Isolated nucleic acid for the production of a vaccine against virus

Abstract

The present invention relates to a nucleic acid for vaccine that has undergone codon optimization for expression in Bombyx mori, a vector comprising the nucleic acid, Bombyx mori comprising the vector, and a method for producing a vaccine in which they are used.

Claims

1. An isolated nucleic acid comprising: a nucleic acid sequence of a virus that has undergone codon optimization for expression in Bombyx mori for the production of a vaccine against that virus in Bombyx mori, wherein the virus is influenza virus, type A, and the nucleic acid sequence encodes influenza virus HA protein which has been modified to attenuate the influenza virus HA protein by replacing the amino acid sequence of SEQ ID NO:7 with that of SEQ ID NO:8.

2. The isolated nucleic acid according to claim 1, wherein the virus is selected from the group consisting of subtypes H5 and H7.

3. The isolated nucleic acid according to claim 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 12 and SEQ ID NO: 18.

4. A vector comprising the isolated nucleic acid according to claim 1.

5. A recombinant baculovirus produced using the vector according to claim 4.

6. A Bombyx mori comprising the recombinant baculovirus according to claim 5.

7. A Bombyx mori comprising the vector according to claim 4.

8. A Bombyx mori comprising: the isolated nucleic acid according to claim 1.

9. A method for producing a vaccine, the method comprising: obtaining the isolated nucleic acid according to claim 1, introducing the obtained nucleic acid according to claim 2 into Bombyx mori to express an encoded protein, and recovering the expressed protein from Bombyx mori.

10. A polypeptide comprising: an amino acid sequence encoded by the isolated nucleic acid according to claim 1.

11. A vaccine comprising: the polypeptide according to claim 10 for vaccinating an animal against a viral infection.

12. The vaccine according to claim 11, which has a virus-like particle structure.

13. The vaccine according to claim 12, wherein the diameter of the virus-like particle structure is 50 nm to 150 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a table showing codon usage frequencies in Bombyx mori obtained by investigating the usage frequencies among 450043 codons in 1180 coding regions (CDS) of Bombyx mori. The table shows the frequencies of occurrence per 1000 of each codon. Numbers in parentheses indicate the total number expressed. Boldface characters indicate the gene codons having the highest usage frequency for each amino acid.

(2) FIG. 2 is a codon correspondence table that has been optimized based on the gene codons having the highest usage frequency shown in FIG. 1. The serine (S) codon is UCA, and the codon of its complementary strand is TGA, which is also the stop codon. Consequently, the complementary strand of serine is the stop codon, and large frames do not appear in the complementary strain.

(3) FIG. 3-1 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) and an amino acid sequence. The base sequence of a synthetic gene was determined based on the base sequence of the HA gene of highly pathogenic avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) in consideration of attenuation, the introduction of a FLAG tag on the C terminal, and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(4) FIG. 3-2 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) and an amino acid sequence. The base sequence of a synthetic gene was determined based on the base sequence of the HA gene of highly pathogenic avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) in consideration of attenuation, the introduction of a FLAG tag on the C terminal, and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(5) FIG. 3-3 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) and an amino acid sequence. The enclosed portion indicates the attenuation site. The base sequence of a synthetic gene was determined based on the base sequence of the HA gene of highly pathogenic avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) in consideration of attenuation, the introduction of a FLAG tag on the C terminal, and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(6) FIG. 3-4 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was determined based on the base sequence of the HA gene of highly pathogenic avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) in consideration of attenuation, the introduction of an FLAG tag on the C terminal, and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(7) FIG. 4 shows the results of analyzing the coding frames of a gene encoding a synthesized vaccine HA protein. N1>-, N2>- and N3>- starting from the bottom indicate the results of analyzing three coding frames for each plus strand. The bar on top indicates the location of the start codon ATG, while the downward protruding bars indicate the locations of stop codons (TAA, TAG, TGA). Although only N1>- constitutes a single large coding frame, the other frames have a large number of downwardly protruding bars, and would be unable to produce a large protein even if they were expressed. N1<-, N2<- and N3<-starting from the fourth result from the bottom to the top indicate the results of analyzing coding frames of the complementary strand. Each of these frames would also be unable to produce a large protein even if expressed.

(8) FIG. 5-1 indicates the alignment between a codon-optimized HA gene DNA sequence and an HA gene cDNA sequence of avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1). Query indicates the codon-optimized HA gene DNA sequence (coding region sequence excluding the FLAG tag), while Sbjct indicates avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) (HA coding region sequence). Those portions where the sequences are the same are indicated with asterisks (*), while - symbols indicate gaps. Although homology is extremely low at 77%, the sequences are designed so as to express the same amino acid sequence with the exception of modifying and deleting the attenuated sequence.

(9) FIG. 5-2 indicates the alignment between a codon-optimized HA gene DNA sequence and an HA gene cDNA sequence of avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1). Query indicates the codon-optimized HA gene DNA sequence (coding region sequence excluding the FLAG tag), while Sbjct indicates avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) (HA coding region sequence). Those portions where the sequences are the same are indicated with asterisks (*), while - symbols indicate gaps. Although homology is extremely low at 77%, the sequences are designed so as to express the same amino acid sequence with the exception of modifying and deleting the attenuated sequence.

(10) FIG. 6 indicates confirmation of expression of HA protein from codon-optimized HA gene of avian influenza virus A/chicken/tufted duck/Fukushima/16/2011 (H5N1) produced using Bombyx mori by western blotting. Marker indicates molecular weight markers. Control indicates uninfected Bombyx mori. Fukushima indicates Bombyx mori infected with codon-optimized HA gene. The arrow indicates a specific band.

(11) FIG. 7 indicates HI activity of HI antibody to various influenza viruses in chickens using an HA solution prepared using pBm-8HA. The antibody reacted with a wide range of naturally-occurring viruses.

(12) FIG. 8 indicates HA activity of various fractions of a sucrose density gradient. HA activity was distributed at a location indicating a lower density than the location where naturally-occurring virus precipitates.

(13) FIG. 9 indicates electron micrographs of sucrose density gradient fractions. A large number of virus-like particles having a diameter of 60 nm to 120 nm were observed.

(14) FIG. 10-1 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/chicken/Sukabumi/2008 (H5N1) and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/chicken/Sukabumi/2008 (H5N1) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(15) FIG. 10-2 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/chicken/Sukabumi/2008 (H5N1) and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/chicken/Sukabumi/2008 (H5N1) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(16) FIG. 10-3 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/chicken/Sukabumi/2008 (H5N1) and an amino acid sequence. The enclosed portion indicates the attenuation site. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/chicken/Sukabumi/2008 (H5N1) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(17) FIG. 10-4 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/chicken/Sukabumi/2008 (H5N1) and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/chicken/Sukabumi/2008 (H5N1) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(18) FIG. 11 indicates confirmation of expression of HA protein from codon-optimized HA gene of avian influenza virus A/chicken/Sukabumi/2008 (H5N1) produced using Bombyx mori by western blotting. Marker indicates molecular weight markers. Control indicates uninfected Bombyx mori. Sukabumi indicates Bombyx mori infected with codon-optimized HA gene. The arrow indicates a specific band.

