WO1995007099A1 - Vaccine and process for producing the same - Google Patents

Abstract

A vaccine containing a vaccinia virus which is a vector having, incorporated thereinto, a base sequence coding for part of the whole of the HA protein of an influenza virus and another base sequence coding for part or the whole of a pathogen-derived substance. It is possible to incorporate into a vector a base sequence obtained, for example, by inserting a base sequence which codes for the envelope protein gp120 or gp160 of an HIV and contains at least 24 base units into a DNA coding for the loop region of the HA protein of an influenza virus. Also provided are a process for producing the vaccine and a method of forming cellular immunity with the vaccine.

Description

 Description Vaccine and its production method

 The present invention relates to a vaccine and a method for producing the vaccine. Background art

 Currently used viral vaccines are live attenuated vaccines or inactivated vaccines. However, when used against human immunodeficiency virus (HIV), which causes frequent mutations, these conventional vaccines clearly had problems in their efficacy. To solve this problem, subunit vaccines, peptide vaccines, and recombinant vaccines that incorporate a part of the viral gene into a vector are being studied.

 It is also known to combine a nucleotide sequence encoding the hemagglutinin (HA) of influenza virus with a nucleotide sequence derived from another pathogen to produce a chimeric protein that can be used as a vaccine (EP -546787-A).

 The use of recombinant viruses as vaccines has also been discussed in the context of the use of recombinant vaccinia virus expressing gp160, the HIV envelope protein (env) (J. Clin. Immunol., 12, 429-436, 1992). The use of recombinant raccoon box viruses that express viral superficial proteins is known (Nature, 354, 520-522, 1991). There is also a commentary on vaccine development in Proteins, Nucleic Acids, Enzymes, 37 (3), 440-454, 1992).

 An object of the present invention is to provide an excellent actin that remarkably induces cell-mediated immunity in addition to humoral immunity. Disclosure of the invention

As a result of repeated research to solve the above-mentioned problems, the present inventor found that the gene encoding the influenza virus HA protein could be derived from pathogens such as HIV-derived peptides. It has been found that the above problem can be solved by inserting a DNA encoding the product into a vector having a strong promoter, particularly a vaccinia virus, and inserting this nucleotide sequence into a vector having a strong promoter. The present invention has been completed based on the above findings. That is, the present invention relates to a vaccine comprising a vaccinia virus, which is a vector into which a nucleotide sequence encoding a part or the entire length of the HA protein of an influenza virus and a nucleotide sequence encoding a part or the whole of a pathogen-derived product are incorporated. Thing.

Further, according to a preferred embodiment of the present invention, in the above-mentioned vaccine, a vaccine in which a nucleotide sequence encoding a pathogen-derived substance is inserted into DNA encoding a loop region of HA protein of influenza virus; and Wakuchin nucleotide sequence encoding a pathogen-derived product is a nucleotide sequence comprising at least 24 bases encoding the E emissions base rope protein g _P 120 or gpl60 of HIV are provided; vaccine but is antigenic material derived from HIV .

 Further, according to another aspect of the present invention, a method for producing the above-mentioned vaccine, a method for immunizing with the above-mentioned vaccine to induce cell-mediated immunity; and (a) immunizing with the above-mentioned vaccine A) further boosting with a chimeric protein expressed in a baculovirus-silkworm system, comprising a portion corresponding to part or all of the HA protein of the influenza virus and a portion corresponding to the antigenic substance desired to be immunized. Immunization methods are provided. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing one example of a vaccine production scheme of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

The vaccine of the present invention includes a nucleotide sequence encoding a part or the full length of the HA protein of influenza virus. According to the vaccine of the present invention, it is preferable to insert a nucleotide sequence encoding a part or the whole of a pathogen-derived substance into DNA encoding the loop region of the HA protein of influenza virus. Serologically, the gene encoding influenza virus HA protein (influenza The HA virus) is divided into 14 subtypes. Antigen mutations are also observed in these subtypes, but this tendency is particularly pronounced in viruses that are endemic in humans. The region in which amino acid mutations frequently occur between strains is a region with few functional restrictions, and an example of this region is an HA loop region. By inserting a foreign gene into the gene region encoding the loop structure, the immunogenicity of the antigen derived from the foreign gene can be increased.

