JP4797149B2 - Vector vaccine against influenza virus - Google Patents

Description

  The present application relates to a novel influenza virus vaccine. Specifically, the present invention relates to a vector vaccine that exhibits high vaccine efficacy against a wide variety of influenza viruses.

  In recent years, the possibility of a human pandemic of H5 avian influenza is emerging, and a swine influenza virus with a mutated H1 is actually causing a pandemic in humans. It is one of the most serious viral infectious diseases that can be prevalent on a global scale.

  Since influenza viruses have a relatively high frequency of antigenic mutations, the fact is that no vaccine has yet been developed that can sufficiently cope with influenza viruses having mutated antigens and can prevent influenza virus infection with a high probability.

  The main vector vaccines against influenza viruses that have been developed and put to practical use are aimed at inducing neutralizing antibodies against hemagglutinin (HA) protein or neuraminidase (NA) protein. However, since HA proteins and NA proteins have a large degree of antigenic variation depending on the virus strain, these vaccines have a drawback in that they may not be fully effective due to insufficient responses to influenza virus mutations. It was.

  Recently, DNA vaccines have been developed due to advances in genetic engineering technology. A DNA vaccine generally uses a recombinant plasmid, and an amino acid having an antigenicity encoded by the DNA in vivo by administering the recombinant plasmid containing the DNA encoding the target antigen in vivo. A sequence is expressed, and this amino acid sequence induces an immune response in vivo. However, even if a specific plasmid DNA vaccine is administered to a living body, the degree of immune reaction that is elicited is not constant, and the degree of immune reaction varies depending on the type of test animal, the type of DNA, and the inoculation method. It is generally difficult to predict whether a DNA plasmid will be useful as a DNA vaccine.

  As a DNA vaccine against influenza virus, it has been reported that plasmid DNA encoding influenza A nucleoprotein (NP) is useful for preventing influenza virus infection (Non-patent Documents 1 and 2). Ulmer et al., Using DNA encoding nucleoprotein (NP), a protein in the virus, as a vaccine, induces cellular immunity with almost no effect on antigenic mutation and achieves a stronger protective effect against infection. Has succeeded. Further, the present inventor has proposed a vector vaccine against influenza virus containing DNA having a base sequence encoding matrix protein of influenza virus or antigenic epitope thereof (Patent Document 1).

  Such DNA vaccines are expected to be practically used as simple and safe vaccines, while DNA vaccines are expected to inevitably increase the number of inoculations due to their low expression level. A recombinant virus vector vaccine has been proposed as a vaccine that can solve this problem. If a recombinant virus vector vaccine is used, it is expected that a sufficient amount of gene expression and an immune reaction obtained therefrom, particularly an antibody production reaction, will be enhanced with a small number of inoculations.

  Various viral vectors for preparing recombinant virus vector vaccines are known, and representative examples include vaccinia virus Ankara strain (MVA) and adenovirus.

  As a recombinant virus vector vaccine for protection against influenza infection, a virus vector vaccine obtained by introducing a nucleic acid encoding an influenza polypeptide into an adenovirus vector has been proposed (Patent Document 2). In Patent Document 2, a nucleic acid encoding hemagglutinin (HA) of influenza virus is used for preparing a vaccine. Patent Document 3 also discloses a recombinant virus vector vaccine obtained by introducing a sequence encoding an avian influenza antigen or immunogen into an adenovirus vector. Patent Document 3 proposes that various proteins may be used as the immunogen, but only the nucleic acid encoding HA is confirmed in the examples. In addition, vector vaccines against influenza viruses introduced with a common consensus sequence of H1-H9 as HA antigen genes have also been proposed (Non-patent Documents 3 and 4).

  On the other hand, the present inventor has proposed a vaccine for protection against HIV infection using a modified adenovirus vector (Patent Document 4). Using an Ad5 / 35 vector in which the gene encoding the non-proliferating type 5 adenovirus fiber protein is replaced with a gene encoding the type 35 adenovirus Faber protein, and using the HIV envelope protein or It is a vector vaccine that has been incorporated so that a gene encoding the mutant having the same function can be expressed. A vaccine prepared using this Ad5 / 35 viral vector has a very strong immunopotentiating ability and has been shown to be useful for protecting against human immunodeficiency virus infection.

JP 2001-151698 A Special table 2008-522621 Special table 2009-512421 gazette JP 2007-37402 A

Ulmer, 1993, Science, 259, 1745-1749 Donnelly, 1995, Nature Med., 1, 583-587 Holman DH, Vaccine 2008, 19; 26 (21): 2627-30 Chen MW, Proc. Natl. Acad. Sci. USA 2008 Sep 9: 105 (36): 13538-43

  An object of the present invention is to provide a vector vaccine against influenza virus, which can protect against a wide variety of influenza virus infections and has a strong immunopotentiating ability.