(19) FIG. 12-1 indicates the correspondence between a core-E1-E2 fused protein DNA sequence codon-optimized for hepatitis C virus and an amino acid sequence. Core protein: DNA sequence positions 1 to 573, E1 protein: positions 574 to 1149, E3 protein: positions 1150 to 2238, FLAG tag: positions 2239 to 2262.

(20) FIG. 12-2 indicates the correspondence between a core-E1-E2 fused protein DNA sequence codon-optimized for hepatitis C virus and an amino acid sequence. Core protein: DNA sequence positions 1 to 573, E1 protein: positions 574 to 1149, E3 protein: positions 1150 to 2238, FLAG tag: positions 2239 to 2262.

(21) FIG. 12-3 indicates the correspondence between a core-E1-E2 fused protein DNA sequence codon-optimized for hepatitis C virus and an amino acid sequence. Core protein: DNA sequence positions 1 to 573, E1 protein: positions 574 to 1149, E3 protein: positions 1150 to 2238, FLAG tag: positions 2239 to 2262.

(22) FIG. 12-4 indicates the correspondence between a core-E1-E2 fused protein DNA sequence codon-optimized for hepatitis C virus and an amino acid sequence. Core protein: DNA sequence positions 1 to 573, E1 protein: positions 574 to 1149, E3 protein: positions 1150 to 2238, FLAG tag: positions 2239 to 2262.

(23) FIG. 12-5 indicates the correspondence between a core-E1-E2 fused protein DNA sequence codon-optimized for hepatitis C virus and an amino acid sequence. Core protein: DNA sequence positions 1 to 573, E1 protein: positions 574 to 1149, E3 protein: positions 1150 to 2238, FLAG tag: positions 2239 to 2262.

(24) FIG. 13 indicates confirmation of expression of core-E1-E2 fused protein from codon-optimized core-E1-E2 fused protein gene of hepatitis C virus produced using Bombyx mori by western blotting. Marker indicates molecular weight markers. Control indicates uninfected Bombyx mori. HCV indicates Bombyx mori infected with codon-optimized core-E1-E2 fused protein gene. The arrow indicates a specific band. This indicates confirmation of expression of core-E1-E2 fused protein produced using Bombyx mori by western blotting.

(25) FIG. 14-1 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/Shanghai/02/2013 and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/Shanghai/02/2013 (H7N9) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(26) FIG. 14-2 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/Shanghai/02/2013 and an amino acid sequence. The enclosed portion indicates the predicted mutation. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/Shanghai/02/2013 (H7N9) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(27) FIG. 14-3 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/Shanghai/02/2013 and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/Shanghai/02/2013 (H7N9) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(28) FIG. 14-4 indicates the correspondence between an HA gene DNA sequence codon-optimized for avian influenza virus A/Shanghai/02/2013 and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the HA gene of avian influenza virus A/Shanghai/02/2013 (H7N9) in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(29) FIG. 15 indicates confirmation of expression of HA protein from codon-optimized HA gene of avian influenza virus A/Shanghai/02/2013 produced using Bombyx mori by western blotting. Marker indicates molecular weight markers. Control indicates uninfected Bombyx mori. H7N9 indicates Bombyx mori infected with codon-optimized HA gene. The arrow indicates a specific band.

(30) FIG. 16-1 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for Japanese encephalitis virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the Pre-M protein gene, M protein gene and E protein gene of Japanese encephalitis virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM/M protein: DNA sequence positions 1 to 501, E protein: positions 502 to 2001, FLAG tag: positions 2002 to 2025.

(31) FIG. 16-2 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for Japanese encephalitis virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the Pre-M protein gene, M protein gene and E protein gene of Japanese encephalitis virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM/M protein: DNA sequence positions 1 to 501, E protein: positions 502 to 2001, FLAG tag: positions 2002 to 2025.

(32) FIG. 16-3 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for Japanese encephalitis virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the Pre-M protein gene, M protein gene and E protein gene of Japanese encephalitis virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM/M protein: DNA sequence positions 1 to 501, E protein: positions 502 to 2001, FLAG tag: positions 2002 to 2025.

(33) FIG. 16-4 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for Japanese encephalitis virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the Pre-M protein gene, M protein gene and E protein gene of Japanese encephalitis virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM/M protein: DNA sequence positions 1 to 501, E protein: positions 502 to 2001, FLAG tag: positions 2002 to 2025.

(34) FIG. 17 indicates confirmation of expression of PreM-M-E fused protein from codon-optimized PreM-M-E fused protein gene of Japanese encephalitis virus produced using Bombyx mori by western blotting. Marker indicates molecular weight markers. Control indicates uninfected Bombyx mori. JEV indicates Bombyx mori infected with codon-optimized PreM-M-E fused protein gene. The arrow indicates a specific band.

(35) FIG. 18-1 indicates the correspondence between a G protein gene DNA sequence codon-optimized for rabies virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the G protein gene of rabies virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(36) FIG. 18-2 indicates the correspondence between a G protein gene DNA sequence codon-optimized for rabies virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the G protein gene of rabies virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(37) FIG. 18-3 indicates the correspondence between a G protein gene DNA sequence codon-optimized for rabies virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the G protein gene of rabies virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(38) FIG. 18-4 indicates the correspondence between a G protein gene DNA sequence codon-optimized for rabies virus and an amino acid sequence. The enclosed portion indicates the FLAG tag. The base sequence of a synthetic gene was designed based on the base sequence of the G protein gene of rabies virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(39) FIG. 19-1 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for West Nile virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the E protein gene of West Nile virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(40) FIG. 19-2 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for West Nile virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the E protein gene of West Nile virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(41) FIG. 19-3 indicates the correspondence between a Pre-M-M-E fused protein gene DNA sequence codon-optimized for West Nile virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the E protein gene of West Nile virus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(42) FIG. 20-1 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(43) FIG. 20-2 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(44) FIG. 20-3 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(45) FIG. 20-4 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(46) FIG. 20-5 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(47) FIG. 20-6 indicates the correspondence between a spike glycoprotein (S protein) gene DNA sequence codon-optimized for MERS coronavirus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of the S protein gene of MERS coronavirus in consideration of the introduction of a FLAG tag on the C terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(48) FIG. 21-1 indicates the correspondence between a VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of VP4, VP2, VP3, VP1, 2A and 3C protein genes of foot and mouth disease virus in consideration of the introduction of a FLAG tag on the N terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(49) FIG. 21-2 indicates the correspondence between a VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of VP4, VP2, VP3, VP1, 2A and 3C protein genes of foot and mouth disease virus in consideration of the introduction of a FLAG tag on the N terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(50) FIG. 21-3 indicates the correspondence between a VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of VP4, VP2, VP3, VP1, 2A and 3C protein genes of foot and mouth disease virus in consideration of the introduction of a FLAG tag on the N terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(51) FIG. 21-4 indicates the correspondence between a VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of VP4, VP2, VP3, VP1, 2A and 3C protein genes of foot and mouth disease virus in consideration of the introduction of a FLAG tag on the N terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

(52) FIG. 21-5 indicates the correspondence between a VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus and an amino acid sequence. The base sequence of a synthetic gene was designed based on the base sequence of VP4, VP2, VP3, VP1, 2A and 3C protein genes of foot and mouth disease virus in consideration of the introduction of a FLAG tag on the N terminal and optimization of the codon usage frequency of Bombyx mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown.