 Examples of the DNA encoding the pathogen-derived product include DNAs derived from various viruses and tumors requiring immunity. More specifically, examples of such DNA include DNA derived from influenza virus, poliovirus, herpes virus, HIV, hepatitis B virus, hepatitis C virus, and DNA derived from tumors such as melanoma. And Plasmodium merozoite surface protein-1 (MSP-1) epitopes: Journal of Immunology, pp.3483-3490, 1994. . Particularly preferred DNAs are those encoding a disease virus that can be effectively treated and prevented by cellular immunity (ie, HIV, hepatitis B virus, hepatitis C virus, etc.).

 The DNA encoding the pathogen-derived product only needs to be long enough to induce immunity. For example, it is appropriate to select a base encoding at least 8 amino acids to induce cell-mediated immunity and 13 or more amino acids to induce humoral immunity. For each pathogen, the site and length most suitable for inducing immunity should be selected according to the purpose of prevention or treatment.

The present invention will be described more specifically when an HIV-derived antigenic substance is used as a pathogen-derived product. As an HIV-derived antigenic substance, for example, gpl60 or gpl20 of the HIV envelope protein (envelope glycoprotein) can be used, and DNA encoding a part or the entire length of the evening protein is replaced with a part of the influenza virus HA protein or What is necessary is just to combine with the base sequence which codes a full length. For example, a tanno having an HIV epitope on the surface of an HA molecule without disrupting the peptide molecule structure containing the HIV epitope by inserting the HIV V3 loop into the loop region of the HA protein. The molecular structure can be constructed. this The HIV loop peptide is suitable because it consists of a region mainly recognized by HIV neutralizing antibodies and cytotoxic T cells, and can be expected to induce both humoral immunity and cellular immunity.

 As the virus, the Xinnia virus, which is more effective against systemic infection, is most suitable, and is particularly advantageous for the formation of cellular immunity. Adenovirus and the like can also be used. It is appropriate to incorporate the foreign gene into a locus encoding a region not essential for vaccinia virus, such as a region encoding vaccinia virus thymidine kinase (ΤΚ) or hemagglutinin (ΗΑ). A multivalent vaccine can be produced by incorporating two or more foreign genes into a viral vector.

 Various vaccinia virus vectors, necessary promoters, and methods for their recombination are known (proteins, nucleic acids, enzymes, 35 (14), 2432-2442, 1990; 37 (3), 440-454, 1990). 1992 etc.), and these known materials and methods may be used. For example, vaccinia virus WR strain, squirrel Yuichi strain and its temperature-sensitive strain, virion maturation-deficient mutant strain and the like available from ATCC and the like can be used. As a preferred vaccinia virus vector, vaccinia virus transfer vectors pSFBs having an ATI (cow pox type A inclusion body) promoter and one to several P7.5 promoters (Fimahashi et al., J. Virology, 65) , 5584-5588, 1991) can be expected to act as a strong promoter and provide good effects.

To produce recombinant Wirusu is yo By carrying out homologous recombination between the viral DNA and © Irusubekuta in Wirusu sensitive cells ₀ and as a means of introducing a D NA to this sensitive cells, whereas calcium phosphate method and electroporation Chillon It is useful to use a mutant strain that is defective in maturation of virions in order to select recombinants efficiently.

 The primary selection of recombinant Exinia virus can be performed by plaque assay using the hemagglutinating ability of Exinnia virus, and a single cloning method can be performed by ordinary cloning methods such as limiting dilution and enzyme-linked immunosorbent assay. You can get the virus.

Use of the vaccine of the present invention induces production of humoral antibodies against various diseases It can be expected to have therapeutic and preventive effects on those diseases. Another major feature is that treatment and prevention by cell-mediated immunity that causes damage or elimination of virus-infected cells by activating cytotoxic T cells can be expected. Vaccination may be based on, for example, experience with smallpox vaccine. While preventive vaccines against HIV are important, it is generally difficult to develop vaccines that respond to severe antigenic variation. According to the present invention, it is intended to amplify a DNA region encoding a specific antigenic site of HIV infecting a patient by using a PCR method or the like, and to prepare a pectin corresponding to a peptide translated from this DNA. Can be. Such vaccines and therapeutic methods using them are important embodiments of the present invention (see Example 4). When a recombinant virus vaccine is used, it is generally not preferable to use the same vaccine for booster immunization. In this case, it is useful to use recombinant silkworm nuclear polyhedrosis virus, produce a large amount of chimeric HA protein using silkworm as a host, and use this chimeric HA protein as an additional antigen (Japan Patent Application Publication No. 108480/1991).