The present invention relates to an Ad5 / 35 adenovirus vector in which a gene encoding a fiber protein of E1-deficient non-proliferating type 5 adenovirus is replaced so that it can be expressed by a gene encoding a fiber protein of type 35 adenovirus.
1) polypeptides derived from influenza virus nucleoprotein (NP) and matrix protein (M), or genes encoding these antigenic epitopes, or 2) influenza virus hemagglutinin (HA), nucleoprotein (NP) and matrix Provided is a viral vector vaccine for protection against influenza virus infection, which is incorporated so that it can express either a polypeptide derived from protein (M) or a gene encoding these antigenic epitopes.

  As the Ad5 / 35 adenoviral vector used in the present invention, a modified Ad5 / 35 adenoviral vector in which the hexon hypervariable region 5 (HVR5) is changed is more preferably used as the Ad5 / 35 adenoviral vector. preferable.

  As the matrix protein (M) used in the present invention, M1 and M2 may be used in combination, or M2 蚤 may be used alone.

  The virus vector vaccine for protection against influenza virus infection according to the present invention is very safe for humans, has a strong immune enhancing ability, and can protect against a wide variety of influenza viruses. Moreover, it can be used for influenza infection protection of vertebrates such as mammals including humans and birds including chickens.

It is a schematic diagram of the modified Ad5 / 35 adenovirus vector used in the present invention. It is a schematic diagram of the vector vaccine with respect to the influenza virus of this invention. The upper row shows the conventional Ad5 / 35 adenovirus vector vaccine, and the lower row shows the modified Ad5 / 35 adenovirus vector vaccine in which the hypervariable region 5 (HVR5) of Ad5 / 35 hexon is changed. The result of the test which compared the antibody production by the anti-HIV vaccine produced using the Ad5 / 35 adenovirus vector used by this invention with the vaccine using a MVA virus vector and a protein antigen is shown. The result of the test which compared the cell-mediated immunity enhancement capability by the anti-HIV vaccine produced using the Ad5 / 35 adenovirus vector used by this invention with the HIV vaccine using a MVA virus vector, and a protein antigen is shown. It is a figure which shows the comparison result of the immunogenicity of the vector vaccine which integrated the nucleic acid which codes HA of influenza virus to Ad5 / 35 adenovirus vector and each modified Ad5 / 35 adenovirus vector. First immunization by combining an Ad5 adenoviral vector, an Ad5 / 35 adenoviral vector, a modified Ad5 adenoviral vector, and a modified Ad5 / 35 adenoviral vector with a vector vaccine incorporating a nucleic acid encoding HA of influenza virus. It is a figure which shows the enhancement ability of cellular immunity at the time of using for booster. It is a figure which shows the gene sequence of the vector vaccine used in Example 1. FIG. It is a figure which shows the infection protection effect of the virus vector vaccine prepared by introduce | transducing various antigens into a modified Ad5 / 35 adenovirus vector. It is a figure which shows the infection protective effect with respect to several subtype influenza viruses of the viral vector vaccine prepared by introduce | transducing various antigens into a modified Ad5 / 35 adenoviral vector. It is a figure which shows the gene sequence of the vector vaccine used in Example 2. The result of Example 2 is shown.

As used herein, the term “vaccine” means a substance that can generate an immune response.
In this specification, the term “influenza” means the influenza virus in the broadest sense unless otherwise specified. Influenza viruses are generally classified into the genus influenza virus (which is further classified into types A and B) and influenza virus type C, although the term “influenza virus” is used herein. In this case, unless otherwise specified, all of them are included, and all mutants of influenza virus are also included.
In the present specification, the “viral vector vaccine” refers to a vaccine obtained by incorporating a nucleic acid encoding an antigenic protein or peptide or an epitope thereof into a viral vector.

  In the present specification, the antigenic epitope of a protein may be a complete protein or its protein as long as it contains a peptide that is an epitope related to the immunogenicity of the protein (for example, an epitope recognized by cytotoxic T cells). It may be a fragment. Therefore, the gene encoding the specific protein or antigenic epitope thereof used in the present invention does not need to encode the full length of the protein, and may encode a partial fragment containing the epitope.

  In the present specification, the terms “matrix protein (M)”, “nucleoprotein (NP)” and “hemagglutinin (HA)” are used to include not only the complete protein but also fragments thereof having an antigenic epitope. May be.