BEST MODE FOR CARRYING OUT THE INVENTION

(53) The nucleic acid according to the present invention contains a nucleic acid sequence of a virus that has been subjected to codon optimization for expression in Bombyx mori cells. More specifically, codon optimization consists of replacing a target nucleic acid sequence using a correspondence table (FIG. 2) of amino acid sequences and gene sequences prepared based on the codon usage (FIG. 1) of Bombyx mori. In this correspondence table, the sequences are designed so as to generate numerous UGA (stop codons) in the complementary strand by using UCA (having the highest usage frequency in Bombyx mori) for the Ser sequence to prevent the synthesis of unexpected by-products as a result of being able to form a long coding frame in the complementary strand.

(54) Although there are no particular limitations on the virus targeted by the vaccine according to the present invention, examples thereof include influenza virus, hepatitis C virus, Japanese encephalitis virus, rabies virus, West Nile virus, MERS coronavirus and foot and mouth disease virus. Among influenza viruses, type A influenza virus is preferable, and subtypes H5 and H7 are more preferable. Among hepatitis C viruses, genotype 1a 1b, 2a, 2b or 3a is preferable.

(55) The virus nucleic acid sequence according to the present invention encodes any arbitrary virus protein provided it can function as a vaccine. In the case of influenza virus, the virus nucleic acid sequence preferably encodes HA protein. In the case of hepatitis C virus, the virus nucleic acid protein preferably encodes core protein, E1 protein, E2 protein or a combination thereof. In the case of Japanese encephalitis virus, the virus nucleic acid sequence preferably encodes PreM protein, M protein and/or E protein. In the case of rabies virus, the virus nucleic acid sequence preferably encodes G protein. In the case of West Nile virus, the virus nucleic acid sequence preferably encodes PreM protein, M protein and/or E protein. In the case of MERS coronavirus, the virus nucleic acid sequence preferably encodes spike glycoprotein (S protein). In the case of foot and mouth disease virus, the virus nucleic acid sequence preferably encodes VP4, VP2, VP3, VP1, 2A and/or 3C protein.

(56) The virus nucleic acid sequence according to the present invention may have been modified to attenuate the influenza virus HA protein. Attenuation can be carried out by an arbitrary modification commonly known among persons with ordinary skill in the art. For example, in the case of the HA protein of influenza virus, the sequence at the cleavage site that binds the HA1 and HA2 molecules that govern pathogenicity is modified. In one mode thereof, the amino acid sequence indicated in SEQ ID NO: 7 in HA protein is replaced with the amino acid sequence indicated in SEQ ID NO: 8.

(57) Thus, the nucleic acid according to the present invention is preferably a nucleic acid that contains or is composed of the nucleic acid sequence of SEQ ID NO: 4, 12, 15, 18, 21, 24, 27, 30 or 33.

(58) In the present invention, a vaccine is produced in Bombyx mori. The method used to produce a vaccine using Bombyx mori can be carried out using any arbitrary method commonly known among persons with ordinary skill in the art. More specifically, the nucleic acid of the present invention is introduced into Bombyx mori and a virus protein encoded by the virus nucleic acid sequence is expressed therein. Although there are no particular limitations thereon, the method used to introduce the nucleic acid is carried out by, for example, inoculating Bombyx mori with a recombinant baculovirus comprising the nucleic acid of the present invention.

(59) Thus, in one embodiment, the present invention is the vector that contains the nucleic acid according to the present invention. The vector of the present invention is an arbitrary vector that enables expression of a protein encoded by the nucleic acid. The vector of the present invention may also be introduced directly into Bombyx mori. The vector of the present invention is preferably a baculovirus transfer vector.

(60) In addition, in one embodiment, the present invention is a recombinant baculovirus that is produced using the aforementioned vector according to the present invention. The method used to produce the recombinant baculovirus can be carried out using any arbitrary method commonly known among persons with ordinary skill in the art. In one embodiment, the recombinant baculovirus of the present invention can be produced by simultaneously introducing the vector of the present invention and DNA extracted from baculovirus into Bombyx mori cells.

(61) Thus, in one embodiment, the present invention is Bombyx mori comprising the nucleic acid, the vector or the recombinant baculovirus according to the present invention. There are no particular limitations on the form in which the vector or recombinant baculovirus is contained, and the vector or recombinant baculovirus may be present independently from the genome of Bombyx mori or may be incorporated in the genome of Bombyx mori.

(62) In one embodiment, the present invention is a polypeptide composed of an amino acid sequence encoded by the nucleic acid according to the present invention.

(63) In one embodiment, the present invention is a method for producing a vaccine that uses the nucleic acid, the vector, the recombinant baculovirus or the Bombyx mori according to the present invention.

(64) In one embodiment, the method for producing a vaccine according to the present invention comprises the following steps:

(65) 1) a step for obtaining the nucleic acid or vector according to the present invention,

(66) 2) a step for introducing the nucleic acid, vector or recombinant baculovirus according to the present invention into Bombyx mori, and

(67) 3) a step for recovering protein from Bombyx mori.

(68) Introduction of the nucleic acid, vector or recombinant baculovirus of the present invention into Bombyx mori can be carried out using an arbitrary method commonly known among persons with ordinary skill in the art. Introduction is preferably carried out using baculovirus. There are no particular limitations on the time at which the nucleic acid, vector or recombinant baculovirus is introduced into Bombyx mori. The time of introduction is preferably the pupal stage.

(69) Recovery of protein from Bombyx mori can use an arbitrary method commonly known among persons with ordinary skill in the art. For example, in the case of HA protein, after homogenizing Bombyx mori in an isotonic buffer solution, HA protein is recovered using immobilized erythrocytes or a sialic acid column (fetuin column).

(70) In one embodiment, the present invention is a vaccine that contains the polypeptide according to the present invention or is produced according to the production method of the present invention for vaccinating an animal against a viral infection.

(71) In one embodiment, the vaccine of the present invention has a virus-like particle structure. A virus-like particle structure refers to a structure in which well-defined HA spikes are densely arranged on the surface of particles having a diameter of 50 nm to 150 nm, and although morphologically resembling virus particles, are naturally non-pathogenic. The virus-like particle structure is preferably a spherical structure that has spikes. The particle diameter of the virus-like particles is preferably 60 nm to 120 nm. An example of the virus-like particle structure of the present invention is shown in FIG. 9.

