# Patent

## Title
Method of preparing live viral vaccines by genetic engineering of viral genome

## Abstract

Disclosed is an attenuation method of an influenza virus, that is, a reassortant virus and a preparation method thereof. The disclosed reassortant virus has, in a ratio of 6:2, genes encoding a recombinant non-toxic protein and a wild type non-toxic protein, and genes encoding toxic proteins, HA (hemagglutinin) and NA (neuraminidase), of an influenza virus, the recombinant non-toxic protein consisting of a substituted caspase recognition sequence without a change of a protein size within the wild type non-toxic protein of the influenza virus. The disclosed attenuated influenza virus shows a high attenuation without a reduction of productivity in a fertilized egg. Accordingly, the method can be used as an economically efficient live vaccine preparation method which has both safety and efficiency and can use a fertilized egg as a production system. Also, since a protein is not removed or modified during attenuation, the method can be used in combination with a conventional attenuated vaccine preparation technology such as cold-adaptation.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims under 35 U.S.C. §119(a) the benefit of Korean Application No. 10-2010-0139734 filed Dec. 31, 2010, the entire contents of which are incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 10, 2014, is named 89159-301264.txt and is 39,731 bytes in size.
TECHNICAL FIELD
The present invention relates to a method for attenuating influenza virus
BACKGROUND ART
Influenza viruses in the form of a flu epidemic or a pandemic occurring every year threaten human health. As the most efficient method for preventing this, a vaccine is used. Vaccines are divided into two types, an inactivated vaccine using a surface protein of a virus as an antigen, and a live vaccine with an attenuated virus. In live vaccine preparation methods developed up to now, a cold adapted attenuated live vaccine has been mainly used as a prevention vaccine (Watanabe, S. et al., Influenza A Virus Lacking M2 Protein as a Live Attenuated Vaccine. J Virol 83, 5947-5950 (2009); Steel, J. et al., Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza. J Virol 83, 1742-1753 (2009); Perez, J. T. et al., MicroRNA-mediated species-specific attenuation of influenza A virus. Nat Biotech 27, 572-576 (2009); Stech, J. et al., A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin. Nat Med 11, 683-689 (2005)). However, live vaccines have safety problems, and thus their use for infants, old people, or some people with a reduced immunity level is restricted (Cox, R. J. et al., Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines. Scandinavian Journal of Immunology 59, 1-15 (2004)).
As a method for improving the safety of a live vaccine, there has been recently suggested a method of employing two or more attenuation methods in one kind of live vaccine. As a conventional technology, there is a method for introducing a cold-adaptation character into a virus, thereby resulting in propagation inhibition at a regular human body temperature of 36 to 37° C. (Monto, A. S. et al., Evaluation of an attenuated, cold-recombinant influenza B virus vaccine. J Infect Dis 145, 57-64 (1982); Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006); Belshe, R. B. et al., Current status of live attenuated influenza virus vaccine in the US. Virus Research 103, 177-185 (2004); Seo, S.-U. et al., Development and characterization of a live attenuated influenza B virus vaccine candidate. Vaccine 26, 874-881 (2008)). As another conventional technology, there is a method for attenuating a virus by removing or modifying a nonstructural protein 1 (NS1) from among influenza virus proteins. Also, there has been developed a method for attenuating a virus by removing M2 ion channel protein, and modifying hemagglutinin (HA) and a protein cleavage site, Further, there has been recently developed a method for reducing gene replication efficiency of a virus within a cell by using a gene silencing mechanism of miRNA.
Meanwhile, virus attenuation often even causes the destruction of propagation capability of a virus in a fertilized egg. Accordingly, in preparation of an attenuated live vaccine, sometimes, it is required to change a preparation system from a fertilized egg to a cell line (such as a MDCK cell line or a Vero cell line). Also, in some cases, an expensive enzyme required for virus propagation has to be added to a cell culture fluid.
Meanwhile, the inventors of the present invention developed X-31 ca as a donor strain of a cold-adapted attenuated live vaccine, which has a strong immunogenicity and a high propagation capability at a replication-competent temperature, and has a high attenuation at a non-replication-competent temperature (Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006)). The X-31 ca virus can be replicated in a lung and an upper airway of a mouse through infection (104 PFU) in spite of its high attenuation character. The infection with a live vaccine in a host cell is apparently directly related to the immunogenicity. However, a long-term survival of a virus in an infected host cell may cause a toxic problem such as spontaneous genetic variation and the virus' reassortment together with another human virus (Cox, R. J., Brokstad, K. A. & Ogra, P. Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines. Scandinavian Journal of Immunology 59, 1-15 (2004)).
