WO2015113188A1 - 流感病毒为载体的HAdV嵌合疫苗的制备及其应用 - Google Patents

流感病毒为载体的HAdV嵌合疫苗的制备及其应用 Download PDF

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WO2015113188A1
WO2015113188A1 PCT/CN2014/000581 CN2014000581W WO2015113188A1 WO 2015113188 A1 WO2015113188 A1 WO 2015113188A1 CN 2014000581 W CN2014000581 W CN 2014000581W WO 2015113188 A1 WO2015113188 A1 WO 2015113188A1
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hadv
gene
influenza virus
virus
cold
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French (fr)
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杨鹏辉
王希良
张绍庚
段跃强
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中国人民解放军第三0二医院
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61K2039/525—Virus
    • A61K2039/5254—Virus 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the preparation and application of a HAdV chimeric vaccine with influenza virus as a carrier.
  • adenovirus AdV
  • AdV adenovirus
  • AdV adenovirus
  • AdV adenovirus
  • adenovirus type 3 and 7 are the main pathogens of adenovirus pneumonia, and the lower respiratory tract infection caused by them accounts for 50% of all adenovirus infections.
  • Adenovirus due to its large variety and wide range of pathogenic diseases, poses a serious threat to human health and life safety, and is receiving more and more attention from the society. At the same time, people will not forget the three epidemic outbreaks in history, and the catastrophic consequences for the world, especially the H5 and H7 subtype avian influenza viruses that have spread across the species barrier in recent years. Humans have once again sounded the alarm. Therefore, the prevention and control of human adenovirus and influenza has become a major problem in today's life sciences.
  • influenza vaccines are the most effective measure to prevent and control human infectious diseases.
  • the development of influenza vaccines has experienced inactivated vaccines, split vaccines, subunit vaccines, and DNA vaccines, which have made important contributions to human health.
  • the live attenuated influenza vaccine developed by the United States and Russia is an attenuated human influenza virus strain that reduces virulence and can grow at optimal temperature.
  • Clinical trials have proven to be safe and effective, and can effectively prevent influenza.
  • the widespread use of this vaccine provides a bright future for humans to conquer the flu in the 21st century.
  • human respiratory adenovirus there is still no vaccine approved for use, but research on vaccines has always been the focus of the international community.
  • adenovirus inactivated vaccines have provided vaccine candidates for the prevention of human respiratory adenovirus, but they have not yet been applied on a large scale. From the study, because there are 51 serotypes of adenovirus, the difference between each type is large, the vaccine induces potential immune overgrowth after immunization, and the immunization can not produce mucosal immunity and the traditional live attenuated vaccine still exists. Stable and potentially over-excited issues pose challenges to the development of vaccines.
  • the third is that the nasal immunization pathway is an important development direction for the human adenovirus vaccine, which is designed to generate mucosal and systemic immune responses against human adenovirus. Therefore, the integration and integration of the above technologies provide a scientific guarantee for the development of safe, effective and practical human adenovirus vaccines.
  • HAV Human adenovirus
  • HAdV capsid protein is composed of 252 shell particles, of which 240 shells constitute 20 faces of protein capsid, and its constituent protein is called hexon (Hexon, 120KD), which is the main envelope.
  • hexon is the main antigenic protein of adenovirus, which stimulates the body to produce antibodies, inhibits the change of viral conformation, and neutralizes virions. Hexaton contains a large number of antigenic determinants, including type-specific and subgenus-specific epitopes and neutralizing epitopes.
  • the pedestal consists of two areas, P1 and P2.
  • the tower area consists of four loops, namely Loop1, Loop2, Loop3, and Loop4.
  • HAdV poses a problem for the prevention and control of HAdV due to its special and complex pathogenic characteristics.
  • Vaccines are the most effective means of controlling infectious diseases. Vaccines play an important role in the fight against disease and infectious diseases, and adenovirus vaccines are no exception.
  • an enteric-coated attenuated adenovirus vaccine was used to immunize adults in the boot camp. It was found that 90% of the adenovirus-induced fever can be reduced, but serum is passed. The test found that the live vaccine induced by this live vaccine has a very low titer.
  • an adenovirus enteric-activated vaccine was used for immunization, and it was found that serum IgM, IgG, and IgA were elevated, but no corresponding IgA type antibody was found in nasal secretions.
  • the live attenuated adenovirus vaccine has a good effect in the US military, there are still ARD outbreaks in the US military. Some studies have confirmed the use of DNA chips and PCR technology. After inoculation of the existing live attenuated adenovirus vaccine, the vaccinators can still Co-infection Other types of adenoviruses demonstrate that existing live attenuated vaccines do not have cross-immunological protection against other types of adenoviruses. Therefore, the HAdV subunit vaccine is duty-bound to become an important development target in this field.
  • adenovirus has been widely used as a gene vector in gene therapy and chimeric vaccine research, and adenovirus infection has greatly hindered the efficacy of this gene therapy, and people will not ignore this chimeric vaccine. Potential side effects.
  • the development of antigen-immunobioinformatics has had a major impact on the design of the primary protective epitope of HAdV hexon, and provided theoretical and technical support for the development of T- and B-cell dominant epitope vaccines for protective antigens.
  • Another scholar compared the amino acid sequence of the adenovirus hexon, combined with the three-dimensional structure of the adenovirus type 2 to reveal the polypeptide exposure and antigenicity prediction analysis, select the hexon protein gene 937 ⁇ 1230, 2166 ⁇ 2607 and 937 ⁇ 2607 coding sequence.
  • the predicted antigenic sites encompassed by this region have high homology between respiratory adenovirus types, may have good exposure on adenoviral particles and may contain intertype or group specific antigenic epitopes.
  • the expression of the epitope of the Loopl region of Ad2 in the coxsackievirus of group B is also used in foreign countries.
  • the L1 loop of the recombinant scorpion hexon protein can be made in HeLa cells. Stable expression, the expressed protein can induce the production of neutralizing antibodies against both adenovirus and coxsackie virus, and then develop multivalent genetic engineering vaccines.
  • influenza virus reverse genetic technology provides a reliable guarantee for the preparation of HAdV chimeric vaccine strains with influenza virus as a vector.
  • influenza virus rescue system of influenza virus has become a mature technology at home and abroad, making it very attractive to express foreign genes with influenza virus as a delivery system.
  • Influenza viruses have many advantages as vectors for the development of vaccines for other infectious disease pathogens: (1) can stimulate the body to produce strong mucosal and systemic immune responses; (2) influenza vaccines need to be produced annually due to antigenic variation; (3) HA and NA surface proteins The structure and function determine the ability to perform genetic manipulation without affecting their function; (4) The influenza virus has developed an efficient reverse genetics operating system; (5) mice and ferrets are candidates for recombinant influenza virus vaccines The study of mucosal immune response and immune protection in the respiratory tract provides a good animal model.
  • influenza viruses do not form DNA intermediates during the replication cycle, and because they do not integrate into the host's chromosomes, they are more secure.
  • Strategies for influenza virus genome manipulation include: Exogenous proteins are chimeric into the surface glycoproteins HA and NA, producing additional gene segments that engineer the non-structural protein NS1.
  • the coding region of this virus is the HA/NA outer functional region of the parainfluenza virus, which can be effectively propagated in chicken embryos but in mice. It is attenuated. When mice are administered intranasally with a recombinant virus, all antibodies against influenza virus and parainfluenza virus are produced. The immunoprotective effect of this bivalent live vaccine is significantly better than the combined vaccine.
  • Zhu Nan Li et al reported that small peptides could be inserted into the "loop" loop region of the influenza virus HA antigen. Subsequent studies showed that the protective antigen (PA) large fragment polypeptide of B. anthracis can be inserted into the influenza virus H3 subtype HA.
  • PA protective antigen
  • the recombinant influenza virus with chimeric HA-PA gene was genetically stabilized after passage through MDCK cells and chicken embryos. Animal experiments showed that the recombinant virus could induce antibody responses induced by HA and PA proteins.
  • Kawaka et al. integrated the GFP gene into a specific location of the NA gene and found that the efficiency of recombinant influenza virion assembly was as high as 91%.
  • Andrej Egorov and other GFP and IL-2 were inserted into the open reading frame of NS1, and the recombinant influenza virus strain was successfully obtained.
  • influenza virus genome is used as a vector for expressing the epitope of HAdV virus, and the HAdV chimeric vaccine for developing influenza virus as a vector is Not reported at home and abroad.
  • the HAdV chimeric vaccine with nasal immunization with influenza virus as a carrier is the cornerstone for improving safety and comprehensive immune protection.
  • influenza trivalent live attenuated vaccine Flumist produced by Medimmune Company of the United States was approved and put into use by the FDA. It is suitable for healthy children aged 2-17 years and healthy adults aged 18-49 years. Clinical trial results show that the vaccine is safe. And effective.
  • the US FDA approved the launch of a four-valent live attenuated influenza vaccine.
  • the vaccine was inoculated through the nasal cavity. The method of use was simple and widely used, and the cold-adapted live attenuated vaccine was used as a delivery system to express foreign genes. The vaccine has opened up new horizons and brought new opportunities.