(72) In one embodiment, the present invention is a method for inoculating an animal with a vaccine against a viral infection, comprising administering to the animal an effective amount of a vaccine comprising the polypeptide according to the present invention or a vaccine that is produced according to the production method according to the present invention.

(73) In one embodiment, the present invention is a method for introducing an immune response to a virus in an animal, comprising administering to the animal an effective amount of a vaccine comprising the polypeptide according to the present invention or a vaccine that is produced according to the production method according to the present invention.

(74) The animal according to the present invention refers to an animal capable of acquiring sufficient humoral immunity or cellular immunity to a virus as a result of being inoculated with a vaccine. The animal according to the present invention is preferably a vertebrate, more preferably a human, bird, pig or horse, and most preferably a human.

(75) An effective amount of vaccine refers to an amount that is sufficient for achieving a biological effect such as inducing sufficient humoral immunity or cellular immunity to a virus. In addition, the administration method includes inhalation, intranasal administration, oral administration and parenteral administration (such as intracutaneous, intramuscular, intravenous, intraperitoneal or subcutaneous administration). The effective amount and administration method are dependent on the age, gender, physical status and body weight of the person undergoing administration. For example, in the case of an influenza vaccine, a vaccine comprising 15 g or more of HA protein per strain in 1 ml is typically injected twice subcutaneously at an interval of about 2 to 4 weeks in an amount of 0.25 ml to subjects age 6 months to under 3 years old or in an amount of 0.5 ml to subjects age 3 to under 13 years old. Subjects age 13 years and older are injected subcutaneously once or twice at an interval of about 2 to 4 weeks in an amount of 0.5 ml.

EXAMPLES

(76) The following provides a more detailed explanation of the present invention by indicating specific examples thereof.

Example 1

Design of DNA for Developing Influenza Vaccine Suitable for Production in Bombyx mori Based on HA Genetic Information of Avian Influenza Virus a Virus/Tufted Duck/Fukushima/16/2011 (H5N1)

(77) Design of Nucleic Acid Sequence of Codon-Optimized HA Gene

(78) HA genetic information of highly pathogenic influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) which was found in wild ducks in 2011 was designed and altered in the manner indicated below.

(79) First, the amino acid sequence (SEQ ID NO: 2) of the hemagglutinin (HA) protein of avian influenza virus A/tufted duck/Fukushima/16/2011 (H5N1) was predicted from the gene sequence (SEQ ID NO: 1) registered with Genbank under Accession No. BAK24078.

(80) Next, the sequence Arg-Glu-Arg-Arg-Lys-Arg (SEQ ID NO: 7) in the predicted amino acid sequence, which is presumed to be associated with high pathogenicity, was replaced with the sequence Arg-Glu-Thr-Arg (SEQ ID NO: 8), and a FLAG tag sequence composed of Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5) was added to the C-terminal to design an attenuated HA protein amino acid sequence (SEQ ID NO: 3).

(81) Codon optimization was carried out for the purpose of improving protein expression using Bombyx mori. A correspondence table between the resulting amino acid sequence and gene sequence (FIG. 2) was prepared based on the codon usage of Bombyx mori obtained from the Codon Usage Database provided by the Kazusa DNA Research Instituted (FIG. 1). In this correspondence table, the sequences are designed so as to generate numerous UGA (stop codons) in the complementary strand by using UCA (having the highest usage frequency in Bombyx mori) for the Ser sequence to prevent the synthesis of unexpected by-products as a result of being able to form a long coding frame in the complementary strand. The designed gene sequence (SEQ ID NO: 4) and the corresponding amino acid sequence are shown in FIG. 3. Large coding frames other than the protein desired to be expressed were confirmed to be unable to be formed (FIG. 4).

(82) Synthesis of Codon-Optimized HA Gene and Vector Construction

(83) Total synthesis of DNA was commissioned to Takara Bio Inc. based on sequence data of the codon-optimized HA gene designed as described above. The synthesized codon-optimized HA gene DNA was inserted into a transfer vector in the form of pBm-8 (Baculotechnologies Co., Ltd.) using an in-fusion method (Clontech Laboratories Inc.) to prepare pBm-8HA.

(84) Infection of Bombyx mori with Baculovirus Introduced with Codon-Optimized HA Gene and Preparation of Emulsion

(85) Recombinant baculovirus for producing a vaccine in a cell supernatant was obtained by simultaneously introducing pBm-8HA and DNA extracted from baculovirus into Bombyx mori cells. Pupas of Bombyx mori were inoculated with the recombinant baculovirus. Namely, a virus stock solution having a virus titer of 110.sup.8 pfu/ml or more was diluted ten-fold with insect cell medium TC-100. Pupas of Bombyx mori were then inoculated with 50 l of this diluted solution by injecting into the abdominal region on the second day of molting followed by breeding the pupas in an incubator at 26 C. 72 hours later, the pupas were cut in half with a scissors on ice and the mesenteron was removed with a forceps followed by homogenizing and emulsifying in phenylthiourea-containing PBS using a Potter-Elvehjem homogenizer. PBS was added to the emulsion to a volume of 50 ml followed by the addition of 5 l of formalin to a final concentration of 0.01% to obtain an emulsion of Bombyx mori pupas.

(86) Recovery of HA Protein and Confirmation of Expression

(87) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment for 5 minutes with a sonicator (UR-20P, Tomy Seiko Co., Ltd.) followed by the addition of immobilized chicken erythrocytes to recover HA protein. Moreover, the HA protein was able to be purified to 95% or more at the protein level by purifying by DEAE ion exchange chromatography. Expression of the purified protein was confirmed by western blotting (primary antibody: anti-FLAG mouse monoclonal antibody, secondary antibody: anti-mouse IgG rabbit polyclonal antibody). A band was confirmed in the vicinity of 65 kDa as shown in FIG. 6.

(88) Evaluation of HA Activity

(89) Influenza virus has the property of agglutinating when mixed with erythrocytes of various animals, which is referred to as hemagglutination, and is the result of formation of a large aggregate by crosslinking of a plurality of erythrocytes through the bonding of hemagglutinin (HA) present on the virus surface with sugar chains of erythrocytes. Since the concentration of virus (HA activity) contained in a stock solution can be calculated by investigating the degree of agglutination when the virus is serially diluted by using this property, this can be used to quantify influenza virus. More specifically, after serially diluting 50 l of a specimen with 50 l of PBS (phosphate-buffered saline) using a 96-well microplate, an equal volume of chicken erythrocytes adjusted to a concentration of 0.5% was added and mixed well followed by allowing to stand undisturbed for 30 minutes to 60 minutes at room temperature. Although HA forms an aggregate with the erythrocytes if HA activity is present in the specimen, in the case the virus is absent, an aggregate is not formed and the erythrocytes settle on the bottom of the plate in the form of a red dot. HA titer is represented in the form of HA agglutination activity based on the dilution factor of the dilution prior to the formation of this red dot.

