses would prevent the ability of live viral vaccines to stimulate the production of neutralizing antibodies. This conclusion is confirmed by the facts that: 1) patients who were previously infected with Japanese encephalitis virus, respond to vaccination with YF17D strain in a similar manner to those patients with prior infection with JE virus was not, and 2) patients who have previously received the vaccine YF17D respond to repeated vaccination increased titers of neutralizing antibodies (Sweet et al., 1962, Amer. J. Trop. Med. Hyg. , 11:562-569). Thus, Hieu natural immunity or vaccination, and can also be reused, or to immunize simultaneously or sequentially along with a few other different designs, including chimeras of yellow fever virus with embedded viruses such as Japanese encephalitis, encephalitis St. Louis or West Nile fever.

For practical vaccination can be used adjuvants which are well known to specialists in this field of technology. Adjuvants that can be used to enhance the immunogenicity of chimeric vaccines include, for example, liposomal preparations, synthetic adjuvants, such as saponins (e.g., QS21), muramyl-dipeptide, monophosphoryl lipid-a or polyphosphazene. Although these adjuvants are commonly used to enhance immune responses to inactivated (killed) vaccines, they can also be used for live vaccines. In the case of the use of chimeric vaccines through the mucous membranes, for example, through the mucous membranes of the mouth, applicable adjuvants can be special adjuvants to mucous membranes, such as thermally stable toxin of E. coli (LT) or mutant derivatives of LT. In addition, genes encoding cytokines, which has active adjuvants may be incorporated into the vectors of the virus of yellow is in/macrophages), IL-2, IL-12, IL-13 or IL-5 (interleukin), can be integrated together with heterologous flaviviruses genes with the aim of obtaining the vaccine, which would have contributed to the strengthening of immune responses, or to modulate the immune system, more specifically directed against the cellular, humoral or mucosal responses. In addition to the use of vaccines, as is clear to a person skilled in the technical field vectors of the present invention can be used in methods of gene therapy with the goal of making "therapeutic gene products in cells of the patient. In these methods, genes encoding therapeutic gene products", are embedded in the composition of vectors, for example, instead of the gene encoding the protein of DDM-that is,

An additional advantage of the vector system of the yellow fever virus is that the replicate of flavivirus are located in the cytoplasm of cells, so the strategy of reproduction of the virus is not associated with the integration of the viral genome into the genome of the host cell (Chambers et al., 1990, "Flavivirus genome organization expression and replication", Ann. Rev. Environ, 44:649-688), which is an important safety factor.

All cited references are included in this text in its entirety.


Claims

1. Chimeric live infeel, coding premembrane and envelope proteins, or demeterova, or shortened, or metirovan so that functional premembrane and envelope proteins of the yellow fever virus is not expressionlist; integrated into the genome of the above-mentioned yellow fever virus nucleotide sequence encoding premembrane and envelope proteins of a second, different, flavivirus, so that premembrane and envelope proteins mentioned second flavivirus expressives, and a signal sequence at the points of connection/DDM and E/NS1 stored in the structure mentioned chimeric flavivirus.

2. Chimeric virus on p. 1 where the above-mentioned second flavivirus is a virus Japanese encephalitis (JE).

3. Chimeric virus on p. 1 where the above-mentioned second flavivirus is the dengue virus selected from the group including dengue virus types 1-4.

4. Chimeric virus on p. 1 where the above-mentioned second virus is selected from the group including virus encephalitis Murray valley virus encephalitis St. Louis, West Nile virus, tick-borne encephalitis virus, the virus fever Kunina, virus, Central European encephalitis virus spring-summer tick anchorage.

5. Chimeric virus under item 1, where the nucleotide sequence encoding premembrane and envelope proteins mentioned second, different, flavivirus, replaces the nucleotide sequence encoding the precursor membrane and envelope proteins of the above-mentioned yellow fever virus.

6. Chimeric virus on p. 1 where the above-mentioned nucleotide sequence encoding the aforementioned premembrane and envelope proteins mentioned second, different, flavivirus, carries a mutation that prevents splitting premembrane protein in the formation of Mature membrane protein.

7. Chimeric live attenuated infectious virus for use for preparing a medicinal product intended for the prevention or treatment flavivirus infection in a patient, where the virus includes yellow fever virus, which has the nucleotide sequence encoding premembrane and envelope proteins, or demeterova, or shortened, or metirovan so that functional premembrane and envelope proteins of the yellow fever virus is not expressionlist; integrated into the genome of the above-mentioned yellow fever virus nucleotide posledovateli premembrane and envelope proteins mentioned second flavivirus expressives, and the signal sequence at the points of connection/DDM and E/NS1 stored in the structure mentioned chimeric flavivirus.

8. Virus on p. 7 where the above-mentioned second flavivirus is a virus Japanese encephalitis (JE).

9. Virus on p. 7 where the above-mentioned second flavivirus is the dengue virus selected from the group including dengue virus types 1-4.

10. The virus under item 7, which referred to the second virus is selected from the group including virus encephalitis Murray valley virus encephalitis St. Louis, West Nile virus, tick-borne encephalitis virus, the virus fever Kunina, virus, Central European encephalitis virus spring-summer encephalitis virus powstancow fever virus kiesanowski forest disease virus Omsk hemorrhagic fever.

11. The virus under item 7, where the nucleotide sequence encoding premembrane and envelope proteins mentioned second, different, flavivirus, replaces the nucleotide sequence encoding the precursor membrane and envelope proteins of the above-mentioned yellow fever virus.

12. Virus on p. 7 where the above-mentioned nucleotide sequence encoding the aforementioned premembrane and obrocea premembrane protein in the formation of Mature membrane protein.

13. The nucleic acid molecule encoding a chimeric live attenuated infectious virus comprising the virus of yellow fever, in which the nucleotide sequence encoding premembrane and envelope proteins, or delegated to, or shorter, or metirovan so that functional premembrane and envelope proteins of the yellow fever virus is not expressionlist; integrated into the genome of the above-mentioned yellow fever virus nucleotide sequence encoding premembrane and envelope proteins of a second, different, flavivirus, so that premembrane and envelope proteins mentioned second flavivirus expressives, and the signal sequence at the points of connection/DDM and E/NS1 stored in the structure mentioned chimeric flavivirus.

14. The molecule of nucleic acid on p. 13 where the above-mentioned second flavivirus is a virus Japanese encephalitis (JE).

15. The molecule of nucleic acid on p. 13 where the above-mentioned second flavivirus is the dengue virus selected from the group including dengue virus types 1-4.

16. The molecule of nucleic acid on p. 13 where the above-mentioned second virus is selected from the group include the CSOs encephalitis, virus fever Kunina, virus, Central European encephalitis virus spring-summer encephalitis virus powstancow fever virus kiesanowski forest disease virus Omsk hemorrhagic fever.

17. The nucleic acid molecule under item 13, where the nucleotide sequence encoding premembrane and envelope proteins mentioned second, different, flavivirus, replaces the nucleotide sequence encoding the precursor membrane and envelope proteins of the above-mentioned yellow fever virus.

18. The molecule of nucleic acid on p. 13 where the above-mentioned nucleotide sequence encoding the aforementioned premembrane and envelope proteins mentioned second, different, flavivirus, carries a mutation that prevents splitting premembrane protein in the formation of Mature membrane protein.

Priority points and features:
28.02.1997 on PP.1-5, 7-10, 11, 13-17;
15.01.1998 on PP.4, 10 and 16 in the second virus presents the tick-borne encephalitis virus;
15.01.1998 on PP.6, 12 and 18.

 

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