Flavivirus with bipartite genome and using it

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. The first flaviviral particle comprises a psudoinfecting viral genome coding cis-active promoter elements required for the RNA replication, coat proteins and a complete kit of non-structural proteins of the flavivrus, and not coding capside proteins of the flavivirus. The second flaviviral particle comprises a complementary genome coding the cis-active promoter elements required for the RNA replication, a capside protein and a complete kit of proteins of the flavivrus, and not coding coat proteins. Since the genetic material of the flavivirus is distributed between two genomes, the flavivirus is replication-deficient and is not able to induce a disease, however it is able to induce an immune response. What is also described is a method for preparing this combination, a pharmaceutical composition and a method for using it. The invention can be used in medicine.

EFFECT: what is presented is a combination of the flaviviral particles.

13 cl, 18 dwg, 14 ex

 

BACKGROUND of INVENTION

The technical field to which the invention relates

The present invention relates to the field of molecular biology, Virology and immunology. More specifically, the present invention relates to deficient for replication flaviviruses and describes their use as vaccines against diseases associated with flaviviruses.

Description of the achieved level

Genus Favivirus of the familyFlaviviridaeincludes many important pathogens of humans and animals, including the viruses of yellow fever, tick-borne encephalitis, Japanese encephalitis, Dengue fever, West Nile fever, classical swine fever, bovine viral diarrhea and hepatitis C. In natural conditions flavivirus circulate between vertebrate animals, as their hosts, and vectors arthropods, mainly represented by many mosquitoes or mosquitoes and various species of ticks. Nearly forty members of this genus, grouped under the classification used in four different antigenic complex that can cause human disease.

Genome flavivirus presents single-stranded RNA of approximately 12 KB with positive polarity. The specified genome encodes a single polypeptide that undergoes the process is Inga during the broadcast after broadcast cellular and viral proteases with the formation of structural proteins of the virus, C, prM/M and E, which form infectious viral particles, as well as nonstructural proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5, which form the enzyme complex required for replication of the viral genome (Lindenbach and Rice, 2001). Genome flavivirus mimics the structure of cellular messenger RNA due to the presence of 5'-methylguanosine cap, which is different from the cellular messenger RNA by the absence of a 3'-terminal poly(A) sequence.

In virions flaviviruses single copy of the viral genomic RNA is packaged through C (capsid) protein in nucleocapsid, surrounded by a lipid membrane with embedded by dimers E and M protein. The mechanism of interaction between the nucleocapsid and the envelope is currently not entirely clear, but, apparently, it is less specific than, for example, the interaction between the nucleocapsid and the envelope at the alpha of the virus, so that the virions flaviviruses can be effectively formed capsid and envelope proteins derived from viruses that belong to different antigenic complexes (Chambers et al., 1999; Lorenz et al., 2002; Monath et al., 2002). In addition, the presence of nucleocapsid is not an absolute requirement for the Assembly of particles, and the formation of virus-like particles and their release from the cells can be achieved by the expression of only the prM and E from a large number of vectors. Obrazuemymi this so-called subversive particles (SVP) do not contain RNA or capsid protein (Mason et al., 1991), but include membrane proteins, organized in the form of dvadtsatiletnih structures containing lipid. These prM/E-built subversive particles are able to induce an effective immune response that protects animals against subsequent infection with replication competent viruses (Konishi and Fujii.2002; Konishi, Fujii, and Mason, 2001; Konishi et al., 1992; Qiao et al., 2004), DNA (Aberle et al., 1999; Colombage et al., 1998; Davis et al., 2001; Kochel et al., 1997; Kochel et al., 2000; Konishi et al., 2000a; Konishi et al., 2000b; Phillpotts, Venugopal, and Brooks, 1996; Schmaljohn et al., 1997). The absence of replication competent RNA, packaged in nucleocapsid, makes use subvirus structures very attractive instrument, as potential vaccines, but necessitates the development of new methods for their large-scale production or delivery of expression constructs. Such prM/E-expressing cassettes can be developed on the basis of viral and non-viral vectors. In the case of viral vectors, there is a problem with either the development or existence of an immune response to used a viral vector. Cassette-based DNA encoding these genes under the control of an effective promoter on the basis of RNA polymerase II, apparently, can be the most preferred. However, their use in clinical practice is still questionable. In this context, vaccination against flaviviruses sun is still achieved mainly through the use of either inactivated, or live attenuated vaccines (INV and LAV, respectively).

The results of recent studies suggest that the structural proteins of flavivirus are optional components for replication of genomic RNA. They can be, entirely or partially, deleterow, and yet such RNAS (replicons) retain the ability or infect other programs and expression is not only structural, but also the remaining structural and/or additional heterologous genes. So, for example, were synthesizedin vitroflavivirus genomes that do not contain functional capsid gene, but including other intact structural genes, and then used directly for immunization. Their replication resulted in the formation of subvirus particles and eventually induced a protective immune response. The modified flaviviruses who are not able to develop productive spreading infection, represents a new way to develop safe and effective, from the point of view of creation of protective immunity, vaccines (Aberle et al., 2005; Kofler et al., 2004). However, to use them you will need, apparently, to improve the delivery system synthesizedin vitroRNA in cells that are sensitive to replication RNA. This can be achieved using the most is close to the natural mechanisms of delivery due to packaging such defective genomes into infectious particles, consisting of structural proteins of the virus.

Despite the growing threat of proliferation associated with flavivirus diseases and the continuing spread of viruses to new areas, antiviral therapeutic tools have not been developed even for such infections, and to date, a very limited number of permitted use of vaccines. Inactivated viral vaccines (INV) were permitted for use for the prevention of tick-borne encephalitis (TBEV) and Japanese encephalitis (JEV). However, as with other inactivated viral vaccines, these vaccines have limited activity, require multiple vaccinations and are expensive in their manufacture. Despite these drawbacks, inactivated viral vaccines against Japanese encephalitis and tick-borne encephalitis confirmed the good safety record, and it was shown that they are not associated with the development of any disease. Only approved vaccine with a live attenuated virus (LAV) for flavivirus is widely used vaccine based on strain 17D yellow fever virus (YFV), which was obtained by serial passirovannye of yellow fever virus strain wild-type Asibi in the tissue of a chicken embryo. Although such a live attenuated vaccine is considered the W as a very safe and effective, there have been instances of development of yellow fever and adverse effects when using such vaccines, including the recently registered a case among the military forces of the United States.

Systems development of reverse genetics applied to flaviviruses opened up the possibility of creating new types of live attenuated vaccines based on rational attenuation of these viruses. Specified a new class of vaccines includes hybrid forms on the basis of YFV 17D in which the prM-E encoding a fragment of the genome of yellow fever virus have been replaced with the prM-E-cassette, derived from the heterologous flaviviruses. A similar approach based on a hybrid virus, has been applied to options on the basis of the viruses of Dengue fever and encephalitis. In most cases, hybrid flavivirus demonstrate vysokointegrirovannyh phenotype, but nevertheless they are able to cause an effective protective immune response and to perform the function of protection against subsequent infection by the same virus structural proteins which are expressed in these hydrides. Vaccination such putative chimeric vaccines not covered pre-existing vector immunity, which is influenced by the activity of recombinant viral vaccine derived from other viral vectors.

Despite the fact that the hybrid is s flavivirus provide, apparently quite versatile approach to produce new vaccines, there are legitimate concerns related to the fact that hybrid forms themselves will have pathogenicity, at least in the case of individuals with reduced immunity, or in connection with the fact that there may be pathogenic hybrid form, because mutations were found in the breeding process those viruses that will be required to receive vaccines.

Thus, the weakness of the level reached in this area is the lack of safe, active and effective vaccines that could be used against pathogens of the genus Flavivirus. The present invention relates to the problem defined in this long-existing need.

A BRIEF DESCRIPTION of the INVENTION

In one embodiment, the present invention relates to flavivirus with two-component genome. This flavivirus includes pseudoinverse viral genome that encodes a CIS-promoter elements necessary for replication of RNA involved in the creation of a protein shell and a full set of non-structural proteins of flavivirus and not encoding capsid protein flavivirus. Additionally, the virus includes complementary gene, which encodes a CIS-promoter elements necessary which are to replicate RNA, create a capsid protein and a full set of non-structural proteins of flavivirus, and does not encode proteins membrane flavivirus.

In another related embodiment, the present invention is treated cell culture system, the victim described above flaviviruses with two-component genome.

In yet another related embodiment, the present invention relates to a method of large-scale breeding of flavivirus with two-component genome. This method involves infection of the cell culture system described above flaviviruses with two-component genome, which is effective towards replication of both genomes in one cell, with the release of two-component flavivirus that leads to large-scale multiplication of flavivirus with two-component genome.

