JP2012191948A - Vaccine and vaccine protein using sendai virus vector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a Sendai virus vector useful for producing an influenza vaccine, a method for producing the influenza vaccine using the vector, an influenza vaccine produced by using the vector and a protein for an immunogen and an immunoassay obtained by using the Sendai virus vector.SOLUTION: A vaccine against a highly-virulent influenza is produced using a recombinant Sendai virus vector to which a hemagglutinin (HA) gene of an influenza virus is transferred. A mouse nasally inoculated with the vaccine exhibits significant resistance to a highly-virulent influenza virus. A protein produced using a Sendai virus vector has strong immunoreactivity and is proved to be suitable for a protein for an immunogen of vaccine use and a protein for immunoassay. Especially, the protein is proved to be useful as an antigen of ELISA and the like for a serodiagnostic agent of HIV infection.

Description

[Technical field to which the invention belongs]
The present invention relates to a Sendai virus vector used for production of an influenza vaccine, a method for producing an influenza vaccine using the vector, and an influenza vaccine produced using the vector. The present invention also relates to an immunogen protein and an immunoassay protein that can be obtained using a Sendai virus vector.

[Conventional technology]
Influenza viruses belonging to the Orthomyxoviridae family are pathogenic viruses that infect many animals including humans and cause respiratory infections (influenza). Infection of the virus can cause fever, headache, pain in every part of the body, including joints, and weakness in a few days, resulting in respiratory symptoms such as cough and sore throat. Occasionally, bronchitis, bacterial pneumonia, otitis media, etc. are often accompanied, and encephalopathy, myositis, myocarditis, etc. may be caused and the disease may become severe. In particular, infection with elderly people, pregnant women, patients with lung disease, heart disease, and kidney disease tends to become more serious and causes a high mortality rate. In Japan, tens of thousands of people are affected every year and nearly 2,000 people die. Historically, the Spanish cold that began in 1918, the Asian cold in 1957, the Hong Kong cold in 1968, the Soviet cold in 1977, and repeated pandemics (outbreaks) from the beginning of this century, Japan also In many countries around the world, it has caused enormous health damage and impact on social activities in terms of the number of deaths and illnesses.

  Among the influenza viruses, those that belong to type A, which are particularly susceptible to pandemics, are currently known as H1-H15 subtypes. Viral hosts are widely distributed in humans, pigs, minks, whales, birds, etc., and many subtypes have been isolated especially in birds. The subtypes that have been prevalent in humans are H1 and H3, and A Hong Kong type (H3N2) and A Soviet type (H1N1) are known as representatives.

  At present, inactivated influenza vaccines are the mainstream as a preventive method against influenza, and its effectiveness has been established epidemiologically. In order to produce an influenza vaccine, the influenza virus is usually inoculated into the chorioallantoic membrane of a developing chicken egg about 10 days after fertilization. Usually, the virus infects only the chorioallantoic membrane, and the virus accumulates in the chorioallantoic fluid. Thereafter, the virus is recovered from the chorioallantoic fluid and concentrated to produce a virus that is a raw material for the vaccine. From this, a virus whole-particle vaccine inactivated with holymarin or the like, an HA subunit vaccine that decomposes the virus and is produced from the HA protein fraction, and the like are produced.

  HA protein refers to hemagglutinin (HA) and is an outer shell spike protein that is present on the surface of influenza virus particles together with neuraminidase (NA).

  HA proteins are prone to mutations, and some mutations are seen even within the same subtype within H1 to H15 (antigen drift). Therefore, in order for influenza vaccines to function most effectively, they must be used as vaccines. It is important that the virus types that were present match. For example, when a new type of influenza virus appears, in order to effectively prevent infection, it is desirable to newly produce a vaccine having exactly the same antigenicity as the virus.

  It is said that the influenza virus undergoes discontinuous mutation at a cycle of 10 to 40 years and a new influenza virus appears (antigen shift). The process by which the new influenza virus emerges and infects humans has not been fully elucidated. Conventionally, it has been said that these influenza viruses can be genetically hybridized with human influenza viruses in the body of pigs, etc., and infect humans as new viruses. Has also been known to directly infect humans (K. Subbarao et al., Science 279: 393-396 (1998)).

  Influenza viruses such as subtype H5 and subtype H7 that use birds as a host are known as virulent types (Y. Kawaoka et al., Virology 158: 218-227 (1987) (Non-Patent Document 2); Walker and Y. Kawaoka, J. General Virol. 74: 311-314 (1993) (Non-Patent Document 3)), there is a concern that these viruses may acquire infectivity to humans. In fact, since May 1997, multiple cases of subtype H5 influenza virus (H5N1), which had been confirmed in birds before but not in humans, were confirmed. It is required to produce vaccines against these influenza viruses as soon as possible.

In order for the influenza virus to infect cells, the HA protein (HA0) must be partially cleaved by trypsin-like proteolytic enzyme and cleaved into HA1 and HA2. When HA1 is adsorbed to the sialic acid receptor on the cell surface, viral particles are taken up into intracellular lysosomes by endocytosis, and membrane fusion occurs under acidic conditions to establish infection. In conventional influenza virus, the C-terminal side of Gln / Arg-X-Arg of HA protein is cleaved and cleaved by trypsin-like proteolytic enzyme that exists only in the lungs and upper respiratory tract, and reinfection is established in adjacent cells Therefore, the site of influenza infection was limited to the airway area (Robert A. Lamb and Robert M. Krug, ' Orthomyxoviridae : The Viruses and Their Replication' in Fields Virology, Third ed., Edited by BN Fields et al. Lippincon-Raven Publishers, Philadelphia, pp.1353-1445, 1996 (Non-Patent Document 4)). On the other hand, some viruses of subtype H5 and subtype H7 found in H5N1 influenza and birds have basic amino acids (Arg-X-Lys / Arg-Arg) side by side at the cleavage site of the HA protein. It is known that infection is systemic and very fatal because it is cleaved by Furin-like enzymes present in a wide range of cell types (Robert A. Lamb and Robert M. Krug, ' Orthomyxoviridae : The Viruses and Their Replication 'in Fields Virology, Third ed., edited by BN Fields et al., Lippincon-Raven Publishers, Philadelphia, pp.1353-1445, 1996 (non-patent document 4)).

  In order to produce a vaccine against such a virus, it is usually necessary to propagate the virus in chicken eggs as described above. However, highly virulent viruses such as subtype H5 and subtype H7, when infected with chicken eggs, the infection spreads to the embryo, and the embryo is lethal early, producing a sufficient amount of virus to produce a vaccine. It was extremely difficult to do. In addition, from the viewpoint of safety, a method for producing a vaccine that does not require direct handling of virulent viruses has been desired.

  So far, an attempt has been made to produce an influenza vaccine strain in which the hyaluronan virus (H5N1) HA gene is attenuated by introducing a mutation at the protease cleavage site (Shuichi Nishimura et al., Japanese Society of Virology 46th) Academic Meeting / General Assembly, IIIE26,1998 (Non-Patent Document 5)). However, since this vaccine strain still contains the genome of avian influenza virus, it is necessary to fully confirm safety to humans.

  In addition, attempts have been made to produce influenza virus proteins without using chicken eggs. For example, there has been a report in which the influenza virus HA gene of subtype H1 has been incorporated into Vesicular stomatitis virus (VSV), the HA protein is expressed, and the effect of the influenza vaccine has been verified (Kretzschmar, E., et al., 1997, J. Virol. 71: 5982-5989 (Non-patent document 6); Roberts, A. et al., 1998, J. Virol. 72: 4704-4711 (Non-patent document 7)) No experiment has been conducted on viruses. In addition, since this vector systemically infects administered mice, there is a concern about the pathogenicity of the vector to the host. Vaccine production of a new influenza virus (H5N1) has also been attempted in a system using baculovirus and silkworm (Nihon Keizai Shimbun, February 14, 1998, morning edition, page 10 (non-patent document 8)), but the vaccine effect It has not been confirmed until now.

  As an example of a vaccine using a non-segmented single-stranded RNA virus, a patent (WO96 / 10400) (Patent Document 1) by G.W. Wertz et al. Is known. However, the optimal vaccine claimed is that the structural protein is derived from a single-stranded RNA virus. In addition, orthomyxovirus is not mentioned among the pathogens that are candidates for vaccine development shown in Table 1 of WO96 / 10400 (Patent Document 1), and influenza A type H5N1 is not claimed. Therefore, the above document only shows the possibility of using a non-segmented single-stranded RNA virus as a general vaccine, and no in vivo effect is shown in the examples.

G.W.Wertz et al., WO96 / 10400

K. Subbarao et al., Science 279: 393-396 (1998) Y. Kawaoka et al., Virology 158: 218-227 (1987) J.A.Walker and Y. Kawaoka, J.General Virol. 74: 311-314 (1993) Robert A. Lamb and Robert M. Krug, 'Orthomyxoviridae: The Viruses and Their Replication' in Fields Virology, Third ed., Edited by B.N.Fields et al., Lippincon-Raven Publishers, Philadelphia, pp.1353-1445, 1996 Shuichi Nishimura et al., The 46th Annual Meeting of the Japanese Society for Virology, IIIE26, 1998 Kretzschmar, E., et al., 1997, J. Virol. 71: 5982-5989 Roberts, A. et al., 1998, J. Virol. 72: 4704-4711 Nihon Keizai Shimbun, February 14, 1998, morning edition, 10 pages

  The present invention uses a Sendai virus vector useful for the production of a vaccine against virulent influenza virus, a method for producing an influenza vaccine using the vector, an influenza vaccine produced using the vector, and a Sendai virus vector. It is an object to provide a protein for immunogen and immunoassay that can be obtained.

  The present inventors have heretofore shown that Sendai virus is very useful as a protein expression vector and a vector for gene transfer into cells and individuals (WO 97/16538 and WO 97 /). No. 16539). Sendai virus vectors have low toxicity and the amount of protein expressed from the introduced gene is extremely high. Moreover, since expression is transient without being introduced into the host chromosome, it is excellent in safety. From these characteristics of Sendai virus vector, the present inventors have found that Sendai virus vector is useful as a vaccine against influenza virus and as a production vector for the vaccine.