Accordingly, in order to eliminate a live vaccine's latent risk raised from the conventional technology and previous research, the inventors of the present invention have tried to develop an attenuation method which can more quickly remove a virus from a virus-infected cell, and can improve stability of a live vaccine without damage to immunogenicity and high productivity, and then thus have completed this invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
SUMMARY OF THE DISCLOSURE
An object of the present invention is to provide a method for attenuating an influenza virus.
Also, another object of the present invention is to provide a method for more quickly removing an influenza virus from an infected cell.
A further object of the present invention is to provide an attenuated virus which can be quickly removed from an infected cell and has a high immunogenicity and a high productivity in a fertilized egg.
A yet further object of the present invention is to provide a method for vaccinating a host by using an attenuated virus which can be quickly removed from an infected cell and has a high immunogenicity and a high productivity in a fertilized egg.
A yet further object of the present invention is to provide a method for preparing a recombinant virus and preparing an attenuated live vaccine, in which the virus is prepared by any one selected from the group consisting of measles virus, mumps virus, rubella virus, poliovirus, common cold virus, rotavirus, yellow fever virus, varicella virus, hepatitis B virus, human papillomavirus, HSV-1, HSV-2, adenovirus and coxsackie virus.
In order to achieve the above objects, the inventors of the present invention completed the present invention by introducing a protease recognition site into a protein within an influenza virus, and using a host's self-defense mechanism in virus attenuation.
The inventors of the present invention noticed that caspase (protease) activated in an influenza virus-infected cell cleaves matrix proteins, resulting in apotopsis of the infected cell. Then, they prepared, as an influenza virus protein, a recombinant protein in which an amino acid residue to be cleaved by caspase is introduced. They found that in a cell infected with a virus having the inventive recombinant protein, the virus's recombinant protein was cleaved by caspase and thus virus toxicity was significantly reduced. Also, the inventive attenuated virus induced the production of a large amount of antibodies in serum and a respiratory organ's mucosal tissue of a mouse. Also, it was observed that the attenuated virus having the inventive recombinant protein can maintain its high propagation capability in a fertilized egg used for mass production of a conventional live vaccine. Accordingly, the preparation of the inventive attenuated virus can employ a conventional live vaccine virus preparation process as it is, and thus is highly advantageous in view of economic efficiency due to there being no increase in an additional production cost. As described above, the present invention is characterized in that through recombination of various proteins within an influenza virus, the virus is subjected to an action of a host's defense mechanism. Thus, unlike the conventional technology, there is no need to remove or modify a specific protein. Accordingly, the present invention in combination with the conventional technology may be used for preparing a live vaccine using an attenuated virus. In this case, according to the characteristics of the conventional technology to be used in combination with the present invention, by adjusting the protein to be cleaved, the number of cleavage sites, etc., it is possible to secure balance of the immunogenicity and the safety of a live vaccine. Especially, in a case of a protease, since the activity is highly sensitive to a temperature, it is possible to inhibit the activity of the enzyme within a fertilized egg by changing a culture temperature of the fertilized egg. This allows a new type of attenuation method to be developed which can keep a live vaccine's productivity as it is.
An attenuated live vaccine has a very similar mechanism to an actual infection mechanism of a virus. It has a higher defense capability than an inactivated vaccine so that it can induce not only a specific antibody response but also a cell immune response. Accordingly, for attenuation of a developed live vaccine through cold-adaptation or subculture in a non-human cell, genetic mutation causing attenuation of a virus was randomly induced, and then, an attenuated virus barely showing any toxicity in a human cell was prepared and used as a live vaccine. However, with the development of molecular virology and the advent of reverse genetic technology, a live vaccine has been recently prepared by directly causing mutation in measles virus, mumps virus, rubella virus, poliovirus, common cold virus, rotavirus, yellow fever virus, varicella virus, hepatitis B virus, human papillomavirus, HSV-1, HSV-2, adenovirus and coxsackie virus (Lauring, A. S., Jones, J. O. & Andino, R. Rationalizing the development of live attenuated virus vaccines. Nat Biotech 28, 573-579 (2010)).