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1

la) Production of the chimeric gene (fragment B):

 The influenza virus A / sw / Ehime / 1 / before and after the nucleotide sequence encoding the 317-323 individual amino acids (HI GPGRA) of the V3 region of gp-120, the envelope protein of the HI V-1 MN strain. A nucleotide sequence encoding 12 amino acids (PNH I GPGRATAA) was synthesized by adding a base sequence encoding an amino acid of HA to 80 (H1N2). The two oligonucleotides 38mer (5 '-CCC AATC AC ATAG GACCAGGCAGAGCAACGGCAGCATG-3,) and 34mer (5'-CT were synthesized (Applied Biosystems) so that the Haelll site was located at the 5 'end and the Sphl site was located at the 3' end. Model 381A DNA synthesizer) ^ These two oligonucleotides were annealed.

The resulting DNA fragment is phosphorylated with T4 DNA kinase and fragmented. Used as B.

lb) Construction of plasmid containing chimeric gene:

 The plasmid pEH-HAl (FERM BP-2585) incorporating the HA gene of influenza virus A / sw / Ehime / 1/80 (H1N2) was cut with BamHI and Haelll, and HAs 1 to 452 Fragment A having the second base sequence was isolated.

 Similarly, the same plasmid was cleaved with Sphl and Accl to isolate fragment C having a base sequence from 492 to 1776 of HA.

 Fragment A and fragment B were ligated using T4 DNA ligase to obtain fragment A + B. This fragment was further ligated with fragment C and T4 DNA ligation, and then treated with Acc I to remove extra terminal bases to produce a chimeric HA gene HA-MN.

 Both cohesive ends of the HA-MN (BamHI site) were converted to blunt ends using T4-DNA polymerase. The HA-MN gene and the vaccinia virus previously cleaved with Smal and dephosphorylated with bacterial alkaline phosphatase '' transfer vector pSFB5 (This transfer vector was obtained from Dr. Shida of the Institute for Virus Research, Kyoto University, Kyoto, Japan. Donated: See Funahashi et al., J. Virology, 65, 5584-5588, 1991. This vector consists of five tandem promoter and ATI (cow pox A-type inclusion bodies). The plasmid contains a 7.5-kDa synthetic promoter and a downstream restriction enzyme site for the transfer of a foreign gene.) To produce plasmid pCE-1 incorporating the HA-MN gene. FIG. 1 shows the above steps in a scheme. The nucleotide sequence of pCE-1 was confirmed by the dideoxy method using a primer (5'-TTTTGGGAAACCCAGAATGTGAA-3 ') corresponding to the HA gene (266 to 287).

lc) Generation of recombinant vaccinia virus E A— 1 R VV:

Plasmid pCE-12 / g containing the chimeric HA gene was co-precipitated with the vaccinia virus WR strain DNA7.5 using the calcium phosphate method, and the resulting DNA was transferred to CV-1 cells (Dainippon Pharmaceutical Co., Ltd.). Company, Osaka, Japan) and homologous recombination was performed in the presence of vaccinia virus IBT ^D mutant (Virology, 155 ^ 97-105 (1986)). Recombinant screening for vaccinia virus The hemagglutination ability of the influenza virus is masked with a specific antibody (anti-A / sw / HK / 1/74 ゥ heron serum), and the plaque that has no hemagglutination ability is used. Was isolated. Furthermore, a single virus was cloned by the limiting dilution method and the enzyme antibody method to obtain a recombinant vaccinia virus EA1-RVV.

 Id) Expression of chimeric HA molecule and its biological properties:

 Erythrocyte adsorption test, enzyme antibody test on EA1-RVV-infected cells, indirect fluorescent antibody test, immunoprecipitation test, EA1-RVV on mice using chimeric HA molecule expressed by An immune response test (HI test) was performed.

Erythrocyte adsorption test: R-13 (Dainippon Pharmaceutical Co., Osaka, Japan) Infect cells with EA1-RVV with moi 1, culture at 37 ° C for 24 hours, and allow cells to adsorb 1% chicken erythrocytes. Was. Cells were negative.