  In order to efficiently produce a vaccine against influenza virus, the present inventors produce recombinants having influenza virus genes using Sendai virus, and use these recombinants to contain influenza virus proteins. Sendai virus was produced. As a result, it was found that by using these recombinants, highly virulent influenza virus proteins were efficiently produced. Furthermore, it has been found that a vaccine using the Sendai virus vector of the present invention can provide an extremely high vaccine effect against highly toxic influenza virus.

  In addition, the present inventors produced a Sendai virus vector that expresses the envelope protein gp120 of human immunodeficiency virus (HIV-1) and examined the immunoreactivity of the serum of HIV-infected persons using the vector. The vector-derived gp120 recombinant protein was found to be very strong with the sera of HIV-infected persons and to react specifically with HIV-1 subtypes.

That is, the present invention can be obtained by using a Sendai virus vector used for production of an influenza vaccine, a method for producing an influenza vaccine using the vector, an influenza vaccine produced using the vector, and a Sendai virus vector. More specifically, with regard to possible immunogenic and immunoassay proteins,
(1) A Sendai virus vector that holds an influenza virus protein or a part thereof in an expressible manner,
(2) Sendai virus vector according to (1), wherein the influenza virus is highly toxic
(3) Sendai virus vector according to (2), wherein the influenza virus is subtype H5 or subtype H7,
(4) The Sendai virus vector according to any one of (1) to (3), wherein the protein is an HA protein of influenza A virus,
(5) A method for producing an influenza vaccine using the Sendai virus vector according to any one of (1) to (4),
(6) (a) inoculating Sendai virus vector into eggs, (b) allowing Sendai virus complex to grow in eggs, and (c) recovering the propagated Sendai virus vector from chicken egg chorioallantoic fluid. Including the method according to (5),
(7) The method according to (6), further comprising a step of inactivating the collected Sendai virus vector,
(8) The method according to (6) or (7), further comprising a step of purifying an influenza virus protein or a part thereof from a Sendai virus vector,
(9) An influenza vaccine comprising the Sendai virus vector according to any one of (1) to (4),
(10) The influenza vaccine according to (9), which is a live vaccine of Sendai virus,
(11) The influenza vaccine according to (9), comprising an inactivated Sendai virus,
(12) An influenza vaccine comprising an influenza virus protein purified from the Sendai virus vector according to any one of (1) to (4) or a part thereof,
(13) A method for vaccination against influenza, comprising administering the vaccine according to any one of (9) to (12) to an intermediate host of an influenza virus other than human,
(14) The method according to (13), wherein the vaccine is administered to the respiratory tract,
(15) The method according to (13) or (14), wherein the vaccine is administered multiple times,
(16) an immunogenic protein that can be obtained by expressing a gene encoding a protein derived from a pathogen that is incorporated so as to be expressible in a Sendai virus vector;
(17) a protein for immunoassay that can be obtained by expressing a gene encoding a protein derived from a pathogen that is incorporated so as to be expressible in a Sendai virus vector;
(18) A kit for immunological analysis of an antibody comprising the protein according to (17),
(19) An ELISA kit for an antibody comprising the protein according to (17).

  A vaccine refers to a composition that induces an immune response. Further, in the present invention, the highly virulent influenza virus refers to a highly toxic influenza virus, unlike the conventionally known subtypes H1N1 and H3N2. Such viruses include subtype H5 virus, subtype H7 virus, viruses that infect embryos when inoculated into chicken eggs, and viruses whose cleavage of HA protein is catalyzed by Furin-like enzymes.

  Influenza A subtypes H5 and H7 viruses are implicated in avian avian toxicity. The reason for this is known to be due to the nature of these types of HA cleaving without the supply of foreign proteases (Fields Virology, vol.1, Chapter 46, p1410, Table2; Nestorowicz, A. et al., Virology). 1987, 160: 411-418). Therefore, it is considered that an influenza virus having a similar cleavage mechanism can exhibit properties as a highly toxic type.

  Influenza subtypes are defined by antigenicity ("Fields Virology, vol.1, Chapter 46, p1399" or "WHO Memorandum A revised system of nomenclature for influenza virus Bull WHO 1980, 58, p585-591 "checking).

  According to the present invention, it has become possible to produce a vaccine against highly toxic influenza, which has been difficult in the past. The Sendai virus vector for producing the influenza vaccine of the present invention has low toxicity unlike the influenza virus itself, and can be produced using chicken eggs in the same manner as conventional influenza vaccines. Therefore, it is possible to produce a safe and simple influenza vaccine. It becomes possible. Moreover, since the pathogen-derived protein produced with the Sendai virus vector of the present invention has high immunoreactivity, it can be suitably used as an immunogen protein or an immunoassay protein.

It is the figure which showed the time course of the intrapulmonary virus growth in a mouse | mouth, the weight change of a mouse | mouth, and the gross lesion | pathological lesion of a lung when avian influenza virus was subcultured with a mouse | mouth. The cross represents that the mouse died. When the mouse was infected with 1 × 10 ⁴ CIU of the avian-derived influenza virus of the egg passage strain (M-0), mouse passage 3 (M-3), and mouse passage 5 (M-5) It is the figure which showed the time course of the intrapulmonary virus multiplication, the weight change of a mouse | mouth, and the gross lesion of the lung. The cross represents that the mouse died. It is a figure which shows the structure of the Sendai virus vector which integrated influenza virus HA gene. A DNA fragment containing the HA gene was incorporated into the NotI site of the Sendai virus vector. “E” and “S” represent Sendai virus transcription termination sequence and transcription initiation sequence, respectively. “N”, “P”, “M”, “F”, “HN”, and “L” represent Sendai virus genes. It is a microscope picture showing the dyeing | staining image of HA recombinant Sendai virus (SeV / tukH5) and its control | contrast (SeV / V (-)) using the anti- H5 <Tern / SA> fluorescent antibody. It is a figure showing the result of the Western blot analysis of the protein extracted from the recombinant Sendai virus production cell using an anti-influenza H5 antibody. It is a figure showing the result of SDS-PAGE and Western blot analysis of the protein extracted from the purified Sendai virus particle. It is a figure which shows the result of having analyzed the reactivity of the chicken antiserum produced by immunizing the purified Sendai virus (SeV / tukH5) particles as an antigen. SeV, SeV / tukH5, and FluV indicate that proteins extracted from SeV / V (−), SeV / V (−), and influenza virus were migrated, respectively. SeV / tukH5 (right) is an immunoelectron micrograph showing the localization of H5 protein in virus particles. SeV / V (-) (left) was also used as a control. It is the figure which showed the time course of the pulmonary virus multiplication in a mouse | mouth when a mouse | mouth is inoculated with a recombinant Sendai virus, the weight change of a mouse | mouth, and the macroscopic lesion of the lung. The cross represents that the mouse died. It is a figure which shows the weight change in inoculation of the recombinant Sendai virus to a mouse | mouth, and the challenge of influenza virus. The top row shows no Sendai virus inoculation, the middle row shows Sendai virus 3 × 10 ⁷ CIU inoculation, and the lower row shows Sendai virus 3 × 10 ⁷ CIU inoculation followed by 5 × 10 ⁷ CIU booster. It is the figure which showed the time course of the intrapulmonary multiplication of the influenza virus in a mouse | mouth when a mouse | mouth was inoculated with the recombinant Sendai virus, and the influenza virus challenge, the weight change of a mouse | mouth, and the macroscopic lesion of the lung. . The anti-HVJ antibody titer (white) and anti-H5 antibody titer (black) are shown in the lower panel. It is a figure which shows construction | assembly of plasmid pSeV / gp120-E for production of the recombinant Sendai virus which expresses HIV-1 subtype E gp120 (SeV / gp120-E). Plasmid pSeV () which amplifies a DNA fragment with SeV transcriptional control signals (E and S) added immediately after the gp120 open reading frame, using a NotI-tagged primer to generate the full SeV genome (15,402 bases). +) It was inserted into the NotI site of 18bV (-). It is a figure which shows expression of recombinant gp120-E from recombinant Sendai virus (SeV / gp120-E) in CV-1 cells. (A) shows the time course of protein production in 100 μl of culture supernatant of CV-1 cells infected with SeV / gp120-E. Recombinant gp120 of HIV-1 subtype ECM strain expressed in a baculovirus system was used as a calibration marker (50 ng to 500 μg). After transfer from the gel to the membrane, Western blot analysis was performed using a pool of sera from 10 Thai patients infected with HIV-1 subtype E as probes. (B) shows the result of determining the amount of SeV / gp120-E in the culture supernatant of CV-1 cells infected with SeV / gp120-E at each time from the Western blot of panel A. (C) is CV− infected with parent Sendai virus (SeV (+) 18bV (−)) (lane “wt”), SeV / gp120-B (lane B), or SeV / gp120-E (lane E). The result of silver staining of the culture supernatant of 1 cell is shown. Each culture supernatant was collected 72 hours after infection. SeV / gp120-E purified through the culture supernatant of CV-1 cells infected with SeV / gp120-E on the affinity column of mAb TQ4B15-2 was also electrophoresed (lane P). MW indicates a prestain size marker. The arrow indicates the position of the recombinant gp120 derived from SeV. It is a figure which shows the Western blot analysis of recombinant gp120 derived from SeV. Serological reactivity of sera from 7 patients infected with HIV-1 subtype E or 4 patients infected with HIV-1 subtype B with recombinant gp120-E or recombinant gp120-B It is the result analyzed by Western blot. Culture supernatant (15 μl) containing about 100 ng of recombinant gp120-E (lane E) or recombinant gp120-B (lane B) was run in each lane and probed with serum diluted 500 times. MW indicates the position of the prestain size marker. The arrow indicates the position of the recombinant gp120 derived from SeV. It is a figure which shows the result of a functional analysis of recombinant gp120-E produced by SeV / gp120-E. (A) shows the binding of recombinant gp120-E to CD4 expressing human T cell line MT4. MT4 cells were incubated with anti-V3 loop mouse mAb TQ4B15-2 made against the HIV-1 subtype E V3 loop peptide and reacted with FITC-conjugated anti-mouse IgG sheep F (ab ') 2 to react with FACScan Detected by. (B) shows inhibition of anti-CD4 mAb (Leu3a) binding to MT4 cells when cells were pre-incubated with the amount of recombinant gp120-E shown in advance. In the figure, “C” is a control using the culture supernatant of cells infected with the parent virus (SeV). It is a figure which shows the serological reactivity of the recombinant gp120 produced by recombinant SeV. (A) shows the reactivity of recombinant gp120-E and recombinant gp120-B using a serum panel. The sera panel includes 88 HIV-1 subtype B positive sera, 76 HIV-1 subtype E infected sera, and 21 HIV-1 seronegative healthy sera. Serum diluted 8,000 times was used for EIA. Reactivity was determined for recombinant gp120-E or recombinant gp120-B by measuring absorbance (OD) at 492 nm. ○ indicates reactivity in each serum sample. The cut-off value (0.3) is almost the same as the average absorbance of 21 negative controls plus 7 times the standard deviation (SD) (dotted line). (B) shows the specificity of EIA using SeV-derived recombinant gp120. The reactivity (OD ₄₉₂ ) of each serum (diluted 8,000 times) against recombinant gp120-E (vertical axis) or recombinant gp120-B (horizontal axis) was plotted two-dimensionally. ○ represents HIV-1 subtype E serum, and Δ represents HIV-1 subtype B serum. The dotted line represents the position when the reactivity of the serum for recombinant gp120-B is the same as for recombinant gp120-E. Each plot was clearly separated on one side of the dotted line, indicating that the EIA system using SeV-derived gp120 can detect subtype-specific antibodies against HIV-1 subtypes E and B. It is a figure which shows the result of having compared the sensitivity of EIA using gp120 derived from SeV with V3 peptide EIA. Serum from a patient infected with HIV-1 subtype B or subtype E or a healthy seronegative person is serially diluted and recombinant gp120-E, recombinant gp120-B, V3 peptide PND-E (HIV-1 V3 peptide derived from type E consensus sequence), or PND-MN (V3 peptide derived from MN strain, an isolate typically found in North America and Europe) (Pau, CP et al., 1993, The reactivity to AIDS 7: 337-340) was investigated. Two sera belonging to each category were arbitrarily selected and the average was plotted. The white circles indicate the reactivity of subtype E serum to recombinant gp120-E, the white triangles indicate the reactivity of subtype B serum to recombinant gp120-B, and the white diamonds indicate the reactivity of subtype E serum to PND-E. The white squares indicate the reactivity of subtype B serum to PND-MN. Black circles, black triangles, and black squares indicate the reactivity of seronegative serum samples with recombinant gp120-E, recombinant gp120-B, and PND-MN, respectively. FIG. 5 shows the serological reactivity of SeV-derived recombinant gp120 to a serum panel containing a portion of known genetic subtypes (HIV-1 subtypes A to H). (A) shows the reactivity (OD ₄₉₂ ) of recombinant gp120-E or recombinant gp120-B using a serum panel. (B) is a two-dimensional plot of EIA reactivity of each serum panel against recombinant gp120-E (vertical axis) or recombinant gp120-B (horizontal axis). The serum of each HIV-1 subtype was represented by the symbol shown in the inset.