  • Influenza virus as a potent vaccine vector can induce systemic and mucosal 1 ⁇ B cell immune responses by delivering different antigens into the nasal tissue.
  • live attenuated vaccines can induce local and systemic immunity through intranasal immunization and thus have a protective effect against upper and lower respiratory tract infections.
  • the immune response induced by a live attenuated vaccine is similar to that of a wild type virus.
  • injection vaccination is not an ideal immune route and is not convenient. Therefore, developing a nasal pathway is the best option for vaccine immunization.
  • the nasal passage is rich in lymphoid tissue and is the target site for nasal vaccination, that is, nasal-associated lymphoid tissue (Li), which is mainly located in the lymphoid tissue ring of the larynx, called the Waldeyer's ring.
  • Li nasal-associated lymphoid tissue
  • the blood flow rate is about 40ml/min, which is larger than that of muscle, brain and liver. In fact, the nasopharynx is a good immune organ.
  • mucosal immunity It is rich in antigen-presenting cells, which can effectively present and process antigens and produce mucosal immunity. And the systemic immune response, and local mucosal immunity, can also cause the mucosal immune response of the respiratory tract, digestive tract, genitourinary tract and other parts of the mucosal immune response, but the strongest respiratory tract.
  • Nasal immunization is an active research field.
  • many companies have developed nasal vaccination, especially respiratory infectious diseases vaccines, such as influenza attenuated live vaccines, which can not only produce local mucosal immunity, but also systemic immunity. And produced with cross-immunity protection. Therefore, the development of HAdV chimeric vaccine replacement injection with influenza virus-inoculated influenza virus as a carrier has great application prospects, because nasal immunity is simple, effective, and very suitable for self-use of the general population, and at the same time, the body can resist HAdV and Influenza is infected by two pathogens, and the type of immune response to HAdV vaccine can be changed, thereby achieving a more safe purpose, providing assurance for immune prevention of HAdV and influenza.
  • the object of the present invention is to provide a preparation of an HAdV chimeric vaccine using influenza virus as a vector and its use.
  • the present invention provides the DNA molecule of SEQ ID No. 1.
  • a recombinant virus is also within the scope of the present invention, and the virus is prepared as follows:
  • the M gene of the attenuated influenza virus and the expression plasmid of the cold-adapted, attenuated influenza virus NS gene, and the expression plasmid containing the HA gene of the target influenza virus and the NA gene of the target influenza virus, respectively, are co-transfected into the host cell, and the culture is recombined. flu virus;
  • the NS gene, and the HA gene and the NA gene of the target influenza virus are located on different expression plasmids; the structural protein gene of the HAdV is the dominant epitope of the hexon protein of HAdV and/or the hexon protein of HAdV gene.
  • the host cell is a cell co-cultured with MDCK, Vero, 293T, COS cells or MDCK/293T, MDCK/COS;
  • the structural protein gene of HAdV is a fusion gene of the dominant epitope gene of Loopl and Loop2 of the hexon protein region of HAdV.
  • the HAdV is a type 3 HAdV or a type 7 HAdV;
  • the type 3 HAdV is specifically a HAdV-3 strain VR-3;
  • the type 7 HAdV is specifically HAdV-7 strain GZ08, GenBank access ion No. GQ47834 L.
  • the dominant epitope gene of Loopl and Loop2 of the hexon protein region of the HAdV The nucleotide sequence of the fusion gene is shown in SEQ ID No. 1.
  • the structural protein gene of the HAdV is inserted in the open reading frame of the cold-adapted, attenuated influenza virus NS gene from the 375th to the 376th nucleus from the 5' end Between the glycosides;
  • the structural protein gene of the HAdV and the 375th nucleotide from the 5' end of the open reading frame of the NS gene of the cold-adapted, attenuated influenza virus are linked with a 5'-UAAUG-3' sequence;
  • the structural protein gene of the HAdV is a substitution of the 223th nucleotide to the 760th nucleotide from the 5' end of the open reading frame of the M gene of the cold-adapted, attenuated influenza virus;
  • the structural protein gene of the HAdV is a nucleotide substitution from the 184th nucleotide to the 1253th nucleotide from the 5' end of the open reading frame of the NA gene of the target influenza virus.
  • the cold-adapted or attenuated influenza virus is a cold-adapted, attenuated A-type influenza virus or a cold-adapted, attenuated influenza B virus;
  • the cold-adapted and attenuated influenza A virus is specifically a cold-adapted, attenuated influenza virus of Roya type, and further a cold-adapted, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2);
  • the target influenza virus is influenza A virus or influenza B virus, and the influenza A virus is specifically any one of HI subtype-H16 subtype, and specifically H1N1 subtype influenza virus, and specifically influenza Virus strain
  • the target influenza virus is a common wild-type influenza virus that has not been subjected to any treatment (e.g., without attenuation, without cold adaptation).
  • the structural protein gene of the HAdV is inserted in the open reading frame of the cold-adapted, attenuated influenza virus NS gene from the 375th to the 376th nucleus from the 5' end
  • the sequence obtained between the glucosinolates is represented by nucleotides 41-1162 of SEQ ID No. 2;
  • the structural protein gene of HAdV is a sequence obtained by replacing the 223th nucleotide to the 760th nucleotide from the 5' end of the open reading frame of the M gene of the cold-adapted, attenuated influenza virus as SEQ Shown at nucleotides 40-759 in ID No. 3;
  • the structural protein gene of the HAdV replaces the nucleotide sequence of the 184th nucleotide to the 1253th nucleotide from the 5' end of the open reading frame of the NA gene of the target influenza virus as SEQ ID No. 4
  • the nucleotides 35_650 are shown.
  • the PB2 gene, the PB1 gene, the PA gene, the NP gene, the M gene, the NS gene, the HA gene or the NA gene are ligated to the 3' NCR sequences of the respective genes at their 5' ends, 5' NCR sequences of the respective genes are linked at their respective 3' ends;
  • the starting plasmid of the expression plasmid is a bidirectional transcription expression vector PAD3000;
  • each gene was inserted into the BsnR site of the bidirectional transcription expression vector PAD3000.
  • a chimeric vaccine prepared by the virus of any of the above is also within the scope of the present invention.
  • any of the above-described viruses for the preparation of a product for preventing and/or treating diseases caused by influenza virus and/or HAdV is also within the scope of the present invention.
  • influenza virus is an influenza A virus or a influenza B virus, and the influenza A virus is specifically any one of the HI subtype-H16 subtype;
  • the HAdV is a type 3 HAdV or a type 7 HAdV;
  • the type 3 HAdV is specifically a HAdV-3 strain VR-3;
  • the ⁇ -type HAdV is specifically HAdV-7 strain GZ08, GenBank access ion No. GQ47834L
  • Figure 1 is a schematic diagram showing the recombination strategy of a HAdV chimeric vaccine constructed by using influenza virus as a vector.
  • Figure 2 shows the morphological identification and particle size and distribution of recombinant virus rFLU/HAdV/NS1.
  • Figure 3 shows the results of antibody titer assay of mouse immunized rFLU/HAdV/NS1.
  • Figure 4 shows that mouse immunization rFLU/HAdV/NS1 produces IFN- ⁇ , IL-4 cellular immune responses.
  • Figure 5 shows murine antibody rFLU/HAdV/NS1 producing mucosal antibody titers against HAdV-3.
  • Figure 6 shows the immunoprotective effect of rFLU/HAdV/NS1 immunized mice against adenovirus strains.
  • PAD3000 is disclosed in the literature "Hoffmann E, Mahmood K, Yang CF, Webster RG, Greenberg HB, Kemble G. Rescue of influenza B virus from eight plasmids. Proc Natl Acad Sci. 2002 ; 99 (17): 11411 - 6.” The public can be obtained from the Academy of Military Medical Sciences of the Chinese People's Liberation Army.
  • mice 6-8 weeks old BALB/c mice were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences.
  • Snow shoals from 10-12 weeks old were purchased from Angora Anglu, Wuxi, China.
  • the 2012/2013 influenza virus epidemic strain A/Cal ifornia/07/2009 (H1N1) was provided by the National Influenza Centre and was available to the public from the Chinese Academy of Military Medical Sciences.
  • the HA and NA genes of the virus strain were amplified and inserted into the ⁇ STBBI site of the bidirectional transcription/expression vector PAD3000 to construct the recombinant plasmids pD-HA and pD-NA.
  • the influenza virus A/PR/8/1934 (PR8) strain is in the literature "Li C, Yang P, Sun Y, Li T, Wang C, Wang Z, Zou Z, Yan Y, Wang W, Wang C, Chen Z, Xing L, Tang C, Ju X, Guo F, Deng J, Zhao Y, Yang P, Tang J, Wang H, Zhao Z, Yin Z, Cao B, Wang X, Jiang C. IX- 17 respon se mediates acute lun A (H1N L) vi rus. Cel l Res. 2012 Mar ; 22 (3) : 528-38. " The public has been publicly available from the Chinese Academy of Military Medical Sciences. The HAdV-3 strain VR-3 was purchased from ATCC.