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:
FIG. 1 shows preparation of an influenza virus attenuated by caspase, in which FIG. 1a shows a schematic view illustrating introduction of a caspase recognition site into NP and NS1 proteins of an A/PR/8/34 virus, FIG. 1b shows the gene configurations of mutant viruses, FIG. 1c shows the western blot results of NP and NS1 protein expressions in MDCK cell lines infected with a wild type virus and mutant viruses, and FIG. 1d shows the results of NP and NS1 protein expressions after treatment of Z-DEVD-FMK as a caspase 3 inhibitor;
FIG. 2 shows an in vitro attenuation characteristic of a mutant virus, and a productivity of the mutant virus in a fertilized egg, in which FIG. 2a shows a propagation speed of the virus in an MDCK cell line under various temperature conditions, FIG. 2b shows a productivity of the virus in a fertilized egg under various temperature conditions, and FIG. 2c shows a comparison of a productivity reduction between a mutant virus and a WT virus in an MDCK cell line and a fertilized egg at respective temperatures, wherein each value is expressed as a log difference between the mutant virus and the WT virus in a maximum value in a MDCK cell line, and an average in a fertilized egg;
FIG. 3 shows the result of a toxicity test on a mouse model of a mutant virus, in which 6 mice in each group were infected with a wild type virus and mutant viruses, and for 2 weeks, weight and a death rate were observed, in which FIGS. 3a to 3d show weight changes after infection of a wild type virus, NP-C, NS1-C, and DM-C, respectively, and FIGS. 3e and 3f show measurement results of a propagation capability of a virus in a mouse, wherein the left graph shows a result in the lung, and the right graph shows a result in the upper airway fluid, and also FIGS. 3e and 3f show the results of a wild type virus and a DM-C virus, respectively, wherein on each marked date, after cervical dislocation, the mouse was dissected and the lung and the upper airway fluid were collected and mixed with PBS;
FIG. 4 shows the test result of an antibody response and a protectiveness after DM-C virus vaccination, in a mouse immunized with or non-immunized with DM-C. FIGS. 4a to 4d show a serum HI titer, a serum IgG titer, an upper airway fluid IgA titer, and a BAL IgA titer, respectively, and FIG. 4e shows a change in the weight of the mouse after infection of a wild type virus; and
FIG. 5 shows attenuation, immunogenicity, and protectiveness of DM-C:H5N1 (a 6:2 reassortant virus of an H5N1), in which FIG. 5a shows a comparison between DM-C and DM-C:H5N1 in the propagation speed in an MDCK cell line, FIG. 5b shows a change in the weight of a mouse after inoculation of DM-C:H5N1, FIGS. 5c and 5d show the measurement results of an antibody response in an inoculated mouse (FIG. 5c: serum HI antibody titer, and FIG. 5d: serum IgG antibody titer), FIG. 5e shows a change in the weight of the mouse after infection of A/Aquatic bird/Korea/W81/05 (ma81) virus 10 LD50 after 4 weeks from inoculation, and FIG. 5f shows the measurement results of a removal speed of an infection virus from a vaccine-inoculated mouse, in the lung and the upper airway fluid, respectively, wherein in the lung (left) and the upper airway fluid (right), the amount of infection virus was measured by using a MDCK cell line through TCID50.



DETAILED DESCRIPTION OF THE DISCLOSURE
Hereinafter, the present invention will be described in more detail.
In the present invention, first, from among 11 proteins encoded by influenza virus, NP (nucleocapsid protein) and NS1 (nonstructural protein) were converted into substrates of a caspase. With several references on three-dimensional structures of the NP protein and the NS1 protein, a position was determined, into which a caspase recognition site can be introduced while the structural modification of these proteins and the productivity reduction in a fertilized egg are minimized (Ye, Q. et al., The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA, Nature 444, 1078-1082, (2006); Boulo, S. et al., Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes, Virus Research 124, 12-21, (2007); Bornholdt, Z. A. et al X-ray structure of influenza virus NS1 effector domain. Nat Struct Mol Biol 13, 559-560, (2006)). The caspase is divided into three sub groups according to a substrate specificity (Nicholson, D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6, 1028-1042, (1999)). In order to effectively deliver an influenza virus protein, a DEVD amino acid sequence (SEQ ID NO: 11), which is a recognition sequence of caspase 3, 7 included in group II, was selected. Through a mutagenesis method using PCR, a DEVD (Asp-Glu-Val-Asp; SEQ ID NO: 11) sequence was inserted into the NP protein and the NS1 protein.
An NP-C virus including a DEVD (SEQ ID NO: 11) sequence inserted into an NP protein, an NS1-C virus including a DEVD sequence inserted into an NS1 protein, and a DM-C virus including a DEVD sequence inserted into both of an NP protein and an NS1 protein were prepared, and these viruses were used for experiments. Specifically, by using a reverse genetics technology, mutant viruses, that is, an NP-C virus, an NS1-C virus, and a DM-C virus, which have DEVD recognition sites at 431st to 434th positions from NP protein's amino terminal end of a human influenza A/PR/8/34 (H1N1) virus (ATCC No. VR-95), and at 101st to 104th positions from NS1 protein's amino terminal end were prepared (Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences of the United States of America 97, 6108-6113 (2000)). Also, a DM-C:H5N1 6:2 reassortant virus was prepared by mixing genes of HA (GenBank accession EU146622), and NA (GenBank accession EU146623) of A/Indonesia/5/2005 (H5N1) virus with 6 genes within a DM-C virus. In an MDCK cell line (ATCC No. CCL-34) infected with the attenuated virus, after 9 hours from infection, cleavage of NP and NS1 proteins was observed. Then, with the lapse of time, the amount of cleaved proteins was increased, and became more than that of the original protein. This supports the fact that the cleavage by caspase is continuously carried out during apoptosis.
According to the present invention, it was determined that in proportion to the number of proteins converted into substrates of caspase, the attenuation level is increased. For example, a DM-C virus (an attenuated virus), which was prepared in such a manner that it has two caspase recognition sites, showed a attenuation character appropriate as a live vaccine in both in vitro and in vivo, and showed a high immunogenicity and a high protectiveness. When the DM-C virus was inoculated with a high concentration of 106 PFU into a mouse, it was found that the virus was restrictively propagated. After 1 day from the inoculation, in the lung, the amount of virus was rapidly reduced up to 1/100 of the inoculation amount, and in the upper airway fluid, for 10 days since the inoculation, no remaining virus was detected. After the infection of cells by an influenza virus, a caspase activated during apoptosis continuously cleaves its substrate proteins until apoptosis of the cells is completed. This vigorous activity allows a DM-C virus protein to be efficiently cleaved, thereby resulting in a rapid reduction of virus. Also, according to results from a recent piece of research, an NS1 protein has an apoptosis inhibiting function, and such a function is inhibited through cleavage by a caspase at the initial stage of infection. This facilitates an apoptosis process, thereby further facilitating the cleavage of NP and NS1. Such a process facilitates the cleavage of proteins of a recombinant virus and a reassortant virus, resulting in a significant decrease of a propagation speed of the viruses. A recombinant virus having a recognition site for any one protein of NP and NS1 proteins, unlike a DM-C virus, showed a lower toxicity than a wild type virus. However, the recombinant virus, when inoculated with a high concentration, resulted in reduction of a mouse weight.
For the attenuated virus according to the present invention, its propagation capability in a fertilized egg was observed. The inventive attenuated virus showed a slightly reduced productivity in a fertilized egg. It is assumed that such a result was caused by the occurring of cleavage of a virus protein in a fertilized egg with caspase activity, or a slight obstruction of a protein's natural structure and function due to the introduction of a caspase recognition site. However, when the culture temperature in a fertilized egg was lowered to 33° C., viruses were propagated up to 108 PFU or more. Thus, there was hardly any difference in productivity between the wild type virus and the inventive attenuated virus. Although the relationship between the culture temperature and the virus productivity in a fertilized egg needs to be examined through detailed research, such a result provided an interesting possibility which allows a fertilized egg itself to be utilized as a production system.
An amino acid sequence of a protein used in the present invention is indicated with sequence Nos 1 to 10: RNA polymerase PA (SEQ ID NO.: 1), RNA polymerase PB1 (SEQ ID NO.: 2, RNA polymerase PB2 (SEQ ID NO.: 3), nucleocapsid protein (NP, SEQ ID NO.: 4), Matrix protein (M) (M1(matrix): SEQ ID NO.: 5, M2 (ion channel): SEQ ID NO.: 6), nonstructural protein (NS) (NS1: SEQ ID NO.: 7, NS2: SEQ ID NO.: 8), HA (hemagglutinin, SEQ ID NO.: 9) and NA (neuraminidase, SEQ ID NO.: 10).
The term “target protein” used in the present invention indicates a protein within an influenza virus, which is subjected to the action of a host enzyme.
The term “attenuated virus” used in the present invention is used jointly with “mutant virus”.
In the present invention, as a protease, a caspase was used. However, it is natural that various enzymes may be used for the object of the present invention. Also, besides NP, NS1, and matrix proteins, membrane proteins such as HA, NA, and M2, or RNA polymerase such as PB2, PB1 and PA may be used as a target protein for the object of the present invention.
The term “caspase recognition sequence” used in the present invention indicates a sequence to be recognized and decomposed by caspase, and includes, but not limited to, DEVD (SEQ ID NO: 11), AEVD (SEQ ID NO: 12), IETD (SEQ ID NO: 13), WEHD (SEQ ID NO: 14), and YVAD (SEQ ID NO: 15; BMC Pharmacology 2004, 4:7, 2004 May, p 8 and Nature, Vol 437|6 Oct. 2005).
The term “influenza virus” used in the present invention indicates a virus which can cause influenza, and includes, but is not limited to, A type, B type, and C type influenza viruses.
The term “wild type non-toxic protein” used in the present invention indicates another protein except for surface proteins causing toxicity in a host, such as hemagglutinin (HA) and neuraminidase (NA), and includes, but is not limited to, three RNA polymerases PA, PB1 and PB2, nucleocapsid protein (NP), Matrix protein (M) and nonstructural protein (NS).
The term “recombinant non-toxic protein” used in the present invention indicates the wild type non-toxic protein substituted with a caspase recognition sequence.
The term “toxic protein surface protein” used in the present invention indicates a surface protein causing toxicity in a host, and includes, but is not limited to, hemagglutinin (HA) and neuraminidase (NA).
All references cited herein are incorporated herein by reference.
All animal experiments in the present invention were approved by Institutional Animal Care and Use Committee (IACUC) of Yonsei Laboratory Animal Research Center (YLARC), and carried out under YLARC IACUC guidelines.
As described above, the influenza virus including the inventive recombinant virus shows a high attenuation characteristic, and does not cause a reduction in productivity in a fertilized egg. Accordingly, the present invention may be developed as an economically efficient live vaccine preparation method which has both safety and efficiency and can use a fertilized egg as a production system. Also, since a protein is not removed or modified during attenuation, the present invention may be used in combination with a conventional attenuated vaccine preparation technology such as cold-adaptation.
EXAMPLES
Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, Preparation Examples and Examples as described below are only for illustrative purposes and are not intended to limit the scope of the invention.
Example 1
Preparation of an Influenza Virus Attenuated by Caspase
By using a reverse genetic technology, mutant viruses, that is, an NP-C virus and an NS1-C virus, were prepared which have DEVD (SEQ ID NO: 11) recognition sites at 431st to 434th positions from NP protein's amino terminal end of a A/PR/8/34 virus, and at 101st to 104th positions from NS1 protein's amino terminal end (see FIGS. 1a and 1b) (Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences of the United States of America 97, 6108-6113 (2000)). Also, a double mutant virus DM-C which has recognition sites for both NP and NS1 was prepared. LipofectamineTM (Invitrogen) was used under the protocol of the manufacturer. The cDNA plasmid of 8 genes of an influenza virus, in an amount of 300 ng, was transfected into human embryonic kidney cell 293T cell line (HEK293T) (ATCC No. CRL-1573), and then after 48 hours, a supernatant was collected. It was determined whether a virus was produced in an MDCK cell line (ATCC No. CCL-34), through plaque assay. The supernatant, in which virus production was found, was inoculated into a fertilized egg (aged 11 days) and cultured. An allantoic fluid (albumen) of the fertilized egg cultured for 3 days was collected and the exact titer of the virus was measured through plaque assay. Also, a DM-C:H5N1 6:2 reassortant virus was prepared by mixing genes HA (hemagglutinin) and NA (neuraminidase) of an H5N1 (A/Indonesia/5/2005) virus with cDNA plasmid of other 6 genes of DM-C virus.
In order to secure the safety of the recombinant virus, a polybasic cleavage site of the HA gene was modified into a monobasic cleavage site (PQRESRRKKRG→PQREKRG; SEQ ID NOs: 16 and 17). In NP and NS1 genes of all of the prepared viruses, sequencing of cDNA obtained through RT-PCR was carried out so as to determine if there exists an undesired mutant.
In order to find the cleavage by caspase in the MDCK cell line, after infection of virus of 1 MOI (see FIG. 1(c)), cells were collected hourly and subjected to western blot. In the western blot, a polyclonal antibody against NP and NS1 proteins of A/WSN/33 (H1N1) virus (ATCC no. VR-219) prepared from a rabbit was used. As a secondary antibody, a goat anti-rabbit IgG monoclonal antibody (Sigma) conjugated with horseradish peroxidase was used with a dilution of 1:10,000. As a result, it was found that both NP 431DEVD434 and NS1 101DEVD104 proteins were cleaved in NP-C and DM-C (see FIG. 1c). After 24 hours from the treatment with Z-DEVD-FMK (BD Biosciences Material Number 550378) as a caspase 3 inhibitor, apoptotic cells were collected and subjected to western blot. As a result, it was found that cleavage was reduced. Thus, it was determined that the cleavage was caused by caspase (see FIG. 1d).
Example 2
Analysis on Propagation Capability in a Cell Line, by Caspase Cleavage
The attenuation character, in vitro, was determined. In order to determine the temperature's effect on virus propagation, a virus propagation speed in an MDCK cell line was measured under various temperature conditions. FIG. 2a shows the result. In order to measure the virus propagation speed according to temperatures, the MDCK cell line was infected with each virus of 0.001 MOI, and the supernatant was collected at an interval of 24 hours. Then, the virus titer in the supernatant was measured through plaque assay in the MDCK cell line. An average value from three experiments was obtained. Under all temperature conditions applied to the experiments, the propagation capability of a mutant virus was reduced, compared to that of a wild type virus. Especially, the propagation speed of the DM-C virus was further reduced compared to those of the NP-C virus or the NS1-C virus. From this result, it can be found that the introduction of caspase cleavage sites into two kinds of proteins further increased the extent of attenuation. Also, compared to the results at other temperatures, the propagation capability of the mutant virus was significantly reduced at a high temperature of 39° C. From this result, it can be found that the mutant virus obtained a temperature sensitivity character.
Example 3
Analysis on Productivity Maintenance in a Fertilized Egg
The high productivity in a fertilized egg (Korean Poultry TS) as well as the attenuation character provides an advantage in that the cost for production of a live vaccine can be reduced. In order to measure the productivity in a fertilized egg, a virus with 100 PFU (plaque forming unit) was inoculated into a fertilized egg, and cultured for 3 days under various temperature conditions. An allantoic fluid of each fertilized egg was collected and then the amount of virus within the fluid was measured (see FIG. 2b). At 37° C. where a wild type virus is most vigorously grown, the productivity of a mutant virus was reduced about 100 times. On the other hand, when the culture temperature was lowered to 33° C. or 30° C., there was hardly any difference in productivity between the wild type virus and the mutant virus (see FIG. 2b). This result can be easily explained in consideration of the fact that caspase used for the attenuation is an enzyme. In other words, an enzyme showing an optimum activity at 37° C. shows a reduced activity at a low temperature. Likewise, at a low temperature such as 33° C. or 30° C., caspase shows a reduced activity, and thus cleavages of NP and NS1 proteins of an influenza virus in a fertilized egg cannot be smoothly carried out. Especially, a growth reduction of a mutant virus in the MDCK cell line was much greater than in the fertilized egg (see FIG. 2c). This result seems to be due to the interspecific difference between the fertilized egg and the MDCK cell line. In other words, it is expected that a series of responses on influenza virus infection in a chorioallantoic membrane (CAM) tissue (where propagation of the influenza virus mainly occurs) in the fertilized egg are different from those occurring in the MDCK cell line (mammalian). Especially, in an apoptosis process, a difference of caspase activation in qualitative and quantitative views allows the productivity of a mutant virus in the fertilized egg to be not reduced compared to a wild type virus. As a result, the DM-C virus having two cleavage sites showed a high growth of 108 PFU/ml (plaque forming units/ml) or more at 33° C. This allows the mass production system using a fertilized egg, as it is, to be utilized.