  The present invention provides a Sendai virus vector that retains an influenza virus protein or a part thereof in an expressible manner. In the present invention, the “Sendai virus vector” includes a complex derived from Sendai virus and having infectivity. In the present specification, the term “infectivity” refers to the ability of a complex to introduce a nucleic acid or the like inside the cell into the cell because it retains the ability to adhere to a cell and membrane fusion. Say that.

  A Sendai virus vector that retains an influenza virus protein or a part thereof in an expressible manner is useful for producing an influenza vaccine or a diagnostic viral antigen. Influenza virus protein refers to the protein encoded by the influenza virus genome. Specifically, in addition to capsid nucleoprotein (NP), matrix (M1), polymerase (PA, PB1, PB2), hemagglutinin (HA), neuraminidase (NA) and other viral particle constituent proteins, NS1, NS2, M2 Etc. are included. For use in the production of an influenza vaccine, an immunogenic protein or a partial peptide thereof is used. All of the above proteins have immunogenicity, and these proteins or parts thereof (partial peptides having immunogenicity) can be expressed using a Sendai virus vector to produce a vaccine. . These proteins may be used alone or in combination. In the present invention, it is particularly preferable to use HA and / or NA which are outer shell spike proteins of influenza virus particles. More preferably, HA is used.

  For example, in order for a virus to replicate autonomously in a Sendai virus vector, it is considered that a protein group made from NP, P / C and L genes is necessary, but the gene encoding the protein group itself is It is not necessarily included in the virus vector of the present invention. For example, the vector of the present invention may be produced using a host cell having a gene encoding the protein group, and the protein group may be supplied from the host cell. In addition, the amino acid sequences of these protein groups may be introduced as mutations or homologous genes of other viruses as long as the activity in introducing the nucleic acid is equal to or higher than that of the natural type, even if it is not the virus-derived sequence as it is. May be substituted.

  In addition, for example, in order for Sendai virus vector to have transmission ability, it is considered that a protein group made from M, F and HN genes is necessary. The gene encoding the protein group itself is the virus of the present invention. Yes, it is not necessarily included in the vector. For example, the vector of the present invention may be produced using a host cell having a gene encoding the protein group, and the protein group may be supplied from the host cell. In addition, the amino acid sequences of these protein groups may be introduced as mutations or homologous genes of other viruses as long as the activity in introducing the nucleic acid is equal to or higher than that of the natural type, even if it is not the virus-derived sequence as it is. May be substituted.

  The influenza virus protein can be expressed by introducing an influenza virus genome gene into a Sendai virus vector. A recombinant virus complex can be obtained by transcribing the recombinant virus genome thus prepared in vitro or in a cell to reconstitute the virus. Such virus reconstitution has already been developed (see WO 97/16539).

  Even if it is not a complete Sendai virus genome, incomplete viruses such as DI molecules (J. Virol. 68, 8413-8417, 1994), synthesized oligonucleotides, etc. may be used as components constituting the complex. Is possible.

  For Sendai virus, a complex containing all of the M, F, and HN genes involved in transmission ability can be used. Here, the “propagating force” means “after the nucleic acid is introduced into the cell by infection or an artificial technique, the nucleic acid present in the cell is replicated to form an infectious particle or a complex equivalent thereto, It means “the ability to propagate to another cell”. However, in order to delete or weaken the transmission power of the natural type, a gene involved in transmission power can be deleted or functionally inactivated from the viral genome contained in the reconstituted complex. In the case of Sendai virus, the gene involved in transmission power is the M, F, and / or HN gene. A method for reconstitution of such a complex has already been developed (see WO 97/16538). For example, in Sendai virus, a vector having a genome in which the F and / or HN gene has been deleted can be prepared from the viral genome contained in the rearranged complex. Such a vector is also included in the vector of the present invention.

  The complex may contain, for example, an adhesion factor, a ligand, a receptor and the like that can adhere to specific cells on the envelope surface. For example, a recombinant Sendai virus may have a modified gene in order to inactivate genes involved in immunogenicity or to increase RNA transcription efficiency or replication efficiency.

  The influenza virus protein to be introduced can be expressed by inserting a gene encoding the protein into an appropriate site of RNA contained in the Sendai virus vector. In Sendai virus RNA, it is desirable to insert a sequence having a base number that is a multiple of 6 between the R1 sequence and the R2 sequence (Journal of Virology, Vol. 67, No. 8, 1993, p. 4822). 4830). The expression level of the inserted foreign gene can be controlled by the position of gene insertion and the RNA base sequences before and after the gene. For example, in Sendai virus RNA, it is known that the closer the insertion position is to the NP gene, the greater the expression level of the inserted gene.

  The influenza virus protein encoded by the RNA contained in the vector can be expressed by introducing the vector into a host cell.

  The vaccine may use one type of influenza virus antigen, but if multiple types of influenza virus are used as antigens, immunity against a wider strain of influenza virus can be acquired. When multiple types of influenza viruses are used as antigens, the combination is not particularly limited, and for example, vaccines can be produced using genes derived from viruses with different HA subtypes, such as subtype H5 and subtype H7. it can. It is also possible to use viruses belonging to the same HA subtype and different NA subtypes. A plurality of influenza virus genes can be mixed with each other after preparing a vaccine by incorporating separate Sendai virus vector genomes, or can be expressed by incorporating a plurality of genes into the same Sendai virus vector genome.

  13 virus strains known as influenza subtype H5 (A / HK / 156/97 (H5N1), A / Ck / PA / 83 (H5N2), A / Ck / Scot / 59 (H5N1), A / Dk / Ir / 83 (H5N8), A / Dk / MI / 80 (H5N2), A / Mall / WC / 75 (H5N3), A / Tern / SA / 61 (H5N3), A / Tk / Eng / 91 ( H5N1), A / Tk / Ir / 83 (H5N8), A / Tk / MN / 95 (H5N2), A / Tk / MN / 81 (H5N2), A / Tk / On / 66 (H5N9), A / Tk When the amino acid sequences of the HA proteins of / WC / 68 (H5N9)) were compared by the Maxim matching method, they all showed 85% or more identity to each other. Similarly, seven types known as subtypes H7 (A / Ck / Japan / 24 (H7N7), A / FPV / Rostock / 34 (H7N1), A / FPV / Weybridge (H7N7), A / Tk / Eng / 63 (H7N3), A / Dk / HK / 293/78 (H7N2), A / Ck / Jena / 87 (H7N7), A / Ck / Victoria / 75 (H7N7)) More than 89% identity. Thus, the amino acid sequence of the HA protein has a high identity among strains of the same HA subtype. Therefore, it is highly possible that immunity against strains of different NA subtypes belonging to the same HA subtype can be obtained to some extent by immunization using the HA protein of one influenza virus belonging to a certain HA subtype as an antigen.