In another related aspect, the present invention relates to immunogenic compositions comprising the above-described flavivirus with two-component genome, adjuvant, a pharmaceutically acceptable carrier or combinations thereof.

In yet another related embodiment, the present invention relates to a method of protecting a subject from infection arising from the impact of flavivirus. This method includes the introduction of immunologist who Cesky effective amount of immunogenic compositions described above, to a subject, where this composition creates an immune response against flavivirus this subject, which leads to the protection of that person from infections associated with exposure of flavivirus.

BRIEF DESCRIPTION of DRAWINGS

Onfiga-1C shows a packaging defective encoding capsid and prM/E YFV genomes with the formation of infectious viral particles. On figa presents a diagram depicting the 5'-end sequences in YFV genomes, deficient in replication. The position signal peptides and transmembrane domains are shown as black rectangles. On FIGU illustrated by the release of viral particles containing the defective gene from cells subjected to joint transfection synthesizedin vitroThe RNA. Environment replace at specified time points and define the titles for the described method.

On figa-2B illustrates the YFV replication with duplicated capsid-specific sequence. On figa shows a schematic illustration of a recombinant YFV genomes. Optimized codons of the sequence encoding the capsid are shown in grey. Alternative open reading frame in the capsid YF/Cfrs/GFP/C genome, which is a result of the introduction of two mutations, leading to a shift in the reading frame, shown as blackened right is gelnika. The titles and the development of CPE assessed 72 hours after transfection using a synthesizedin vitroThe RNA. On FIGU illustrates the results of the analysis of the released recombinant viruses. Synthesizedin vitroRNA transferout in cells. Environment replace at specified time points and viral titers determined by the procedure of analysis belascoaran.

On figa-3B illustrates the process of selecting variants containing YF/C/GFP/C gene, which is capable of efficient replication and identification of adaptive mutations. On fegashows a schematic representation of the genome YF/C/GFP/C and deletions identified in effectively can replicate options. Numbers indicate positions of deletions in the amino acid sequence of the capsid and GFP proteins. On FIGU illustrated reconstructed replication mutants with a deletion in BHK-21 cells. Synthesizedin vitroRNA transferout in the cells and the medium replaced at the specified time point. Determine the titers of released viruses according to the method of analysis belascoaran according to the present description. The dotted line indicates the detection limit.

Fig 4A-4C illustrates the replication of the recombinant YFV genomes that encode heterologous gene against the direction of reading polyprotein. On figa demonstrated diagram depicting recomb nanny genome and sequence of the open reading frames, located against the direction of reading GFP gene. Optimized codons of the sequence encoding the capsid are shown in grey. Arrows indicate the start of the GFP-coding sequence. Capital letters indicate mutations introduced into the capsid and GFP sequence. On FIGU illustrated replication created YFV options in BHK-21 cells. Synthesizedin vitroRNA transferout in the cells and the medium replaced at the specified time point. Determine the titers of released viruses according to the method of analysis belascoaran given in this description. The dotted line indicates the detection limit. On figs shows the titers of recombinant YFmut/GFP virus after serial passage in BHK-21 cells.

On figa-5F illustrate the results of analysis of virus replication with two-component genome. On figa is a diagram depicting the genomes encoding the capsid and the prM/E YFV, which is capable of transcontinental replication in a single cell. Optimized codons of the gene encoding the capsid are shown in grey. On FIGU demonstrated the release of viral particles containing the defective gene from cells that were transliterowany synthesizedin vitroThe RNA. Medium was replaced at the specified time points and determined the titers of infectious virus cha is TIC, containing each of these genomes. On figs illustrated replication component of the YFV genome when passirovannye with a multiplicity of infection equal to ~10 infectious units/cell. Environment replace at specified time points to determine the titers of released infectious particles containing each as defined in the present description genomes. On fig.5D illustrated replication component of the YFV genome after infection of cells with different indicators of multiplicity of infection. Environment replace at specified time points to determine the titers of released infectious particles using the above method. On file illustrated replication both defective genomes in infected cells. Cells BHK-21 infected two YFV genome with a multiplicity of infection equal to ~1 infectious unit/cell, and evaluated the replication of the genomes of 48 hours after infection. Panel (a) depicts cells that can replicate YF/Cherry/Co; panel (b) shows the cells can replicate the genome YF/GFP/prME, and panel (c) shows the overlay. On fig.5F shows the results of the analysis of the release of infectious virus and the VLP from cells transfected with the different YFV-specific RNA. Cells BHK-21 was transfusional specified RNA. 24 hours after infection, the medium was replaced Bassington the th environment and after 24 hours were collected derived cells. Particles collected by ultracentrifugation and then analyzed in a continuous sucrose gradient, as will be described below. The presence of YFV-specific proteins in the fractions were identified by the method of Western blotting using D1-4G2 MAB that recognize viral protein E.

On figa-6C illustrates the packaging YFV replicon, lost all structural genes, in line packing cells. On figa shows a schematic illustration of YFV replicon encoding a fluorescent marker and Cherry instead of the structural proteins. On FIGU shows a schematic illustration of the previously described VEE replicon encoding the C-prM-E and its new version. Shows the titles Packed Yfrep/Cherry created in packing cell lines using both VEEV replicons. On figs illustrated by the release of infectious viral particles containing the gene Yfrep/Cherry, cells containing VEErep/GFP-C-prM-E/Pac, provided they are specified YF replicon or during infection with the same particles in the following passage. Medium was replaced at the specified time points and determined the titles released packaged replicons, as described in the present description.

7 shows the proposed strategy to achieve the replication of the virus with a two-component genome in the case of high and low multiplicity of infection. At high multiplicity of infection about the genome, genome PIV (encoding the prM/E) and complementary gene (encoding the capsid), delivered in the same cell and produce a full set of proteins required for virus replication. Cells produce virus with two-component genome, which is then subjected to passages in ascending order. At low multiplicity of infection of cells received only one of the two genomes, and those who were infected with PIV, create SVP, do not contain genetic material and nucleocapsid.

DETAILED description of the INVENTION

The aim of the present invention is to develop a new type of flavivirus, scarce replication, which could be used as a prophylactic vaccines against diseases associated with flaviviruses. In this regard, the present invention relates to flavivirus with two-component genome, for example, describes the yellow fever virus (YFV), where the genome of flavivirus divided into two genomes. Both genomes are deficient in the expression of at least one of the proteins required for productive replication (capsid or prM/E), but is able to complementaly functions of each other when delivered in the same cell. Such replication defective flavivirus, if their products on an industrial scale, could further find use as vaccines against infections, is caused by flaviviruses, because they are infectious and able to carry out a single cycle of infection in infected animals. Such animals cells infected with particles that contain only one of the genomes produce viral nonstructural proteins and incomplete set of structural proteins. Synthesized prM/E proteins form only subversive particles that do not contain genetic material, but function as effective immunogen. Thus, these defective flavivirus can be combined with inactivated vaccines, the advantage of which is their safety, as well as with live attenuated vaccines, which are characterized by efficiency and amenable to scaling. Additionally, these defective flavivirus able not only to SVP products, but also to the expression of heterologous proteins.

Currently flavivirus remain one of the most serious public health problems. They are widely distributed in both hemispheres of the globe and cause many human diseases. In this safe and effective vaccine produced against hundreds flavivirus infections. Vaccines can be described as live attenuated or inactivated. The only widespread allowed live attenuated vaccine against flavivirus is and based on the yellow fever virus, strain 17D, which was obtained by serial passirovannye strain of yellow fever virus wild-type Asibi in the tissues of chicken embryos. Live attenuated vaccines have been received against JEV, TBEV and YFV, but were not received approved products against other flaviviruses, such as Dengue virus and WNE. Live vaccines, apparently, are more effective products than vaccines based on inactivated virus or subunit vaccines. However, the obvious security issues continue to be relevant because of the possibility of reversion to a pathogenic phenotype. The use of inactivated vaccines usually require multiple vaccinations and obtain large quantities of material, and a hard-equipped systems for the propagation of virulent viruses used to create inactivated products. Thus, despite the promising options for obtaining flavivirus vaccines both types, there is no universal approach to their development.

A distinctive feature of flaviviruses is the ability of their protein shell to form the so-called subversive particles (SVP). Such particles can be effectively produced by eukaryotic cells that contains the standard vectors expressing the prM/E glycoproteins, or defective flavivirus gene is we, containing deleteriously capsid gene. These virus-like particles lose genetic material and complete nucleocapsid, but operate as an effective immunogen and induce a protective immune response against subsequent infection by the competent for replication flaviviruses. Defective flavivirus genomes, invalid encoding the capsid sequence, can be delivered into cells in the form of RNA or in the form Packed in infectious viral particles using packing cell lines, in which the capsid is presented in the TRANS-form, for example, using persistence can replicate alpha-viral replicons encoding the capsid gene of flavivirus under control subgenomic promoter. When infection "untrained" cellsin vitroandin vivothese pseudoinverse flavivirus demonstrate the ability to replicate and SVP products, but no they can not cause spreading of productive infection. In this regard, their application does not lead to the development of the disease, so they represent an interesting intermediate form between live and inactivated viruses. They perform a single cycle of infection, leading to the induction of an effective immune response, and received, in this regard, the name pseudoinverse viruses (PIV).