Enzyme antibody test: RK-13 cells (3 × 10Vdish) were infected with EA1-RVV at 100 plaque / dish and cultured at 37 ° C. for 48 hours. Purified influenza virus A / sw / HK / 1/74 in incomplete Freund's adjuvant and emulsion was administered subcutaneously and intramuscularly to an antiserum (diluted 1: 100) from a heron as a primary antibody, HRP-labeled anti-heron Plaques were developed using an IgG (H + L) antibody (diluted 1: 100 in Cappel Laboratories) as a secondary antibody and 4-cloth-1-naphthol as a substrate. As a result, all plaques were stained purple-blue and positive.

Indirect fluorescent antibody test: CV-1 cells (Dainippon Pharmaceutical Co., Ltd.) were cultured on an 18 × 18 round cover glass, and infected with EA1-RVV at moi 10 and cultured at 37 ° C. for 24 hours. 100-fold diluted anti-A / sw / HK / 1/7474 egret serum was used as a primary antibody, and 200-fold diluted FITC-labeled anti-Peagle IgG (H +) (Cappel Laboratories) was used as a secondary antibody. Fluorescence microscopy showed strong fluorescent findings in the form of dots on the infected cell surface, indicating that the chimeric HA molecule was expressed on the cell surface. This fact suggests that the chimeric HA molecule is normally transported to the cell membrane, and then the hydrophobic part near the C-terminal is buried and fixed in the membrane. It also suggests that a protein having the same biological activity as the HA molecule of natural influenza virus is expressed. Immunoprecipitation test: CV-1 cells were infected with EA1-RVV at moi 10. Four hours later, the virus-constituting protein was cultured and labeled with ³⁵ S-methionine, and then the infected cells were treated with a detergent and the lysate was subjected to anti-A / sw / HK / 1/74. Absorbed using serum. The absorbed chimeric HA-antibody binding molecule was immunoprecipitated with Protein A. The precipitate was separated by SDS-polyacrylamide electrophoresis and subjected to autoradiography. As a result, a band was detected at a position corresponding to a molecular weight of 76 K. It was confirmed that the molecular weight at 76 K was almost the same as the molecular weight of the HA molecule of A / sw / HK / 1/74 and had the same size.

Immune response test in mice: 4-week-old female ddY mice (n = 10) were infected with lxlO ⁵ PFU I mouse EA 1—RVV or vaccinia virus as a control from the tail vein for infection. The HI antibody titer of the mouse immune serum was 32 after 1 week, 64 after 2 weeks, and 128 after 3 to 4 weeks, indicating an efficient increase in antibody. On the other hand, HI titers of control-immunized mice infected with the wild-type vaccinia virus were 32 or less.

 Based on the above results, it is possible to incorporate a foreign gene (a gene encoding seven amino acids in the HIV-MN strain V3 region) into the influenza virus HA gene. It was clarified that HA retained the same biological properties as those of HA.

 The negative hemagglutination ability is presumed to have caused a subtle change in the tertiary structure of the protein due to the number and sequence of amino acids translated from the inserted gene. Example 2

 2a) Production of chimeric gene (fragment D):

 Add the Haelll and Sphl restriction enzyme sites at both ends of the nucleotide sequence encoding the 15 amino acids 315 to 329 (RI QRGPGRAFVT I GK) of the V3 region of the envelope protein gp-120 of the HIV IIIB strain, and add 17 ( 53-nucleotide oligonucleotide (5'-CCC) encoding the amino acid of PR I QRGPGRAFV TI GKA)

ATAGGTAAGGCATG-3 ') and complementary strand consisting of 49 bases (5'-CCT GGATTCTGGG-3 ′) was synthesized, and the obtained nucleotide was further annealed. The obtained DNA fragment was phosphorylated with T4 DNA kinase to obtain fragment D.

 2b) In the same manner as in lb) of Example 1, a plasmid was prepared in which a chimeric HA gene HA-IIIB containing fragment D and the HA gene of influenza virus was incorporated.

 2c) Recombinant vaccinia virus EA2-RVV was produced in the same manner as in lc). 2d) The chimeric HA molecule was expressed by EA2-RVV, and this protein was subjected to erythrocyte adsorption test, enzyme antibody test on EA2-RVV infected cells, and fluorescent antibody test. The same results as in Example 1 were obtained.