  As long as the virus complex is reconstituted from the Sendai virus vector, the host cell for producing the vector used for reconstitution is not particularly limited. For example, cultured cells such as monkey kidney-derived CVI cells, LLCMK2 cells, and hamster kidney-derived BHK cells can be used to reconstitute Sendai virus complexes. However, in order to obtain a complex in a large amount, it is preferable to amplify the obtained complex using a growing chicken egg. As shown in the Examples, the recombinant Sendai virus of the present invention expressing highly virulent influenza virus protein has low toxicity and does not exhibit significant cytotoxicity. Therefore, unlike the case of using the highly toxic influenza virus itself, it is possible to produce a large amount of influenza vaccine using chicken eggs. A method for producing a vector using chicken eggs has already been developed (Nakanishi et al., (1993), “Advanced Technology Protocol III for Neuroscience Research, Molecular Neuronal Physiology”, Koseisha, Osaka, pp.153-172) . Specifically, for example, fertilized eggs are placed in an incubator and cultured at 37-38 ° C. for 9-12 days to grow embryos. Sendai virus vector is inoculated into the chorioallantoic cavity and eggs are cultured for several days to propagate the viral vector. Conditions such as the culture period may vary depending on the recombinant Sendai virus used. Thereafter, chorioallantoic fluid containing virus is collected. Separation and purification of Sendai virus from chorioallantoic fluid can be performed according to a conventional method (Tatsuro Tashiro, “Virus Experiment Protocol”, supervised by Nagai and Ishihama, Medical View, pp. 68-73, (1995)).

  The collected Sendai virus vector can be used as a live vaccine. In the present invention, a live vaccine refers to a vaccine in which a Sendai virus vector grows in an administered individual and acquires immunity. As shown in the Examples, the highly toxic influenza vaccine using the Sendai virus vector of the present invention has a low cytotoxicity and is therefore preferably used as a live vaccine. There is no restriction | limiting in the object which inoculates such a live vaccine, All animals which can be infected with influenza viruses, such as a human, a bird, a pig, a horse, a cow, are included. Since the influenza virus may infect humans from non-human animals such as pigs and birds, it is also effective to use the vaccine of the present invention against non-human animals. In addition, if a Sendai virus vector lacking the above-mentioned transmission ability is used, a vaccine in which the vector is not transmitted can be produced even if it is a live vaccine.

  The collected Sendai virus can also be used as an inactivated whole particle vaccine. Inactivation means that the original function of the virus is lost and normal growth does not occur. This avoids the risk of virus growth within the vaccinated individual. The method for inactivation is not particularly limited, and examples include UV irradiation and formalin treatment.

  Sendai virus vectors can also be fragmented to reduce allergenicity and increase tolerance compared to whole particle vaccines. The fragmentation method is not particularly limited, and examples thereof include treatment with a solvent such as ether or chloroform and / or an ionic or nonionic surfactant.

  Moreover, the expressed influenza virus protein can be separated and purified from the Sendai virus vector to obtain a vaccine. Since Sendai virus vectors contain only a limited number of types of proteins, for example, influenza virus proteins expressed in cells using an expression vector or the like are much more purified than separating them from whole cell extracts. Easy. Well-known separation techniques can be used for protein purification. For example, it is possible to purify by an immunoaffinity column as described in Example 8 using an antibody against influenza H5 protein. By using the purified protein as a vaccine, it can be expected that the frequency of fever and local reactions after inoculation will be suppressed compared to live and inactivated vaccines.

  The pathogen protein obtainable by the present invention can be expected to have stronger antigenic activity than the natural pathogen protein from which the protein is derived. That is, as confirmed with the HIV env antigen protein gp120 in the examples, when the Sendai virus vector was used as an expression vector according to the present invention, the expression product was more than the synthetic peptide constituting the natural gp120 epitope. It has been confirmed that the reactivity with the antibody is improved. Influenza virus antigens were able to achieve strong immunostimulation while keeping the cytotoxic activity low, which is presumed to be due to the enhanced antigenicity by using Sendai virus vectors. The mechanism by which the pathogen protein obtainable by the present invention enhances antigen activity is not clear. However, since the amino acid sequence should not be mutated as long as the same gene is used, the enhancement of antigenicity may be caused by changes in the sugar chain structure or differences in the three-dimensional structure of the protein.

  In any case, the pathogen protein obtainable by the present invention is useful as an immunogenic protein or an immunoassay protein for detecting an antibody. When the protein according to the present invention is used as an immunogen, it can be expected as a raw material of a vaccine excellent in safety and effectiveness by improving immunogenicity.

  The vaccine may contain a pharmacologically acceptable carrier or vehicle (saline, vegetable oil, suspending agent, surfactant, stabilizer, etc.) as necessary. Preservatives and other additives can also be added. Moreover, in order to improve immunogenicity, immunostimulants, such as cytokine, cholera toxin, salmonella toxin, can also be added.

  Vaccination can be percutaneous, intranasal, transbronchial, intramuscular, intravenous, or oral. The dose may vary depending on the vaccine form, administration method, etc., but those skilled in the art can appropriately select an appropriate dose. When influenza virus grows in the cells of the upper respiratory tract, symptoms such as fever are immediately induced. Therefore, it is preferable that local antibodies produced by vaccination work in this area, that is, the intranasal mucosa and the upper respiratory tract. For example, vaccination by subcutaneous injection can produce a large amount of antibody in the blood, but the amount of antibody that exudes to the intranasal mucosa and upper respiratory tract is small, so it is effective in preventing the severity of pneumonia, etc. It is thought to fade. For this reason, it is considered effective to inoculate the respiratory tract with an influenza vaccine by nasal spray or the like.

  Moreover, since it is considered that there is no basic immunity especially against new influenza, it is considered effective to obtain sufficient immunity by inoculating the vaccine twice. For humans, the interval between two inoculations is usually 2-4 weeks.

  Vaccinated animals include any host that has an immune system and is infected with an influenza virus, including humans, mice, rats, rabbits, pigs, cows, horses, monkeys, birds, and the like.

  If the pathogen protein according to the present invention is used as an antigen for antibody detection, a more specific and sensitive immunoassay system can be constructed. In the present invention, examples of the pathogen protein include a viral glycoprotein antigen. The viral glycoprotein includes many important antigens for immunoassay, such as influenza virus HA antigen and HIV envelope protein gp120. An antibody against a pathogen protein can be measured using the protein according to the present invention. Antibodies against a pathogen are an important clinical indicator of experience of infection with that pathogen. The protein according to the present invention can be applied to known immunological analysis techniques. Among them, a technique typified by ELISA using an enzyme label is desirable because it can establish a highly sensitive analysis system at a low cost. In general, antibody analysis by ELISA involves contacting a sample with a solid phase sensitized with an antigen, and detecting the antibody binding to the antigen with an enzyme-labeled anti-IgG antibody. Alternatively, after capturing all IgG in a sample using protein A or the like, only an antibody against a pathogen can be detected with an enzyme-labeled antigen. Solid phase antigens and enzyme-labeled antibodies necessary for ELISA can be combined in advance and supplied as a kit.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of mouse acclimation strain of avian influenza virus Avian influenza virus A / whistling swan / Shimane / 499/83 isolated from chicken eggs in order to obtain a virus that can be passaged in mice and exhibits pathogenicity The H5N3) strain was passaged by nasal inoculation of mice. Until the 3rd generation of the mouse passage, the maximum viral load at that time was inoculated, and the lung homogenate showing the highest infectious titer on the 1st to 3rd day of infection was passed to the next generation. For the virus, the egg passage strain was named “M-0”, and the mouse passages 1 to 5 were named “M-1” to “M-5”, respectively.

With the passage of mice, the virus quickly adapted to the mice and proliferated well, and the pathogenicity became stronger, and mice that died under these conditions were also observed. That is, with the passage of mice, the ability of the virus to grow increased and the lung lesions also became stronger. Therefore, the virus inoculation amount was set to 1 × 10 ⁴ CIU / mouse after the fourth generation. From the fourth generation onwards, the virus titer reached a plateau. FIG. 1 shows the time course of intrapulmonary virus growth, the change in body weight of the mouse, and the gross lesion of the lung.

  Intrapulmonary virus growth was expressed as the virus titer contained in 10% of the homogenate obtained by grinding the lung in PBS. For gross lesions of the lung, the ratio of the hyperemic area to the whole lung is measured, <1 if 25%, 2 if 25-50%, 3 if 50-75%, 4 if> 75%, 4 death In this case, it was set to 5 (A. Kato et al., EMBO J. 16: 578-587 (1997)).

Three strains of M-0, M-3, and M-5 were inoculated intranasally with 1 × 10 ⁴ CIU / mouse, and changes in body weight, gross lesion of the lung, and time course of intrapulmonary virus growth were measured. The result is shown in FIG. All strains showed rapid growth of pulmonary virus, maximizing on the first or second day of infection, and gradually decreasing thereafter. However, M-0 clearly showed a weaker growth of intrapulmonary virus compared to the subcultured mouse strains, the lung lesions were mild, there was almost no weight loss, and none of the mice died. . Although there was no significant difference in virus growth between M-3 and M-5, the extent of lung lesions and weight loss was stronger in M-5.

Next, in order to examine the change in mouse pathogenicity accompanying passage in mice, LD ₅₀ for mice was measured for three strains M-0, M-3 and M-5. The results are shown in Table 1. “Dead mice / inoculated mice” in the table is the mortality rate on the 13th day of infection. M-0 did not kill any mice at 4 × 10 ⁶ CIU inoculation, so LD ₅₀ => 1.26 × 10 ⁷ CIU / mouse for convenience. On the other hand, both passage strains of mice showed strong pathogenicity, and the LD ₅₀ of M-3 and M-5 was calculated as <31.6 CIU / mouse and 12.6 CIU / mouse, respectively.