  • HAdV-7 strain GZ08 (GenBank accession No. GQ478341) in the literature "Qiu H, Li X, Tian X, Zhou Z, Xing K, Li H, Tang N, Liu W, Bai P, Zhou R. Se: n) Ty pe— spec if ic Virol. 2012 Aug; 86 (15): 7964-75. "Disclosed in public, the public can be obtained from the Academy of Military Medical Sciences of the People's Liberation Army.
  • the hexon protein tower region of HAdV is composed of four loops, namely, Loo Loop2, Loop3, and Loop4 regions.
  • L1 and L2 represent Loop1 and Loop2, respectively.
  • Experimental Example 1 Preparation of influenza virus as a chimeric HAdV-Hexon-Ll/L2 dominant epitope HAdV chimeric vaccine rFLU-HAdV/NS1 preparation
  • HAV-Hexon-Ll/L2 The dominant epitope sequence of Hexon-Ll/L2 (HAdV-Hexon-Ll/L2) in HAdV of type 3 and type 7 is shown in SEQ ID No. 1. It is verified by animal experiments and clinical patient serum, which is in line with the next step. Experimental requirements.
  • the NS1 gene fragment of cold-adapted and attenuated influenza virus strain A/AA/6/60 was used as a target for inserting the HAdV-Hexon-Ll/L2 dominant epitope gene, and the recombinant plasmid pHex 0n -Ll was constructed by molecular biological method. /L2-NSl, the specific strategy is shown in Figure 1A.
  • the HAdV-Hexon-L1/L2 dominant epitope gene was inserted into the coding gene of the first 125 amino acids of the NS1 gene open reading frame (0RF) of the cold-adapted, attenuated influenza virus strain A/AA/6/60.
  • l inker 5 ' -UAAUG-3 '
  • l inker has both a terminator and a promoter
  • the gene encoding the NEP protein is named pHexon-Ll/L2. -NSl.
  • the DNA molecule shown in SEQ ID No. 2 is synthesized, and the SEQ ID No. 2 is the cleavage site from the 1st to the 14th position from the 5' end, and the NS1 gene 3 is from the 15th to the 41st position.
  • positions 41 to 415 are the 1st to 375th nucleotides from the 5' end of the cold-adapted, attenuated influenza virus strain A/AA/6/60 NS gene open reading frame
  • 416 to 420 are l inker
  • positions 421 to 699 are HAdV- Hexon-Ll/L2 dominant epitope genes
  • positions 700 to 1162 are from the 5' end of the NS gene open reading frame.
  • From position 376 to position 838, from 1163 to 1191, the NS gene is 5' NCR, and positions 1192 to 1203 are cleavage sites.
  • Recombinant plasmid pD-PB2 constructed by recombinant plasmid pHexon-Ll/L2-NS1 and cold-adapted and attenuated influenza virus strain A/AA/6/60 internal viral gene backbone PB2, PB1, PA, NP and M, respectively , pD-PB pD-PA, pD-NP, pD-M, and the recombinant plasmid pD-HA, pD-NA constructed by the HA and NA genes of influenza virus strain A/California/07/2009 (H1N1) 8 plasmids each 0.
  • the test result of HA hemagglutination titer was 1: 256 -512, HAdV chimeric vaccine (rFLU-HAdV/NSl) which obtained a chimeric HAdV-Hexon-L1/L2 dominant epitope.
  • the chimeric vaccine strain obtained in the third step was identified, and the virus morphology was observed by electron microscopy. The results are shown in Fig. 2.
  • Figure 2 shows that the vaccine strain conforms to the typical morphological characteristics of influenza virus, and the virus particle size is between 80-120 nm.
  • rFLU-HAdV/NS1 Inoculate rFLU-HAdV/NS1 into 9-11 day old SPF chicken embryos (purchased from Beijing Experimental Animal Research Center), take the second generation chicken embryo allantoic fluid to extract viral RNA, and amplify PB2 by RT-PCR. A total of 8 gene fragments, PB1, PA, NP, HA, NA, M and NS, were sent to the company for sequencing, and the results were consistent with the expected gene sequence.
  • RFLU-HAdV/NSl is cultured in large quantities in chicken embryos, concentrated by ultrafiltration, purified by sucrose gradient centrifugation, and run.
  • rFLU-HAdV/NS1 was inoculated into MDCK cells or 9-11 day old chicken embryos, and cultured at 25, 33, 37 and 39 °C for 3 days, and the cell supernatant or chicken embryo allantoic fluid was collected to determine the virus titer. The results showed that the vaccine strain had a ts, ca phenotype. At the same time, the vaccine strain exhibited an attenuated phenotype in BALB/c mice and 10-12 week old ferrets.
  • mice Eight, rFLU-HAdV/NSl immune effect in mice
  • the rFLU-HAdV/NS1 was purified by ultrafiltration and purified by sucrose density gradient centrifugation to prepare a live attenuated vaccine dosage form of the vaccine.
  • mice 6-8 weeks old BALB/c mice were selected and divided into vaccine group and control group, with 20 rats in each group.
  • Vaccine group The vaccine was intranasally immunized with BALB/c mice, immunized twice, at intervals of 2 weeks, and the immunization dose was set to 10 4 TCID 5 . , 10 5 TCID 5 . Two dose groups with a volume of 20 ⁇ 1.
  • Control group PBS was used instead of vaccine, and the immunization mode, immune volume and immunization time were consistent with the vaccine group.
  • the vaccine group and the control group were collected from the tail vein of the mice 2 weeks after the initial immunization and the booster immunization, and the serum of each group of mice was collected by centrifugation. Determination of wild-type influenza virus A/California/07/2009 in serum using the HI method (HIND antibody titer, while using a micro-neutralization method to detect the specific antibody production in the serum against the HAdV-3 virus strain VR-3, the results are shown in Figure 3, in Figure 3, * represents the comparison with the control group , p ⁇ 0. 05; ** represents p ⁇ 0.01 compared with the control group.
  • Figure 3A shows the results of antibody titers against wild type influenza A/California/07/2009 (H1N1) in serum.
  • Fig. 3B shows the results of measurement of specific antibody production against the HAdV-3 strain VR-3 in serum.
  • Figure 3 shows that the HI neutralization titer of the experimental group against wild-type influenza virus A/California/07/2009 (H1N1) and the neutralizing antibody titer against the HAdV-3 strain VR-3 were compared with the PBS group.
  • Significantly elevated indicating that the influenza virus-mediated HAdV chimeric vaccine (rFLU-HAdV/NS1) immunized animals, high levels of specific antibody titers against HAdV and influenza virus were detected in the serum, indicating that the vaccine immunized animals
  • the body can then be induced to produce a dual immune response against HAdV and influenza virus.
  • Figure 4 shows that after intranasal immunization with rFLU-HAdV/NS1, the mouse body can produce IFN- ⁇ and IL-4 cellular immune responses against influenza virus PR8 and HAdV_3 virus strain VR-3, and the difference from PBS group is extremely significant (p ⁇ 0. 01 ), indicating that the recombinant virus can induce a better cellular immune response in the body.
  • step (3) intranasally immunize BALB/c mice with rFLU-HAdV/NS1, and set a control. Two weeks after booster immunization, the lung and nasal lavage fluids of mice were detected by ELISA.
  • the slgA antibody titer of HAdV-3 strain VR-3 was shown in Fig. 5A and Fig. 5B, respectively, in Fig. 5, * represents p ⁇ 0.05 compared with the control group; ** represents the control group Ratio, p ⁇ 0.01.
  • Figure 5 shows that the rgLU-HAdV/NS1 intranasal immunization of mice showed a significant increase in slgA antibody in the nasal and lung lavage fluid, and the difference from the PBS group was extremely significant (p ⁇ 0.01), indicating that the recombinant virus can induce the body to produce Good mucosal immune response.
  • BALB/c mice were immunized intranasally with rFLU-HAdV/NS1 using the method of step eight, and a control was set up. Two weeks after booster immunization, the clinically isolated strain HAdV-3 (VR-3 HAdV-7 (GZ08) was used. BALB/c mice were challenged with an infection dose of 2 X 10 9 VPs. The lung tissues of each group of mice and the lung tissues of normal mice were subjected to pathological examination. The results are shown in Fig. 6A.
  • Figure 6A shows that the lung pathological changes of the mice in the PBS group are obvious, pulmonary edema can be seen under the microscope, and a large number of inflammatory cells and lymphocytes are seen. Ba cells infiltrated, alveolar interstitial thickening, and normal mouse lung tissue morphology intact.
  • the lung pathological lesions of the rFLU-HAdV/NS1 group were significantly improved compared with the PBS group, of which 10 5 TCID 5 .
  • the high dose group was superior to 10 4 TCID 5 .
  • Low dose group was superior to 10 4 TCID 5 .
  • the intrapulmonary virus copy number of each group of mice was detected by qPCR method, and the results are shown in Fig. 6B and Fig. 6C respectively.
  • * represents p ⁇ 0.05 compared with the control group.
  • Figures 6B and 6C show that the replication numbers of HAdV-3 (VR-3) and HAdV-7 (GZ08) in the lungs of the rFLU-HAdV/NS1 mice were significantly lower than those in the PBS group.
  • mice immunized with the recombinant vaccine rFLU-HAdV/NS1 obtained immunoprotective antibodies against HAdV.
  • Experimental Example 2 Incorporation of influenza virus as a vector HAdV-Hexon-Ll/L2 dominant epitope of HAdV chimeric vaccine rFLU-HAdV/M preparation
  • HAdV-Hexon-Ll/L2 dominant epitope sequence in HAdV of type 3 and type 7 is verified by the method of step 1 in Example 1 as shown in SEQ ID No. 1, which meets the requirements of the next experiment.
  • the M gene fragment of cold-adapted and attenuated influenza virus strain A/AA/6/60 was used as a target for insertion of the HAdV-Hexon-Ll/L2 dominant epitope gene, and the recombinant plasmid pHex 0n -Ll was constructed by molecular biological method. /L2-M, the specific strategy is shown in Figure 1B.
  • the HAdV-Hexon-L1/L2 dominant epitope gene was inserted into the 222 nucleotides of the open reading frame (0RF) of the cold-adapted, attenuated influenza virus strain A/AA/6/60 and Between the 222 nucleotides, the constructed recombinant plasmid was named pHexon-L1/L2-M.
  • the DNA molecule shown in SEQ ID No. 3 is synthesized, and the first to the fifteenth position from the 5' end in the SEQ ID No. 3 is the restriction enzyme site, and the 16th to the 40th position is the M gene 3 'NCR, positions 40 to 261 are nucleotides from position 1 to position 222 from the 5' end of the cold-adapted, attenuated influenza virus strain A/AA/6/60 M gene open reading frame.
  • 262th to 537th are HAdV-Hexon-Ll/L2 dominant epitope genes, and 538th to 759th are cold-adapted, attenuated influenza virus strain A/AA/6/60 M gene open reading frame From the 5' end, from the 761th to the 982th nucleotide, the 760th to the 782th are the M gene 5' NCR, and the 783th to the 794th are the cleavage sites.
  • Recombinant plasmid pD-HA, pD- constructed by HA, NA gene of PB2, pD-PBK pD-PA, pD-NP, pD-NS, and influenza virus A/Cal if ornia/07/2009 (H1N1) NA, 8 plasmids were co-transfected into MDCK cells according to the method of step 3 in Example 1, 37 ° C, 5% CO 2 , cultured for 48-60 h, cell suspension was obtained, and the cell suspension was inoculated into 9-day-old SPF chicken.
  • HAdV chimeric vaccine strain (rFLU-HAdV/M) of chimeric HAdV-Hexon-Ll/L2 dominant epitope.
  • the vaccine strain obtained in the third step was identified, and the virus morphology was observed by electron microscopy. The results showed that the vaccine strain conformed to the typical morphological characteristics of the influenza virus.
  • rFLU-HAdV/M Inoculate rFLU-HAdV/M with 9-11 days old SPF chicken embryo (purchased from Beijing Experimental Animal Research Center), take the second generation chicken embryo allantoic fluid to extract viral RNA, and amplify PB2 by RT-PCR. Eight gene fragments, PB1, PA, NP, HA, NA, M and NS, were sent to the company for sequencing, and the results were consistent with the expected gene sequence.
  • rFLU-HAdV/M Sixth, rFLU-HAdV/M was cultured in large quantities in chicken embryos, concentrated by ultrafiltration, purified by sucrose gradient centrifugation, and run on SDS-PAGE. The main components of the antigen were present.
  • rFLU-HAdV/M was inoculated with MDCK cells or 9-11 day old chicken embryos, and cultured at 25, 33, 37 and 39 °C for 3 days, and the cell supernatant or chicken embryo allantoic fluid was collected to determine the virus titer. The results showed that the vaccine strain had a ts, ca phenotype. At the same time, the vaccine strain exhibited an attenuated phenotype in BALB/c mice and ferrets.
  • mice Eight, rFLU-HAdV/M immune effect in mice
  • the rFLU-HAdV/M was purified by ultrafiltration and purified by sucrose density gradient centrifugation to prepare a live attenuated vaccine formulation of the vaccine.
  • mice 6-8 weeks old BALB/c mice were selected and divided into vaccine group and control group, with 20 rats in each group.
  • mice were immunized with rFLU-HAdV/M according to the method of Step 8 of Example 1, and a control group was set.
  • the vaccine group and the control group were collected from the tail vein of the mice two weeks after the initial immunization and the booster immunization, and the serum of each group of mice was collected by centrifugation.
  • the antibody titer against wild-type influenza virus A/California/07/2009 was determined by HI method, and the specific antibody production against HR-3 of HAdV-3 strain VR-3 was detected by micro-neutralization method. The results are shown in Table 2.
  • the second dose ⁇ 10 ⁇ 10 Table 2 shows that after immunizing animals with the influenza virus-based HAdV chimeric vaccine (rFLU-HAdV/M), antibody titers against HAdV and influenza virus can be detected in the serum, indicating that the vaccine Immunization of animals can induce the body to produce a dual immune response against HAdV and influenza virus.
  • Example 3 Preparation of influenza virus as a vector HAdV chimeric vaccine of HAdV-Hexon-Ll/L2 dominant epitopes Preparation of rFLU-HAdV/NA
  • HAdV-Hexon-Ll/L2 dominant epitope sequence in HAdV of type 3 and type 7 is verified by the method of step 1 of Example 1 as shown in SEQ ID No. 1, which meets the requirements of the next experiment.
  • NA gene fragment of A/Cal if ornia/07/2009 was used as a target for insertion of the HAdV- Hexon - L1/L2 dominant epitope gene, and the recombinant plasmid pHex 0n -Ll/L2- was constructed by molecular biological method. NA, the specific strategy is shown in Figure 1C.
  • the HAdV-Hexon-L1/L2 dominant epitope gene was inserted into the first 183 nucleotides of the NA gene open reading frame (0RF) of the current influenza virus strain H1N1 subtype A/Cal if ornia/07/2009.
  • the constructed recombinant plasmid was named pHexon-Ll/L2-NA between and after nucleotides 157.
  • the DNA molecule shown in SEQ ID No. 4 is synthesized, and the first to the 15th position from the 5' end in the SEQ ID No. 4 is the restriction enzyme site, and the 16th to the 35th position is the NA gene 3 'NCR, the 35th to the 217th are the first to the 183th nucleosides from the 5' end of the NA gene open reading frame of the influenza virus strain A/Cal if ornia/07/2009 (H1N1) acid, at position 218 through 493 of HAdV-Hexon-Ll / L2 epitopes of the gene, the 494 through 650 of the NA gene open reading frame from the 5 'end of 1254 through 1410 Nucleotides, from position 651 to position 681, are 5 ' NCR of the NA gene, and positions 682 to 693 are cleavage sites.
  • the DNA molecule shown by SEQ ID No. 4 is digested with d al to obtain a gene fragment; BsnBl is digested with pAD3000 to obtain a large fragment of the vector; and the gene fragment is ligated with a large fragment of the vector to obtain a recombinant plasmid, which is named as P Hexon-Ll/L2-NA, the recombinant plasmid was sent to the sequencing result correctly.
  • Recombinant plasmid pD-HA constructed by pD-PB2, pD-PB pD-PA, pD-NP, pD-M and pD-NS, and HA gene of influenza virus current strain A/Calif ornia/07/2009 (HlNl) 8 plasmids were co-transfected into MDCK cells according to the method of step 3 in Example 1, 37 ° C, 5% CO 2 , cultured for 48-60 h, cell suspension was obtained, and the cell suspension was inoculated into 9-day-old SPF chicken embryos.
  • the chicken embryo allantoic fluid was harvested at 33 °C for 72 h to obtain a HAdV chimeric vaccine strain (rFLU-HAdV/NA) with a chimeric HAdV-Hexon-Ll/L2 dominant epitope.
  • the vaccine strain obtained in the third step was identified, and the virus morphology was observed by electron microscopy. The results showed that the vaccine strain conformed to the typical morphological characteristics of the influenza virus.
  • rFLU-HAdV/NA Inoculate rFLU-HAdV/NA with 9-11 day old SPF chicken embryos (purchased from Beijing Experimental Animal Research Center), pass the second generation chicken embryo allantoic fluid to extract viral RNA, and amplify PB2 by RT-PCR.
  • the rFLU-HAdV/NA was cultured in large amounts in chicken embryos, concentrated by ultrafiltration, purified by sucrose gradient centrifugation, and run on SDS-PAGE. The main components of the antigen were present.
  • the rFLU-HAdV/NA was inoculated with MDCK cells or 9-11 day old chicken embryos, and cultured at 25, 33, 37 and 39 °C for 3 days, respectively, and the cell supernatant or chicken embryo allantoic fluid was collected to determine the virus titer.
  • the results showed that the vaccine strain had a ts, ca phenotype. At the same time, the vaccine strain exhibited an attenuated phenotype in BALB/c mice and ferrets.
  • the rFLU-HAdV/NA was purified by ultrafiltration and purified by sucrose density gradient centrifugation to prepare a live attenuated vaccine formulation of the vaccine.
  • mice 6-8 weeks old BALB/c mice were selected and divided into vaccine group and control group, with 20 rats in each group.
  • mice were immunized with rFLU-HAdV/NA according to the method of Step 8 in Example 1, and a control group was set.
  • the vaccine group and the control group were collected from the tail vein of the mice two weeks after the initial immunization and the booster immunization, and the serum of each group of mice was collected by centrifugation.
  • the antibody titer against wild-type influenza virus A/California/07/2009 (HlNl) was determined by HI method, and the specific antibody production against HR-3 of HAdV-3 strain VR-3 was detected by micro-neutralization method. The results are shown in Table 3.
  • the second dose ⁇ 10 ⁇ 10 Table 3 shows that the antibody titer against HAdV and influenza virus can be detected in the serum after immunization of the animal with the HAdV chimeric vaccine (rFLU-HAdV/NA) with the influenza virus as the vector, indicating that the vaccine is immunized. Animals can then induce the body to produce a dual immune response against HAdV and influenza virus.
  • the HAdV chimeric vaccine of the invention can cover the pathogen of HAdV infectious diseases, protect more people from influenza virus and HAdV infection, and lays a foundation for realizing "one seedling dual purpose” or "one seedling multipurpose”.

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Abstract

本发明公开了一种流感病毒为载体的HAdV嵌合疫苗的制备及其应用。本发明还公开了SEQ ID NO.1所示的DNA分子。

Description

流感病毒为载体的 HAdV嵌合疫苗的制备及其应用 技术领域
本发明涉及一种流感病毒为载体的 HAdV嵌合疫苗的制备及其应用。
背景技术
呼吸道传染病至今仍然是世界上导致死亡的主要原因之一， 而腺病毒 (Adenovirus , AdV)和流感病毒则是重要的呼吸道病原体。众所周知， 腺病毒在自然界 分布十分广泛， 是儿童呼吸道疾病的常见病原之一。 在 3岁以下小儿急性上呼吸道感 染中约 5%-10%是由腺病毒引起的。 从我国北方和南方各地住院病儿的临床调查结果， 证实 3、 7型腺病毒是腺病毒肺炎的主要病原， 由它们所导致的下呼吸道感染占所有腺 病毒感染的 50%。 而腺病毒因其型别多、 致病范围广， 给人类健康和生命安全造成了 严重威胁， 越来越受到社会关注。 与此同时， 人们不会忘记历史上的三次流感暴发流 行， 给全世界带来的灾难性后果， 特别是近年来出现的 H5、 H7亚型禽流感病毒跨越种 属屏障感染人的事件， 给人类再次敲响了警钟。 因此人腺病毒和流感的防控已成为当 今生命科学的重大问题。
目前， 疫苗接种是预防和控制人类传染病的最有效措施。 在过去的 50 多年中， 流感疫苗的发展经历了灭活疫苗、 裂解疫苗、 亚单位疫苗、 DNA 疫苗， 已经为人类健 康作出了重要贡献。 近年来美国和俄罗斯研制的流感减毒活疫苗， 是一种降低了毒力 并能在最佳适应温度下生长的减毒人流感病毒株， 临床实验证实是安全、 有效的， 可 以有效预防流感，该疫苗的广泛应用为人类在 21世纪征服流感提供了美好前景。然而， 对于人类呼吸道腺病毒而言， 至今还没有被批准使用的疫苗， 但对于疫苗的研究一直 是国际社会关注的重点。 尽管腺病毒疫苗发展迅速， 研制的腺病毒灭活疫苗、 亚单位 疫苗和减毒活疫苗， 为人类呼吸道腺病毒的免疫预防提供了候选疫苗， 但尚未大规模 应用。从研究看因为腺病毒有 51个血清型之多， 各个型别之间的差异较大， 疫苗免疫 后诱发机体产生潜在免疫过激， 并且注射免疫不能产生黏膜免疫以及传统减毒活疫苗 还存在不稳定和潜在免疫过激等问题， 给疫苗的发展提出了挑战。
近年来， 一是抗原免疫生物信息学技术迅速发展为针对腺病毒六邻体主要保护性 抗原表位设计产生了重大影响， 为实现提高 B细胞产生中和抗体滴度、减低免疫过激、 提升全面保护的腺病毒疫苗研制提供了理论基础和技术支持； 二是反向遗传技术快速 发展， 为研制安全、 有效、 多价的减毒活疫苗提供了先进技术， 使得流感病毒作为递 送系统显现了良好的发展前景， 同时为嵌合病毒达到 "一苗两用"或 "一苗多用" 的 目的提供了理论依据； 三是喷鼻免疫途径为嵌合病毒针对人腺病毒设计出具有产生黏 膜和系统免疫应答、 实现免疫交叉保护， 成为人腺病毒疫苗的重要发展方向。 因此， 以上技术集承、 整合为研制安全、 有效、 实用的人腺病毒疫苗提供了科学保证。
一、 抗原免疫生物信息技术的迅速发展， 为腺病毒疫苗的研发提供了新策略 早在 50年代中期， 人们试图用手术切除的扁桃体、 增殖体建立细胞培养系时， 发现有一种传染性因子可使培养的上皮细胞出现退变， 1956年这种因子被正式命名为 腺病毒 (Adenovirus)。 它能引起人类呼吸道、 胃肠道、 泌尿系及眼的疾病。 人类腺病 毒（human adenovirus, HAdV)属于哺乳动物 AdV属， 为无包膜的双链 DNA病毒， 呈二 十面体结构， 直径为 70〜90nm。 根据物理、 化学、 生物学性质分为 A〜G 7组， 每一 组包括若干血清型， 共 51型， 其中 1-8， 19， 21， 35血清型都与严重急性呼吸道疾病 相关， 而 3、 7型是致病性最高的两种。 国外也有不少文献报道： 3、 7型腺病毒引起 急性的呼吸道疾病在美国军队新兵训练营中流行较为严重。 从病毒结构看， HAdV衣壳 蛋白由 252个壳粒组成，其中 240个壳粒组成蛋白衣壳的 20个面，其组成蛋白被称为 六邻体 (Hexon, 120KD)， 是主要的包膜蛋白； 12个壳粒位于 20面体的顶角， 被称为 五邻体 (penton, 82KD)； 还有三聚体蛋白的纤维（fiber, 62KD)。 其中， 六邻体是腺病 毒的主要抗原蛋白， 刺激机体产生抗体， 抑制病毒构象的改变， 从而中和病毒体。 六 邻体含有大量的抗原决定簇， 包括型特异性和亚属特异性抗原表位以及中和性抗原表 位。 基底（pedestal)包含两个区域 P1和 P2区， 塔区由 4个环（loop)构成， 即 Loopl、 Loop2、 Loop3、 Loop4 区。 近年来， 由于腺病毒六邻体蛋白的结构特异性， 编码中和 抗原表位， 参与免疫反应， 成为国内外学者研究的焦点。 由此可见， HAdV由于其特殊、 复杂的病原学特性， 给 HAdV的防控提出了难题。
疫苗是控制传染病最有效的手段。 在人类战胜疾病和传染病的斗争中， 疫苗发挥 了重要作用， 腺病毒疫苗也不例外。 美国早在六十年代已采用包有肠溶衣的腺病毒减 毒活疫苗对新兵训练营的成年人进行免疫实验， 发现可以减少百分之九十的腺病毒引 起的发热症状，但是通过血清学检测发现这种活疫苗诱导产生的中和抗体滴度十分低。 七十年代使用腺病毒肠溶活疫苗进行免疫， 发现血清中 IgM、 IgG、 IgA升高， 但是在 鼻分泌物中没有发现相应的 IgA型抗体。 腺病毒减毒活疫苗虽然在美军中使用效果良 好， 但是美军中仍存在着 ARD爆发的现象， 有研究采用 DNA芯片以及 PCR技术证实， 接种现有腺病毒减毒活疫苗后， 接种者仍能共感染 （Co-infection) 其它型别的腺病 毒， 证明现有减毒活疫苗的对其它型别的腺病毒没有交叉免疫保护能力。 因此， HAdV 亚单位疫苗责无旁贷成为本领域的重要发展目标。 在过去数年中， 腺病毒作为一种基因载体被广泛应用于基因治疗和嵌合疫苗研究 中， 而腺病毒感染大大阻碍了这种基因治疗的疗效， 同时人们不会忽视这种嵌合疫苗 带来的潜在副反应。近年来，抗原免疫生物信息学的发展对 HAdV六邻体主要保护性抗 原表位设计产生了重大影响， 为实现保护性抗原的 T、 B细胞优势表位疫苗研发提供了 理论技术支持。 有文献报道应用噬菌体展示短肽技术探索腺病毒表位， 用 3型腺病毒 中和抗体筛选噬菌体随机十五肽库， 经三轮筛选后， 随机挑取了 22个噬菌体克隆， 分 别加入 3型腺病毒感染的 Hela细胞。结果发现 8 I 22个克隆菌体短肽对 3型腺病毒感 染 Hela细胞具有保护作用。另有学者通过计算机比较腺病毒六邻体氨基酸序列， 结合 2 型腺病毒三维结构图提示的多肽暴露情况以及抗原性预测分析， 选择六邻体蛋白基 因的 937〜1230， 2166〜2607及 937〜2607编码序列。 此区域涵括的预测的抗原位点 在呼吸道腺病毒型间具有很高的同源性， 在腺病毒颗粒上可能具有较好的暴露性并可 能含有型间或组特异性抗原表位。 国外也有人应用 Ad2的 Loopl区的抗原决定簇在 B 组柯萨奇病毒中的表达， 以柯萨奇病毒作为载体， 重组 Π型腺病毒六邻体蛋白的 L1 环可以使其在 HeLa细胞内稳定地表达，所表达的蛋白可以诱导产生既抗腺病毒又抗柯 萨奇病毒的中和抗体， 进而研制多价的基因工程疫苗。
二、 流感病毒反向遗传技术的发展， 为制备流感病毒为载体的 HAdV嵌合疫苗病 毒株提供了可靠保证
目前， 反向遗传学技术在流感病毒领域的广泛应用和迅速发展， 流感病毒的 8质 粒病毒拯救系统在国内外已经成为成熟的技术， 使得以流感病毒作为递送系统表达外 源基因成为很有吸引力的研究方向， 重组呼吸道传染病疫苗的研究热点集中在流感病 毒身上。流感病毒作为开发其它传染病病原体疫苗的载体有许多优势： （1)能够刺激机 体产生强烈的黏膜和系统免疫应答； （2)流感疫苗由于抗原变异需要每年生产； （3) HA 和 NA表面蛋白的结构和功能决定了能够对它们进行基因操作而不影响它们的功能； (4) 流感病毒已经形成了高效的反向遗传学操作系统；（5)小鼠和雪貂为重组流感病毒疫苗 候选株呼吸道黏膜免疫应答和免疫保护的研究提供了很好的动物模型。 与其他载体如 腺病毒， 逆转录病毒相比， 流感病毒在复制周期不会形成 DNA中间产物， 此外由于它 不会整合进宿主的染色体， 使得其具有更高的安全性。 用于流感病毒基因组操作的策 略有： 外源蛋白嵌合入表面糖蛋白 HA和 NA， 产生另外的基因片段， 改造非结构蛋白 NS1。
鉴于流感病毒反向遗传技术取得的突破性进展， 已有大量文献报道将流感病毒作 为递送系统成功制备了能够达到有效免疫保护的、 多价的嵌合疫苗候选株， 为疫苗的 研究拓宽了发展前景。 2004年 Horimoto等构建了嵌合体病毒 (A/B)， 该病毒含有嵌合 体 (A/B)的 HA和 B型病毒全长 NA片段。该研究为从单一宿主菌株开发减毒活疫苗提供 了一种新的思路。 2005年 Maeda Y等通过反向遗传学技术产生一种重组的 A型流感病 毒， 该病毒的编码区是副流感病毒的 HA/NA外功能区， 能够在鸡胚中有效繁殖但在小 鼠体内却是减毒的。 当用重组病毒鼻内给药小鼠后， 全部都能产生抵抗流感病毒和副 流感病毒的抗体。这种双价的活疫苗的免疫保护效果明显优于联合疫苗。同年 Zhu Nan Li 等报道小肽能够插入流感病毒 HA 抗原的 " loop"环区域， 随后的研究显示， B. anthracis的保护性抗原 (PA)大片段多肽能被插入流感病毒 H3亚型 HA的功能区， 嵌 合 HA-PA基因的重组流感病毒经 MDCK细胞和鸡胚传代后，遗传稳定性好，动物实验结 果表明， 重组病毒能够引起 HA和 PA蛋白诱导的抗体反应。 2003年 Kawaka等将 GFP 基因整合入 NA基因的特定位置后，结果发现重组流感病毒粒子组装的效率可高达 91%。 2005年 Andrej Egorov等先后将 GFP和 IL-2插入 NS1的开放阅读框， 结果成功得到 了重组的流感病毒株。
以上所述是目前将流感病毒作为递送系统表达外源基因的研究现状， 而国内外， 将流感病毒基因组作为表达 HAdV病毒抗原表位的载体， 用于研制流感病毒为载体的 HAdV嵌合疫苗在国内外还未见报道。
三、 喷鼻免疫流感病毒为载体的 HAdV嵌合疫苗， 是提升安全、 全方位免疫保护 的基石
2003年 6月，美国 Medimmune公司生产的流感三价减毒活疫苗 Flumist被 FDA批 准和投入使用，适用于 2-17岁健康少年儿童以及 18-49岁健康成人， 临床实验结果显 示该疫苗是安全和有效的。 到 2012年 2月， 美国 FDA批准流感四价减毒活疫苗上市， 该疫苗通过鼻腔接种， 使用方法简便， 应用范围广泛， 使得以冷适应流感减毒活疫苗 为递送系统表达外源基因研制其他疫苗开辟了新的天地， 带来了新的机遇。 流感病毒 作为一种强有力的疫苗载体通过递送不同的抗原进入鼻组织能够诱导产生系统和黏膜 的1\ B细胞免疫应答。 此外， 减毒活疫苗通过鼻内免疫， 可以诱导局部和全身免疫， 因而对上呼吸道和下呼吸道感染有防御作用。 减毒活疫苗诱生的免疫应答类似于对野 生型病毒的应答。对于呼吸道黏膜感染的 HAdV疫苗来说，注射接种不是理想的免疫途 径， 也不方便。 因此， 发展鼻腔途径是疫苗免疫的最佳选择。
鼻道富有淋巴样组织， 是鼻腔接种疫苗的靶部位， 即鼻相关淋巴样组织 (丽）， 它主要位于喉部的淋巴样组织环， 称为 Waldeyer 氏环。 研究表明， 鼻腔黏膜面积约 150cm2, 上皮细胞间隙较大并与毛细血管紧密相连， 血管和淋巴结丰富， 鼻黏膜的单 位血流量约 40ml/min， 比肌肉、 脑、 肝脏的都大， 实际上鼻咽部是良好的免疫器官， 有丰富的抗原提呈细胞， 能对抗原有效的提呈、加工， 产生黏膜免疫和系统免疫应答， 并且局部的黏膜免疫， 还能引起呼吸道、 消化道、 泌尿生殖道等部位黏膜免疫应答的 相通性， 但以呼吸道最强。
喷鼻免疫是目前研究活跃的领域， 近年也有许多企业开发鼻腔接种疫苗， 特别是 呼吸道传染性疾病疫苗， 如流感减毒活疫苗通过鼻腔接种， 不仅可以产生局部黏膜免 疫， 还可以产生系统免疫， 并且产生具有交叉免疫保护。 因此， 开发鼻腔接种的流感 病毒为载体的 HAdV嵌合疫苗替代注射具有很大的应用前景，这是由于鼻腔免疫具有简 单、有效、非常适于广大人群的自我使用， 同时使得机体能够抵抗 HAdV和流感两种病 原体感染， 并且对 HAdV 疫苗免疫应答类型可以转变， 由此达到更加安全的目的， 为 HAdV和流感的免疫预防提供保证。
发明公开
本发明的目的是提供一种流感病毒为载体的 HAdV嵌合疫苗的制备及其应用。 本发明提供 SEQ ID No. 1所示的 DNA分子。
一种重组病毒也属于本发明的保护范围， 该病毒按照如下方法制备：
将分别含有冷适应、 减毒流感病毒的 PB2基因、 冷适应、 减毒流感病毒的 PB1基 因、 冷适应、 减毒流感病毒的 PA基因、 冷适应、 减毒流感病毒的 NP基因、 冷适应、 减毒流感病毒的 M基因和冷适应、减毒流感病毒的 NS基因的表达质粒， 以及分别含有 靶标流感病毒的 HA基因和靶标流感病毒的 NA基因的表达质粒共转染宿主细胞， 培养 得到重组流感病毒；
在如下所述基因中的至少一种基因的开放读码框中的任意位置插入或替换为 HAdV的结构蛋白基因：
( 1 ) 冷适应、 减毒流感病毒的 PB2基因；
( 2 ) 冷适应、 减毒流感病毒的 PB1基因；
( 3 ) 冷适应、 减毒流感病毒的 PA基因；
( 4) 冷适应、 减毒流感病毒的 NP基因；
( 5 ) 冷适应、 减毒流感病毒的 M基因；
( 6 ) 冷适应、 减毒流感病毒的 NS基因；
( 7 ) 靶标流感病毒的 HA基因；
( 8 ) 靶标流感病毒的 NA基因；
所述冷适应、 减毒流感病毒的 PB2基因、 PB1基因、 PA基因、 NP基因、 M基因和 NS基因， 以及靶标流感病毒的 HA基因和 NA基因位于不同的表达质粒上； 所述 HAdV的结构蛋白基因为 HAdV的六邻体蛋白全基因和 /或 HAdV的六邻体蛋白 的优势抗原表位基因。
上述重组病毒中， 所述宿主细胞为 MDCK、 Vero、 293T、 COS细胞或 MDCK/293T、 MDCK/COS共培养的细胞；
所述 HAdV的结构蛋白基因为 HAdV的六邻体蛋白塔区 Loopl和 Loop2的优势抗原 表位基因的融合基因。
上述任一所述的重组病毒中， 所述 HAdV为 3型 HAdV或 7型 HAdV;
所述 3型 HAdV具体为 HAdV-3病毒株 VR-3 ;
所述 7型 HAdV具体为 HAdV- 7病毒株 GZ08 , GenBank access ion No. GQ47834 L 上述任一所述的重组病毒中，所述 HAdV的六邻体蛋白塔区 Loopl和 Loop2的优势 抗原表位基因的融合基因的核苷酸序列如 SEQ ID No. 1所示。
上述任一所述的重组病毒中， 所述 HAdV的结构蛋白基因插在所述冷适应、减毒流 感病毒的 NS基因的开放读码框的自 5 ' 末端起第 375位至第 376位核苷酸之间；
所述 HAdV的结构蛋白基因与所述冷适应、 减毒流感病毒的 NS基因的开放读码框 的自 5 ' 末端起第 375位核苷酸之间连有 5 ' - UAAUG -3 ' 序列；
和 /或，
所述 HAdV的结构蛋白基因是将所述冷适应、减毒流感病毒的 M基因的开放读码框 的自 5 ' 末端起第 223位核苷酸至第 760位核苷酸替换；
禾口 /或，
所述 HAdV 的结构蛋白基因是将所述靶标流感病毒的 NA基因的开放读码框的自 5 ' 末端起第 184位核苷酸至第 1253位核苷酸替换。
上述任一所述的重组病毒中， 所述冷适应、 减毒流感病毒为冷适应、 减毒 A型流 感病毒或冷适应、 减毒 B型流感病毒；
所述冷适应、 减毒 A型流感病毒具体为羅亚型冷适应、 减毒流感病毒， 再具 体为冷适应、 减毒流感病毒株 A/Ann Arbor/6/60 (H2N2)；
所述靶标流感病毒为 A型流感病毒或 B型流感病毒，所述 A型流感病毒具体为 HI 亚型 -H16 亚型中的任意一种， 再具体为 H1N1 亚型流感病毒， 再具体为流感病毒株
A/Cal ifornia/07/2009 ( H1N1 ) ;
所述靶标流感病毒为未经过任何处理的 （如未经过减毒、 未经过冷适应） 的普通 野生型流感病毒。 上述任一所述的重组病毒中， 所述 HAdV 的结构蛋白基因插在所述冷适应、 减毒 流感病毒的 NS基因的开放读码框的自 5 ' 末端起第 375位至第 376位核苷酸之间所得 序列为 SEQ ID No. 2中第 41-1162位核苷酸所示；
所述 HAdV的结构蛋白基因是将所述冷适应、 减毒流感病毒的 M基因的开放读码 框的自 5 ' 末端起第 223位核苷酸至第 760位核苷酸替换所得序列为 SEQ ID No. 3中 第 40-759位核苷酸所示；
所述 HAdV的结构蛋白基因将所述靶标流感病毒的 NA基因的开放读码框的自 5 ' 末端起第 184位核苷酸至第 1253位核苷酸替换所得序列为 SEQ ID No. 4中第 35_650 位核苷酸所示。
构建所述表达质粒时， 上述 PB2基因、 PB1基因、 PA基因、 NP基因、 M基因、 NS 基因、 HA基因或 NA基因， 均在各自的 5 ' 端连接有各自基因的 3 ' NCR序列， 均在各 自的 3 ' 端均连接有各自基因的 5 ' NCR序列；
所述表达质粒的出发质粒为双向转录表达载体 PAD3000 ;
构建所述表达质粒时，均将各基因插在双向转录表达载体 PAD3000的 BsnR 位点。 上述任一所述的病毒制备得到的嵌合疫苗也属于本发明的保护范围。
上述 DNA分子在制备预防和 /或治疗流感病毒和 /或 HAdV引起的疾病的产品中的 应用也属于本发明的保护范围；
和 /或，
上述任一所述的病毒在制备预防和 /或治疗流感病毒和 /或 HAdV 引起的疾病的产 品中的应用也属于本发明的保护范围；
和 /或，
上述嵌合疫苗在制备预防和 /或治疗流感病毒和 /或 HAdV 引起的疾病的产品中的 应用也属于本发明的保护范围；
所述流感病毒为 A型流感病毒或 B型流感病毒， 所述 A型流感病毒具体为 HI亚 型 -H16亚型中的任意一种；
所述 HAdV为 3型 HAdV或 7型 HAdV;
所述 3型 HAdV具体为 HAdV-3病毒株 VR-3 ;
所述 Ί型 HAdV具体为 HAdV- 7病毒株 GZ08， GenBank access ion No. GQ47834L 附图说明
图 1为构建流感病毒为载体的 HAdV嵌合疫苗的重组策略示意图。
图 2 为重组病毒 rFLU/HAdV/NSl的电镜形态学鉴定以及颗粒大小和分布情况。 图 3 为小鼠免疫 rFLU/HAdV/NSl的抗体效价测定结果。
图 4为小鼠免疫 rFLU/HAdV/NSl产生 IFN- γ、 IL-4细胞免疫应答。
图 5为小鼠免疫 rFLU/HAdV/NSl产生针对 HAdV-3的黏膜抗体效价。
图 6 为 rFLU/HAdV/NSl免疫小鼠对腺病毒株攻击的免疫保护效果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明， 均为常规方法。
下述实施例中所用的材料、 试剂等， 如无特殊说明， 均可从商业途径得到。
PAD3000在文献 "Hoffmann E， Mahmood K, Yang CF， Webster RG, Greenberg HB， Kemble G. Rescue of influenza B virus from eight plasmids. Proc Natl Acad Sci . 2002 ; 99 (17)： 11411 - 6. "中公开过， 公众可从中国人民解放军军事医学科学院获 得。
6-8周龄 BALB/c小鼠购自军事医学科学院实验动物中心。
10-12周龄的雪貂购自中国无锡 Angora安哥鲁公司。
2012-2013年流感病毒当年流行株 A/Cal ifornia/07/2009 ( H1N1 ) 由国家流感中 心提供， 公众可从中国人民解放军军事医学科学院获得。 分别扩增该病毒株的 HA、 NA 基因，再分别插入到双向转录 /表达载体 PAD3000的 ^STBBI位点，构建得到该病毒株的 重组质粒 pD-HA、 pD-NA。
冷适应、减毒流感病毒株 A/Ann Arbor/6/60 (H2N2) (简写 A/AA/6/60 )在文献 "Yang P, Duan Y, Wang C, Xing L, Gao X, Tang C, Luo D, Zhao Z, Jia W, Peng D, Liu X, Wang X. Immunogenicity and protect ive efficacy of a l ive attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets. Vaccine. 2011 Jan 17 ; 29 (4) : 698-705. " 中公开过， 公众可从中国人民解放军军事医学科学院获得。 该 病毒株的 6个内部基因？82、？81、？ 、 、^、¾1，分别插入到双向转录/表达载体 403000 的 A?7BBI位点， 构建得到该病毒的重组质粒 pD-PB2、 pD-PB pD-PA、 pD-NP、 pD-NS、 pDHVL
流感病毒 A/PR/8/1934 (PR8)病毒株在文献 " Li C, Yang P， Sun Y， Li T， Wang C, Wang Z, Zou Z, Yan Y, Wang W, Wang C, Chen Z, Xing L, Tang C, Ju X, Guo F, Deng J, Zhao Y, Yang P, Tang J, Wang H, Zhao Z, Yin Z, Cao B, Wang X, Jiang C. IX- 17 respon se mediates acute lun injury induced by the 2009 pandemic influenza. A (H1N L) vi rus. Cel l Res. 2012 Mar ; 22 (3) : 528-38. " 中公开过， 公众 可从中国人民解放军军事医学科学院获得。 HAdV-3病毒株 VR-3,购自 ATCC。
HAdV- 7病毒株 GZ08 (GenBank accession No. GQ478341)在文献 "Qiu H， Li X, Tian X, Zhou Z, Xing K, Li H, Tang N， Liu W, Bai P, Zhou R. Se:n)ty pe— spec i f ic
Figure imgf000010_0001
Virol. 2012 Aug ; 86 (15) : 7964-75. " 中公开过， 公众可从中国人民解放军军事医学科学院获得。
HAdV的六邻体蛋白塔区由 4个环（loop)构成， 即 Loo Loop2、 Loop3、 Loop4 区， 下述实施例中 Ll、 L2分别表示 Loopl和 Loop2。 实验例 1、 流感病毒为载体的嵌合 HAdV-Hexon-Ll/L2优势抗原表位的 HAdV嵌合 疫苗 rFLU-HAdV/NSl的制备
一、 3型和 7型的 HAdV中 Hexon-Ll/L2 (HAdV-Hexon-Ll/L2 ) 优势抗原表位序列 如 SEQ ID No. 1所示， 经动物实验和临床病人血清验证， 符合下一步实验要求。
二、 构建重组质粒 pHexon - L1/L2- NS1
以冷适应、减毒流感病毒株 A/AA/6/60的 NS1基因片段为插入 HAdV-Hexon-Ll/L2 优势抗原表位基因的靶点， 利用分子生物学方法构建重组质粒 pHex0n-Ll/L2-NSl， 具 体策略如图 1A所示。