(90) Results of Evaluation

(91) The resulting HA protein solution was evaluated under the aforementioned conditions. As a result, HA activity was demonstrated to be 2,097,152. As a result, total activity produced in 30 Bombyx mori pupas was 2,097,15250 ml=104,857,600, and the amount of HA activity per pupa was 3,495,253. This value of HA activity per recombinant Bombyx mori individual is roughly 11.4 times of HA activity of 307,160 per Bombyx mori individual when HA gene of human-derived highly pathogenic avian influenza virus A/HK/483/97 (H5N1) was expressed in Bombyx mori in 1998, and is roughly 340 times of HA activity of 10,240 produced from a single embryonated chicken egg, thereby demonstrating a remarkable increase in the amount of HA activity.

(92) Immunization of Chickens with HA Protein Solution

(93) Chickens were immunized using the aforementioned HA protein solution. Chickens were inoculated with 0.5 ml of HA protein solution adjusted to an HA activity value between 4,096 and 8,192 into leg muscle on two occasions at an interval of 16 days. Blood samples of 5 ml each were collected from the chickens at 33 days, 40 days, 47 days and 50 days after the initial inoculation.

(94) Evaluation of HI Activity of Antibody Produced by Immunized Chickens

(95) The aforementioned chicken blood samples were each allowed to stand at room temperature followed by centrifuging at room temperature to obtain chicken serum. The hemagglutination inhibitory activity (HI activity) of each sample of chicken serum was examined using the method indicated below. First, a 1:2 serial dilution series of the serum was prepared that contained 25 l per well using a 96-well plate. Next, 25 l of the aforementioned HA protein (adjusted to 8 HA with PBS) were added to each well. After allowing the plate to stand undisturbed for 30 minutes at room temperature, 50 l of chicken erythrocytes at a concentration of 0.5% in PBS were added to each well. One hour later, the hemagglutination pattern was observed and the largest dilution factor at which agglutination of erythrocytes was observed was taken to be the HI activity value. Serum at 33 days after initial inoculation demonstrated HI activity of 8,192.

(96) Evaluation of HI Activity for Various Influenza Viruses

(97) In addition, the presence or absence of HI activity was examined for various influenza viruses using chicken serum obtained at 40 days after initial inoculation. HI activity was measured under the same conditions as previously described using each of the viruses of A/chicken/Legok/2004 (H5N1) and A/chicken/West Java/2009 (H5N1). The results are shown in FIG. 7. The chicken serum demonstrated HI activity against all viruses.

(98) Fractionation of HA Protein Solution by Sucrose Density Gradient

(99) In addition, the HA protein solution was fractionated using the sucrose density gradient method. The HA activity of each liquid fraction was examined using the same method as previously described. The results are shown in FIG. 8. Fraction 4, having a buoyant density of naturally-occurring virus of 1.18 g/l, did not show HA activity. On the other hand, HA activity was observed in fractions 7 to 9 demonstrating smaller buoyant densities.

(100) Observation of HA Protein Solution Fraction with an Electron Microscope

(101) Fraction 7 of the aforementioned sucrose density gradient was observed with an electron microscope. The results are shown in FIG. 9. A virus particle-like structure in the shape of a sphere having spikes and having a particle diameter of 60 nm to 120 nm was observed. As a general rule, naturally-occurring viruses have a spherical shape of 60 nm to 150 nm, a long, narrow fibrous shape of 100 nm to 1000 nm or an annular shape of 80 nm to 200 nm. In contrast, as shown in FIG. 10, the HA protein derived from Bombyx mori exhibited a diverse shape in the manner of naturally-occurring viruses, and the shape was determined for the first time in this research to have a spherical shape of 60 nm to 150 nm, a fibrous shape of about 200 nm and an annular shape of about 60 nm to 150 nm. These morphological characteristics are thought to be reflected in the aforementioned previously unobserved strong immunity of HA protein.

(102) In this manner, the reason for the activity level of HA protein of avian influenza virus A/tufted duck/Fukushima/16/2001 being high in pupas of Bombyx mori is thought to be due to modification of the design of DNA based on genetic information and the effectiveness of genetic manipulation of synthetic DNA on the basis thereof. Moreover, since the target gene was highly expressed using the pupal stage of Bombyx mori, the resulting vaccine is through to have shown a virus particle-like structure. In this manner, it is thought that this novel method for producing a vaccine that utilizes design modification and synthesis of DNA based on genetic information will be used in numerous fields in the future.

(103) In conclusion, the greatest significance of vaccine production utilizing Bombyx mori by design-modified DNA is that an amount of vaccine equivalent to 340 embryonated chicken eggs can be produced with a single Bombyx mori pupa.

Example 2

Design of DNA for Developing Influenza Vaccine Suitable for Production in Bombyx mori Based on HA Genetic Information of Avian Influenza Virus a Virus/Chicken/Sukabumi/2008 (H5N1)

(104) Since viruses frequently mutate over time in both humans and animals, apprehension remains with respect to only using DNA of the avian influenza virus A/tufted duck/Fukushima/16/2011 strain. Therefore, DNA for developing a vaccine for influenza virus (SEQ ID NO: 12) was designed in the same manner as Example 1 based on amino acid sequence data (SEQ ID NO: 10) predicted from the HA gene sequence of influenza virus A/chicken/Sukabumi/2008 (H5N1) (SEQ ID NO: 9) isolated in Indonesia in 2008. The corresponding amino acid sequence (SEQ ID NO: 11) is shown in FIG. 10. In the same manner as Example 1, a recombinant baculovirus comprising the aforementioned DNA for development was inoculated into Bombyx mori followed by synthesis and recovery of HA protein and confirmation of the expression thereof by western blotting (FIG. 11). Evaluation of HA activity yielded an amount of HA activity per pupa of 419,430.

Example 3

Design of DNA for Developing Hepatitis C Virus Vaccine Suitable for Production in Bombyx mori

(105) The particle structure of hepatitis C virus is such that it is covered with a lipid layer comprising E1 and E2 glycoproteins, with the virus gene being contained within the particle with a nuclear protein or a protein referred to as the core protein. It has been clearly determined that the E1 protein and E2 protein play an important role in order for the virus to initiate infection, while conversely, the E1 protein and E2 protein are also protective antigens against infection, and therefore have an important function as vaccine proteins as well. In actuality, E1 protein and E2 protein have been used to prepare a test vaccine, and that vaccine has been reported to demonstrate preventive effects as well as mild, albeit inadequate, protective effects in chimpanzees. However, more highly concentrated E1 protein and E2 protein are required in order to demonstrate more adequate preventive effects.