[Table 1]
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Inoculation volume Dead mouse LD ₅₀
Virus (CIU / mouse) / Inoculated mouse (CIU / mouse)
────────────────────────────────
M-0 4 × 10 ⁶ 0/5 => 1.26 × 10 ⁷
────────────────────────────────
M-3 1 × 10 ⁵ 5/5
1 × 10 ⁴ 5/5 <31.6
1 × 10 ³ 5/5
1 × 10 ² 5/5
────────────────────────────────
M-5 1 × 10 ⁴ 5/5
1 × 10 ³ 5/5 12.6
1 × 10 ² 5/5
1 × 10 ¹ 2/5
────────────────────────────────
Non-infected (-) 0/5
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

In other words, the avian influenza virus A / whistling swan / Shimane / 499/83 (H5N3) strain can be rapidly propagated in the mouse lung by being passaged in the mouse lung, and the pathogenicity to the mouse is rapidly increased. The LD ₅₀ was reduced to 1 / 1,000,000 in the fifth generation. As for M-3, all mice were killed with 10 ² CIU, so LD ₅₀ <31.6 CIU was set for convenience. However, M-3 may show the same pathogenicity as M-5.

[Example 2] Production of recombinant Sendai virus vector From influenza virus strain (A / turkey / Ireland / 1378/83 (H5N8)) (Kawaoka Y. et al., Virology, 1987, 158: 218-227), literature Template DNA was prepared according to “Kawaoka Y. et al., Virology, 1987, 158: 218-227”. Primer HKH5-F (5'-aag cgg ccg ctc tgt caa aat gga gaa aat-3 ') (SEQ ID NO: 1) and primer HKH5-R (5'-aag cgg ccg cga tga act ttc acc cta agt ttt tct tac “NT / 60/5/4” (JA Huddleston and GG Brownlee, 1982, Nucleic Acids Res. 10: 1029-) using tac ggc gta cgt taa atg caa att ctg cat t-3 ′) (SEQ ID NO: 2) 1038) The above HA gene was amplified by a standard PCR method. The base sequence of the amplified fragment is shown in SEQ ID NO: 3. PSeV (+) 18bV (-) digested with NotI and 1758-base fragment digested with NotI (Experimental Medicine Vol.15 No.19 (extra) 1997; Kato A., et al., ENBO J. 16: 578-587 (1997)) (FIG. 3). Next, this is transformed into E. coli, the DNA of each colony of E. coli is extracted by the “Miniprep” method, and clones that have been confirmed to contain DNA fragments of the expected size are selected. A plasmid was obtained. The resulting plasmid was purified by cesium chloride density gradient centrifugation.

This plasmid and a control plasmid without insertion were introduced into LLCMK2 cells according to known methods to reconstitute Sendai virus particles, and the produced recombinant Sendai virus particles were further inoculated into chicken eggs and amplified (International publication). 97/16539 and WO 97/16538). The collected virus particles were diluted in PBS, and the virus titer was measured. As a result, it was 3 × 10 ⁹ CIU (average value).

  LLCMK2 cells and CV-1 cells infected with the recombinant virus (referred to as "SeV / tukH5") and the parental Sendai virus (referred to as "SeV / V (-)") Since there was no significant difference in morphology, cytotoxicity due to the expression of subtype H5 HA was not observed.

[Example 3] Immunochemical analysis using anti-H5 antiserum 3-1) Preparation of influenza virus subtype H5 antiserum A / Tern / South Africa / 61 (H5N3) strains grown in growing chicken eggs 1 day after infection, the cells are mixed with PBS to make a 10% emulsion, inactivated with formalin, inoculated subcutaneously into mice, and mouse anti-H5 <Tern according to conventional methods. / South Africa> Antiserum was prepared.

  In addition, A / Tern / South Africa / 61 (H5N3) strain propagated in growing chicken eggs was precipitated by ultracentrifugation (30,000 rpm, 90 minutes), and this was used as an antigen to immunize chickens according to conventional methods, and chicken anti-H5 <Tern / South Africa> antiserum was prepared.

3-2) Analysis by indirect fluorescent antibody method A CV1 cell monolayer was prepared on a chamber slide glass. It was infected with SeV / V (-) (control without insertion) or SeV / tukH5 as the parent strain at moi1. After infection, the cells were cultured overnight in a medium without serum. After removing the culture solution, the cells were washed once with PBS. Cells were fixed with 0.5% formalin / PBS for 5 minutes at room temperature. After washing once with PBS, a PBS solution containing 0.2% NP-40 was added, and the cell membrane was permeabilized at room temperature for 5 minutes. Next, 0.1 ml of 200-fold diluted mouse anti-influenza virus A / Tern / SA was treated at room temperature for 1 hour. Thereafter, the plate was washed 5 times with PBS, and finally 0.1 ml of an antibody (commercial product, Cappel) in which FITC was bound to anti-mouse Ig (H + L) was diluted 100 times and allowed to act at room temperature for 1 hour. Thereafter, the plate was washed 5 times with PBS, covered with 80% glycerin / PBS, and microscopically examined.
As a result, fluorescent images of the cells were observed specifically for SeV / tukH5-infected cells (FIG. 4).

3-3) Western blot analysis of infected cells
BHK cells were cultured in a monolayer on a 6-well tissue culture plate. Next, the cells were infected with SeV / V (−) or SeV / tukH5 at moi 10. After about 24 hours, the cells were scraped with a rubber policeman and centrifuged at 6,000 rpm for 5 minutes to collect the cells as a precipitate. This precipitate was dissolved by adding 0.1 ml of 2 × SDS sample buffer in the same manner as 0.1 ml of PBS, heated at 90 ° C. for 5 minutes, and then subjected to SDS-PAGE.

  The concentration of the gel was 12.5%, and after electrophoresis, it was blotted electrophoretically on the PVDF membrane by the semi-dry method. After transfer, the transfer membrane is stained as it is with Coomassie Brilliant Blue (CBB), and the rest is mixed with 3% skim milk for 1 hour at room temperature and mixed with mouse anti-H5 antiserum diluted 500 times. The mixture was further reacted at room temperature for 1 hour. Thereafter, the antiserum was extracted and washed 4 times with a washing solution comprising 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20. After washing, peroxidase-labeled anti-mouse IgG (commercial product, Cappel) was diluted 250 times and added, and the mixture was allowed to act at room temperature for 1 hour in the same manner. Finally, after washing again 4 times with the washing solution, color was developed according to the method of Konica Immunostain Kit.

  As a result, BHK cells infected with recombinant Sendai virus showed that HA0 was cleaved by HA0 into HA1 and HA2 in the absence of trypsin (FIG. 5).

3-4) Analysis of purified Sendai virus particles
The urine fluid of embryonated chicken eggs inoculated with SeV / V (-) or SeV / tukH5 was collected on the third day. The liquid was collected and centrifuged at 9,000 rpm for 15 minutes in a cooling high-speed centrifuge to remove blood cells and cell components. The supernatant was collected and then subjected to ultracentrifugation (30,000 rpm, 90 minutes), and the virus was recovered as a precipitate. Several milliliters of PBS was added to the precipitate and completely suspended using an ultrasonic crusher. The suspension was gently placed on top of two layers of 60% and 20% sucrose and subjected to density gradient centrifugation at 27,000 rpm for 90 minutes. After centrifugation, the virus solution that came between the 20% and 60% layers was collected.

  The purified virus particles thus recovered were subjected to SDS-PAGE, and Western blotting was performed using mouse anti-H5 antiserum against influenza virus. As a result, in SeV / tukH5, it was observed as a result of Coomassie staining and immunoblotting that the HA protein of the influenza virus in the purified virus particles was cleaved into HA1 and HA2 (FIG. 6). Therefore, it was concluded that a part of influenza virus HA was taken up into SeV particles.

  In addition, using this purified SeV / tukH5 virus particle and SeV purified in the same manner as a control as an antigen, chickens were immunized according to a conventional method to produce anti-SeV / tukH5 antiserum and anti-SeV antiserum, respectively. In order to confirm whether the obtained anti-SeV / tukH5 antiserum reacts with influenza virus of subtype H5, immunoblot was performed using influenza virus (A / whistling swan / Shimane / 499/83 (H5N3) strain) as an antigen. went. As a result, it was confirmed that the antiserum raised against SeV / tukH5 virus particles has reactivity with the HA molecule of subtype H5 (FIG. 7). This also proves that influenza virus HA is incorporated into SeV particles.

[Example 4] Analysis by immunoelectron microscope
In order to examine whether the H5 protein is localized on the surface of the Sendai virus in SeV / tukH5 virus particles, analysis by immunoelectron microscopy was performed. SeV / tukH5 virus particles were collected by centrifuging the urine fluid of embryonated chicken eggs inoculated with SeV / tukH5 by ultracentrifugation (28,000 rpm, 30 minutes) and suspended in distilled water. 5 μl of this virus suspension (1 × 10 ⁹ particles / ml) was dropped onto a microgrid with a supporting membrane and dried. Pretreatment with PBS containing 0.1% BSA for 30 minutes, and then suck the pretreatment solution with filter paper, add mouse anti-H5 antiserum diluted 200-fold with PBS containing 0.1% BSA to the grid, and in a moisturized state, 60 Reacted for 1 minute. After washing 6 times with PBS, absorb excess water with filter paper, add gold colloid-labeled anti-mouse IgG (ISN) diluted 20-fold with PBS containing 0.1% BSA to the grid, and react for 60 minutes in a moisturized state. It was. After washing 6 times with PBS, it was further rinsed twice with distilled water and allowed to air dry. Stained with 4% uranium acetate for 3 minutes and allowed to air dry. The images were observed and photographed with an electron microscope JEM-1200EXII (JEOL) (FIG. 8).

  As a result, gold colloid was observed on the surface of Sendai virus particles, indicating that in SeV / tukH5 virus particles, H5 protein was expressed on the surface of virus particles.

[Example 5] Immunization of mice with recombinant Sendai virus 5-1)
The mice were lightly anesthetized with ether, and the diluted recombinant Sendai virus vectors (wild type, SeV / V (−) strain, and SeV (−) / tuk-H5) (1 × 10 ⁷ CIU / 25 μL) were added to the mice. Immunized by nasal inoculation. As the mouse, ICR / Crj (CD-1) strain, a 3-week-old male (8-10 g) was used. Lung virus was measured on days 0, 1, 3, 5, 7, and 9 after inoculation with Sendai virus. In addition, antibody production in serum was measured after 0, 5, 7, 10, 14, and 28 days. We also attempted to isolate the virus from the spleen, liver and blood at 0, 1, and 3 days after infection. Heparinized blood was used. For blood collection, the mouse was lightly anesthetized with ether, then the skin of the groin was cut small, the femoral artery and vein were cut with scissors, and the bleeding blood was collected with a capillary pipette. In collecting heparin blood, 20 μl of heparin was placed in an Eppendorf tube in advance, and 180 μl of blood was collected with a micropipette and mixed.