The development of systems reverse enetica for flaviviruses opened up the possibility of new types of vaccines based on live attenuated viruses using rational system attenuation of these viruses. This new class of vaccines includes hybrids on the basis of yellow fever virus 17D, in which the genomic fragment encoding the prM-E of the yellow fever virus have been replaced with the prM/E cassette, derived from the heterologous flaviviruses. These chimeric flavivirus, apparently, are a reasonable universal approach to the development of new vaccines. However, there are concerns related to the fact that such hybrid forms may themselves be pathogenic, at least for individuals with reduced immunity, and pathogen hybrids can occur when replicating the immunized vertebrate animals, or recombinant, replication competent flavivirus will be transferred by mosquitoes, mosquitoes or ticks.

Another promising direction in the development of vaccines is based on the creation of non-restorable deletions in the genome of flaviviruses, which allows to achieve productive replication of the virus in the vaccinated organism-host or lesser efficiency, or it becomes impossible event. In the latter case, viral genomes containing full replicative mechanism, but lost, for example, encoding section, can be delivered in the case of immunizationin vivoin the form of synthesizedin vitroRNA, reproduce or infect other programs. It was shown, Thu is a direct immunization using synthesized in vitrodefective RNA genomes that define the products subvirus particles (SVP) in the absence of a full cycle of viral replication, is a safe and effective method from the point of view of creation of protective immunity. However, there are serious obstacles to obtaining vaccines on the basis of RNA-defined synthesis, stability and delivery to the tissues. Thus, the available methods development flavivirus vaccines, based on receiving or inactivated viral vaccines are very safe, but have limited activity and require multiple vaccinations, or based on live, attenuated vaccines have great potential from the point of view of reversion towards pathogenic variant of the wild type and the transmission of arthropods as vectors.

In addition, the use of PIV for vaccination requires large-scale production and development of cell lines that would allow packaging of defective genomes with the formation of viral virions demonstrated this possibility. PIV can be passaged in cell packing lines, but not in the "trained" cells, in ascending order. However, apparently, this is not the only way large-scale breeding. Products flaviviruses according to the present invention does not require the development is aka cell lines, while the method described in the present invention, however, leads to efficient production PIV. Additionally, flavivirus, replication defective, according to the present invention, are not only safe to use, but in addition they are capable of replication in tissue culture, in ascending, suitable for industrial scale application and to the expression of additional genes. Thus, these flavivirus are replication defective and not able to cause productive spreading infection in humans and animals.

Mainly genetic material required for viral replication, has been isolated from two genomes capable of transcontinental existing nedostatochnosty. Both of them encode CIS-active promoter elements necessary for replication of RNA, and a full set of non-structural proteins that form the replicative enzyme complex. Thus, both RNA genome reproduce or infect other programs, but one of them encodes the capsid, but the genes encoding the proteins of the membrane, it deleterows, and the other encodes genes shell, but not capsid.

When delivered in the same cell genomes produce a complete set of structural proteins and cells release high titers of infectious viral particles containing each of these genomes. The next is assage "untrained" cells can be infected with viruses with this indicator, multiplicity of infection, which allows both genomes delivered to a single cell. This leads to the achievement of productive replication and release of infectious viral particles containing each of these genomes. Thus, this system allows to propagate recombinant viruses in ascending order. Inoculation in the body of the animal (which contains a large number of sensitive cells)F. each of the genomes is delivered in different cells, spreading the infection reaches an unacceptable level, and cells infected with virions that contain genomes that encode membrane proteins, produce non-infectious, virus-like particles that have lost genetic material, but which serve as effective immunogenic and induce a protective immune response against following infection with wild-type virus (Fig.7).

To enhance efficient replication and complementaly both defective genome require 5'UTR and more than 60 nucleotides (element 1) in the following natural sequence that encodes a protein represented by the amino-terminal fragment of the capsid, in the case of most flaviviruses or Nprothe gene for the members of the genus pestivirus. This sequence is followed by a sequence ubicacin or protease 2A, specific to FMD virus (FAMDV), merged with another consistently is thew, encoding the capsid or protein shell. This combination of fused genes is required for replication of both genomes and their packaging with equal efficiency in viral particles.

The use of artificial, optimized codon sequences encoding viral structural proteins, eliminates the possibility of recombination between two defective viral genomes, which might potentially lead to the formation of replication competent of flaviviruses. Both defective genome can be used for the expression of additional genes and therefore serve as vectors for the generation of the immune response to heterologous proteins. These additional genes can be cloned between the sequence of element 1 and ubication or FAMDV 2A protease.

As mentioned above, the use of such flaviviruses with two-component genome for vaccination does not lead to the development of productive spreading infection in immunized animals and humans. Because humans and animals have many cells, these cells are infected with a very low multiplicity, which leads to infection only one genome. Such cells are able to produce only the so-called viruspositive particles that lose nucleocapsid and any genetic material. The last frequent the hospitals serve as an effective immunogen, but they are not able to perform the following cycles of infection. According to the present description of these defective viral genomes with complementarity functions can Express the additional genetic information and to serve as multivalent vaccines.

Specifically, the present invention relates to the use of genetic material from the virus of yellow fever for the purposes of demonstrating the effectiveness of the described method. In this regard, it should be noted that the genetic material of the virus of yellow fever is divided between the two viral genomes capable of transcontinental of nedostatochnosty from each other. Each originally developed defective YFV genomes encode a full replication of RNA and one of them contained a deletion of almost the complete capsid gene and the second gene does not encode prM/e To track the replication of each of the genomes in tissue culture and to determine the titers of infectious particles used genomes encoding different fluorescent markers, GFP and Cherry. Their expression in the cells indicated the level of infection and replication of a particular genome. When delivered in the same cells was expected YF/GFP/prME and YF/C/Cherry will produce a complete set of structural proteins of the virus and eventually packaged into infectious virions. However, it has been unexpectedly discovered that when p is pout attempts to establish a productive replication, that is impossible due to the high cytotoxicity determined by the replication YF/C/Cherry. It was producirovanie very high levels of fluorescent protein, but also created hard CPE, which led to low level of release of infectious viral particles.

For further investigation of this phenomenon was developed YFV genome, which encodes two copies of the capsid gene, where one of them could be used for a wide array of genetic manipulations. This virus is also characterized by unusual cytotoxicity and replicated with low ranges. After modifications of the sequences encoding the capsid, it was shown that increasing the cytotoxicity associated with the very capsid protein (when he expressively not within the C-prM-E cassette), and not to possible changes in the secondary structure of RNA (figure 2). In addition, the virus YF/C/GFP/C, containing two copies of the capsid gene in the genome, could continue to develop and create solutions for growth with higher titles, but at low levels of viral CPE development. To date, the exact mechanism of the effect of expression of YFV capsid viruses YF/C/Cherry or YF/C/GFP/C in the induction of CPE remains unclear.

Sequencing options YF/C/GFP/C, adapted to the higher level of release of viruses, provides approaches to create a modified INF is czynnik viruses, capable of stable expression more different heterologous proteinsin vivoandin vitro. However, the most important is the fact that the identified spontaneous deletions provide the ability to modify originally developed replication defective virus genome YF/C/Cherry to YF/Cherry/Co, which led to a different strategy encode proteins and created the possibility of effective transcontinental replication YF/GFP/prME. Cells that were subjected to joint transfection synthesizedin vitroRNA from both genomes were produced viral proteins, in which the genomes encoding and the capsid, prM/E, were presented in the same concentration, and this unusual virus could be further passaged in the "untrained" cells in ascending order.

Infection of cells with a low multiplicity of infection clearly demonstrated that both genomes are packaged into viral particles, and therefore, such YFV, containing two genomes with complementary functions, cannot be designated as the segmented genome of the virus (which involves the imposition of all genomic fragments, Packed into one virion), but most likely it is a virus with two-component genome. This type of virus containing both genomic segment, Packed separately, have been previously described in plants. Far is its use of such viruses to immunize fraught with problems, identify potential recombination between genomes, which could lead to the formation of infectious, full, replication competent viruses. In this regard, despite the fact that it is highly improbable event, capsid gene in RNA YF/Cherry/C was presented in the form of synthetic, optimized codons version, lost cyclization sequence. In many experiments using YFV with two-component genome was not detected education infectious YFV, containing not fragmented genome. However, it is possible to further reduce the probability of recombination due to the use of different pairs of sequences cyclization in self-replicating fragments encoding the capsid and prME.

Interestingly, modification of the coding strategies C-prM-E in the construction of genome based on YFV, led to a sharp increase efficiency packaging YFV vectors, which do not encode structural proteins. These cell lines producing C-GFP-Copt-prM-E with 25 amino acids from persistence can replicate VEErep/GFP-C-prM-E/Pac, created Packed YFV the replicons with a significantly higher titers than the same cell line expressing only the C-prM-E tape. Packed YFV the replicons not only released with higher titers in excess of 10 infective units/ml, but could also passivates in packing cell lines without reducing the titles. Thus, a simple modification subgenomic RNA encoding C-prM-E by cloning 25 codons specific for the capsid, against the direction of reading structural polyprotein, had a pronounced positive effect on the release of infectious particles and could expand the number of vectors on the basis of YFV for delivery and expression of heterologous genetic information. It is believed that developed the unusual strategy of expression of C-prM-E leads to another compartmentalization translated structural proteins that enhance the formation of infectious virions.

In conclusion, it should be noted that discussed above results allow us to suppose that YF PIV capable of expression of prM/E, and most likely PIV obtained from other flaviviruses, can be passaged in tissue culture using a different replication defective flavivirus genome, producing the capsid (Fig.7). When you replicate in the same cell, the two defective genome produce a complete set of viral structural proteins, which then effectively packaged with the formation of individual infectious viral particles, which can be characterized as a virus with two-component genome. As the decrees of ALOS above, PIV serve as an effective immunogen, and in this regard, the virus with a two-component genome can be used to develop recombinant vaccines specific to flavivirus (Fig.7). These vaccines will be cheaper than inactivated vaccines, and safer than vaccines based on live attenuated viruses. The expression of the capsid of the YFV genome containing deleteregvalue prM/E genes requires additional modification of the 5'-terminal sequence and cloning additional sequence-specific capsid. Applying the same kind of modifications in case the competent YFV replication has led to the development of the virus, which is not capable of expression of genetic information. Modification of YFV C-prM-E expression cassettes in the VEEV replicons used to create a packing cell lines, dramatically improves packaging vectors on the basis of YFV. The separation sequence that encodes the capsid, and promoter elements in the YFV genome or replication defective YFV in the genome, encoding the capsid, allows the expression of the structural genes derived from the heterologous flaviviruses, regardless of the signal cyclization, and represents a possible tool for studying the mechanism of the packaging process.

Further, the present invention describes the process of obtaining and evaluating new ti is s designs RepliVAX, which includes, without limitation, the following provisions:

(1) Production of particles with two-component genome (including destruction of reporter genes from existing structures) and system testing. System RepliVAX can be replicated using either stable cell lines, or by using a unique system with a two-component genome. In the case of YFV, the system was developed with two defective genomes, where one of the genomes encodes binding With gene and red fluorescent protein (Cherry), and the other gene encodes the prM and E protein and green fluorescent protein (GFP). Cells subjected to joint transfection synthesizedin vitroRNA from both genomes were produced viral particles, With or prME-encoding genomes represented in the same concentration, and such a virus with two-component genome can be further passaged in the "untrained" cells in ascending order, if the multiplicity of infection than 1 infectious unit/cell.

To strengthen this system deleteroute reporter genes in both YFV genomes (RepliVAX YF and helper) to create viruses with two-component genome applicable to animal testing and to develop cell lines that can be used for the quantitative analysis of particles containing each of the genomes. In this izobreteny is also considered a similar approach in the development of WNV. Modified C - prM/E encoding genomes synthesizedin vitroand transferout them in Vero cells. Have been developed for quantitative determination of the content of the particles of each of the genomes (C or prM/E) diluted at such a joint two-component crops, followed by assessment of cell lines that Express or prM/E proteins. Particles with RepliVAX genome, encoding the prM/E, is able to form foci in producing cladach and-producing "helper" genomes will form foci in S-producing cell line.

(2) the Creation of TBE hybrids. Use RepliVAX encoding TBEV prM/E, on the basis of YFV. Cassette encoding the prM/E, synthesized on the basis of oligonucleotides and sequencing optimize to achieve the most effective expression through the use of frequency codons defined in assessing the most efficiently transmitted human mRNA. Based on preliminary data are replaced TBEV signal peptide in the prM YFV-specific amino acid sequences, as according to the results of preliminary experiments it was assumed that such substitution will be significant extent, increase the production of viral particles. These RepliVAX genomes subjected to (i) assess their ability to produce SVP and (ii) their ability to be Packed in the TBEV obolos who have both packing cell lines Vero, expressing TBEV the capsid and the packaging system based on two-component genome, where the second defective gene expresses TBEV WITH optimized codons.

Additionally, the present invention relates to systems development transylavania. The present invention also relates to systems development transparancy for YFV RepliVAX platforms. Was developed universal packaging system RepliVAX YF in the shell, which will be the most effective from the point of view of infection of dendritic cells and, consequently, from the point of view of antigen presentation. This package does not depend on the glycoproteins of flavivirus encoded by genomes of RepliVAX. Specified transaqua system is expected to help overcome possible inefficient infection arising from the use of DEN glycoproteins, as well as the difference in immune response induced by the genomes of RepliVAX encoding the glycoprotein shell, which were obtained from different DEN viruses.

In the present invention are considered: (i) different strategies packing (packing cell line in comparison with the two-component genomes) to achieve the most efficient production of infectious particles; (ii) proteins derived from different DEN viruses (DENl and DEN2); (iii) the effectiveness of large-scale products Packed RepliVAX genomes in Vero cells; (iv) the stability of the DEN to set subsequent passirovannye; and (v) the effectiveness of the immune response induced in mice against DENl and 2 after immunization RepliVax.

The present invention also relates to packaging TBEV prM/E encoding RepliVAX YF in homologous YFV structural proteins. Preliminary data largely testify in favor of the fact that such packaging is efficient and its further development may eventually lead to universal packing systems for RepliVAX genomes that encode any heterologous prM/E cartridge.

The present invention relates to flavivirus with two-component genome, including pseudoinverse viral genome that encodes a CIS-active promoter elements necessary for replication of RNA, envelope proteins and a full set of non-structural proteins of flavivirus, and not encoding capsid proteins flavivirus; and complementary gene that encodes a CIS-active promoter elements necessary for replication of RNA, capsid protein and a full set of non-structural proteins of flavivirus, and not encoding proteins membrane flavivirus. Additionally, pseudoinverse viral genome and complementary genome must encode the 5'- UTR and amino-terminal fragment of the open reading frame for the capsid protein, which contains a sequence cyclization, required for replication of the RNA, in Addition, pseudo is picirywi viral genome or complementary gene may include ubicacin or 2A protease, specific to FMD virus (FAMDV), merged with a sequence that encodes a protein shell or capsid protein. In addition, pseudoinverse viral genome and complementary genome may also include additional genetic material, including structural genes from other viruses, bacteria or parasites, where the expression of these genes induces an immune response against infections caused by viruses, bacteria or parasites. Representative examples of flavivirus may include, without limitation, the yellow fever virus, West Nile virus, Dengue virus, tick-borne encephalitis virus, the virus encephalitis San Louis, Japanese encephalitis virus, the virus encephalitis Murray valley virus classical swine fever or hepatitis C.

The present invention also relates to a cell culture system, infected described above flaviviruses with two-component genome. Representative examples of cell culture systems include, without limitation, Vero cells, BHK-21, C7/10 or other cells of vertebrates and mosquitoes and mosquitoes.

The present invention also relates to a method of large-scale breeding of flavivirus with two-component genome, including: infection of the cell culture system flaviviruses with two-component genome, as described in the above description, which is effective to achieve the replication of both genomes in the same cell; and the release of flavivirus with two-component genome that allows you to achieve a large-scale reproduction of flavivirus with two-component genome. Mainly cell culture system to infect flaviviruses with two-component genome with a multiplicity of infection of more infective than 1 unit/cell. Additionally, flavivirus defective in replication is replication defective and unable to cause disease, infection and is capable of a single round infectionin vivo.

The present invention also relates to immunogenic compositions, comprising: the above flavivirus with two-component genome, adjuvant, a pharmaceutically acceptable carrier or combinations thereof.

The present invention also relates to a method of protecting a subject from infection resulting from exposure flavivirus, including: the introduction of immunologically effective amount described immunogenic compositions of this subject, where this composition induces an immune response against flavivirus the subject, which leads to the protection of the subject from infections resulting from exposure flavivirus. Such introduction can be carried out by nutriplus the frame, intradermal, subcutaneous, intramuscular, oral or intranasal route of administration. Examples of flavivirus may include, without limitation, the yellow fever virus, West Nile virus, Dengue virus, tick-borne encephalitis virus, the virus encephalitis San Louis, virus, Japanese encephalitis virus, Murray valley virus, classical swine fever or hepatitis C.