(106) Design of Nucleic Acid Sequence of Codon-Optimized Core-E1-E2-Fused Protein Gene

(107) Therefore, the inventors of the present invention designed genetic information for expressing a fused protein consisting of hepatitis C virus-like core protein, E1 protein and E2 protein in order to develop a hepatitis C virus vaccine. Here, a fused protein was designed since a virus particle-like protein can be expected to be synthesized by expressing simultaneously. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence from the core protein to E1 protein and E2 protein (SEQ ID NO: 13) in the amino acid sequence of HCV gene registered with Genbank under Accession No. ACK28185 to obtain an amino acid sequence of a fused protein based on the nucleic acid design (SEQ ID NO: 14). The nucleic acid sequence of a codon-optimized Core-E1-E2 fused protein was then designed in the same manner as Example 1 based on the amino acid sequence of this fused protein (SEQ ID NO: 15). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 12.

(108) DNA Synthesis of Codon-Optimized Core-E1-E2 Fused Protein Gene and Vector Construction

(109) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the codon-optimized core-E1-E2 fused protein gene followed by insertion into pBm-8 vector to produce pBM-8Core-E1-E2.

(110) Infection with Baculovirus Introduced with Codon-Optimized Core-E1-E2 Fused Protein Gene

(111) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8Core-E1-E2 followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(112) Recovery of Core-E1-E2 Fused Protein and Confirmation of Expression Thereof

(113) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment for 5 minutes with a sonicator (UR-20P, Tomy Seiko Co., Ltd.) followed by purifying the Core-E1-E2 fused protein using the sucrose density gradient method (sucrose concentration: 10% to 50%, 100,000 G, 24 hours). Expression of the purified Core-E1-E2 fused protein was confirmed by western blotting in the same manner as Example 1. As shown in FIG. 13, a band was confirmed in the vicinity of 60 kDa.

(114) Evaluation of Activity

(115) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

(116) Discussion of Results

(117) Since a molecular weight of 83 kDa or more is predicted to be demonstrated as a result of linked expression of Core-E1-E2 fused protein, a molecular size of 60 kDa was suggested to indicate that the Core-E1-E2 protein was subjected to intracellular processing by protease resulting in synthesis of mature E2. This fact clearly indicates that the vaccine protein designed based on DNA was accurately produced without any significant difference in molecular size, and judging from the strong protein band as well, hepatitis C vaccine was suggested to be efficiently synthesized.

Reference Example 1

Design of DNA for Developing Influenza Vaccine Suitable for Production in Bombyx mori Based on HA Genetic Information of Avian Influenza Virus a Virus/Shanghai/2/2013 (H7N9)

(118) DNA for developing a vaccine for influenza virus (SEQ ID NO: 18) was designed in the same manner as Example 1 based on amino acid sequence data (SEQ ID NO: 16) of the HA gene sequence of avian influenza virus A/Shanghai/2/2013 (H7N9) registered with GISAID under Accession No. EPI1439502 that was isolated in China in 2013. The corresponding amino acid sequence (SEQ ID NO: 17) is shown in FIG. 14. Since the 199th Glu of HA protein is predicted to mutate to Asp and the 234th Gly is predicted to mutate to Asp when influenza caused by H7N9 influenza virus becomes epidemic in humans, those mutations were introduced. A recombinant baculovirus comprising the aforementioned DNA for development was inoculated into Bombyx mori pupas followed by synthesis and recovery of HA protein in the same manner as Example 1 (FIG. 15). The HA activity thereof was then evaluated in the same manner as Example 1.

Reference Example 2

Design of DNA for Developing Japanese Encephalitis Virus Vaccine Suitable for Production in Bombyx mori

(119) Japanese encephalitis virus is an encephalitis virus that is mainly transmitted by Culex triaeniorhynchus, and is currently prevalent in the regions of Southeast Asia, India and China. Although mouse brain infected with the same virus has been used as a vaccine for its prevention, viruses for vaccine use have more recently come to be cultured in cultured cells. However, there is the need to develop a new vaccine in response to demands for enhanced vaccine immunity, reduction of adverse side effects and lower production costs, and considerable expectations are being placed on improvement of vaccine quality.

(120) Design of Nucleic Acid Sequence of PreM-M-E Fused Protein Gene Codon-Optimized for Japanese Encephalitis Virus

(121) Therefore, the inventors of the present invention designed genetic information for expressing the E protein of Japanese encephalitis virus in order to develop a vaccine against Japanese encephalitis virus. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence from the PreM protein to the M protein and E protein (SEQ ID NO: 19) in the amino acid sequence registered with Genbank under Accession No. ABQ52691 to obtain an amino acid sequence of the Japanese encephalitis virus PreM-M-E fused protein based on the nucleic acid design (SEQ ID NO: 20). The nucleic acid sequence of a PreM-M-E fused protein codon-optimized for Japanese encephalitis virus was then designed in the same manner as Example 1 based on the amino acid sequence of this Japanese encephalitis virus PreM-M-E fused protein (SEQ ID NO: 21). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 16.

(122) DNA Synthesis of PreM-M-E Fused Protein Gene Codon-Optimized for Japanese Encephalitis Virus and Vector Construction

(123) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the PreM-M-E fused protein gene codon-optimized for Japanese encephalitis virus followed by insertion into pBm-8 vector to produce pBM-8JevpMME.

(124) Infection with Baculovirus Introduced with PreM-M-E Fused Protein Gene Codon-Optimized for Japanese Encephalitis Virus

(125) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8JevpMME followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(126) Recovery of Japanese Encephalitis Virus PreM-M-E Fused Protein and Confirmation of Expression

(127) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment and purification in the same manner as Example 3. Expression of the purified Japanese encephalitis virus E protein was confirmed by western blotting in the same manner as Example 3 (FIG. 17).

(128) Evaluation of Activity

(129) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

Reference Example 3

Design of DNA for Developing Rabies Virus Vaccine Suitable for Production in Bombyx mori

(130) Although rabies virus is distributed on a global scale and results in numerous deaths, resulting in the international need for the development of an effective, safe and inexpensive vaccine, the development of such a vaccine is proceeding slowly. Therefore, if it were possible to develop a vaccine that is safe, inexpensive and demonstrates potent immunity, the demand for such a vaccine is thought to be on a global scale. Vaccines currently in use are derived from rabbit, goat and mouse brain, while human diploid cells and chicken embryonic cells are also used. In the future, it will be desirable to develop a component vaccine that can be used stably both in terms of supply and costs, and the successful development of a component vaccine using Bombyx mori is thought to result in use of that vaccine on a global scale.

(131) Design of Nucleic Acid Sequence of G Protein Gene Codon-Optimized for Rabies Virus

(132) Therefore, the inventors of the present invention designed genetic information for expressing the G protein of rabies virus in order to develop a vaccine against rabies virus. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence registered with Genbank under Accession No. ABX46657 (SEQ ID NO: 22) to obtain an amino acid sequence of the rabies virus G protein based on the nucleic acid design (SEQ ID NO: 23). The nucleic acid sequence of a G protein codon-optimized for rabies virus was then designed in the same manner as Example 1 based on the amino acid sequence of this rabies virus G protein (SEQ ID NO: 24). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 18.