  Anti-SeV ELISA IgG antibody titer (anti-Sendai virus ELISA IgG antibody titer) was measured using purified virus particles from HVJ Hamamatsu strain as an antigen, and anti-H5 ELISA IgG antibody titer was measured using A / whistling swan / Shimane / 499. Measurement was performed by ELISA using / 83 (H5N3) purified virus particles as an antigen (Table 2).

5-2)
Sendai virus vector-free vaccine (mock vaccination), Sendai virus wild strain (10 ⁷ CIU), recombinant Sendai virus without insertion (SeV / V (-)) (10 ⁷ CIU) (A. Kato et al., EMBO J. 16: 578-587 (1997)), and SeV (-) / tuk-H5 (10 ⁷ CIU), respectively, when mice were inoculated nasally, body weight changes, lung gross lesions, and lungs The measurement results of the internal virus growth are shown in FIG. There was no significant change in body weight throughout the observation period. SeV (-) / tuk-H5 infected mice showed virus growth on the order of 10 ⁶ to 10 ⁷ until the first day after infection, but they were rapidly eliminated from the lung after the second day, and completely after the seventh day. No longer detected. Macroscopic lesions of the lung were not so severe. In addition, virus isolation from organs other than lung and blood was negative in all cases.

5-3)
Table 2 shows the time course of antibody production in the immunized mice. As the SeV (-) / tuk-H5 infection progressed, the antibody titers against Sendai virus and H5 protein (the columns of “anti-HVJ” and “anti-H5” in the table, respectively) increased. Antibody responses to both viral antigens were confirmed in all measured mice.

[Table 2]
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
After infection Mouse SeV / V (-) SeV (-) / tuk-H5
ELISA-IgG antibody titer ELISA-IgG antibody titer Days Number ────────── ──────────
Anti-HVJ Anti-H5 Anti-HVJ Anti-H5
────────────────────────────────────
0 1 10 <10 <10 <10
2 10 <10 <10 <10
3 10 <10 <10 <10
────────────────────────────────────
5 1 400 <10 20 <10
2 800 <10 10 <10
3 400 <10 <10 <10
────────────────────────────────────
7 1 2,000 <10 800 200
2 2,000 <10 800 20
3 1,000 <10 800 40
────────────────────────────────────
9 1 800 <10 1,000 10
2 4,000 <10 800 100
3 4,000 <10 800 40
────────────────────────────────────
14 1 20,000 <10 8,000 800
2 20,000 <10 10,000 4,000
3 10,000 <10 4,000 400
────────────────────────────────────
21 1 20,000 <10 ND ND
2 20,000 <10 ND ND
────────────────────────────────────
28 1 80,000 <10 80,000 1,000
2 20,000 <10 80,000 2,000
────────────────────────────────────
Positive control-1 (anti-HVJ antiserum) 8,000 <10
Positive control-2 (anti-H5 antiserum) <10 20,000
Negative control (normal serum) <10 <10
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

[Example 6] Measurement of protective ability of immunized mice against influenza virus challenge with recombinant Sendai virus Mice (ICR / Crj (CD-1) strain, 3-week-old male (8-10 g)) were lightly anesthetized with ether The diluted recombinant Sendai virus vector (SeV (−) / tuk-H5) (3 × 10 ⁷ CIU / 25 μL) was immunized by nasal inoculation. Mice were weighed daily. Blood samples were collected 2 weeks after Sendai virus inoculation, and antibody production in the serum was measured. Half of the mice were boosted by nasal inoculation with a recombinant Sendai virus vector (SeV (−) / tuk-H5) (5 × 10 ⁷ CIU / mouse), and the other half of the mice were raised as they were. In the second week of the booster, challenged (infection) with avian influenza virus-derived strain (M-5) 1 × 10 ⁴ CIU acclimatized to the mouse in Example 1 nasally, 0, 1, 2, 3, and Intrapulmonary virus growth after 5 days was measured.

  Anti-SeV ELISA IgG antibody titer was measured using purified virus particles of HVJ Hamamatsu strain as antigen, and anti-H5 ELISA IgG antibody titer was measured using purified virus particles of A / whistling swan / Shimane / 499/83 (H5N3). Was measured by ELISA as an antigen.

  The change in body weight after Sendai virus inoculation is shown in FIG. 10, and the change in weight after challenge with influenza virus, the gross lesion of the lung, and the time course of intrapulmonary virus growth are shown in FIG.

  Similar to Example 5, Sendai virus vaccination did not significantly affect body weight changes throughout the observation period until influenza virus challenge (FIG. 10). Moreover, the influence on the weight gain by a booster was not seen.

Regarding the protective effect against influenza virus challenge, in non-immunized mice, 10 ⁷ CIU virus was detected on the first day of challenge, further proliferated on the second day, decreased after the third day, and decreased on the fifth day. Was around 10 ^5.5 . Correspondingly, a decrease in body weight was observed from the second day, a gross lesion of the lung also appeared from the third day, and one mouse died on the fourth day.

  In contrast, in the immunized mice (non-booster), the amount of virus in the lungs on the first day of challenge was almost the same level as that in the non-immunized mice, but on the second day, there was already a tendency to decrease. Weight loss was observed in 1 out of 3 animals, but apparently weaker than that of non-immunized mice, and pulmonary lesions appeared on day 5 but no dead mice were seen. . In the immunized mice subjected to the booster, virus growth was almost completely suppressed on the first day of the challenge. From the second day onwards, virus growth was allowed, but the degree was around 1/10 that of non-immunized mice. There was no weight loss and the lung lesions were very slight.

  Table 3 shows the time course of production of anti-SeV antibody and anti-H5 antibody (the columns of “anti-HVJ” and “anti-H5” in the table, respectively) along with the time course of lung gross lesions and influenza virus growth.

[Table 3]
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Mouse ELISA-IgG antibody titer of pulmonary virus days ───────── Gross lesion (CIU / ml)
Number Anti-HVJ Anti-H5
────────────────────────────────────
Second week of immunization -1 20,000 4,000
(Before booster) -2 20,000 40
-3 20,000 4,000
-4 20,000 20
-5 20,000 1,000
-6 10,000 2,000
────────────────────────────────────
4th week of immunization Non-immune -1 ND
(Booster -2 ND
2 weeks later, -3 ND
(Before challenge) ───────────────────────────
Booster (-) -1 40,000 400 0 <20
-2 40,000 8,000 0 <20
-3 40,000 1,000 0 <20
────────────────────────────
Booster (+) -1 160,000 4,000 0 <20
-2 80,000 8,000 0 <20
-3 80,000 1,000 0 <20
────────────────────────────────────
Challenge Non-immune -1 <10 <10 0 6.34x10 ⁶
Day 1 -2 <10 <10 0 1.07x10 ⁷
-3 <10 <10 0 4.35x10 ⁶
────────────────────────────
Booster (-) -1 100,000 2,000 0 5.01x10 ⁶
-2 100,000 2,000 0 5.81x10 ⁶
-3 200,000 2,000 0 4.49x10 ⁶
────────────────────────────
Booster (+) -1 200,000 2,000 0 4.00x10 ¹
-2 400,000 4,000 0 4.00x10 ¹
-3 200,000 8,000 0 4.00x10 ¹
────────────────────────────────────
Challenge Non-immune -1 <10 <10 0 3.10x10 ⁶
Day 2 -2 <10 <10 0 2.15x10 ⁷
-3 <10 <10 0 1.46x10 ⁷
────────────────────────────
Booster (-) -1 100,000 200 0 2.13x10 ⁵
-2 100,000 100 0 1.98x10 ⁶
-3 400,000 800 0 1.38x10 ⁶
────────────────────────────
Booster (+) -1 200,000 2,000 0 4.99x10 ⁵
-2 200,000 8,000 0 5.68x10 ⁵
-3 100,000 2,000 0 3.91x10 ⁶
────────────────────────────────────
Challenge Non-immune -1 <10 <10 0 1.82x10 ⁶
Day 3 -2 <10 <10 0 8.26x10 ⁵
-3 <10 <10 1 1.58x10 ⁶
────────────────────────────
Booster (-) -1 160,000 2,000 0 2.75x10 ⁵
-2 160,000 2,000 0 3.80x10 ⁵
-3 160,000 2,000 0 5.44x10 ⁵
────────────────────────────
Booster (+) -1 80,000 200 0 2.48x10 ⁵
-2 40,000 1,000 1 3.29x10 ⁵
-3 80,000 8,000 1 2.48x10 ⁵
────────────────────────────────────
Challenge Non-immune -1 <10 <10 0 1.57x10 ⁵
Day 5 -2 <10 <10 5 4.03x10 ⁵
-3 <10 <10 3 3.80x10 ⁵
────────────────────────────
Booster (-) -1 80,000 8,000 3 1.98x10 ⁵
-2 80,000 8,000 2 2.06x10 ⁵
-3 80,000 200 1 6.73x10 ⁵
────────────────────────────
Booster (+) -1 160,000 2,000 1 5.68x10 ⁵
-2 40,000 200 0 2.99x10 ⁵
-3 80,000 2,000 0 4.62x10 ⁵
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[Example 7] Construction of recombinant Sendai virus having HIV-1 subtype E gp120 gene 7-1) Plasmid construction SeV vector expressing human immunodeficiency virus (HIV-1) envelope protein gene (env) Was built. The HIV-1 subtype E gp120 gene used for the construction of the SeV expression plasmid is pNH2a-, which contains the entire env gene derived from NH2 (HIV-1 _NH2 ), an R5 strain of HIV-1 subtype E identified in Japan. 1 (Sato, H. et al., 1997, AIDS 11: 396-397). The SeV-based HIV-1 subtype E gp120 expression plasmid is described in the literature “Kato, A. et al., 1997, EMBO J. 16: 578-587, Yu, D. et al., 1997, Genes Cells 2 : 457-466 "(FIG. 12). Briefly, an HIV-1 subtype E env gp120 gene (1,515 bp) was added to a primer pair NH2SU501A [5'-AAgcggccgcAAGACAGTGGAAATGAGAGTGAAGGAGACACAGATG-3 '/ SEQ ID NO: 4 (sense strand)] and NH2SU502B [5' -TTgcggccgcGATGAA CTTTCACCCT AAG TTTTTCTTACT ACGGCGTACG tca TCTTTTTTCTCTCTCC-3 '/ SEQ ID NO: 5 (antisense strand)] was used for amplification by polymerase chain reaction (PCR). Lowercase letters without underscores in the sequence represent NotI recognition sites. The underlined capital letters represent the new S and E signals of SeV linked via the conserved intervening sequence 3'-GAA-5 '. The underlined lowercase letters represent stop codons inserted immediately after the open reading frame of the gp120 gene. The E (termination) and S (initiation) signals each have a function of terminating transcription of the inserted gp120 gene and initiating transcription of the downstream N gene (FIG. 12). The expression of gp120 gene was designed to be initiated by S signal of N gene of SeV vector. PCR was performed using ExTaq polymerase (Takara Shuzo Co., Japan). The amplified fragment was digested with NotI and then pSeV (+) 18bV (-) (Kato, A. et al., 1997, EMBO J. 16: 578-587) was directly inserted into the NotI site to obtain Sendai virus vector pSeV / gp120-E in which gp120 was incorporated. The pSeV-based expression plasmid with HIV-1 subtype B (pNL432) env gp120 (Yu, D. et al., 1997, Genes Cells 2: 457-466) was named pSeV / gp120-B, Used for experiments.