  In the present invention, an Ad5 / 35 adenovirus vector in which the gene encoding the fiber protein of E1-deficient non-proliferating type 5 adenovirus is replaced with a gene encoding the fiber protein of type 35 adenovirus is substituted. Use. Here, it is specified as an Ad5 / 35 adenovirus vector in which the gene encoding the fiber protein of E1-deficient non-proliferating type 5 adenovirus is replaced so that it can be expressed by the gene encoding the fiber protein of type 35 adenovirus. Is incorporated so that it can express any of the proteins, peptides or genes encoding the antigenic epitopes of the protein ", and the final viral vector vaccine configuration encodes the fiber protein of the E1-deficient non-proliferating adenovirus. If the gene to be expressed is replaced so that it can be expressed by a gene encoding the fiber protein of type 35 adenovirus, and the gene is incorporated so that a specific protein or the like can be expressed. It may be prepared by a simple procedure. For example, an E1-deficient Ad5 / 35 adenovirus vector in which the gene encoding the fiber protein of E1-deficient non-proliferating type 5 adenovirus is replaced so that it can be expressed by the gene encoding the fiber protein of type 35 adenovirus. After the preparation, the gene encoding the desired protein is incorporated and prepared. After the gene encoding the desired protein is incorporated into the E1-deficient Ad5 adenovirus vector, or at the same time, the gene encoding the fiber protein is replaced. A procedure such as preparation is conceivable. A method for preparing an adenovirus vector vaccine is known, and for example, it may be performed according to the method described in JP-A-2007-37402.

  Specifically, the gene encoding the fiber protein of type 5 adenovirus from which the early gene E1 has been removed is the gene encoding the fiber protein of type 35 adenovirus, and the gene encoding the fiber protein of type 35 adenovirus is Substitute for expression. This replacement may be performed by a conventional method of gene recombination, and this method is described in, for example, JP-A-2007-37402. The gene encoding the type 35 adenovirus fiber protein and its amino acid sequence are already known, Mei, YF et al., Virology 206 (1), 686-689, 1995: Dmitry M. et al., J Virology, Mar. 2000, 2567-2583; NCBI U10272; NCBI AAA66361.1. The fiber protein is composed of a shaft and a knob. In the present invention, a gene encoding the fiber protein composed of these is usually used. In the present invention, more preferably, an E1 / E3-deficient Ad5 / 35 adenovirus vector in which the E3 region is also deleted in addition to the E1 region is used. For example, a viral vector vaccine may be prepared using the pAdHM4 plasmid (Mizuguchi, H., Kay, M. A. 1998. Human Gene Therapy. 9: 2577 2583) which is E1 / E3 region-deficient and contains the full-length Ad5 / 35 genome.

  In the present invention, as the Ad5 / 35 adenoviral vector, it is more preferable to use a modified Ad5 / 35 adenoviral vector in which the hyper variable region (HVR) 5 of the hexon is changed. Examples of this modified Ad5 / 35 adenovirus vector include introduction of a spacer into a native HVR5 part consisting of 14 amino acids, partial mutation of amino acids, and the like. Examples of the modified Ad / 35 adenovirus vector include those described in J. Gene Med. 2009: 11: 570-579. For example, 269TTEATAGNAGNDNTP282 is changed to TTAATAGAGANLTP or LGSHHHHHHLGS is preferably used. Is not to be done.

  The modified Ad5 / 35 adenoviral vector preferably used in the present invention can avoid most of the antibodies against Ad5 type existing in humans and can give strong immunogenicity. In addition, the affinity of Ad5 / 35 to human dendritic cells is enhanced and it is very immunogenic. Ad5 / 35 adenovirus vectors have the advantage that they are very safe because they do not propagate in the body.

The vector vaccine against influenza virus of the present invention is an Ad5 / 35 adenovirus vector. 1) Polypeptides derived from influenza virus nucleoprotein (NP) and matrix protein (M), or genes encoding these antigenic epitopes, or 2) Incorporate and prepare an influenza virus nucleoprotein (NP), matrix protein (M) and hemagglutinin (HA) derived polypeptide, or a gene encoding these antigenic epitopes.