(133) DNA Synthesis of G Protein Gene Codon-Optimized for Rabies Virus and Vector Construction

(134) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the G protein gene codon-optimized for rabies virus followed by insertion into pBm-8 vector to produce pBM-8rvG.

(135) Infection with Baculovirus Introduced with G Protein Gene Codon-Optimized for Rabies Virus

(136) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8rvG followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(137) Recovery of Rabies Virus G Protein and Confirmation of Expression

(138) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment and purification in the same manner as Example 3. Expression of the purified rabies virus G protein was confirmed by western blotting in the same manner as Example 3.

(139) Evaluation of Activity

(140) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

Reference Example 4

Design of DNA for Developing West Nile Virus Vaccine Suitable for Production in Bombyx mori

(141) West Nile virus is prevalent in the US, Eastern Europe and Europe, and there is the risk of its propagation to Japan in the near future. In addition to its ecocycle from mosquitoes to chickens and back to mosquitoes, this cycle also includes propagation to humans or horses. Although there are currently no vaccines available, there is an urgent global demand for the development of a vaccine. Although vaccine research and development is being actively conducted in the US and Europe, those efforts have yet to be successful. Therefore, the inexpensive, large-scale production of a vaccine against West Nile fever would enable its use on a global scale.

(142) Design of Nucleic Acid Sequence of PreM-M-E Fused Protein Gene Codon-Optimized for West Nile Virus

(143) Therefore, the inventors of the present invention designed genetic information for expressing the PreM-M-E fused protein of West Nile virus in order to develop a vaccine against West Nile virus. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence from the PreM protein to the M protein and E protein (SEQ ID NO: 25) in the amino acid sequence registered with Genbank under Accession No. AAT95390 to obtain an amino acid sequence of the West Nile virus PreM-M-E fused protein based on the nucleic acid design (SEQ ID NO: 26). The nucleic acid sequence of a PreM-M-E fused protein codon-optimized for West Nile virus was then designed in the same manner as Example 1 based on the amino acid sequence of this West Nile virus PreM-M-E fused protein (SEQ ID NO: 27). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 19.

(144) DNA Synthesis of PreM-M-E Fused Protein Gene Codon-Optimized for West Nile Virus and Vector Construction

(145) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the PreM-M-E fused protein gene codon-optimized for West Nile virus followed by insertion into pBm-8 vector to produce pBM-8wnvpMME.

(146) Infection with Baculovirus Introduced with PreM-M-E Fused Protein Gene Codon-Optimized for West Nile Virus

(147) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8wnvpMME followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(148) Recovery of West Nile Virus PreM-M-E Fused Protein and Confirmation of Expression

(149) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment and purification in the same manner as Example 3. Expression of the purified West Nile virus PreM-M-E fused protein was confirmed by western blotting in the same manner as Example 3.

(150) Evaluation of Activity

(151) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

Reference Example 5

Design of DNA for Developing MERS Coronavirus Vaccine Suitable for Production in Bombyx mori

(152) Design of Nucleic Acid Sequence of Spike Glycoprotein (S Protein) Gene Codon-Optimized for MERS Coronavirus

(153) The inventors of the present invention designed genetic information for expressing the S protein of MERS coronavirus in order to develop a vaccine against MERS coronavirus. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence registered with Genbank under Accession No. AGN52936 (SEQ ID NO: 28) to obtain an amino acid sequence of the MERS coronavirus S protein based on the nucleic acid design (SEQ ID NO: 29). The nucleic acid sequence of an S protein codon-optimized for MERS coronavirus was then designed in the same manner as Example 1 based on the amino acid sequence of this MERS coronavirus S protein (SEQ ID NO: 30). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 20.

(154) DNA Synthesis of S Protein Gene Codon-Optimized for MERS Coronavirus and Vector Construction

(155) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the S protein gene codon-optimized for MERS coronavirus followed by insertion into pBm-8 vector to produce pBM-8mcvS.

(156) Infection with Baculovirus Introduced with S Protein Gene Codon-Optimized for MERS Coronavirus

(157) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8mcvS followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(158) Recovery of MERS Coronavirus S Protein and Confirmation of Expression

(159) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment and purification in the same manner as Example 3. Expression of the purified MERS coronavirus S protein was confirmed by western blotting in the same manner as Example 3.

(160) Evaluation of Activity

(161) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

Reference Example 6

Design of DNA for Developing Foot and Mouth Disease Virus Vaccine Suitable for Production in Bombyx mori

(162) Design of Nucleic Acid Sequence of VP4-VP2-VP3-VP1-2A-3C Fused Protein Codon-Optimized for Foot and Mouth Disease Virus

(163) The inventors of the present invention designed genetic information for expressing the VP4-VP2-VP3-VP1-2A-3C fused protein of foot and mouth disease virus in order to develop a vaccine against foot and mouth disease virus. First, a FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence obtained by binding from the N-terminal side towards the C-terminal side an amino acid sequence of VP4, Vp2, VP1, 2A and 3C proteins in an amino acid sequence predicted from the nucleic acid sequence registered with GenBank under Accession No. HV940030 (SEQ ID NO: 31) to obtain an amino acid sequence of the foot and mouth disease virus based on the nucleic acid design (SEQ ID NO: 32). The nucleic acid sequence of a VP4-VP2-VP3-VP1-2A-3C fused protein codon-optimized for foot and mouth disease virus was then designed in the same manner as Example 1 based on the amino acid sequence of this foot and mouth disease virus VP4-VP2-VP3-VP1-2A-3C protein (SEQ ID NO: 33). Correspondence between the designed nucleic acid sequence of the chimeric synthetic DNA and the amino acid sequence is shown in FIG. 21.

(164) DNA Synthesis of VP4-VP2-VP3-VP1-2A-3C Fused Protein Gene Codon-Optimized for Foot and Mouth Disease Virus and Vector Construction

(165) Full-length gene DNA was synthesized in the same manner as Example 1 based on the designed nucleic acid sequence of the VP4-VP2-VP3-VP1-2A-3C fused protein gene codon-optimized for foot and mouth disease virus followed by insertion into pBm-8 vector to produce pBM-8fmdvP.

(166) Infection with Baculovirus Introduced with VP4-VP2-VP3-VP1-2A-3C Fused Protein Gene Codon-Optimized for Foot and Mouth Disease Virus

(167) A recombinant baculovirus was obtained in the same manner as Example 1 using pBm-8fmdvP followed by inoculation of Bombyx mori pupas to obtain an emulsion of Bombyx mori pupas.