7-2) Reconstruction of recombinant Sendai virus expressing HIV-1 gp120s The stock of recombinant Sendai virus (SeV) is according to the literature (Kato, A. et al., 1996, Genes Cells 1: 569-579). Prepared and determined virus titer. Briefly, monkey kidneys on a 60 mm diameter plastic plate using minimal essential medium (MEM) containing 10% fetal calf serum (FCS) and antibiotics (100 units / ml penicillin G and 100 μg / ml streptomycin) Cell line LLCMK2 is cultured to 70-80% confluent (2 × 10 ⁶ cells) and recombinant vaccinia virus expressing T7 polymerase vTF7-3 (Fuerst, TR et al., 1986, Proc. Natl. Acad Sci. USA 83: 8122-8126, Kato, A. et al., 1996, Genes Cells 1: 569-579) were infected with 2 PFU / cell. One hour after infection, 60 μg of pSeV / gp120-E was transformed into plasmids (24 μg of pGEM-N, 12 μg of pGEM-P, and 24 μg of pGEM that express the trans-acting viral proteins essential for full-length Sendai virus genome generation. -L) (Kato, A. et al., 1996, Genes Cells 1: 569-579) and transfected by the lipofection method (DOTAP, Boehringer-Mannheim, USA). Transfected cells were cultured in serum-free MEM containing 40 μg / ml cytosine arabinoside (araC) (Sigma, USA) and 100 μg / ml rifampicin (Sigma, USA). To minimize toxicity and maximize virus recovery (Kato, A. et al., 1996, Genes Cells 1: 569-579). Forty-eight hours after transfection, the cells were collected, and freeze-thaw was repeated three times to disrupt the cells. Thereafter, the cells were inoculated into the chorioallantoic membrane of 10-day-old embryonated chicken eggs. Three days later, the urine fluid was collected and the virus titer was determined. The titer of the collected Sendai virus was 10 ⁸ to 10 ⁹ PFU / ml, and the vaccinia virus vTF7-3 contained together was 10 ³ to 10 ⁴ PFU / ml. Dilute to ^10-6 and re-amplify with eggs to remove vaccinia virus. The recombinant virus obtained in the second passage was stocked and named SeV / gp120-E or SeV / gp120-B, respectively, and used for the subsequent experiments. Plaque-forming ability of virus stock was determined by infecting LLCMK2 cells, and hemagglutination activity (HA) was determined by “endo-point dilution method” (Kato, A. et al., 1996, Genes Cells 1: 569-579). Were determined. Virus stocks of recombinant viruses that had been passaged twice in chicken eggs generally had titers of 10 ⁹ PFU / ml or 10,240 HA unit / ml.

[Example 8] Expression of recombinant gp120 of HIV-1 subtype E For expression and preparation of recombinant gp120, monkey kidney cell line CV-1 cells were treated with 10% fetal calf serum (FCS) and antibiotics (100 units). / ml penicillin G and 100 μg / ml streptomycin). Recombinant Sendai virus (pSeV / gp120-B or pSeV / gp120-E) was infected with 10 PFU / cell for 1 hour at 37 ° C, washed once with PBS, and then cultured in serum-free DMEM (Yu, D. et al., 1997, Genes Cells 2: 457-466). Culture supernatants were collected at various times and gp120 production levels were analyzed by Western blot using pools of 10 sera from patients infected with either HIV-1 subtype B or E. In other words, the culture supernatant was mixed with 3 volumes of ethanol, cooled at -80 ° C for 1 hour, centrifuged at 12,000g for 30 minutes, and the precipitate was aliquoted with 2% SDS-containing sample buffer for PAGE (Daiichi Pure Chemical Co., Japan) ). Samples were subjected to SDS-PAGE, electrophoretically transferred to a membrane, and a serum pool of a patient infected with HIV-1 subtype E or B diluted 500-fold and incubated, ¹²⁵ I-labeled protein A (NEX146, Dupnt, USA) was added, and autoradiography was performed using BAS2000 (Fujix, Japan). Quantification of gp120 includes known concentrations of subtype E reference protein (HIV-1 subtype E gp120 from CM strain expressed by baculovirus vector; “National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Programn, Lot No. 4-96196) was serially diluted and subjected to Western blot analysis together with the sample.

  For silver staining, the culture supernatant (200 μl) of CV-1 cells infected with recombinant SeV was mixed with 3 volumes (600 μl) of ethanol, cooled at −80 ° C. for 1 hour, and then distant at 12,000 g for 30 minutes. The precipitate was dissolved in an equal amount of 2% SDS-containing PAGE sample buffer (Daiichi Pure Chemical Co., Japan). A 5 μl sample was electrophoresed on a 4-20% polyacrylamide gradient gel containing SDS and silver stained using a kit (BIO-RAD, USA).

  As a result of Western blot analysis, a band corresponding to HIV-1 envelope gp120 was detected at an apparent molecular weight of about 110-120 kDa (FIG. 13A). Production of recombinant gp120-E was detected 24 hours after infection and was maximal 72 hours after infection. Its kinetics were similar to that of recombinant gp120-B with pSeV / gp120-B (Yu, D. et al., 1997, Genes Cells 2: 457-466) (FIGS. 13A and B). Recombinant gp120-E detected in the culture supernatant is a major protein with an apparent molecular weight of about 100-120 kDa. From analysis of the protein image by silver staining, about 10-20% of the total protein is obtained. It was thought to occupy (FIG. 13C). The apparent molecular weight of the recombinant gp120-E seems to vary depending on the degree and pattern of glycosylation. Recombinant gp120-E produced in CV-1 cells by SeV vector system was transferred to gp120 of HIV-1 subtype E CM strain expressed in Sf-9 cells by baculovirus system (provided by NIH AIDS Reagent Program) In comparison, the mobility of electrophoresis on SDS-PAGE was slow. This seems to be due to the difference in the degree and pattern of glycosylation between mammalian cells and insect cells. The amount of recombinant gp120-E secreted into the culture supernatant of CV-1 cells was determined by comparison of the CM gp120 baculovirus product with a known concentration of protein by Western blot analysis, based on the culture of a 6-well plate. Then, it reached about 2 μg / ml at 72 hours after infection (FIG. 13A), and in 50 ml bottle culture, it reached 6 μg / ml at 72 hours after infection. In contrast, commercially available gp120 quantification kits (including HIV-1 gp120 Antigen Capture Kit (Advanced Biotechnologies, USA) and gp120 Capture ELISA kit (Immuno Diagnostics, Inc. USA)) Quantification was not possible. These kits are designed to detect HIV-1 subtype B gp120, likely due to differences in the antigens of HIV-1 subtype E and B gp120.

  Next, a single step affinity column chromatography to which a monoclonal antibody (mAb) TQ4B15-2 (Emini, EA et al., 1992, Nature 355 (6362): 728-730) against the V3 loop of HIV-1 subtype E was bound. The homogeneous recombinant gp120-E was purified from the culture supernatant by chromatography. The culture supernatant of CV-1 cells infected with SeV / gp120-E was collected 72 hours after infection. 50 ml of the collected culture supernatant was passed through an immunoaffinity column (Affi-Gel Hz Immunoaffinity Kit (Bio Rad, USA)) bound with 5 ml of mAb TQ4B15-2 and equilibrated with PBS. The column was washed twice with 10 ml PBS and the bound protein was eluted with 10 ml 0.2 M glycine-HCl buffer pH 4.0. The protein fraction was neutralized with 1M Tris-HCl pH 9.5 to pH 7.4, stored at −80 ° C. and used for experiments. The recovery rate with the affinity column was about 60%. A silver-stained image of the purified protein is shown in FIG. 13C (lane P).

Example 9 Serological Reactivity of Recombinant gp120 to Patient Serum To verify the antigenic differences between recombinant gp120-E and recombinant gp120-B, sera from patients infected with HIV-1 subtype E The reactivity of each protein against the pool (7 sera) or the serum pool (4 sera) of patients infected with subtype B was analyzed by Western blot. As shown in FIG. 14, subtype E serum and subtype B serum reacted specifically with the corresponding subtype of recombinant gp120 (FIG. 14).