  The sequences of M (M1 and M2), NP, HA of many subtypes of influenza virus are known. As such a sequence, a sequence obtained from a subtype of influenza virus to be protected may be used, but preferably a consensus sequence conserved among many subtypes of influenza virus is used. By using such sequences, vector vaccines against influenza viruses that can protect against a wider range of influenza viruses can be obtained. As the consensus sequence, those reported up to now may be adopted, or a new search may be performed. As a result of actually examining the sequence of about 200 kinds of viruses reported so far, such as birds, humans and pigs, NP was 92-95%, M was 92-97% (M1; 95- 97%, M2; 92-95%), and it has been confirmed that those that have been separated and reported so far have almost identical amino acid sequences. The consensus sequence for HA is also reported in, for example, Chen MW et al., Proc Natl Acad Sci US A. 2008 Sep 9; 105 (36): 13538-43. Just choose.

  The gene encoding M (M1 and / or M2), NP, and HA of the present invention or an antigenic epitope thereof may be an influenza virus genomic DNA isolated from any influenza virus subtype. It may be. In addition, in the present invention, when preparing a vector vaccine against influenza virus in order to protect human influenza infection, a gene encoding M (M1, M2), NP and HA or an epitope thereof has its DNA codon as human. It is preferable that it is a type. Humanization of DNA sequences is well known (eg Nakamura Y et al., Nucleic Acids Res. 2000 28; 292, or www.kszusa.or.jp/codon/), and appropriate software based on such information is available. Can be obtained by using.

  The sequence of the matrix protein (M) of influenza virus is disclosed in, for example, LAMB, RA., 1981, Virology, 112, 746-751 and the like. As the matrix protein used in the present invention, either M1 or M2 may be used, or a combination of both may be used. Preferably, M2 alone or a combination of M1 and M2 is used. As a sequence including the epitope region of M2 protein, for example, MSLLTEVETPTRNGWGCSCSDSDLILWILDRLFFKCIYRRRLKY can be used.

  NP proteins and their expression genes are known, and NP expression genes have been used in DNA vaccines by Ulmer et al. In 1993 (Ulmer et al., Science 1993, 259, 1745,, Fu et al. J. Virology 1997 71; 2715). NP protein is known to induce many cellular immunity-inducing epitopes (Townsend et al. J. Exp. Med .. 1984, 160; 552, Yewdwll et al. PNAS 1985 82; 1785) and high antibody titers against influenza virus (Chen et al., J. Virology, 2001, 75; 11630).

  There are many reports on the HA protein used in the present invention and its expressed gene, and production of a vaccine using a consensus sequence is disclosed in, for example, (Chen et al. J. Virology, 2001, 75; 11630).

  As an example, there are amino acid sequences of HA, M and NP used in the following examples, and a gene encoding an amino acid sequence having one or more substitutions, deletions and additions in these sequences and showing equivalent efficacy For example, but not limited to.

  In the present invention, the specific gene of the present invention may be incorporated into the Ad5 / 35 adenovirus vector or the modified Ad5 / 35 adenovirus vector by a standard gene recombination method, and is not particularly limited. In the present invention, a gene encoding an antigen and a promoter sequence can be arranged in a vector so that transcription of the gene encoding each antigen can be commanded by a unique promoter specific to each antigen. Alternatively, the gene encoding the antigen and the promoter sequence may be arranged so that one promoter drives the expression of a gene encoding two or more antigens connected in-frame to each other and expresses the fusion protein. Good. The type of promoter and the positional relationship between the gene sequence encoding the antigen and the promoter can be appropriately set by those skilled in the art.

  In the present invention, those designed to drive the expression of the gene encoding the antigen, preferably having one promoter connected in frame, are particularly preferably used. A schematic diagram of a vector vaccine against influenza virus used in the present invention is shown in FIG. The upper diagram is a conventional A5 / 35 adenovirus vector vaccine, and the lower diagram is a modified A5 / 35 adenovirus vector vaccine.

  Moreover, when manufacturing the vector vaccine of this invention, the order which distribute | arranges NP and M or HA, M, and NP is not specifically limited, What is necessary is just to set it as arbitrary orders. For example, the promoter sequence is followed by HA-M-NP, NP-M, HA-NP-M2, etc. in that order. In addition, an enhancer moiety that enhances expression may be further introduced between the sequences if desired.

  Examples of the method for preparing the vector vaccine of the present invention include the following procedures. An expression cassette containing the CAG promoter is inserted between the SalI-HindIII or SalI-PstI sequences of the shuttle vector pHM5, and further inserted between the PI-SceI-I-CeuI sequences of pAdHM34, which is the DNA cosmid of the Ad5 / 35 type virus vector, The obtained DNA was purified and transfected into HEK293 cells carrying E1 gene using FuGene 6 (Roche Diagnostics, Indianapolis, USA), and Ad5 / 35 type virus vector obtained therefrom was infected into HEK293 cells. Incubate in large quantities. Next, after the cells infected with the Ad5 / 35 virus are disrupted by an ultrasonic method, the large cell debris is centrifuged at 1500 rpm to remove large cell debris. Thereafter, the supernatant is purified by an ultracentrifugation cesium chloride concentration gradient method, dialyzed to remove cesium chloride, and then appropriately diluted with PBS to obtain a vector vaccine solution.

  Alternatively, instead of the Ad5 / 35 viral vector, the modified Ad5 / 35 plasmid gene pAdHM62 in which the XbaI site of the hexon region is replaced so that the HVR5 region of the hexon of Ad5 / 35 can be replaced with, for example, a gene encoding LGSHHHHHHLGS Thus, the expression cassette may be inserted into the I-CeuI / PI-SceI site.

  The plasmid gene thus obtained was transformed into embryonic kidney (HEK293) carrying the human E1 gene by using FuGENE6T gene insertion reagent (Roche Diagnostics, Indianapolis, USA) (Microbix Biosystems Inc., Toronto, Canada). , Ontario, Canada) and can be propagated.

  The grown cells are disrupted by an ultrasonic method, and then centrifuged at 1500 rpm to remove large cell debris. Thereafter, the supernatant can be purified by an ultracentrifugation cesium chloride concentration gradient method and dialyzed to remove cesium chloride to obtain a vector vaccine. Dilute appropriately with PBS to make a vector vaccine solution

  The vector vaccine against influenza virus of the present invention is administered to vertebrates including mammals including humans and birds such as chickens, and immunity against influenza occurs in the administered animals. Influenza virus-derived protein encoded by the DNA used in the present invention or its epitope peptide is used as an antigen to activate cytotoxic T cells to stimulate cellular immunity. Differentiates into production cells and humoral immunity is also aroused), resulting in immunity against influenza. Immunization using the vector vaccine against the influenza virus of the present invention is useful not only for preventing the onset of influenza but also for reducing the symptoms caused by influenza infection.

  As a method for administering the vaccine of the present invention, for example, the vaccine of the present invention is dissolved in a suitable solvent, for example, a buffer solution such as PBS, physiological saline, sterilized water, etc., and then sterilized by filtration with a filter or the like as necessary. Then, the injection is prepared by filling into a sterile container and administered by injection. A conventional carrier or the like may be added to the injection as necessary. The vector vaccine against the influenza virus of the present invention can be administered to veins, muscles, abdominal cavity, nasal cavity, skin and the like.

  The present invention also provides a pharmaceutical composition comprising a vector vaccine against the influenza virus of the present invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is not particularly limited, and may contain any of the components blended in a normal injection as long as it does not inhibit the efficacy of the vaccine of the present invention. Other components such as adjuvants, stabilizers, pH adjusting agents, preservatives and the like may be included.

The dose of the vector vaccine against the influenza virus of the present invention is the amount of the vector vaccine effective for inducing an effective immune response to protect the host from influenza virus infection. It depends on the form. Usually in the range of 10 ⁹ to 10 ¹³ virus particles per adult, preferably in the range of 10 ¹¹ to 10 ¹² virus particles.

  What is necessary is just to determine suitably the frequency | count of administration of the vector vaccine of this invention so that it may be effective in inducing an effective immune response in order to protect a host from influenza virus infection. The vector vaccine of the present invention has a high immunity enhancing ability, and a high effect is expected when administered once or twice.

  The vector vaccine against influenza virus of the present invention may be used in combination with other vaccines. In this case, the second vaccine was modified in the same manner as the modified Ad5 / 35 vector used in the present invention, for example, the E1 deficient adenovirus vector Ad5 or the hexon HVR5 of the E1 deficient adenovirus vector. It may be a vector vaccine prepared using a modified Ad5 vector, a vector used for the production of other known vector vaccines, or an antigen vaccine such as an inactivated vaccine. In addition, vector vaccines against the influenza virus of the present invention may be prepared separately using vectors Ad5 / 35 and modified Ad5 / 35, and these may be used in combination.

  When two types of vector vaccines against the influenza virus of the present invention are used in combination, or when the vaccine of the present invention is used in combination with the vector vaccine against the second influenza virus, the order of administration is not particularly limited, and either one is administered first. May be.

  The vector vaccine against the influenza virus of the present invention and the second vaccine are prepared as separate pharmaceutical compositions without being combined in a single pharmaceutical composition, and are administered simultaneously or sequentially or simultaneously to a subject. Can do. That is, the vector vaccine against the influenza virus of the present invention can be used in combination with a vector vaccine against a second influenza virus different from the vector vaccine against the influenza virus of the present invention. In this case, it can also be provided in the form of a vaccine kit comprising a vector vaccine against the influenza virus of the present invention and a vector vaccine against the second influenza virus. When the vector vaccine against the influenza virus of the present invention is used in combination with the vector vaccine against the second influenza virus, the order of their administration is not limited, and either may be administered first. Further, the second vaccine may be either a DNA vaccine or an antigen-based vaccine (ie, a recombinant protein or a whole-death pathogen). In one preferred embodiment, the second vaccine is an antigen-based vaccine and the vector vaccine of the invention is administered concurrently with the antigen-based second vaccine. Alternatively, in another embodiment, by first administering a vector vaccine of the present invention to sensitize an immune response and then after 2 to 8 weeks, administering an antigen-based second vaccine, An immune response can be enhanced. Other examples of the second vaccine to be combined with the vaccine of the present invention include protein vaccines, M2 and NP synthetic vaccines prepared by the MAP method having strong antigenicity, and formalin inactivated vaccines. Such a second vaccine may be boosted for several weeks as described above, or boosted several years later.

  The vector vaccine against influenza virus of the present invention will be described in more detail based on reference examples and examples. In the following reference examples and examples, influenza viruses A / Puerto Rico / 8/1934 strains H1N1 and A / SWN are provided by Yokohama City Institute of Public Health, Kanagawa Prefecture, and other H3N2 viruses are provided by National Institute for Infectious Diseases, Virus Department. Was used.

  In the following Reference Examples and Examples, vector vaccines were prepared using DNAs encoding HA, NP, M1 and M2 sequences derived from influenza A virus A / Puerto Rico / 8/1934 strain (H1N1). Each amino acid sequence encoded by the gene used in the examples is as follows.

[Reference Example 1]
Properties of the modified Ad5 / 35 adenoviral vector:
The vector vaccine prepared by the modified Ad5 / 35 adenovirus vector used in the present invention was compared with the vaccinia (MVA) vector vaccine often used for vector vaccines. In this reference example, HIV-1 Env antigen protein was introduced into both vectors to prepare vector vaccines, and their immunity was compared. The modified Ad5 / 35 adenoviral vector vaccine was prepared using the modified Ad5 / 35 plasmid gene pAdHM62 in which the XbaI site of the hexon region was replaced so that the HVR5 region of the hexon of Ad5 / 35 could be replaced with, for example, a gene encoding LGSHHHHHHLGS. .

  A modified Ad5 / 35 adenovirus vector, a vector vaccine in which an HIV-1 Env antigen was incorporated into a vaccinia vector, and an HIV-1 Env protein antigen were used. Each group of 6 rhesus monkeys was immunized twice with each vector vaccine at 0 and 8 weeks. FIG. 3 shows the antibody production ability and cellular immunity enhancement ability. As is clear from FIG. 3, the vector vaccine prepared using the modified Ad5 / 35 adenovirus vector showed extremely strong immunity as compared with the vaccine using MVA.

[Reference Example 2]
Comparison of immunity between Ad5 / 35 adenoviral vector and modified Ad5 / 35 adenoviral vector:
A vector vaccine was prepared using the Ad5 / 35 adenovirus vector and the modified Ad5 / 35 adenovirus vector, respectively, so that the HA antigen gene could be expressed. The HA antigen gene was a sequence obtained from influenza virus A / Puerto Rico / 8/1934 strain H1N1 (hereinafter referred to as A / PR / 8), and a gene encoding the amino acid sequence shown in SEQ ID NO: 1 was used.

  The Ad5 / 35 adenoviral vector vaccine incorporates an HA protein expression cassette between the SalI-HindIII or SalI-PstI sequences of the shuttle vector pHM5, and also the PI-SceI-I of pAdHM34, the DNA cosmid of the Ad5 / 35 type viral vector. -Incorporated between CeuI sequences, the resulting plasmid was purified, introduced into HEK293 cells carrying the E1 gene, and obtained from proliferating cells.

  The modified Ad5 / 35 adenoviral vector vaccine will incorporate the HA protein expression cassette between the SalI-HindIII or SalI-PstI sequences of the shuttle vector pHM5 and replace the HVR5 region of the Ad5 / 35 hexon with a gene encoding LGSHHHHHHLGS The plasmid was incorporated into a modified Ad5 / 35 plasmid gene pAdHM62 in which the XbaI site of the hexon region was replaced, and the resulting plasmid was purified, introduced into HEK293 cells, and proliferated.

The grown cells were disrupted by an ultrasonic method, and then a large cell debris was removed by centrifuging at 1500 rpm. Thereafter, the supernatant was purified by an ultracentrifugation cesium chloride concentration gradient method and dialyzed to remove cesium chloride to obtain a vector vaccine. A vector vaccine solution was prepared by appropriately diluting with PBS. The OD260nm was measured with an absorption clock, and the virus concentration was determined at 1OD260nm = 10 ¹² vp (virus particles) / ml.

Balb / C mice were each immunized with 1 × 10 ¹¹ VP / mouse at 0 week each with Ad5 / 35 virus vector vaccine, and after 2 weeks as booster, the same amount of Ad5 / 35 virus vector vaccine or modified Ad5 / 35 virus vector vaccine The anti-HA antibody titer after 8 weeks, 14 weeks and 24 weeks was measured. The HA antigen was prepared from A / PR8 by genetic manipulation, and the antibody titer was measured by ELISA using the antigen. The results are shown in FIG.
As shown in FIG. 4, when immunized with Ad5 / 35HA and boosted with modified Ad5 / 35HA (black circle), the strongest antibody titer was obtained.

[Reference Example 3]
Comparison of the ability to enhance cellular immunity between Ad5 / 35 adenoviral vector and modified Ad5 / 35 adenoviral vector:
In the same manner as in Reference Example 2, an Ad5 virus vector vaccine, a modified Ad5 virus vector vaccine, an Ad5 / 35 virus vector vaccine and a modified Ad5 / 35 vector virus vaccine, which were incorporated so as to express a gene encoding HA, were prepared. Balb / C mice were administered the Ad5 viral vector vaccine as the first immunization, and the modified Ad5 / 35 viral vector vaccine or the Ad5 / 35 viral vector vaccine was administered as a boost after 2 weeks. After 28 weeks, spleen cells were taken from each mouse, and the antigen (antigen is a protein of HA) was cultured for 48 hours simultaneously with the spleen cells, and then specifically reacted INF-γ SFC (INF-γ spot forming cells) was measured. Then, its ability to activate cellular immunity was measured.
As shown in FIG. 5, the vector vaccine obtained using the modified Ad5 / 35 virus vector showed strong antibody production. It is known that Ad5 type antibodies possessed by a large number of humans have a very high immunosuppressive response to Ad5 / 35 or Ad5, compared to previous reports, but the modified Ad5 / 35 viral vector used in the present invention is in vivo. It is considered that the antibody action against Ad5 present in the rat exerts a strong immune activation ability without much influence.

A vector vaccine was prepared by inserting A / PR / 8 (H1N1) genes encoding NP, M2, and HA into pAdHM62 containing the modified Ad5 / 35 vector DNA, or inserting a gene encoding NP-M2, respectively. . Specifically, an expression cassette containing a CAG promoter is inserted between the SalI-HindIII or SalI-PstI sequences of the shuttle vector pHM5, inserted between the PI-SceI-I-CeuI sequences of pAdHM62, and the resulting DNA is purified. HEK293 cells carrying the E1 gene were transfected with FuGene 6 (Roche Diagnostics, Indianapolis, USA), and the modified Ad5 / 35 type virus vector obtained therefrom was infected with HEK293 cells and cultured in large quantities. Next, after the cells infected with the modified Ad5 / 35 type virus are disrupted by an ultrasonic method, the large cell debris is centrifuged at 1500 rpm to remove large cell debris. Thereafter, the supernatant was purified by an ultracentrifugation cesium chloride concentration gradient method, dialyzed to remove cesium chloride, and diluted with PBS to obtain a virus solution. The OD260nm was measured with an absorption clock, and the virus concentration was determined at 1OD260nm = 10 ¹² vp (virus particles) / ml. The gene structure of the vector vaccine of Example 1 is shown in FIG.

The obtained vaccine was immunized by intranasal administration of 1 × 10 ¹¹ VP (virus particles) to 10 Balb / C mice in each group. Ten days later, 10 LD ₅₀ of A / PR / 8 (H1N1) virus was anesthetized with Nenbutanol and then administered intranasally to cause infection. Survival after infection was examined. The results are shown in FIG.

In addition, Balb / C mice similarly immunized with each vector vaccine were subjected to LD ₅₀ dose of influenza virus A / PR / 8 (H1N1), A / WSN / 33 (H1N1) or A / Udron / 72 (H3N2). Infected nasally, the survival rate after 15 days was observed. The results are shown in FIG. The vector vaccine in which the gene encoding NP-M2 was inserted into the modified Ad5 / 35 showed high protection against not only A / PR / 8 but also A / SWN (H1N1) and A / Udron (H3N2) On the other hand, the vector vaccine introduced with only HA did not have such a broad protective ability.