 In this test, the expression of the HIV gene inserted into EA2-RVV was further confirmed by an enzyme antibody test and an indirect fluorescent antibody test using a synthetic peptide immune serum.

Preparation of synthetic peptide immune serum:

 A peptide having the same sequence as the 15 amino acids derived from HIV encoded by the inserted gene fragment D was synthesized, and a conjugate was prepared by covalently linking hemocyanin (KLH) to the peptide. The conjugate was administered to 2.5 kg female egrets. For the first administration, complete Freund's adjuvant was used, and a 1.2 mg equivalent of KLH conjugate peptide was intradermally administered. For the second and third doses, one week and two weeks later, the same amount of peptide as the first dose was administered intradermally using incomplete Freund's adjuvant. Serum samples were collected every other week after the first administration, and the increase in antibody titer was confirmed by ELISA.

Detection of expressed protein by the inserted HIV gene:

 RK-13 cells were infected with EA2-RVV. Serum obtained by peptide immunization (serum 3 weeks after the first immunization) was used as a primary antibody, HRP-labeled anti-Peagle IgG (H + L) antibody as a secondary antibody, and 4-cloth-1-naphthol as a substrate. The plaques formed were subjected to the reaction. As a result, all plaques were stained purple-blue and positive.

In addition, using a negative control, which is normal sperm serum, the same primary antibody as above, and a secondary antibody, which is a FITC-labeled anti-pea sera IgG antibody, the indirect fluorescent antibody method was used. As a result, specific fluorescence for the peptide immune serum was observed on the surface of the infected cells.

 From the above results, it was confirmed that the HIV gene inserted into the influenza virus HA gene expressed an antigenic peptide in EA2-RVV-infected cells.

Example 3

Cytotoxicity (cellular immunity) test:

 The CTL inducibility of the recombinant vaccinia virus EA2-RVV was determined according to the method of Takahashi et al. (Pro Natl. Acad. Sci. USA, 85, pp. 3105-3109, 1988). 3a) EA2—RVV sensitization:

BALB / c (H-2 ^d ) mice were immunized with 10 ⁷ / PFU / mouse of EA2-RVV administered via the tail vein. Five weeks later, lymphocytes were collected from the spleen (5 xlO ⁶ cells / ml), and the lymphocytes were added together with BALB / c 3T3 cells (2,5 xlO ⁵ cells / ml) into which the gp160 gene had been introduced in vitro. By culturing for 6 days, effector cells activated by secondary stimulation were obtained.

3b) CTL Atsushi:

BALB / c 3T3 cells transfected with the gp160 gene (15-12) and BALB / c 3T3 cells (18 IIIBXTownsend) labeled with the synthetic peptide of the V3 region (RI QRGPGRAFVT I GK) of the HIV IIIB strain were used as target cells. Cell, 44, 959-968, 1986) and unlabeled BALB / c 3T3 cells were used as controls. The above effector cells (E) were added to these target cells ((T): 5 xlO ³ cells / well) (EZT ratio 20: 1, 40: 1. 80: 1), and the cytotoxicity was reduced to ⁵¹ Cr -Measured by the release method (Zweerink et al., Eur. J. Immunol., 7, pp. 630-635, 1977). The value was calculated by the following equation. percent specinc release =

 test release-spontaneous release

 100 maximum release-spontaneous release

lo As shown in Table 1, lymphocytes (cytotoxic T cells) sensitized by EA2-RVV were labeled with 15-12 cells presenting ^ 11 ¥ 61 ^ protein or 18 II labeled with ぺ pitide. High cytotoxic activity against IB cells. On the other hand, the lymphocytes did not show activity against BALB / c 3T3 used as a control.

 The pectin of the present invention, which is effective in this experiment, is effective in preventing the onset of disease and treating it by eliminating infected cells. Table 1. CTL activity of EA2—RVV

% specific lysis

 Immunogen E / T ratio

 15-12 18IIIB control

 EA2-RVV 80: 1 60.4 49.4 0.3

〃 40: 1 56.7 45.0 3.0

 〃 20: 1 43.2 33.8 0.7 Example 4

Construction of foreign gene recombination vector:

 By incorporating DNA encoding the epitope region amplified by the PCR method or the like, a vector capable of expressing the HA chimeric protein was produced.