Example 4
Analysis of Attenuation Characteristic of a Mouse Model
In order to determine the safety as a live vaccine candidate in an animal model, toxicity of a mouse model was tested. A BABL/c female mouse (aged 6 weeks) (from Orientbio) was anesthetized with ketamine, and nasally administered with virus (50 ul). For the mice, a change in the weight and the death rate were measured daily. The toxicity tests on a wild type virus, mutant viruses, and a DM-C:H5N1 reassortant virus were carried out by using 6 mice in each group. The mice were infected with the mutant virus (103 ̃106 PFU), while as a control group, the wild type virus (104 PFU) was inoculated (see FIGS. 3a to 3d). In the control group, mice showed a rapid weight reduction and then were subsequently all dead on the 7th day from the infection (see FIG. 3a). Meanwhile, in all mice inoculated with the mutant viruses, the virus toxicity was reduced. When the NP-C virus (103 PFU) and the NS1-C virus (104 PFU) were inoculated, there was no reduction in the weight of mice. However, at high concentrations of 105 and 106 PFU (NP-C virus and NS1-C virus, respectively), slight toxicity was shown (see FIGS. 3b and 3c). Like the test result of propagation capability in the MDCK cell line (see FIG. 2a), the toxicity of the DM-C virus was reduced compared to each single mutant virus. In the group inoculated at 103 and 104 PFU, weight reduction was not shown at all, and even at high concentrations of 105 and 106 PFU, a weight reduction of only less than 10% was observed (see FIG. 3d). In order to quantify the attenuation of the DM-C virus, virus propagation in the lung and the upper airway of an infected mouse was observed. In order to measure the propagation extent of the DM-C virus in the lung and the upper airway fluid of the mouse, the lungs and the upper airway fluids of 4 mice in each group were collected on the 1st, 3rd, 5th, 7th and 10th days from the infection. In the mouse infected with the wild type virus (103 PFU), virus propagation increased immediately after the infection, and within 7 days, a considerable amount of virus was continuously propagated in the lung and the upper airway. Meanwhile, after the infection of the DM-C virus at high concentrations of 105 and 106 PFU, on the 1st day, virus propagation was already reduced in the lung (see the left side of FIG. 3f), and on the 7th day, the virus propagation was reduced up to less than the limit of determination (see FIG. 3e). Especially, in the upper airway, from 1st day to 10th day, no virus was detected (see the right side of FIG. 3f). From this result, it can be found that the propagation capability and the toxicity of the DM-C virus were significantly reduced in an animal model as well as a cell line.
Example 5
Analysis of Genetic Stability
Since a gene of an influenza virus is RNA, a mutation may frequently occur during virus propagation. During a subculture process in a production system of a DM-C virus, in order to test the genetic stability of a caspase recognition site, the virus was subjected to subculture ten times in a fertilized egg and an MDCK cell line, and then it was found that the base sequence of the caspase recognition site was not changed. In order to measure the genetic stability of the DM-C virus, the virus was subjected to subculture ten times in a fertilized egg and an MDCK cell line. During a subculture process in a fertilized egg, 10 fertilized eggs were inoculated with a virus (100 PFU), and after 3 day-culture, an allantoic fluid was collected. Then, chicken red blood cells were used to carry out HA assay. From among these, a sample with the highest titer was selected, subjected to measurement of a virus titer through plaque assay in the MDCK cell line, and was used for the following subculture. For subculture using the MDCK cell line, the MDCK cell line was infected with the virus (103 PFU) obtained from the previous subculture. After culture at 37° C. for 48 hours, the amount of virus within the supernatant was measured through plaque assay. From the virus obtained after subcultures 10 times in the fertilized egg and the MDCK cell line, the RNA was extracted, and then sequencing of NP and NS genes was carried out so as to determine if a mutation had occurred.
Example 6
Test on Antibody Induction and Protectiveness
After 3 weeks from DM-C virus infection, from a serum of a mouse, the amounts of IgG antibody and hemagglutinin inhibition (HI) antibody were measured and from the nasal wash and the lower respiratory tract (BALF), the amount of secretory antibody IgA was measured. The amount of the antibody was expressed as an inverse number of a dilution multiple, in which by the dilution multiple, an O.D value measured at 450 nm through ELISA assay is 0.1 or more. As the amount of infection was increased, the amounts of the antibodies were increased (see FIGS. 4a to 4d). From the non-infected mice used as a control group, no antibody response was detected. In order to test the protectiveness of infection, after 4 weeks from vaccine inoculation, 10 LD50 A/PR/8/34 or A/Aquatic bird/Korea/W81/2005 (ma81) virus (Song, M.-S. et al., The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model. J. Virol. 83, 12325-12335 (2009)) was nasally administered. In order to test the protectiveness, after 4 weeks from vaccination, a wild type virus of 10LD50 was infected. As a result, non-vaccinated mice underwent rapid weight reduction and subsequently all died. Meanwhile, in vaccinated mice, even in mice vaccinated with the lowest concentration of 103 PFU, no infection signs such as weight reduction were observed at all. From this result, it can be found that vaccination using a DM-C virus induced a powerful immune response (see FIG. 4e).