  A gene encoding HA-NP-M2 of A / PR / 8 (H1N1) was inserted into pAdHM62 containing the modified Ad5 / 35 vector DNA (Example 3), or genes encoding HA, NP and M2, respectively. A vector vaccine was prepared in the same manner as in Example 1. The gene structure of the vector vaccine of Example 3 is shown in FIG.

  As an inactivated vaccine, A / PR / 8 (H1N1) influenza virus cultured in embryonated chicken eggs was reacted with about 1% formalin for 24 hours at room temperature and washed twice with physiological saline. We used what we did.

1 × 10 ¹¹ VP of each vector vaccine (when viral particles, 2 or 3 types of vector vaccines are administered, 1/2 and 1/3, respectively, are administered simultaneously) into Balb / C mouse muscle I was immunized. Ten days later, 10LD ₅₀ influenza virus A / PR / 8 (H1N1) or A / Guizuhou-X (H3N2) was infected nasally, and the survival rate after 15 days was measured. The results are shown in FIG.

  The vaccine of Example 3 in which the gene encoding HA-NP-M2 was inserted into the modified Ad5 / 35 showed 100% protection against both types of influenza virus. However, such an effect was not observed in a vaccine prepared only with HA or an inactivated vaccine.

In the above examples, HA / NP / M2 sequences derived from A / PR / 8 (H1N1) were used, but the sequences were optimized based on common gene sequences found in many types. In particular, a gene sequence encoding the following amino acid sequence, particularly preferably a humanized gene sequence, is used as HA, NP, M1 and M2, and this is based on the procedure of Example 1 in the order of HA-M2-M1-NP below the promoter. Introduce a modified Ad5 / 35 vector vaccine. The vector vaccine of this example can be widely used not only for A / PR / 8 (H1N1) but also for other types of influenza viruses.

M2 1-97 SEQ ID NO: 6
MSLLTEVETPTRNGWECRCSDSSDPLVIAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTEGVPESMREEYRQEQQNAVDVDDGHFVNIELE

M1 1-252 SEQ ID NO: 7
MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLYKKLKREITFHGAKEVALSYSTGALQSCDSLIYNRMGTVQQ

NP 1-228 SEQ ID NO: 8
RASVGRMVGGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNKYLEIRRIWAAVKGVGTMVMELVRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGSTEGVQQ

Claims (9)

To the Ad5 / 35 adenovirus vector in which the gene encoding the fiber protein of E1-deficient non-proliferating type 5 adenovirus is replaced with the gene encoding the fiber protein of type 35 adenovirus, hemagglutinin of influenza virus (HA), the gene encoding the matrix Sutanpaku protein (M) and nucleoprotein (NP) polypeptide derived from or their antigenic epitopes, following the promoter HA-NP-M incorporates to allow expression in this order made, vector vaccine against influenza virus. The amino acid of the hexon hypervariable region 5 (HVR5) of the Ad5 / 35 adenoviral vector is a polypeptide derived from influenza virus hemagglutinin (HA), matrix protein (M) or nucleoprotein (NP) or an antigenic epitope thereof. The vector vaccine against influenza virus according to claim 1, which is modified with an amino acid.   The vector vaccine against influenza virus according to claim 1, wherein 269TTEATAGNAGNDNTP282 of hexon protein hypervariable region 5 (HVR5) of Ad5 / 35 adenovirus vector is replaced with TTAATAGAGANLTP or LGSHHHHHHLGS. Polypeptides derived from influenza virus hemagglutinin (HA), matrix protein (M) and nucleoprotein (NP) or their antigenic epitopes are derived from influenza A and are used to protect against influenza A The vector vaccine with respect to the influenza virus in any one of Claims 1-3.   The vector vaccine against influenza virus according to claim 4, wherein the influenza A virus is an influenza A virus H1N1 type. At least one of the genes encoding influenza virus hemagglutinin (HA), nucleoprotein (NP) and matrix protein (M) or genes encoding these antigenic epitopes is a consensus sequence in multiple types of influenza viruses, The vector vaccine with respect to the influenza virus in any one of Claims 1-5. The polypeptide derived from influenza virus hemagglutinin (HA), matrix protein (M) and nucleoprotein (NP) or at least one gene encoding these antigenic epitopes is a humanized gene. A vector vaccine against the influenza virus according to any one of 6. The vector vaccine against influenza virus according to any one of claims 1 to 7, wherein HA-NP-M2 is introduced in this order following the promoter. The pharmaceutical composition containing the vector vaccine with respect to the influenza virus in any one of Claims 1-8 .

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