 Plasmid PUC119 (Takara Shuzo) was digested with KpnI and SphII, and both ends of the obtained fragment were blunt-ended with T4 DNA polymerase. Separately, the plasmid pEH-HAl (FERM-BP-2585) was digested with BamHI to extract the HA gene of influenza virus A / sw / Ehime / 1/80 (H1N2), and both ends were T4 DNA polymerase. And blunt ends.

The resulting two DNA fragments were ligated into plasmid pSK (-), which was then infected with M13K07phage to prepare single-stranded DNA. The single-stranded DNA was annealed with a synthetic oligonucleotide GATATTCCCCGGGAC AAGTTCG having a SmaI site, and then treated with DNA polymerase to produce a double-stranded circular plasmid pAC-1. If this plasmid pAC-1 is digested with Sraa I and Sph I and blunt-ended with T4 DNA polymerase, various foreign genes can be inserted into plasmid pAC-1 to obtain a plasmid containing chimeric genes. I can do it. If this plasmid is treated with Puv II, a chimeric gene can be obtained, which is useful for introducing the chimeric gene into a pixia virus transfer vector or a baculovirus transfer vector. Industrial applicability

 The use of the vaccine of the present invention is expected to be effective in the treatment and prevention of various diseases by the production of humoral antibodies. In addition, the vaccine of the present invention is useful because an effect by cell-mediated immunity can be expected.

Claims

The scope of the claims 1. A vaccine containing a vaccinia virus, which is a vector in which a nucleotide sequence encoding a part or the entire length of the influenza virus HA protein and a nucleotide sequence encoding a part or the whole of a pathogen-derived product are incorporated.  2. The actin according to claim 1, wherein a nucleotide sequence encoding a pathogen-derived product has been inserted into DNA encoding a loop region of HA protein of influenza virus.  3. The vaccine according to claim 1, wherein the pathogen-derived product is an antigenic substance derived from HIV.  4. The vaccine according to claim 3, wherein the nucleotide sequence encoding the pathogen-derived product is a nucleotide sequence encoding the HIV envelope protein gpl20 or gpl60 and containing at least 24 bases.  5. A method for producing a vaccine, comprising a step of incorporating a nucleotide sequence encoding a part or the entire length of an influenza virus HA protein and a nucleotide sequence encoding a part or the entirety of a pathogen-derived product into a vaccinia virus as a vector.  6. The method according to claim 5, comprising a step of inserting a nucleotide sequence encoding a pathogen-derived product into DNA encoding a loop region of HA protein of influenza virus.  7. The method according to claim 5 or 6, wherein the pathogen-derived substance is an antigenic substance derived from HIV.  8. The method according to claim 7, wherein the nucleotide sequence encoding the pathogen-derived product is a nucleotide sequence encoding the HIV envelope protein gpl20 or gpl60 and containing at least 24 bases.  9. A step of immunizing with a pectin containing vaccinia virus, which is a vector in which a nucleotide sequence encoding a part or the entire length of the influenza virus HA protein and a nucleotide sequence encoding a part or the whole of a pathogen-derived product are incorporated. A method for forming cell-mediated immunity, comprising: 10. The nucleotide sequence encoding the pathogen-derived product is the HA protein of influenza virus. 10. The method according to claim 9, comprising a step of immunizing with a vaccine inserted into DNA encoding a loop region of cloning.  11. The method according to claim 9 or 10, comprising a step of immunizing with a vaccine whose pathogen-derived substance is an antigenic substance derived from HIV.  12. The method according to claim 11, comprising a step of immunizing with a vaccine wherein the nucleotide sequence encoding the pathogen-derived product is a nucleotide sequence encoding the HIV envelope protein gpl20 or gpl60 and containing at least 24 bases. .  13. The following steps:  (a) A step of immunizing with a vaccine containing a vaccinia virus, which is a vector in which a nucleotide sequence encoding a part or the entire length of the HA protein of influenza virus and a nucleotide sequence encoding a part or the whole of a pathogen-derived product are incorporated. ;as well as (b) a step of further boosting with a chimeric protein expressed in a baculovirus-silkworm system, including a portion corresponding to a part or all of the HA protein of influenza virus and a portion corresponding to an antigenic substance to be immunized; ;  An immunization method comprising: