WO2014077096A1 - Procédé de prévention d'infections à l'aide d'une association d'un vaccin vecteur et d'un vaccin vivant - Google Patents

Procédé de prévention d'infections à l'aide d'une association d'un vaccin vecteur et d'un vaccin vivant Download PDF

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WO2014077096A1
WO2014077096A1 PCT/JP2013/078719 JP2013078719W WO2014077096A1 WO 2014077096 A1 WO2014077096 A1 WO 2014077096A1 JP 2013078719 W JP2013078719 W JP 2013078719W WO 2014077096 A1 WO2014077096 A1 WO 2014077096A1
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vaccine
protein
virus
vector
chicken
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Japanese (ja)
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清剛 末永
正士 坂口
宏 岡村
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一般財団法人化学及血清療法研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a method for preventing infectious diseases by the combined use of a vector vaccine and a live vaccine. Specifically, a method for preventing non-human infectious diseases, characterized by using a vector vaccine comprising a recombinant vector virus in which an infection protective antigen derived from an infectious agent is incorporated, and a live attenuated vaccine of the infectious agent, And a vaccine comprising the vector vaccine and the live attenuated vaccine as active ingredients.
  • a virus vector that expresses the protective antigen of Mycobacterium tuberculosis with a vaccinia virus vector does not show sufficient efficacy by itself, so it is based on a conventional live BCG (Bovine tuberculosis) live vaccine. Priming is required (Non-patent Document 1).
  • Non-patent document 2 In chicken vaccines, recombinant viruses that express protective antigens against Newcastle disease (ND) and infectious bursal disease (IBD) using Marek's disease virus type 1 (MDV1) or turkey herpesvirus (HVT) vectors (Patent Document 1, Non-patent document 2) has been put to practical use as a vaccine. In this case, sufficient effectiveness is achieved by itself, but it takes time for immunity to rise.
  • ND Newcastle disease
  • IBD infectious bursal disease
  • MDV1 Marek's disease virus type 1
  • HVT turkey herpesvirus
  • Non-patent Document 3 In the case of chicken infectious bronchitis (IB), since there are multiple serotypes, it is necessary to administer multiple live vaccine strains (Non-patent Document 3).
  • virus vectors have made it possible to set a more labor-saving vaccination program than before, but there is still room for improvement at present.
  • An object of the present invention is to provide a recombinant vector virus (hereinafter sometimes referred to as “vector vaccine”) obtained by integrating an infection protective antigen derived from an infectious agent that causes an infectious disease in a vector virus genome, A vaccine obtained by attenuation (hereinafter also referred to as “live vaccine”), a method for preventing infectious diseases other than humans by using these vaccines in combination, and a vaccine comprising the vector vaccine and the live vaccine as an active ingredient It is to provide.
  • vector vaccine obtained by integrating an infection protective antigen derived from an infectious agent that causes an infectious disease in a vector virus genome
  • live vaccine A vaccine obtained by attenuation
  • a vaccine comprising the vector vaccine and the live vaccine as an active ingredient It is to provide.
  • the inventors of the present application have improved the rise of the antibody by the vector vaccine by administering the viral vector vaccine and the live vaccine simultaneously or in combination, and additional administration Has led to the development of vaccines or vaccination programs that do not require any.
  • vaccines or vaccination programs that do not require any.
  • infectious diseases with multiple serotypes antibodies against one serotype are raised by vector vaccine, and antibodies against multiple serotypes required by administering another serotype are raised.
  • a labor-saving vaccination program was established.
  • a vaccine against chicken infection is shown below as an example. That is, a mixture of a recombinant Marek's disease virus (vector vaccine) into which an F protein, which is one of the protective antigens of Newcastle disease virus (NDV), is inserted and a live vaccine of Newcastle disease (ND) is added to a commercial chicken chick.
  • vector vaccine a recombinant Marek's disease virus
  • F protein which is one of the protective antigens of Newcastle disease virus (NDV)
  • ND Newcastle disease
  • the vector vaccine and live vaccine of the present invention and the administration method using these vaccines in combination can be effectively used for the prevention of infectious diseases in animals. Therefore, the present invention is as follows. [1] A method for preventing infectious diseases other than humans, which comprises using a recombinant vector virus (vector vaccine) incorporating an infection protective antigen of an infectious agent and the attenuated infectious agent (live vaccine). [2] The preventive method according to [1], wherein the vector vaccine and the live vaccine are mixed vaccines. [3] The prevention method according to [1], wherein the vector vaccine and the live vaccine are two independent vaccines. [4] The preventive method according to [3], wherein the vector vaccine and the live vaccine are mixed and administered.
  • vector vaccine recombinant vector virus
  • live vaccine live vaccine
  • Infection protective antigen of the chicken infectious agent is Newcastle disease virus F protein or HN protein, chicken infectious bronchitis-derived S1 protein, Marek's disease virus gB protein, chicken infectious bursal disease virus VP2 protein , Chicken infectious laryngotracheitis virus gB protein, turkey rhinotracheitis virus F protein, avian leukemia virus Env protein, reticuloendotheliosis virus Env protein, chicken anemia virus VP1 + VP2 protein, avian influenza virus
  • the prophylactic method according to [10] which is selected from the group consisting of HA protein, p29 protein of Mycoplasma gallisepticum, and MSA1 protein of Leucochitozone Cowleri.
  • a vaccine comprising as an active ingredient a recombinant vector virus (vector vaccine) incorporating an infection protective antigen of an infectious agent and the attenuated infectious agent (live vaccine).
  • vector vaccine a recombinant vector virus
  • live vaccine a recombinant vector virus incorporating an infection protective antigen of an infectious agent and the attenuated infectious agent
  • live vaccine a recombinant vector virus incorporating an infection protective antigen of an infectious agent and the attenuated infectious agent
  • live vaccine live vaccine
  • the vaccine according to [13] wherein the vector vaccine and the live vaccine are mixed vaccines.
  • the vaccine of [14], wherein the mixing ratio of the vector vaccine and the live vaccine is 1: 1-10.
  • the vaccine according to [13], wherein the vector vaccine and the live vaccine are two independent vaccines.
  • the infectious agent is a factor that infects chickens (chicken infectious agent).
  • Infection protective antigen of the chicken infectious agent is Fcastle protein or HN protein of Newcastle disease virus, S1 protein derived from chicken infectious bronchitis, gB protein of Marek's disease virus, VP2 protein of chicken infectious bursal disease virus , Chicken infectious laryngotracheitis virus gB protein, turkey rhinotracheitis virus F protein, avian leukemia virus Env protein, reticuloendotheliosis virus Env protein, chicken anemia virus VP1 + VP2 protein, avian influenza virus
  • the vaccine according to [19] which is selected from the group consisting of HA protein, Mycoplasma gallisepticum p29 protein, and Leucochitozone Cowleri MSA1 protein.
  • the vaccine according to [19], wherein the protective antigen of the chicken infectious agent is Newcastle disease virus F protein or HN protein, or S1 protein derived from chicken infectious bronchitis.
  • vector vaccine a recombinant vector virus incorporating an infection protective antigen derived from an infectious agent and the attenuated infectious agent (live vaccine)
  • live vaccine a recombinant vector virus incorporating an infection protective antigen derived from an infectious agent and the attenuated infectious agent
  • the method of the present invention is useful for preventing NDV infection during the period when the transferred antibody decreases.
  • the protective antibody against the vaccine strain can be rapidly introduced.
  • the protective antibody is also effective against various virus strains of different serotypes.
  • an antibody against Marek's disease virus is induced and has the effect as a bivalent vaccine, it is possible to formulate an efficient vaccine administration program for various chicken infections such as reducing the number of vaccinations.
  • FIG. 1-1 is a drawing showing a procedure for constructing a plasmid (pUC-nTK-gpt-BAC) for inserting a BAC sequence into MDV1 (CVI988 strain).
  • FIG. 1-2 is a drawing showing a procedure for constructing a plasmid (pUC-nTK-gpt-BAC) for inserting a BAC sequence into MDV1 (CVI988 strain).
  • FIG. 1-3 is a drawing showing a procedure for constructing a plasmid (pUC-nTK-gpt-BAC) for inserting a BAC sequence into MDV1 (CVI988 strain).
  • FIG. 1-1 is a drawing showing a procedure for constructing a plasmid (pUC-nTK-gpt-BAC) for inserting a BAC sequence into MDV1 (CVI988 strain).
  • FIG. 1-4 is a drawing showing a procedure for constructing a plasmid (pUC-nTK-gpt-BAC) for inserting a BAC sequence into MDV1 (CVI988 strain).
  • FIG. 2-1 shows the procedure for constructing a plasmid (pKA4BPKmI-LgAsGC292Ftm) for inserting the chicken infectious bronchitis virus S1 protein (IBV-S1) gene into MDV1 genomic DNA.
  • FIG. 2-2 shows a procedure for constructing a plasmid (pKA4BPKmI-LgAsGC292Ftm) for inserting the chicken infectious bronchitis virus S1 protein (IBV-S1) gene into MDV1 genomic DNA.
  • FIG. 2-1 shows the procedure for constructing a plasmid (pKA4BPKmI-LgAsGC292Ftm) for inserting the chicken infectious bronchitis virus S1 protein (IBV-S1) gene into MDV1 genomic DNA
  • FIG. 3-1 is a graph showing the transition of neutralizing antibody titers against IBV strains (TM86 strain, AK01 strain, Nerima strain, C78 strain) in an IBV-S1-expressing rMDV1 immunization test.
  • A Antibody response when IBV strain AK01 was additionally administered.
  • b Antibody response when AKV strain IB01 is administered alone.
  • FIG. 3-2 is a drawing showing the transition of neutralizing antibody titer against each IBV strain (TM86 strain, AK01 strain, Nerima strain, C78 strain) in an IBV-S1-expressing rMDV1 immunization test.
  • A Antibody response when IBV Nerima strain is additionally administered.
  • FIG. 3-3 is a graph showing the transition of neutralizing antibody titer against each IBV strain (TM86 strain, AK01 strain, Nerima strain, C78 strain) in an IBV-S1-expressing rMDV1 immunoassay.
  • A Antibody response when IBV strain C78 was additionally administered.
  • B Antibody response when IBV strain C78 is administered alone.
  • FIG. 3-4 is a graph showing changes in neutralizing antibody titers against IBV strains (TM86 strain, AK01 strain, Nerima strain, C78 strain) in an IBV-S1-expressing rMDV1 immunization test.
  • FIG. 4 is a graph showing changes in neutralizing antibody titers against IBV (TM86 strain) in the rMDV1 + AK01 strain administration group and the AK01 strain administration group in an IBV-S1-expressing rMDV1 immunization test.
  • FIGS. 5-1 to 5-4 are graphs showing the transition of neutralizing antibody titers against IBV strains (TM86 strain, AK01 strain, Nerima strain, C78 strain) in chickens administered IBV-S1-expressing rMDV1. .
  • FIG. 5-1 shows the transition of neutralizing antibody titer when AKV strain AK01 was additionally administered at the age of 4 days after administration of IBV-S1-expressing rMDV1.
  • FIG. 5-2 shows the change in neutralizing antibody titer when IBV strain AK01 was additionally administered at the age of 21 days after administration of IBV-S1-expressing rMDV1.
  • FIG. 5-3 shows the transition of neutralizing antibody titer when only IBV-S1 expressing rMDV1 was administered.
  • FIG. 5-4 shows the transition of neutralizing antibody titer when IBV strain AK01 was administered alone at 4 days of age without administration of IBV-S1 expressing rMDV1.
  • the present invention provides a method and vaccine for preventing infectious diseases, and is a recombinant vector obtained by incorporating an infection protective antigen derived from an infectious agent such as a virus or microorganism causing an infectious disease in an animal into a vector virus. It is characterized by the combined use of a virus (vector vaccine) and the attenuated infectious agent (live vaccine).
  • combination in the present invention refers to a case where one vaccine mixed with the vector vaccine and the live vaccine is administered, and two independent vaccines of the vector vaccine and the live vaccine are used to prevent infection. It means the case of administration at the same time or different time in the National program.
  • the vector vaccine and live vaccine constituting the present invention can be obtained by the following method.
  • a vaccine against chicken infection will be described as an example, but the vaccine is not limited to chicken infection.
  • the live vaccine of the present invention uses an attenuated chicken infectious factor (an infectious agent causing chicken infection) that has low toxicity and side effects on chickens.
  • Chicken infectious agents are isolated from chickens infected with the chicken infectious agents, and are inoculated and cultured in growing chicken eggs, chicken primary cells (eg, CEF, kidney cells), cell lines (LMH, DT40), etc. Is obtained.
  • the chicken infectious agent is inoculated into an 8-12 day-old growing egg and then cultured at 34-37 ° C. for 2-4 days.
  • chicken infectious agents obtained by the above method include Newcastle disease virus (NDV), chicken infectious bronchitis virus (IBV), Marek's disease virus (MDV), chicken infectious bursal disease virus (IBDV), and chicken infection Laryngotracheitis virus (ILTV), turkey rhinotracheitis virus (TRTV), avian leukemia virus (ALV), reticuloendotheliosis virus (REV), chicken anemia virus (CAV), avian influenza virus (AIV), avian leo Virus (ARV), Pigeonpox virus (PPV), Fowlpox virus (FPV), Turkey herpesvirus (HVT), EDS-76 virus (EDS), Chicken encephalomyelitis virus (AEV), EDS-76 virus ( EDS), Salmonella prolum (SP), Salmonella enteritidis (SE), Abibacterium paragalinalum (H.pg), Mycoplasma gallicepticum (Mg), Examples include Mycoplasma sinobiae (MS), Eimeria protoz
  • a method of repeating passage of the chicken infectious agent-inoculated cells or a method of mutating or removing a gene encoding a protein showing toxicity is used. It is done. Cell passage is performed using the same method and conditions as the above cell culture, but the culture temperature is often low at 30-37 ° C. Attenuation of infectious agents is determined based on the infectivity of chickens, attenuation of diseases, etc. At this time, markers such as temperature dependency, proliferative power, restriction enzyme cleavage pattern and genome base sequence in the growth of infectious agents are included. Be an indicator. When a gene responsible for pathogenicity is clarified, the gene is mutated or removed by a genetic recombination technique described later.
  • chicken infectious agents whose pathogenicity has been attenuated, chicken infectious factors that have been attenuated at the isolation stage, and chicken infectious factors from which genes involved in pathogenicity have been removed are used as active ingredients of live vaccines.
  • the produced attenuated chicken infectious agent is formulated and stored by the following method. After growing and cultivating the attenuated chicken infectious agent according to the same method as above, the culture is disrupted, and various centrifuges (coarse centrifugation, ultracentrifugation), filtration, etc. are repeated to remove cells from the disrupted product. Separation and recovery operations are performed.
  • the obtained attenuated chicken infectious agent is formulated and stored by adding a stabilizer (for example, lactose) or a buffer and maintaining the effectiveness by freezing or lyophilizing under vacuum. Strongly cell-dependent attenuated chicken infectious agents (for example, Marek's disease virus) are stored in a frozen preparation with the infected cells sealed in ampoules after growth in cultured cells. .
  • vector vaccine of the present invention a recombinant vector virus in which an infection protective antigen derived from a chicken infectious agent is incorporated into a vector virus is used. Attenuation of the vector virus takes the same way as attenuation of live vaccines. MDV, fowlpox virus, avian adenovirus, and the like have been reported as viruses that can be used as vector viruses (see Non-Patent Document 2), and any virus can be used in the present invention.
  • Attenuated MDV for example, CVI-988 strain, 61-554 strain (JP-A-6-22757), Md11 / 75C strain (RL Witter; AVIAN DISEASE 31: 752-765, 1987) is used.
  • an infection protective antigen derived from a chicken infectious agent is incorporated into a vector virus
  • a DNA fragment encoding the infection protective antigen is inserted into a specific region in the genome that does not affect the replication of the vector virus.
  • Such a specific region is not particularly limited as long as it is in a non-translated region that is not involved in either transcription or translation.
  • Even when a foreign gene is inserted into the open reading frame for example, when using MDV, US10 gene (patent No. 3428666), when using HVT, TK gene (Ross, J Gen Virol., 1993) , Etc. may be used.
  • NDV F protein, HN protein, IBV S1 protein, MDV gB protein, IBDV VP2 protein, ILTV gB protein, TRTV F protein, ALV Env protein Env protein of REV, VP1 + VP2 protein of CAV, HA protein of AIV, p29 protein of Mycoplasma gallicepticum, MSA1 protein of Leucochitozone and Cowleri see Non-patent Document 2
  • these proteins have been reported.
  • a part thereof can be used as an infection protective antigen derived from the chicken infectious agent of the present invention.
  • a combination of a recombinant vector virus (vector vaccine) expressing an infection protective antigen derived from the above infectious agent and a live vaccine in which the infectious agent is attenuated can be used in the present invention.
  • a combination of a live vaccine of IBV and a recombinant vector virus in which the S1 protein of IBV is incorporated, and a recombinant vector vaccine in which a live vaccine of NDV and an F protein of NDV are incorporated.
  • the above recombinant vector virus is prepared by a general gene recombination technique described by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Laboratory Press, NY, 1989).
  • various gene manipulations developed based on this technology include extraction and cloning of nucleic acid fragments (DNA, RNA), introduction of mutations into nucleic acid fragments, introduction of nucleic acid fragments into a host, and cultivation of a host. Since kits are commercially available, these commercial products are used.
  • (A) Preparation of vector virus genome First, preparation of a vector virus by a method similar to that for a live vaccine, extraction of the virus genome, insertion of the virus genome into a plasmid having a BAC sequence (BACmid; New England Biolabs), And cloning and mass production of the BACmid in E. coli. Next, the BACmid is extracted from Escherichia coli and cultured in a BAC sequence-cleaving enzyme-producing cell, thereby extracting a vector virus genome from which the BAC sequence has been removed. This series of operations is performed according to the attached protocol. The vector virus genome thus obtained is used for the preparation of recombinant vector viruses.
  • BACmid New England Biolabs
  • the MDV genome of the present invention is prepared according to the method disclosed in the report of Messerle et al. (Proc Natl Acad Sci USA, 1997), Wussow et al. (PLoS One, 2009) and the like.
  • a gene encoding an infection protective antigen derived from a chicken infectious agent (hereinafter sometimes referred to as “infection protective gene”) is prepared from a vector virus genome. After extracting the genome of a chicken infectious agent by the same method as described above, for example, it is obtained by amplifying and cloning an infection protection gene by PCR using the genome as a template. At this time, promoters such as early SV40, late SV40, cytomegalovirus IE, ⁇ -actin, etc.
  • the base sequence of the insertion site of the vector virus genome (hereinafter also referred to as “homologous recombination sequence region”) is added to both ends of the protective antigen gene so that homologous recombination of the protective antigen gene is performed.
  • an enhancer sequence, a signal sequence, a restriction enzyme recognition sequence, a transmembrane region may be added or a host-dependent base sequence may be substituted. is there.
  • the site-directed mutagenesis method is generally used for the substitution (mutation) of the base sequence.
  • the IBV S1 protein is disclosed in the literature (Song et al., J Gen Virol., 1998)
  • the NDV F protein is disclosed in the literature (Morgan et al., Avian Disease, 1992), etc.
  • Confirmation of the target DNA fragment is performed by determining the base sequence with a DNA sequencer (ABI Prism 377 Applied Biosystems) and comparing it with the existing base sequence.
  • the existing sequences referred to here can be easily obtained by searching a database such as NCBI (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/nuccore). it can.
  • the recombinant vector virus of the present invention can be obtained by causing homologous recombination between the above-described vector virus genome and a plasmid into which an infection protective antigen gene has been inserted.
  • CEF cells inoculated with MDV are cultured in a medium containing a plasmid in which the NDV-F protein is inserted, so that homologous recombination occurs in the MDV genome homologous recombination sequence region, and the NDV F protein in the MDV genome.
  • a recombinant vector virus in which is incorporated is produced.
  • the culture method and conditions the method and conditions used for obtaining the above chicken infectious agent are used.
  • the recombinant vector virus is formulated and stored as a lyophilized preparation or a frozen liquid preparation in accordance with the properties of the recombinant vector virus in the same manner as the live vaccine. At this time, if the effectiveness and properties of the vector vaccine and the live vaccine are not affected by each other, the vaccine may be formulated and stored in a mixed form.
  • a vector vaccine comprising a recombinant vector virus in which an infection protective antigen derived from an infectious agent thus obtained is incorporated into a vector virus as an active ingredient and a live vaccine comprising the attenuated infectious agent as an active ingredient can be obtained by any known method, for example, It can be introduced into chickens by injection into muscles, intraperitoneally, intradermally or subcutaneously, or by inhalation through the nasal cavity, oral cavity or lung, instillation, or oral administration.
  • the live vaccine and vector vaccine of the present invention are administered to chicken eggs (in ovo)
  • the urine membrane, amniotic cavity and fetus are inoculated by injection, but when the vaccine is administered to chicken chicks, it is instilled.
  • the method by nasal spray, spray inoculation, and drinking water administration is preferable.
  • the dose is appropriately selected according to the type of active ingredient, administration route, administration subject, body weight, symptom, and other conditions, and a single dose is as follows.
  • the live vaccine of the present invention varies depending on the type of vaccine strain, and is used in a minimum effective amount of 1 dose or more described in the formulation standard of each vaccine.
  • the vector vaccine of the present invention is used at a minimum effective dose of 1 dose or more described in the MD vaccine formulation standard.
  • 1000-10000 PFU (plaque forming units) / feather Preferably, it is used in the range of 2000-6000 PFU / feather, in the case of inoculation into the chick, 1000-10000 PFU / feather, preferably 2000-6000 PFU / feather.
  • the vector vaccine and the live vaccine may be administered simultaneously, or may be administered separately at different times.
  • both vaccines are administered simultaneously, the above-described mixed vaccine may be used, or two independent vaccines may be mixed and administered immediately before use.
  • two independent vaccines are provided as a kit or set.
  • simultaneous administration is also meant for administration in the same period in an independent state.
  • either the live vaccine or the vector vaccine may be administered first, but preferably the live vaccine is administered after the vector vaccine is administered.
  • the live vaccine has a sufficient effect by a single administration, but may be further administered.
  • the live vaccine is administered for 1 to 200 days after the vector vaccine is administered. Preferably between 4 and 126 days.
  • the serum is separated from the immunized chicken and then the serum is separated, and the antibody titer against the live vaccine of the present invention and the neutralizing antibody titer against the chicken infectious agent in the serum are determined.
  • measurement is performed by administering a virulent strain of a chicken infectious agent to the immunized chicken, and then observing the life and death of the immunized chicken, the disease state, and the like.
  • ELISA, PHA, plaque assay, etc. are commonly used for antibody measurement in in vitro systems.
  • Vaccines administered to chickens vary depending on the situation at each breeding place (contamination status of pathogens, surrounding chicken disease epidemic situation, egg-laying chicken or meat chicken, breeder chicken, or practical chicken). A nation program is organized.
  • the live vaccine and vector vaccine of the present invention may be formulated as a vaccine program to be administered together with other chicken infection vaccines.
  • a chicken infection vaccine in the case of a live vaccine, it can be combined with at least one vaccine selected from a live vaccine group for ARV, NDV, IBV, IBDV, MDV and AEV.
  • ARV, NDV, IBV, EDS, IBDV bacteria (eg, E.
  • the method for preventing chicken infection by using the vector vaccine and the live vaccine of the present invention in combination is a method that can be widely applied generally, and is not limited to chickens, but also for infections of birds and animals other than chickens. The effect is expected. Therefore, the prevention method of the present invention can be used for animals other than humans, and the vaccines comprising the vector vaccine and the live vaccine of the present invention as active ingredients can be widely used for animals.
  • influenza virus, Marek's disease virus (herpes virus), Newcastle disease virus, etc. are not limited to chickens and turkeys, and are widely used for birds.
  • Birds other than chickens include birds bred for commercial or non-commercial purposes, examples of which include pheasants (eg chickens, quails, turkeys), ducks (eg ducks, geese), plovers Eyes (eg, gulls, quail, plover), pigeons (eg, pigeons), ostriches (eg, ostriches), sparrows (eg, crows, finch, sparrows, sterling, swallows), parrots (eg, parrots) , Hawks (for example, eagle, hawk), owls (for example, owl), penguins (for example, penguin), parrots (for example, parakeet, parrot).
  • Preferred are turkey, duck, quail and duck.
  • herpes viruses When applied to animals other than chickens, herpes viruses, paramyxoviruses, rhabdoviruses, poxviruses, adenoviruses and the like can be used as vector viruses. Any of these may be used.
  • infection protection antigens of animal infectious agents other than chicken infectious agents such as G protein gene of rabies virus (Esposito et al., Virus Genes, 1987) and HA protein gene of influenza virus (Tang et al., Arch Virol., 2002)
  • a vector vaccine for chicken infection is obtained by encoding a gene, a vector vaccine incorporating the gene, and a live vaccine obtained by attenuating the animal infectious agent. And it can produce by the method similar to the method of producing a live vaccine.
  • rMDV1-BACmid recombinant Marek's disease virus type 1 genomic DNA having BAC sequence
  • pUC-nTK-gpt-BAC for inserting BAC sequence into MDV1 (CVI988 strain) gpt (xanthine) downstream of the thymidine kinase (TK) gene by the following steps (a) to (e)
  • a plasmid pUC-nTK-BAC was constructed by inserting a -guanine phosphoribosyl transferase) gene and a BAC sequence downstream thereof into pUC119.
  • a fragment containing the entire thymidine kinase (TK) gene was amplified by PCR using the MDV1 genomic DNA as a template and the primers of SEQ ID NOs: 1 and 2.
  • 3 'side: CCC AAGCTT CTCCCCGGCCAATCATACA underlined part is HindIII recognition sequence
  • BACmid pBeloBAC11 (New England Biolabs, product code; E4154) containing the BAC sequence was cleaved with NotI and then cloned into pBlueScriptII KS + (Stratagene) previously cleaved with the same restriction enzyme to obtain plasmid pBSII-BAC It was.
  • BACmid plasmid that seals the BAC sequence is sometimes referred to as “BACmid”.
  • C PCR was performed using Escherichia coli genomic DNA as a template and the primers of SEQ ID NOs: 5 and 6 to amplify a fragment containing the entire gpt (xanthine-guanine phosphoribosyl transferase) gene.
  • (E) PCR was carried out using the pBSII-gpt-BAC of (2) above as a template and the primers of SEQ ID NOs: 7 and 8, and an about 8.7 kbp fragment containing the gpt gene and the BAC sequence was amplified.
  • MDV1-infected cells were cultured in a medium supplemented with GPT Selection Reagent (Chemicon International) containing 25 ⁇ g / mL mycophenolic acid, and cells retaining cyclic rMDV1-BACmid were selected.
  • GPT Selection Reagent Cemicon International
  • the MDV1 genomic DNA in rMDV1-BACmid was identified by comparing the restriction enzyme (EcoRI, BamHI, HindIII) cleavage patterns of the genomic DNA of the rMDV1-BACmid and the parent strain MDV1. As a result, the cleavage patterns of both were identical except for the BACmid insertion region, and insertion of the full-length MDV1 genome into BACmid was confirmed.
  • EcoRI EcoRI
  • BamHI HindIII
  • rMDV1 genomic DNA rMDV1-BACmid / S1 containing chicken infectious bronchitis virus S1 protein (IBV-S1) gene and BACmid ⁇
  • a codon optimized DNA fragment was obtained so that the GC content in the AT-rich region on the IBV-S1 gene was high.
  • the pKA4BP-LgAsGC292Ftm was partially cleaved with SnaBI in advance and amplified by PCR using pACYC177 (Nippon Gene, product code: 310-02191) as a template and primers of SEQ ID NOs: 13 and 14. An about 1.0 kbp fragment containing the kanamycin resistance gene sequence was inserted to obtain a plasmid pKA4BPKmI-LgAsGC292Ftm having the kanamycin resistance gene (FIG. 2-2).
  • the culture temperature was shifted from 32 ° C. to 42 ° C., and competent cells were prepared according to a standard method in a state where ⁇ -Red recombinase was expressed.
  • the competent cell was introduced with pKA4BPKmI-LgAsGC292Ftm of (1) previously linearized with restriction enzymes KpnI and BglI, cultured in a medium supplemented with chloramphenicol and kanamycin, and inserted with an IBV-S1 expression plasmid E. coli harboring rMDV1-BACmid (GS1783 / rMDV1-BACmid / S1 + Km) was cloned (Wussow et al., PLoS One, 2009).
  • the GS1783 / MDV1-BACmid / S1 + Km was cultured in an arabinose-added medium having a final concentration of 1.6%, and then the culture temperature was shifted from 32 ° C. to 42 ° C. and incubated for 30 minutes. Subsequently, the cells were cultured in a chloramphenicol-added medium or a kanamycin-added medium, and clones that grew only on the chloramphenicol-added medium were selected (Wussow et al., PLoS ⁇ One, 2009).
  • Escherichia coli (GS1783 / rMDV1-BACmid / S1) carrying the recombinant MDV genomic DNA (rMDV1-BACmid / S1) in which the IBV-S1 expression plasmid was inserted into rMDV1-BACmid was obtained.
  • rMDV1 genomic DNA 24 / gBGC292Ftm having only the IBV-S1 gene from which the gpt-BAC sequence was removed was obtained.
  • the expression of IBV-S1 by 24 / gBGC292Ftm was confirmed by a fluorescent antibody method using a monoclonal antibody against IBV-S1.
  • rMDV1, rMDV1-S1 a vector vaccine (hereinafter sometimes referred to as “rMDV1, rMDV1-S1”) containing 24 / g BGC292Ftm (3200 PFU / feather) obtained in Example 3 as an active ingredient was purchased from an hatchery.
  • Inoculate chicken eggs (Julia, Tsuboi breeding hatchery, Yamaga City) (5 birds in each group), and three IBV live vaccines (AK01; Chemistry and Serum Therapy Research Institute, Nerima) at 18 weeks of age after hatching Strain; Chemo-Serum Therapy Laboratory, C78 strain; Nissei Laboratories Co., Ltd.)
  • AK01 Chemistry and Serum Therapy Research Institute, Nerima
  • Chemo-Serum Therapy Laboratory C78 strain
  • Nissei Laboratories Co., Ltd. the group inoculated only with rMDV1-S1 (5 birds per group), the group administered live vaccine only to chicks at 18 weeks of age after hatching (4 birds each group), the non-immunized group (4 groups per group) Feather) was set.
  • the neutralizing antibody titer was maintained for at least 22 weeks after the booster immunization for the TM strain examined for the persistence of the antibody titer (FIG. 4).
  • a group in which only rMDV1-S1 was inoculated in the ovum and a group in which only the live vaccine was administered to chicks at the age of 4 days after hatching were set (5 birds in each group). Blood was collected over time and evaluated by examining the neutralizing antibody titer against each IBV strain (TM86 strain, AK01 strain, Nerima strain, C78 strain).
  • a vector vaccine comprising the recombinant vector virus 207 strain (Journal of Virology, 2000, 74, 3217-26) in which the F protein (NDV-F) of Newcastle disease (ND) virus is inserted in the US10 gene region of MDV1 And ND live vaccine B1 strain (Chemistry and Serum Therapy Research Institute) were mixed at the rate shown in Table 2, and 0.2 ml / wing (21 birds per group) was purchased from the hatchery. Inoculated subcutaneously in the neck of Tsuboi breeder hatchery, Yamaga City). Blood was collected over time, and the antibody titer against NDV-F was measured by ELISA for the obtained serum.
  • the NDV Ishii strain was dissolved in 1% octyl gorcoside (DOJINDO), diluted with PBE (-), and about 100 HA (red blood cell agglutination) / 100 ⁇ L per well was dispensed into a 96-well microplate. Then, it was left to stand at 4 ° C. overnight to be solid-phased.
  • Chicken serum diluted 100-fold with diluted solution (5% skim milk powder and 0.5% polysorbate 20 in PBS) was reacted at room temperature for 1 hour, washed, and then monoclonal antibody against NDV-F labeled with HRP.
  • the group vaccinated with vector vaccine and ND live vaccine showed a higher positive rate than the group administered vector vaccine (207 strains) alone after 4 weeks of age when the titer of the transfer antibody decreased.
  • the vector vaccine in which the infection protective antigen derived from the infectious agent of the present invention is incorporated, the live attenuated vaccine of the infectious agent, and the method of using these in combination are effective for vaccination for preventing non-human infectious diseases. Can be used.

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Abstract

La présente invention concerne une méthode de prévention d'infections à l'aide d'une association d'un vaccin vecteur et d'un vaccin vivant. L'invention concerne ainsi un virus vecteur recombiné (un vaccin vecteur) qui est produit en intégrant un fragment d'ADN codant pour un antigène protégeant des infections dérivé d'un facteur infectieux animal d'un virus, d'une bactérie ou similaires dans un génome de virus par une technique de recombinaison génique ; un facteur infectieux atténué (un vaccin vivant) qui est produit en atténuant le facteur infectieux ; une méthode de prévention des infections chez un animal non humain, qui est caractérisée par l'utilisation d'une association du vaccin vecteur et du vaccin vivant ; et un vaccin qui est caractérisé en ce qu'il est produit en utilisant le vaccin vecteur et le vaccin vivant comme principes actifs.
PCT/JP2013/078719 2012-11-15 2013-10-23 Procédé de prévention d'infections à l'aide d'une association d'un vaccin vecteur et d'un vaccin vivant WO2014077096A1 (fr)

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JP2019509260A (ja) * 2016-12-01 2019-04-04 普莱柯生物工程股▲ふん▼有限公司 ワクチン組成物及びその製造方法と使用
JP2019510497A (ja) * 2016-04-05 2019-04-18 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． Ｒｓｖに対するワクチン
WO2021029385A1 (fr) 2019-08-09 2021-02-18 Ｋｍバイオロジクス株式会社 Virus de la vaccine oncolytique
US11759514B2 (en) 2016-05-30 2023-09-19 Janssen Vaccines & Prevention B.V. Stabilized pre-fusion RSV F proteins
US11998597B2 (en) 2015-07-07 2024-06-04 Janssen Vaccines & Prevention B.V. Vaccine against RSV

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CN110188538B (zh) * 2019-04-26 2021-07-20 奇安信科技集团股份有限公司 采用沙箱集群检测数据的方法及装置

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11998597B2 (en) 2015-07-07 2024-06-04 Janssen Vaccines & Prevention B.V. Vaccine against RSV
JP2019510497A (ja) * 2016-04-05 2019-04-18 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． Ｒｓｖに対するワクチン
JP7233928B2 (ja) 2016-04-05 2023-03-07 ヤンセン ファッシンズ アンド プリベンション ベーフェー Ｒｓｖに対するワクチン
US11801297B2 (en) 2016-04-05 2023-10-31 Janssen Vaccines & Prevention B.V. Vaccine against RSV
US11759514B2 (en) 2016-05-30 2023-09-19 Janssen Vaccines & Prevention B.V. Stabilized pre-fusion RSV F proteins
JP2019509260A (ja) * 2016-12-01 2019-04-04 普莱柯生物工程股▲ふん▼有限公司 ワクチン組成物及びその製造方法と使用
WO2021029385A1 (fr) 2019-08-09 2021-02-18 Ｋｍバイオロジクス株式会社 Virus de la vaccine oncolytique

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