Example 7
Possibility as Attenuated Live Vaccine Parent Strain
Possibility as a live vaccine parent strain was tested. For this, a 6:2 DM-C:H5N1 reassortant virus was prepared by combining antigen genes HA and NA of A/Indonesia/5/2005 (H5N1) virus with 6 genes of DM-C virus. The propagation speed of the reassortant virus in an MDCK cell line was similar to that of DM-C virus (see FIG. 5a). Also, even in mice infected with a high concentration of 103 ̃106 PFU, no toxicity was observed. From this result, it can be found that a recombinant virus also obtained an attenuation character by the gene transferred from the DM-C virus (see FIG. 5b). Also, from the capability of inducing a considerable amount of serum IgG antibody and anti-hemagglutinin antibody, it was found that in spite of the high attenuation, the reassortant virus has a high immunogenicity (see FIGS. 5c and 5d). After 4 weeks from vaccination of mice by the reassortant virus, the mice were infected with A/Aquatic bird/Korea/W81/05 (ma81) (H5N2) virus (Song, M.-S. et al., The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model. J. Virol. 83, 12325-12335 (2009)) at a concentration of 10 LD50, and then their protectiveness was tested. The control group mice administered with PBS were all dead on the 7th day, while all of vaccinated mice survived (see FIG. 5e). Even the lowest amount (103 PFU) of inoculated vaccine resulted in a perfect protectiveness. From this result, it can be found that although the types of a vaccine strain (H5N1) and an infection strain (H5N2) do not completely correspond to each other, a high cross-protectiveness can be achieved. From mice inoculated with vaccine including DM-C:H5N1 reassortant virus, a removal speed of infection virus was measured. For this, 4 mice in each group, after 4 weeks from vaccine inoculation, were infected with 10 LD50 A/Aquatic bird/Korea/W81/05 (ma81) virus. In order to precisely determine the propagation and the removal efficiency of infection virus in vaccinated mice, on the 3rd, 5th, and 7th days from infection, the lung and the upper airway fluid of mice were collected, and the amount of infection virus was measured in an MDCK cell line through TCID50 method (see FIG. 5f).
For 3 days after infection, from the lungs of mice in all groups, infection virus was observed. Meanwhile, from the upper airway fluid, virus was observed in only a group vaccinated with 103 PFU while in other groups vaccinated with greater than 103 PFU, no virus was observed (see FIG. 5f). On the 5th day from infection, in the lung, virus was detected only in a 103 PFU-vaccinated group, while in the upper airway fluid, no virus was detected in all groups (see FIG. 5f). On the 7th day from infection, in the lung and the upper airway of mice in vaccinated groups, no infection virus was detected at all. In other words, complete virus removal was carried out.
Although several exemplary embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

## Claims

1. A reassortant influenza virus comprising genes encoding PA, PB1, PB2, NP (nucleocapsid protein), M (matrix protein) and NS1 (nonstructural protein 1), and genes encoding HA (hemagglutinin) and NA (neuraminidase);
wherein amino acids 431 to 434 of NP having the sequence of SEQ ID NO: 4 or amino acids 101 to 104 of NS1 having the sequence of SEQ ID NO: 7 is substituted with a caspase recognition sequence selected from the group consisting of DEVD, AEVD, IETD, WEHD, and YVAD (SEQ ID NO: 11 to SEQ ID NO: 15, respectively).

2. The reassortant influenza virus as claimed in claim 1, wherein the amino acid sequence of the caspase recognition sequence is DEVD (SEQ ID NO: 11).

3. The reassortant influenza virus as claimed in claim 1, wherein the influenza virus is an A-type influenza virus.

4. An attenuated vaccine composition comprising the reassortant influenza virus as claimed in any one of claims 1, 2, or 3.