(168) Recovery of Foot and Mouth Disease Virus VP4-VP2-VP3-VP1-2A-3C Fused Protein and Confirmation of Expression

(169) The resulting emulsion of Bombyx mori pupas was subjected to ultrasonic treatment and purification in the same manner as Example 3. Expression of the purified foot and mouth disease virus VP4-VP2-VP3-VP1-2A-3C fused protein was confirmed by western blotting in the same manner as Example 3.

(170) Evaluation of Activity

(171) Immune reaction can be predicted from the amount of protein produced, and the effect in a neutralization test was estimated using antibody produced as a result thereof.

(172) The nucleic acid according to the present invention demonstrated the remarkable effects indicated below.

(173) 1) Protein derived from a dangerous virus is obtained by using artificially synthesized DNA without having to handle the dangerous virus. (Since dangerous viruses can be handled without requiring P4 or P3 facilities, there is no possibility of the virus escaping to the outside as a result of infecting people or adhering to clothing, thereby making it safe.)

(174) 2) An amino acid sequence having low pathogenicity can be designed from the start based on bioinformatics.

(175) 3) Amino acid sequences can be designed from the start while predicting future amino acid mutations based on bioinformatics and evolutionary analyses.

(176) 4) Although a gene sequence is determined from an amino acid sequence, and the amino acid sequence is completely identical to the original amino acid sequence with the exception of the virulent site and FLAG tag in an HA protein, for example, when a gene sequence is obtained therefrom, the homology thereof is only 77% (see, for example, FIG. 5), thereby making it possible to easily distinguish from the virus gene.

(177) 5) Since a FLAG tag sequence is attached to the C-terminal, even if the virus should happen to infect cells, there is a high degree of safety since there is no interaction between virus RNP and M1 protein.

(178) 6) Since a gene sequence is artificially designed using a codon usage optimized for Bombyx mori cells followed by incorporating in a vector, the expression level thereof in the Bombyx mori cells is significantly higher than that using a sequence derived from virus gene, and antigenicity is completely identical to that of the original virus.

(179) 7) As a result of using Bombyx mori cells, the sugar chain structure is not complex and antigenicity masking action attributable to sugar chains is extremely weak. Consequently, the increase in antibody titer when used as a vaccine is extremely high at 340 times that of purified HA protein derived from virus grown in chicken eggs.

(180) 8) The use of a FLAG tag facilitates confirmation of expression and purification.

(181) The production of a vaccine in Bombyx mori by using this novel technology of the present application makes it possible to produce a component vaccine comprising only the active component at low cost while also allowing the vaccine to be produced in a shorter period of time. In addition, this also simultaneously solves the problem of egg allergies. Moreover, this technology also makes it possible to considerably reduce the amount of time required to produce seed viruses for rapid proliferation in embryonated chicken eggs, which in combination with the other aforementioned advantages, clearly makes this technology superior.

INDUSTRIAL APPLICABILITY

(182) The nucleic acid of the present invention, which is artificially synthesized based on the design of a nucleic acid sequence codon-optimized for expression in Bombyx mori, is useful for producing a large amount of vaccine as previously described.

SEQUENCE LISTING FREE TEXT

(183) SEQ ID NO: 1: HA gene DNA sequence of A/tufted duck/Fukushima/16/2011 (H5N1) avian influenza virus

(184) SEQ ID NO: 2: HA amino acid sequence of A/tufted duck/Fukushima/16/2011 (H5N1) avian influenza virus

(185) SEQ ID NO: 3: Modified HA amino acid sequence of A/tufted duck/Fukushima/16/2011 (H5N1) avian influenza virus

(186) SEQ ID NO: 4: HA gene DNA sequence codon-modified for A/tufted duck/Fukushima/16/2011 (H5N1) avian influenza virus

(187) SEQ ID NO: 5: FLAG tag amino acid sequence

(188) SEQ ID NO: 6: FLAG tag DNA sequence

(189) SEQ ID NO: 7: Highly pathogenic amino acid sequence

(190) SEQ ID NO: 8: Lowly pathogenic amino acid sequence

(191) SEQ ID NO: 9: HA gene DNA sequence of A/chicken/Sukabumi/2008 (H5N1) avian influenza virus

(192) SEQ ID NO: 10: HA amino acid sequence of A/chicken/Sukabumi/2008 (H5N1) avian influenza virus

(193) SEQ ID NO: 11: Modified HA amino acid sequence of A/chicken/Sukabumi/2008 (H5N1) avian influenza virus

(194) SEQ ID NO: 12: HA gene DNA sequence codon-optimized for A/chicken/Sukabumi/2008 (H5N1) avian influenza virus

(195) SEQ ID NO: 13: Core-E1-E2 fused protein amino acid sequence of hepatitis C virus

(196) SEQ ID NO: 14: Modified Core-E1-E2 fused protein amino acid sequence of hepatitis C virus

(197) SEQ ID NO: 15: Core-E1-E2 gene DNA sequence codon-optimized for hepatitis C virus

(198) SEQ ID NO: 16: HA amino acid sequence of A/Shanghai/02/2013 (H7N9) influenza virus

(199) SEQ ID NO: 17: Modified HA amino acid sequence of A/Shanghai/02/2013 (H7N9) influenza virus

(200) SEQ ID NO: 18: HA gene DNA sequence codon-optimized for A/Shanghai/02/2013 (H7N9) influenza virus

(201) SEQ ID NO: 19: PreM-M-E fused protein amino acid sequence of Japanese encephalitis virus

(202) SEQ ID NO: 20: PreM-M-E fused protein+FLAG tag amino acid sequence of Japanese encephalitis virus

(203) SEQ ID NO: 21: PreM-M-E fused protein gene DNA sequence codon-optimized for Japanese encephalitis virus

(204) SEQ ID NO: 22: G protein amino acid sequence of rabies virus

(205) SEQ ID NO: 23: G protein+FLAG tag amino acid sequence of rabies virus

(206) SEQ ID NO: 24: G protein gene DNA sequence codon-optimized for rabies virus

(207) SEQ ID NO: 25: PreM-M-E fused protein amino acid sequence of West Nile virus

(208) SEQ ID NO: 26: PreM-M-E fused protein+FLAG tag amino acid sequence of West Nile virus

(209) SEQ ID NO: 27: PreM-M-E fused protein gene DNA sequence codon-optimized for West Nile virus

(210) SEQ ID NO: 28: S protein amino acid sequence of MERS coronavirus

(211) SEQ ID NO: 29: S protein+FLAG tag amino acid sequence of MERS coronavirus

(212) SEQ ID NO: 30: S protein gene DNA sequence codon-optimized for MERS coronavirus

(213) SEQ ID NO: 31: VP4-VP2-VP3-VP1-2A-3C fused protein amino acid sequence of foot and mouth disease virus

(214) SEQ ID NO: 32: VP4-VP2-VP3-VP1-2A-3C fused protein+FLAG tag amino acid sequence of foot and mouth disease virus

(215) SEQ ID NO: 33: VP4-VP2-VP3-VP1-2A-3C fused protein gene DNA sequence codon-optimized for foot and mouth disease virus