[Example 10] Functional analysis of recombinant gp120-E
In order to analyze the function of SeV-derived recombinant gp120-E, the binding activity to CD4 was measured by FACS using the crude culture supernatant of CV-1 cells infected with SeV / gp120-E. Specifically, human CD4 + T cell line MT4 cells (2 × 10 ⁶ ) were incubated at room temperature for 1 hour in MEM containing 5 μg / ml recombinant gp120-E and 10% FCS. After washing with PBS, the cells were reacted with the mouse mAb (TQ4B15-2) against HIV-1 subtype E V3-loop at 4 ° C. for 45 minutes. Bound antibody was reacted with FITC-conjugated sheep F (ab ') 2 fragment (Pharmaceuticals Inc., USA) against mouse IgG for 4 minutes at 4 ° C and leveled. The difference in fluorescence intensity was measured using FACScan (Becton Dickinson, USA), and the average fluorescence intensity was determined. In another experiment, MT4 cells were incubated with various recombinant gp120-E for 1 hour at room temperature and further with anti-CD4 mAb Leu-3a (Becton Dickinson, USA) for 45 minutes at 4 ° C. Labeled with FITC-bound F (ab ′) as described above, FACS analysis was performed.

  As a result, it was found that the recombinant gp120-E expressed with the SeV vector can bind to the surface of MT4 cells expressing CD4 (FIG. 15A). To the cell surface of anti-CD4 antibody (Leu3a) (Yu, D. et al., 1997, Genes Cells 2: 457-466) known to sterically compete with the binding of gp120 to the cell surface. Binding was inhibited in a dose-dependent manner with recombinant gp120-E (FIG. 15B). In a control experiment, the culture supernatant of CV-1 cells infected with the parent virus SeV showed neither CD4 binding activity nor competition for Leu3a binding (“C” in FIGS. 15A and 15B). Therefore, gp120-E was shown to have the activity of specifically recognizing CD4, which is the main receptor for HIV-1, similar to natural gp120 (Yu, D. et al., 1997 Genes Cells 2: 457-466).

[Example 11] Analysis of serological reactivity of EIA
The serological reactivity of SeV-derived recombinant gp120 in enzyme immunoassay (EIA) was verified by comparison with V3 loop peptide EIA (Pau, CP et al., 1993, AIDS 7: 337-340).

  HIV-1 infection in Thailand (n = 20), Vietnam (n = 44), Malaysia (n = 3), Cambodia (n = 9), and Japan (n = 88) to obtain HIV-seropositive sera A total of 164 HIV-seropositive sera were collected from the patients. Twenty-four HIV negative control sera were also collected from healthy seronegative Japanese. These sera were assayed for antibodies against HIV-1 subtype E or B gp120 by the V3-peptide enzyme immunoassay (EIA) (Pau, CP et al., 1993, AIDS 7: 337-340). ). In addition, the 324 base sequence of the HIV-1 envelope C2 / V3 region was amplified by PCR from PBMC or serum according to the literature “Kusagawa, S. et al., 1998, AIDS Res. Hum. Retroviruses 14: 1379-1385”. The sequence was determined. A phylogenetic analysis was performed by the nearest neighbor method using the nucleotide sequence of the env C2 / V3 region to determine the genetic subtype. All 76 samples from Southeast Asian countries were confirmed to be infected with HIV-1 subtype E. All 88 HIV-positive sera in Japan are derived from hemophilia patients infected by transfusion of blood products from North America in the early 1980s. They were infected with HIV-1 subtype B (Hattori, T. et al., 1991, AIDS Res. Hum. Retroviruses 7: 825-830, Komiyama, N. et al., 1989, AIDS Res. Hum. Retroviruses 5: 411-419, Shimizu, N. et al., 1992, J. Mol. Evol. 35: 329-336). These 164 HIV positive sera (subtype E 76 sera, subtype B 88 sera) and 21 control negative sera were used.

According to preliminary experiments, the conditions in which SeV-derived recombinant gp120 was immobilized at a concentration of 15 ng / well and the serum was diluted 1: 8,000 were optimal for EIA using SeV-derived recombinant gp120. In the case of EIA using V3-peptide, it was carried out under conditions using HIV-1 subtype E and B V3 loop peptide (14mer) 1 μg / well and 200-fold diluted serum (Pau, CP et al., 1993, AIDS 7: 337-340). Recombinant gp120 or V3 loop peptide was immobilized on a microtiter EIA plate (Immulon II microtiter plate, Dynatech Laboratories, USA) by overnight incubation at 4 ° C. Blocked with PBS containing 5% skim milk and 0.3% Tween 20 (milk buffer), washed with PBS containing 0.05% Tween 20, and diluted 8,000-fold serum [HIV-1 subtype E infected Thai people (n = 11) , HIV-1 subtype B infected Japanese hemophilia patients (n = 21) or healthy individuals (n = 20)] The horseradish peroxidase-conjugated goat anti-human IgG (Bio-Rad, USA, Catalog No. 172-1001) was incubated at 37 ° C. for 1 hour. A substrate (GENEVAVIA MIX, Sanofi Diagnostic Pasteur, France) containing o-phenyldiamine dihydrochloride and H ₂ O ₂ was added, and color was allowed to develop at room temperature for 6 minutes. The reaction was terminated by adding 1N H ₂ SO ₄ . The reactivity was determined by measuring the absorbance at 492 nm (FIG. 16).

  In the figure, a value (0.3) obtained by adding a value 7 times the standard deviation to the average absorbance of the negative control was defined as a cutoff value (FIG. 16A). As shown in FIG. 16B, subtype-specific antibodies are detected with high specificity in EIA (EIA / SeV) using recombinant gp120-E and B expressed in the SeV system (FIG. 16B). The results were 100% consistent with the genetic data. EIA using recombinant gp120-E and recombinant gp120-B had almost 1,000 times higher sensitivity than V3 peptide EIA (FIG. 17). These were able to detect subtype-specific antibodies with 100% sensitivity (76 in 76 sera for subtype E samples and 88 in 88 sera for subtype B samples), whereas HIV-1 The sensitivity of peptide EIA (PEIA) using V3 loop peptides of type E and MN strains was 90.8% (69 out of 76) and 76.1% (67 out of 88), respectively. Recombinant gp120-E was also tested against seven serum samples that were genetically HIV-1 subtype E but were not reactive with PEIA using HIV-1 subtype E V3 loop peptide. Showed specific reactivity without exception. Similarly, 21 serum samples that did not show reactivity with PEIA using the V3 loop peptide of MN strain but showed serological reaction with recombinant gp120-B were genetically and HIV-1 subcellular. It was confirmed that it was type B.

  The serological reactivity of SeV-derived recombinant gp120 was further verified using a different serum panel. Serum panels consisted of WHO panel G (n = 8) and 20 sera collected through the “UNAIDS Network for HIV isolation and characterization” (Dr. Osmanov, WHO, Switzerland, Dr. Harvey Holmes, NISHC, and Dr. Johnathan Weber , Provided by UK). This serum panel (n = 28) consists of HIV-1 subtype A sera (5 sera from Rwanda, 1 sera from Uganda), subtype B 7 sera (from Brazil), subtype C 4 sera (from Brazil) ), Subtype D 4 sera (from Uganda), subtype E 6 sera (from Thailand), subtype F 1 sera (from Brazil).

  FIG. 18B is a two-dimensional representation of EIA reactivity of recombinant gp120-E (vertical axis) or recombinant gp120-B (horizontal axis) against sera from these countries. As can be seen, HIV-1 subtype E and subtype B sera reacted specifically with the same subtype of recombinant gp120. In contrast, sera from other subtypes react only at low levels with recombinant gp120-E and recombinant gp120-B, reflecting that the subtype is nonspecific, There was no trend.

Claims (26)

A Sendai virus vector capable of expressing an immunogenic protein of influenza virus or a partial peptide thereof having immunogenicity. The Sendai virus vector according to claim 1, wherein the protein is an influenza virus HA protein or NP protein. The Sendai virus vector according to claim 2, wherein the protein is an HA protein of influenza virus. The Sendai virus vector according to claim 2, wherein the HA protein or NP protein of influenza virus is retained so as to be expressible. The Sendai virus vector according to claim 4, wherein the HA protein of influenza virus is retained so that it can be expressed. The Sendai virus vector according to any one of claims 1 to 5, wherein the influenza virus is highly toxic. The Sendai virus vector according to claim 6, wherein the influenza virus is subtype H5 or subtype H7. The Sendai virus vector according to any one of claims 1 to 7, wherein the protein is an HA protein of influenza A virus. The manufacturing method of the influenza vaccine using the Sendai virus vector in any one of Claim 1 to 8. The method according to claim 9, wherein the Sendai virus vector according to claim 2 is used. The method according to claim 9, wherein the Sendai virus vector according to claim 3 is used. The method according to claim 9, wherein the Sendai virus vector according to claim 4 is used. The method according to claim 9, wherein the Sendai virus vector according to claim 5 is used. (A) inoculating a chicken egg with a Sendai virus vector, (b) propagating the Sendai virus complex in the egg, and (c) recovering the propagated Sendai virus vector from the chorioallantoic fluid of the egg. A method according to any of claims 9 to 13. The method according to claim 14, further comprising a step of inactivating the collected Sendai virus vector. The method according to claim 14 or 15, further comprising the step of purifying an influenza virus protein or a part thereof from a Sendai virus vector. An influenza vaccine comprising the Sendai virus vector according to any one of claims 1 to 8. The vaccine according to claim 17, comprising the Sendai virus vector according to claim 2. The vaccine according to claim 17, comprising the Sendai virus vector according to claim 3. The vaccine according to claim 17, comprising the Sendai virus vector according to claim 4. The vaccine according to claim 17, comprising the Sendai virus vector according to claim 5. The influenza vaccine according to any one of claims 17 to 21, which is a live vaccine of Sendai virus. The influenza vaccine according to any one of claims 17 to 21, comprising an inactivated Sendai virus. 24. A method for vaccination against influenza, comprising administering the vaccine according to any one of claims 17 to 23 to an intermediate host of a non-human influenza virus. 25. The method of claim 24, wherein the vaccine is administered to the respiratory tract. 26. The method of claim 24 or 25, wherein the vaccine is administered multiple times.

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