The use of forms of the indefinite article in combination with the term "comprising" in the claims and in the description text can mean "one", but can also correspond to a value of "one or more", "at least one" and "one or more than one." Some variants of the present invention can comprise or consist essentially of one or more elements of the stages of implementation of the method and/or methods according to the present invention. In the framework of the present invention, it is assumed that any method or any composition described in the present description may be implemented with respect to any other method or composition according to the present description. In the claims used the term "or" should be understood as meaning "and/or"if there is no guidance on alternatives, or alternatives are mutually exclusive, although in the present description, the definition that the apply is to only alternatives and "and/or".

In the context of the present description, the term "immunologically effective amount" refers to an amount which leads to improvement or cure the symptoms of the disease or condition due to the induction of an immune response. For specialists in this area it is obvious that the effective amount may improve the condition of the patient or subject, but may not lead to complete recovery from the disease and/or condition. In the context of the present description, the term "adjuvant" is used to identify substances, which when included in a vaccine composition specifically enhances the immune response to an antigen.

Immunogenic composition according to the present description can be entered either by themselves or in combination with another drug, compound or antibiotic. This drug, compound or antibiotic can be administered simultaneously or sequentially relative to the immunogenic composition according to the present description. The joint effect of the introduction with the immunogenic composition is to reduce the dosage of drugs, compounds or antibiotics, in which the norm is required to achieve at least the minimum pharmacological or therapeutic effect, known, in relation to the disease that is being treated. If e is ω toxicity of the medicinal product, compounds or antibiotic for normal cells, tissues or organs is reduced, without reducing, mitigating, removing, or otherwise influence on the cytotoxic, cytostatic, apoptotic or other therapeutic effect associated with the destruction or inhibition characteristic of drug, compound or antibiotic.

In the present description, composition, drug, compound or antibiotic can be entered independently in the system or local mode, using any of the traditional to this area of the method, for example, by subcutaneous, intravenous, parenteral, intraperitoneal, intradermal, intramuscular, local, enteral, rectal, nasal, transbukkalno, vaginal injection or administration by inhalation, using a pump for injecting drugs or using a transdermal patch or an implant. Dosage forms of the composition according to the present invention can include conventional non-toxic physiological or pharmaceutically acceptable carriers or excipients suitable for the selected method of administration.

Immunogenic composition according to the present description and the drug, compound or antibiotic may be administered independently one or more times, for the achievements the tion, maintain or improve therapeutic effect. Specialists in this field can determine dosage or whether a sufficient dosage of the immunogenic compositions and medicaments, the compounds or antibiotic, which is contained in a single dose or in doses that are given several times.

As is well known in this area, a specific level of dosing such immunogenic compositions for any particular patient will depend on many factors including the activity of the specific compound, the age, body weight, health status, dietary habits of the patient, time of administration, route of administration, excretion rate, combination of drugs, and the severity of the particular disease to be treated. The person responsible for the introduction, should determine the appropriate dose relation to a particular subject. In addition, in the case of the introduction of human drugs must meet the requirements of sterility must meet accepted standards for progenote, General safety and purity, as defined by the Federal Administration on drug monitoring of biological components.

The introduction of immunogenic compositions according to the present invention, the subject must conform to the General protocols, the principles of the tym for the introduction of therapeutic agents, used to treat bacterial infections, where it is taken into account the toxicity, if they are natural components in immunogenic compositions, and/or the presence of a combination therapy, the toxicity of the antibiotic. It is expected that the cycles of treatment will be repeated, if necessary. Also examines various standard approaches to therapy, and the possibility of surgical intervention, which can be used in combination with the described therapeutic approaches.

As is well known to specialists in this field, the immunogenic compositions according to the present invention can be administered together with any known pharmaceutically acceptable carriers. Additionally, the immunogenic composition may be injected into the mode of any of the known routes of administration, such as subcutaneous, intranasally or slimy way. In addition, the dosage of the input composition can be determined by experiments, known to specialists in this field.

Below are the examples that are given to illustrate various embodiments of the present invention and are not to be understood as limiting in any way the present invention. For specialists in this sphere, it is obvious that the present invention can be well adapted for the of stijene goals and receive these benefits and solutions and other features, objectives and advantages inherent in the present invention. When this changes, and other uses that are consistent with the principles of the present invention defined in the claims, will be obvious to experts in this field.

EXAMPLE 1

Cell culture

Cells BHK-21 were received from Paul Olivo (Washington University, St. Louis, Mo). These support cells at 37°C in alpha-minimum essential medium (αMEM) with the addition of 10% fetal calf serum (FCS) and vitamins.

EXAMPLE 2

Plasmid construction

Were used the standard techniques of recombinant DNA for all plasmid constructions. Source nicocodine plasmid pACNR/FLYF-17Dx containing infectious cDNA of the YFV genome, strain 17D, was previously described (Bredenbeek et al., 2003) and provided by Dr. Charles rice (Dr. Charles Rice (Rockefeller University, New York)).

pYF/GFP/prME contains a defective gene YFV (YF PIV), in which the fragment encoding amino acids in 26-100 capsid gene GFP was replaced with optimized codons gene derived from pEGFP-Nl (Clontech). The indicated plasmid was developed in the framework of previous studies, where it was designated as pYF/PIV. The indicated plasmid pYF/C/Cherry encoding a full capsid protein, which should signal peptide prM and six and is inoculat of prM, merged with a sequence that encodes a Cherry (one of the red fluorescent protein). The last specified gene was fused in reading frame with the rest of the YF ORF that begins from the transmembrane domain protein E.

Plasmid pYF/C/GFP/C contains the YFV genome, which sequence encoding a capsid protein with a length of 101 amino acids was fused with GFP, followed by protease 2A virus (FAMDV 2A), optimized codons capsid protein and the remaining part of the sequence encoding the prM-NS5 from polyprotein YF. These pYF/Cfrs/GFP/C and pYF/Chyb/GFP/C had essentially the same design (figa), but in pYF/Cfrs/GFP/C single nucleotide was built after nucleotide 202, and the nucleotide 422 was deleterule and pYF/Chyb/GFP/C the sequence between nucleotides 201 and 422 has been replaced by a synthetic gene coding for the same amino acid sequence, but when using other codons.

pYF/DC/GFP/C and pYF/C/DGFP/C are derived pYF/C/GFP/C that contain deletions in the sequences encoding the capsid and GFP, respectively, and which were identified in the selected mutants with a deletion (see figa, which describes the relevant details). pYF/GFP contains YFV genome, in which the 5'UTR should open reading frame encoding a 25 amino acids of the capsid YFV, which was fused with GFP and FAMDV 2A, and full polyprotein C-N5 from YFV, where capsid gene is presented in a version optimized codons (figa). pYF/GFPmut had essentially the same design, but the fragment that encodes a 25 amino acids of the capsid contained 3 single nucleotide insertions and point mutations at the beginning of the GFP - coding sequence.

pYF/Cherry/Co contains a defective gene YFV, in which 75 nucleotides capsid were fused with the gene Cherry, followed by a sequence encoding a 2A protease FAMDV optimized according to the codon of the capsid with the prM signal peptide, 6 amino acids prM, 49 carboxy-terminal amino acids of the E protein and the residual part of the polypeptide YFV (figa). pYFrep/Cherry contains YFV replicon in which the structural genes were replaced by a sequence that encodes a protein Cherry. At the amino-end Cherry was flushed with 25 amino acids of the capsid YFV, and the carboxy - end followed by FAMDV 2A, and then the signal peptide NSl and the residual part of polyprotein YFV (figa).

Plasmid pVEErep/C-prM-E/Pac was widely described. Plasmid pVEErep/GFP-C-prM-E/Pac containing the VEEV replicon, where subgenera RNA encoding a 25 amino acids of the capsid YFV, was fused with GFP, followed by the genes of FMDV 2A protease, optimized codons capsid YFV and prM/E. Second subgenomic the promoter provides the expression of the Pac, parameterizedproperty. All recombinant viral genomes or replicons were cloned under the control of promote is and RNA polymerase SP6.

EXAMPLE 3

RNA transcription

Plasmids purified by centrifugation in CsCl gradients. Before carrying out the reaction transcription YFV genome or replicon-containing plasmids linearized when processing Xhol. Plasmids with VEEV replicons linearized when processing the Mlul. RNA was synthesized using RNA SP6 polymerase in the presence of the cap analog, as described earlier. The yield and integrity of the transcripts were analyzed when conducting gel electrophoresis in adenocarinoma conditions. Aliquots of the quota taken from the transcription reactions were used for electroporation without additional purification.

EXAMPLE 4

Transfection RNA

Electroporation of cells BHK-21 was performed in a previously described conditions (Liljestrom et al., 1991). To establish the packing cell cultures to Wednesdays added Pur at a concentration of 10 mg/ml 24 hours after electroporation of VEEV replicons. Transfection of synthesizedin vitroYF PIV genome was performed after 5 days, when the replicon-containing cells was achieved growth.

EXAMPLE 5

Definition titles infectious viral particles containing the defective YFV genomes

To determine the titers of released virions containing various defective genomes, cells BHK-21 were sown in sectionarea tablets Costar in a concentration of 5×105to etoc/cell. Four hours later the cells were infected samples at different dilutions. After one hour incubation at 37°C in an incubator containing 5% CO2the cells were layered with 2 ml of αMEM medium with addition of 10% FTS. The number of infected cells was determined by counting GFP - and Cherry-positive cells under UV microscope with inversion after 36 hours of incubation. The fraction of infected cells relative to the seed number was determined by counting fluorescent cells in a specific area of the microscope. Counts for 5 different fields were averaged and counted the titles corresponding to each of the investigated serial dilutions.

Viral titres, competent replication was determined by the standard test for analysis of belascoaran using samples of BHK-21 cells (Lemm et al., 1990). After three days of incubation at 37°C, the monolayers were fixed by treatment with 2.5% formaldehyde and stained with crystal violet.

EXAMPLE 6

The passage of viruses

Packing cell lines detected by transfection synthesizedin vitroRNA VEEV replicon with subsequent selection on Pur. These cell lines were either subjected to transfection synthesizedin vitroRNA YFV replicon, or were infected previously packaged replicons. Samples were collected in the ass the major time points, specified in the description of drawings, replacing the environment. The passage YF viruses with two-component genome was performed by infection of cells with the above values of multiplicity of infection. Samples were collected at specified time points indicated on the drawings when replacing the used media, and credits of the particles containing the defective genomes were determined by the above procedure. Viruses, competent in replicia were subjected to passage through infection "untrained" cells BHK-21 by adding 100 μl of virus harvested at the stage preceding passage. Samples were collected at 72 hours after infection and titers were determined in the analysis of belascoaran.

EXAMPLE 7

Analysis of the product YF SVP

Cells BHK-21 were subjected to transfection using 8 mg synthesized in vitro viral genomes YFV 17D or YF/GFP/prME or by transfection of genomes YF/Cherry/Co and YF/GFP/prME. After 24-hour incubation in 10 ml of αMEM medium with addition of 10% FTS specified medium was replaced with 10 ml Bassington environment VP-SF (Invitrogen), which was collected after 24 hours for analysis of the level of release of SVP. Collected VP-SF samples were osvetleni when performing low-speed centrifugation (5000 rpm, 10 min, 4°C) and then concentrated by ultracentrifugation in 2 ml of 10% sucrose prepared on the FBI using rotor SW-41 at a speed 39000 rpm, so is the temperature value 4°C for 6 hours.

Precipitated material was then analyzed in the density gradient of sucrose as described previously (Schalich et al., 1996). In General, the procedure consisted in the fact that 0.5 ml of the samples was made in a continuous sucrose ingredient (1.5 ml 50%, 1.5 ml of 35% and 1.5 ml of 10% sucrose prepared for the FBI). Centrifugation is performed using rotor SW-55 with a speed of 45,000 rpm at 4°C for 4 hours. After fractionation, the samples were diluted three times with the use of the FBI and besieged SVP by centrifugation using a rotor TLP-55 with a speed of 45,000 rpm at 4°C for 1 h in an ultracentrifuge Optima MAX (Beckman). The precipitate was dissolved in the buffer used for electrophoresis in LTO-polyacrylamide gel, without β-mercaptoethanol (to keep in touch with Dl-4G2 MAB), and then analyzed by the method of Western blotting. After transfer of the protein nitrocellulose membranes were processed using D1-4G2 MAB and secondary protivorechivyh antibodies ass, conjugated with horseradish peroxidase (HRP)obtained from Santa Cruz Biotechology. The presence of HRP was detected using luminal-reagent for Western blotting according to the manufacturer's instructions (Santa Cruz Biotechnology). Tested by comparing the gradient zones obtained with the use of YFV (2×108The COMBAT), according to the same procedures that were described above in relation to the SVP, Poluchenie YFV-PIV.

EXAMPLE 8

Experimentsinvivo

6-day-old mice (outbred mouse Swiss Webster, Harlan) were infected with recombinant YFV in the doses given in the description, by intracranial (and/R) injection (volume 20 ml). Further, within 8 days, the mice were observed for possible signs of disease and for the registration of deaths, after which the mice were killed and determined the titers in the brain according to the method of analysis belascoaran.

EXAMPLE 9

The YFV genomes defective in the expression of the capsid and the prM/E, significantly differ in replication efficiency

In the previous study, a system was developed for transcontinental defects in YFV replication and packaging of defective genomes with the formation of the YF infectious viral particles. To achieve this, cell lines were engineered to contain the VEEV replicons, producing or capsid YFV or full structural polyprotein, which will complementary replication of the capsid-deficient YFV genomes. However, the use of alpha-viral replicons is not an absolute condition for transcontinental. Functional capsid can be obtained using other tapes, capable of production at a level sufficient for packaging flaviviruses genome. In this regard, reference was made to pop the TCI to exclude any heterologous expression vectors of the packing system and get the capsid based on the second YFV genome, which does not contain the structural genes other than the gene encoding the capsid.

PIV genome (YF/GFP/prME) contains a deletion of almost the complete sequence that encodes the capsid, and the second complementary genome (YF/C/Cherry) contains depletirovannoi sequence encoding a prM/E and capsid gene remains intact. To analyze the nature of the replication of both genomes in tissue culture, two different fluorescent protein, GFP and Cherry, cloned in their open reading frames (figa). It was expected that both of the genome will not be able to cause productive spreading infection due to their inability to produce a complete set of structural proteins. However, they could produce all the proteins needed for the formation of viral particles, replication in the same cage.

Synthesizedin vitroRNAS were subjected to joint transfection cells BHK-21, and the expression of their markers, GFP and Cherry, has confirmed that their replication. Unexpectedly it was found that the titers of released infectious viral particles containing either the gene encoding the capsid, or a gene encoding the prM/E, were below the expected level, approaching the value of 106infective units/ml, and this could indicate that TRANS-complementation was ineffective (Fig. 1B). Comparison of the nature of the expression of GFP and Cherry indicates that encodes caps the d genome is replicated much more efficiently than the genome, producing prM/e After electroporation expression Cherry reached significant levels for 18-24 hours earlier than expressing GFP defective gene, but that we were more important, its replication is also caused cell death within 2-3 days after transfection. The rapid development of CPE was not a side effect of expression of Cherry protein, because the same cassette, in which Cherry was replaced by GFP, showed a high cytopathogenicity (data not shown).

In another set of additional experiments, replication of genomes expressing GFP and Cherry, were compared using previously developed cell lines, in which the precursor of the full structural polyprotein YFV, C-prM-E is expressed on the basis of the VEEV replicon. With the above data is consistent and the fact that replication of the genome YF/C/Cherry expressing the capsid, had a harmful effect on the cells and essentially all transfetsirovannyh cells were killed at 96 hours after transvestie (figs), and infectious virus particles were released with lower titers than was found in the environments for the same cells transfected with the prM/E - expressing design YF/prME/GFP.

As noted previously (Mason, Shustov, and Frolov, 2006), cells containing YF/prME/GFP, do not demonstrate the development of CPE (figs) and continue to release Packed VI is asnie genomes even after the passage of cells. Thus, the results of the above experiments indicate that either the sequence that encodes the capsid in the genome YF/C/Cherry, or the expression of the capsid protein (or both combined) contributed significantly to cytopathogenicity this can replicate RNA, and has created marked differences in replication of the genome, producing the capsid and the prM/E that could be causing inefficient transcontinental. In addition, transfetsirovannyh cells more likely to die before the release of high titers of infectious virus particles.

EXAMPLE 10

The effect of capsid protein on the replication of the genomic RNA of YFV

To distinguish differences in the effects of capsid and capsid-coding sequence replication defective YFV-specific RNA was developed set of recombinant YFV genomes, which were separated by a sequence encoding the full polyprotein included all structural and non-structural genes and the 5'-terminal sequence containing the promoter, the RNA elements required for replication, were divided. To achieve this goal, natural capsid gene in polyprotein was replaced by its optimized codons version (Ceo), which contained the mutated sequence cyclization, which was not able to function is activated in the replication of RNA. Then was cloned sequence of the 5'UTR YFV against the direction of reading information Ceo, followed by a sequence encoding a natural capsid without prM-specific signal peptide, fused with GFP genes and 2A protease FAMDV. Thus, the capsid gene against the direction of reading the information contained signal cyclization, required for replication of the RNA, as well as meinenemy codon initiation of replication.

In the finished design YF/C/GFP/C open reading frame begins with the specified initiator AUG codon and continues throughout polyprotein. Amino acid sequence Ceo differed from natural YFV capsid only in the presence of Proline as the first amino acid, because it was necessary for FAMDV 2A-specific processing. The specified experimental system allows a large number of manipulations on the 5'-end, including extensive modifications in the amino-terminal, natural capsid-coding part of the open reading frame, without affecting the expression of a functional capsid protein (Ceo). In this regard, you have used a different cassette YF/Cfrs/GFP/C, where the first capsid contained the insertion of 1 nucleotide after nucleotide 202 and a deletion of 1 nucleotide after nucleotide 421. These modifications were designed in such a way as to preserve the possibility of a computer is arizonasonora prediction of secondary structures at the 5'end of the viral genome and the 3'end of the negative RNA strand, however, they changed the sequence of the peptide of 73 amino acids, which covered the full capsid fragment.

In the third design YF/Chyb/GFP/C the first capsid gene was a hybrid between natural and optimized codon sequences. It encodes a protein of the wild type, but the RNA sequence in the direction of the read information signal cyclization starting from nucleotide 202, differed from the corresponding sequence in the genome of wild-type YFV. Thus, the 5'end of the recombinant viral genomes encode either : (i) natural capsid gene, fused with GFP (YF/C/GFP/C), or (ii) almost natural RNA sequence (containing only two framework mutations), but significantly modified protein (YF/Cfrs/GFP/C), or (iii) a modified RNA sequence, but natural protein (YF/Chyb/GFP/C). All three RNA and RNA in the genome of YF 17D were synthesizedin vitroand an equal number of them were transliterowany cells BHK-21.

Analysis of the release of infectious virus showed that only design, expressing mutated first capsid YF/Cfrs/GFP/C, was able to effectively replicated to titers comparable to titers obtained in the case of YFV 17D (pigv). However, differences in the rates of replication were even more noticeable. YF/Chyb/GFP/C and in particular YF/C/GFP/C, both of the genome, ekspressiruyushchikhsya wild-type, fused with GFP, showed viscozitatii phenotype and a sharp decline titles released infectious virus, despite the fact that they expressed GFP at higher levels than YF/Cfrs/GFP. Combined results of these experiments and the results presented in the previous section indicate that YFV the capsid, expressed in terms other than the natural environment has a strong effect on cytopathogenicity recombinant viruses and, therefore, their growth in tissue culture. In an additional series of experiments it was shown that the cytotoxicity of structures does not depend on the expression of the capsid in a fusion with GFP-form or free-form (data not shown). The only noticeable effect between GFP expressed in fused or free-form notes in the picture intracellular distribution.

EXAMPLE 11

The choice of YFV variants with reduced cytopathogenicity

In the above-described experiments, the virus YF/C/GFP/C, containing two copies of the capsid gene, showed a highly unusual for replication (pigv), which was characterized by a very inefficient release of infectious virus during the first three days after transfection synthesizedin vitroRNA and the destruction of the greater part of the cell population. However, a small% of the NT GFP-positive cells survived, continuing to grow and in the period of 72 hours after transfection, and they have produced a more effective virus than in the earlier period after transfection (pigv). On day 5 titres reached 108infective units/ml these data suggest that accumulation of mutations in viral genomes, which may affect such viscozitatii phenotype and lead to prolonged, more effective release of infectious virus.

To identify these adaptive changes have been carried out sequencing of the 5'-UTR, amino-terminal fragment encoding the capsid and the GFP - coding fragment in randomly selected mutants. They contained large deletions in the reading frames in the capsid gene (amino acids 29-66) or in the gene for GFP (amino acids 3-121), or both deletions together. (Figa). It is interesting to note that deletions were observed between the very short repeats (UAAA; SEQ ID NO: 1) and were located in the capsid sequences (loops predicted by computer calculation of secondary structure) and replays UGGUGA (SEQ ID NO: 2) in the gene for GFP. Obtained during the sequencing results were insufficient for a full understanding of what has exerted a decisive positive impact on viral replication. In this regard, both deletions specific to GFP and capsid were separately Clonie is owani in the genome YF/C/GFP/C (pigv). Synthesizedin vitroRNAS were subjected to transfection into cells BHK-21, and a single deletion in the capsid had a positive effect on the yield of infectious virus. Recombinant YF/DC/GFP/C, but not YF/C/DGFP/C showed a growth rate similar to the growth rate characteristic of YFV 17D (pigv). Thus, the experimental results suggest that the modification of the first sequence that encodes the capsid, can be a very effective approach to change the replication efficiency of the virus and design options, capable of efficient replication in tissue culture.

EXAMPLE 12

Development of YFV capable of expression of heterologous genes.

For testing in an experimental embodiment, the possibility of developing YFV, capable of efficient replication and stable expression of heterologous genes, were generated two recombinant YFV genome, YF/GFP and YFmut/GFP. Fragment length 75 nucleotide sequence that encodes the capsid, was cloned against the direction of reading the information in the genes GFP and FAMDV 2A, followed by a sequence encoding a full YFV polyprotein (figa)containing opensymphony the codons capsid gene. YF/GFP did not contain any other changes in the 5'-terminal sequence, and in YFmut/GFP had the following additional modificati is: (i) sequence UGGUGA (SEQ ID NO: 2) in GFP was replaced with the sequence UCGUCA (SEQ ID NO: 3), which has not changed sequence encoding a protein, but have modified one of the repeats, which, as shown, are used in the formation of deletions in the genomes of YF/C/DGFP and YF/DC/DGFP; (ii) a short fragment localized between cyclization sequence and GFP was modified by introducing three nucleotide insertions.

GFP-specific mutations were introduced in order to further reduce the possibility of recombination in the GFP gene, leading to deletions in the coding sequence. These changes in the sequence that encodes the capsid, were introduced in order to avoid the possibility of recombination between the residual sequence in 75 nucleotides in length at the beginning of the open reading frame for optimized codons capsid gene, localized in the direction of reading information for GFP. Synthesizedin vitroRNA was plunged transfection into cells BHK-21. Essentially, all cells showed very similar levels of GFP expression, which was detected within 18 hours after transfection, which allows to assume that both viruses were viable and did not require additional adaptation for replication. Viruses YF/GFP and YFmut/GFP were less cytopathic than YFV 17D wild-type and GFP-positive cells continued to grow to a full merger. However, despite reduced cytopathic the face, both viruses were capable of efficient replication and accumulated in the medium to titres exceeding 5×108infective units/ml (pigv).

To assess the stability of the GFP insert one of the concentrated virus samples YFmut/GFP were subjected to a blind version 5-fold passage in cells BHK-21. Found no significant change in titer among samples (figs). After 5 passages 11% of lesions were GFP-negative, but was colored with YFV-specific antibodies. These GFF options were still not able to form plaques, which indicates that mutations, apparently, was accumulated in the GFP gene due to a lack of functional protein as the result of positive selection, but not as a result of selection on the basis of best virus can replicate. Analysis of proceduree PCR also showed no fragments, which would be a noticeable extent were shorter than expected. Thus, if you used a cassette expressing heterologous genes other than GFP, were not found differences in the production of a protein.

In another experiment conducted intracranial inoculation of 6-day mice 5×106and 5×105infective units YF/GFP. All mice developed clinical signs of encephalitis, and they were euthanized on day 8 after infection. All mice was detected the GFP-expressing virus in the brain at a concentration of 2.46±0,68×l0 8infective units/ml did Not identify variants with increased replication, which expressed higher levels of GFP or showed more cytopathic phenotype. Eight days after infection the virus samples isolated from the brain, also contained less than 3% of GFP-negative variants. These data indicate that the strategy has been developed modification of the YFV genome, aimed at separating the functional sequence that encodes polyprotein, and promoter elements opens the possibility of stable expression of heterologous proteins. For YF/GFP and YFmut/GFP revealed very similar characteristics replication, but nevertheless vectors based YFmut, apparently, are more preferable for studies requiring long-term experiments and/or repeated passages.

EXAMPLE 13

TRANS-complementaly between two defective YFV genomes

Based on the results of the above experiments was developed replication defective YFV genome, YF/Cherry/Co (figa). The specified genome was capable of the expression of the capsid gene and contained a deletion in the sequence encoding the prM/e It contains the sequence of the 5'UTR YFV, followed by the sequence for the capsid gene with a length of 25 amino acids, Cherry, FAMDV 2A, Co with prM-specific signal is determined as being the peptide and carboxy-terminal protein fragment E, for the proper processing and compartmentalization created NS1-5 polyprotein. Synthesizedin vitroYF/Cherry/Co and its TRANS-complementary component genomes YF/GFP/prME were transliterowany cells BHK-21. They complementarily failure from each other in the synthesis of structural proteins, and cells were effectively created an infectious viral particles containing the defective genomes, encoding the capsid or prM/E, is capable of expression Cherry or GFP, respectively (pigv). It is important to note that both of the genome were Packed to achieve a very similar title, which was 108infective units/ml, They were not effectively caused CPE and easily established a persistent infection. The cells continued to grow and was produced viruses not only 4-5 days after infection, but also after passages.

To test the possibility of large-scale production of concentrated samples of the virus in the cells BHK-21 further passively in the "untrained" BHK-21 cells and the titers of particles containing each of the genomes, has reached the value of 108infective units/ml. in Addition, it was necessary to spend passage at high multiplicity of infection. Cells infected with a multiplicity of infection equal to ~1 infectious unit/cell, released Packed genomes as effectively as cell INFI is new with a multiplicity of infection of ~10 infectious units/cell. However, the additional reduction of multiplicity of infection of ~0,1 infectious units/cell resulted in a marked reduction credits (fig.5D). In cell monolayers infected with a multiplicity of infection of 1, could be easily identified cells expressing only 1 marker GFP or Cherry. However, a very large fraction of these cells expressed both markers (fige). Analysis of the density of the virus in the density gradient of sucrose showed that cells transfetsirovannyh RNA from YF/GFP/prME, released only viral particles of low density, which corresponded to the so-called subbaratnam units containing prM/E (SVP), which has lost nucleocapsid and RNA. However, infection with viruses containing both defective TRANS-complementary genome, led to the release of particles and low and high density, which were characterized by the same pattern of distribution of density of sucrose, and samples of wild-type virus YF 17D. The data additionally indicate that the virus with the two-component genome is characterized by a performance close to that of natural YFV.

EXAMPLE 14

Packaging YFV of replicons, lost structural genes

In the previous study was derived cell line expressing YFV C-prM-E cassette-based persistence can replicate VEEV replicon. The specified glue the full-line function effectively when packaging YF/prME/GFP defective viral genome, and this activity was indicated that the capsid protein was producyrovtsa and accordingly subjected to processing for genomic inkapsulirovanie. However, the same cell line was ineffective in the packaging process of YF replicons, not coding for structural proteins. As a result, the titers of packaged replicons were always below 107infective units/ml Reason for such a low level packaging unclear, but the data obtained correlate with previously published results of another study in which the replicons of Sindbis virus, producing a tape YF C-prM-E, packaged similarly YF the replicons also inefficient.

To test the possibility of packaging YF of replicons with achievement of higher titles, authors created the VEEV replicons encoding YFV C-prM-E in the same protein fusions, as in the case of the viral genome YF/GFP/Co. One of subgenomic RNA encoded by an open reading frame, which began with a 25 amino acid capsid protein, also covered GFP gene, gene FAMDV 2A protease sequence encoding the capsid with the optimization of the codons and the prM/E-coding sequence. Second subgenera RNA directed the expression of the PAC gene encoding parameterizedthreadstart, which gives the cells a resistance to the action of puromycin in the environment, the presence of which in the medium blocked the et broadcast. Synthesizedin vitroRNA VEErep/GFP-C-prM-E/Pac was transliterowany cells BHK-21 cell line Purrwas installed in a few days when selection puromycin. Then the cells were transfusional the YFV replicon (YFrep/Cherry), in which all the structural genes were replaced by Cherry-coding sequence (figa). As shown in figv, cell line was Packed last replicon achieving significantly higher titers and continued to produce infectious particles within a few days without the development of pronounced CPE (figs). Cells containing YF the replicon, continued to grow, and usually the experiments were over, because cells expressing and GFP and Cherry reached confluence, which caused their death. When conducting multiple experiments did not reveal packaging of VEEV replicons in YFV structural protein. An additional advantage of cell lines containing VEErep/GFP-C-prM-E/Pac, was the possibility of using it to further passages of YFV replicons. These cells could be infected previously Packed structures, and this situation has led to the development of spreading infection and the release of the replicon-containing particles with titers reaching 108infective units/ml

In the present description cited the following works:

Aberle et al. (1999).J Immunol163(12), 6756-61.

Aberle et al. (2005).J Virol79(24), 15107-13. Chambers et al. (1999)J Virol73(4), 3095-101.

Colombage et al. (1998)Virology250(1), 151-63.

Davis et al. (2001)Journal of Virology75(9), 4040-4047.

Kochel et al. (1997)Vaccine15(5), 547-52.

Kochel et al. (2000).Vaccine18(27), 3166-3173. Kofler et al. (2004)Proc Natl Acad Sci USA101(7), 1951-6.

Konishi and Fujii (2002)Vaccine20(7-8), 1058-67.

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Lindenbach and Rice (2001).Flaviviridae:The Viruses and Their Replication. Fourth ed. In "Fields Virology" (D. M. Knipe, P. M. Howley, D. G. Griffin, R. A. Lamb, M. A. Martin, and B. Roizman, Eds.), Vol. 1, pp. 991-1041. 2 vols. Lippincott Williams & Wilkins, Philadelphia.

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1. The combination flavivirus particles to induce an immune response, including:
first flavivirus particle, including the first pseudoinverse viral genome that encodes a CIS-active promoter elements necessary for replication of RNA, proteins, membranes and a complete set of non-structural proteins of flavivirus; and not encoding capsid proteins flavivirus; and
second flavivirus particle comprising a second complementary gene that encodes a CIS-active promotionnelle, necessary for replication of RNA, capsid protein and a full range of non-structural proteins of flavivirus and not encoding proteins membrane flavivirus,
where specified pseudoinverse viral genome and complementary genome encode the 5'UTR and amino-terminal fragment of the open reading frame for the capsid protein, which contains a sequence cyclization, required for replication of RNA.

2. The combination flavivirus particles according to claim 1, characterized in that the specified pseudoinverse viral genome or specified complementary genome include the sequence ubicacin or protease 2A, specific to FMD virus (FAMDV), merged with a sequence that encodes a protein shell or capsid protein.

3. The combination flavivirus particles according to claim 1, characterized in that the specified pseudoinverse the viral genome and the specified complementary genome further include:
additional genetic material, including structural genes of other viruses, bacteria or parasites, where the expression of these genes induces an immune response against infections caused by these viruses, bacteria or parasites.

4. The combination flavivirus particles according to claim 1, characterized in that the specified flavivirus is a yellow fever virus, West Nile virus, Dengue virus, the virus is lesiolo encephalitis, the virus encephalitis San Louis, Japanese encephalitis virus, the virus encephalitis Murray valley virus classical swine fever or hepatitis C.

5. A host cell for reproduction of flaviviruses with two-component genome, infected combination flavivirus particles according to claim 1.

6. A host cell according to claim 5, characterized in that specified a host cell is a Vero cell, KSS-21, C7/10 or other cell of vertebrate animals or mosquitoes.

7. The way large-scale reproduction of flaviviruses with two-component genome, including:
infection of cells by a combination of flavivirus particles according to claim 1 or their genomes, effective to achieve the replication of both genomes in one cell, and
the release of the above flavivirus with two-component genome that allows you to achieve a large-scale reproduction of flavivirus with two-component genome.

8. The method according to claim 7, characterized in that the specified cell to infect combination flavivirus particles with a multiplicity of infection of more than 1 infectious unit/cell.

9. The method according to claim 7, characterized in that the specified flavivirus, deficient in replication, is a virus that is replication defective, unable to cause disease, infectious and capable of a single round of infection in vivo.

10. Immunogenic composition is La inducing an immune response against flavivirus, including:
an effective amount of a composition flavivirus particles according to claim 1, adjuvant, a pharmaceutically acceptable carrier or combinations thereof.

11. The way to protect the subject from infection caused by the impact of flavivirus, including:
introduction immunologically effective amount of the immunogenic composition of claim 10, subject, where this composition induces an immune response against flavivirus the subject to protect the subject from infection caused by the impact of the specified flavivirus.

12. The method according to claim 11, characterized in that the said introduction is carried out intraperitoneal, intradermal, subcutaneous, intramuscular, oral or intranasal method.

13. The method according to claim 11, characterized in that the specified flavivirus is a yellow fever virus, West Nile virus, Dengue virus, tick-borne encephalitis virus, the virus encephalitis San Louis, Japanese encephalitis virus, the virus encephalitis Murray valley virus, classical swine fever, or hepatitis C.



 

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