具体是将 HAdV- Hexon -L1/L2优势抗原表位基因插入到冷适应、 减毒流感病毒株 A/AA/6/60 的 NS1基因开放阅读框 （0RF) 的前 125氨基酸的编码基因处， 中间加入 l inker ( 5 ' -UAAUG-3 ' ) 连接， l inker 既有终止子作用， 又有启动子作用， 最后是 NEP蛋白的编码基因， 将构建好的重组质粒命名为 pHexon-Ll/L2-NSl。
步骤如下：
(一） 合成 SEQ ID No. 2所示的 DNA分子， SEQ ID No. 2中自 5 ' 末端起第 1位至 第 14位为酶切位点， 第 15位至第 41位为 NS1基因 3 ' NCR, 第 41位至第 415位为冷 适应、 减毒流感病毒株 A/AA/6/60 NS基因开放读码框的自 5 ' 末端起第 1位至第 375 位核苷酸，第 416位至第 420位为 l inker,第 421位至第 699位为 HAdV- Hexon-Ll/L2 优势抗原表位基因，第 700位至第 1162位为 NS基因开放读码框的自 5 ' 末端起第 376 位至第 838位核苷酸， 第 1163位至第 1191位为 NS基因 5 ' NCR, 第 1192位至第 1203 位为酶切位点。
(二） 酶切 SEQ ID No. 2所示的 DNA分子，得到基因片段； 酶切 pAD3000， 得到载体大片段； 将基因片段与载体大片段连接， 得到重组质粒， 将其命名为 PHexon-Ll/L2-NSl , 将重组质粒送测序结果正确。
三、将重组质粒 pHexon-Ll/L2-NSl与冷适应、减毒流感病毒株 A/AA/6/60的内部 病毒基因骨架 PB2、 PB1、 PA、 NP、 M分别构建的重组质粒 pD-PB2、 pD-PB pD-PA、 pD-NP、 pD-M， 及流感病毒当年流行株 A/California/07/2009 (H1N1 ) 的 HA、 NA基因 构建的重组质粒 pD-HA、 pD-NA, 共 8种质粒各 0. 2μ§, 等量混合加入 1(^L转染试剂 (Effectene, 购自 Qiagen公司） 室温作用 10min， 共转染 MDCK细胞， 33°C， 5% C02， 培养 48-60h，得细胞悬液， 将细胞悬液接种 9日龄 SPF鸡胚， 33°C培养 72h，收获鸡胚 尿囊液测定 HA血凝效价， HA血凝效价的检测结果是 1： 256-512，获得嵌合 HAdV- Hexon -L1/L2优势抗原表位的 HAdV嵌合疫苗 （rFLU- HAdV/NSl )。
四、对步骤三获得的嵌合疫苗株进行鉴定， 电镜观察病毒形态， 结果如图 2所示。 图 2表明该疫苗株符合流感病毒典型形态特征， 病毒颗粒大小在 80-120nm之间。
五、将 rFLU-HAdV/NSl接种 9-11日龄 SPF鸡胚（购自北京实验动物研究中心）传 代， 取第二代鸡胚尿囊液提取病毒 RNA，经过 RT-PCR, 扩增出 PB2、 PB1、 PA、 NP、 HA、 NA、 M和NS共 8个基因片段，分别将基因片段送公司测序，结果与预期基因序列一致。
六、 将 rFLU-HAdV/NSl 经鸡胚大量培养， 超滤浓缩、 蔗糖梯度离心纯化后， 跑
SDS-PAGE电泳， 凝胶染色、 脱色后， 可检测到相应大小的 NP、 HA1、 HA2、 NEP蛋白， 表明抗原的主要成分没有丢失。
七、 rFLU-HAdV/NSl 的温度每夂感 ( temperature sensitive, ts )、 冷适应 ( cold adapted, ca) 和减毒 (attenuated, att ) 表型检测
将 rFLU-HAdV/NSl接种 MDCK细胞或 9-11 日龄鸡胚， 分别于 25、 33、 37和 39 °C 条件下培养 3天， 收集细胞上清或鸡胚尿囊液测定病毒滴度。 结果表明， 该疫苗株具 有 ts、 ca表型。 同时， 该疫苗株在 BALB/c小鼠和 10-12周龄的雪貂上表现减毒表型。
八、 rFLU-HAdV/NSl在小鼠体内的免疫效果
(一） 将 rFLU-HAdV/NSl经超滤浓缩、 蔗糖密度梯度离心纯化后， 制备该疫苗的 减毒活疫苗剂型。
选用 6-8周龄 BALB/c小鼠， 分为疫苗组和对照组， 每组 20只。
疫苗组： 将该疫苗滴鼻免疫 BALB/c小鼠， 免疫 2次， 间隔 2周， 设置免疫剂量为 104TCID5。， 105TCID5。两个剂量组， 体积为 20μ1。
对照组： PBS代替疫苗， 同时免疫方式、 免疫体积及免疫时间与疫苗组一致。 将疫苗组和对照组分别于初次免疫后、 加强免疫后 2周， 经小鼠尾静脉采血， 离心收集 各组小鼠的血清。 采用 HI方法测定血清中针对野生型流感病毒 A/California/07/2009 (HIND 的抗体效价， 同时采用微量中和方法检测血清中针对 HAdV-3病毒株 VR-3的特 异性抗体产生情况， 结果如图 3所示， 图 3中， *代表与对照组相比， p<0. 05; **代表与 对照组相比， p<0. 01。
图 3A为血清中针对野生型流感病毒 A/California/07/2009 (H1N1 ) 的抗体效价 测定结果。
图 3B为血清中针对 HAdV-3病毒株 VR-3的特异性抗体产生情况测定结果。
图 3表明， 实验组针对野生型流感病毒 A/California/07/2009 (H1N1 ) 的 HI中 和效价及针对 HAdV-3病毒株 VR-3的中和抗体效价分别与 PBS组比效价显著升高， 表 明流感病毒为载体的 HAdV嵌合疫苗 （rFLU-HAdV/NSl ) 免疫动物后， 血清中可检测到 针对 HAdV和流感病毒的高水平的特异性抗体效价，表明该疫苗免疫动物后可诱导机体 产生针对 HAdV和流感病毒的双重免疫应答。
(二） 采用步骤 （一） 相同的方法用 rFLU-HAdV/NSl分别滴鼻免疫 BALB/c小鼠， 并设置对照， 于加强免疫后 2周， 处死动物， 分离脾淋巴细胞， ELISP0T方法检测疫 苗株免疫动物后产生 IFN- γ和 IL-4细胞免疫应答， 结果分别如图 4A和图 4B所示， 图 4中， **代表与对照组相比， p<0. 01。
图 4表明， rFLU-HAdV/NSl滴鼻免疫后小鼠机体可产生针对流感病毒 PR8和 HAdV_3 病毒株 VR-3的 IFN- γ、 IL-4细胞免疫反应， 与 PBS组比差异极显著（p<0. 01 )， 表明 重组病毒能够诱导机体产生较好的细胞免疫反应。
(三）采用步骤（一）相同的方法用 rFLU-HAdV/NSl滴鼻免疫 BALB/c小鼠， 并设 置对照，于加强免疫后 2周， ELISA法检测小鼠肺、鼻灌洗液中针对 HAdV-3病毒株 VR-3 的 slgA抗体效价，结果分别如图 5A和图 5B所示，图 5中， *代表与对照组相比， p<0. 05； **代表与对照组相比， p<0. 01。
图 5表明， rFLU-HAdV/NSl滴鼻免疫后小鼠鼻、 肺灌洗液中 slgA抗体明显升高， 与 PBS组比差异极显著（p<0. 01 )，表明重组病毒能够诱导机体产生较好的黏膜免疫反 应。
九、 动物保护性实验
采用步骤八的方法用 rFLU-HAdV/NSl滴鼻免疫 BALB/c小鼠， 并设置对照， 于加强 免疫后 2周， 分别用临床分离的病毒株 HAdV-3 (VR-3 HAdV-7 (GZ08)攻击 BALB/c 小鼠，感染剂量为 2 X 109VPs，取各组小鼠的肺组织以及正常小鼠的肺组织进行病理学 检测， 结果如图 6A所示。
图 6A表明， PBS组小鼠的肺病理变化明显， 镜下可见肺水肿， 大量炎性细胞、 淋 巴细胞浸润， 肺泡间质增厚， 而正常小鼠肺组织形态完好。 rFLU-HAdV/NSl 组小鼠肺 组织病理病变较 PBS组明显改善， 其中 105TCID5。高剂量组优于 104TCID5。低剂量组。
采用 qPCR方法对各组小鼠的肺内病毒拷贝数进行检测，结果分别如图 6B和 6C所 示， 图 6中， *代表与对照组相比， p<0. 05。
图 6B和 6C表明， rFLU-HAdV/NSl组小鼠肺内病毒 HAdV- 3 (VR- 3)、 HAdV- 7 (GZ08 ) 的复制数均较 PBS组明显降低。
结果表明，重组疫苗 rFLU-HAdV/NSl免疫后小鼠获得了针对 HAdV的免疫保护性抗 体。 实验例 2、 流感病毒为载体的嵌合 HAdV-Hexon-Ll/L2优势抗原表位的 HAdV嵌合 疫苗 rFLU-HAdV/M的制备
一、 3型和 7型的 HAdV中 HAdV-Hexon-Ll/L2优势抗原表位序列如 SEQ ID No. 1 所示， 按照实施例 1中步骤一的方法验证， 符合下一步实验要求。
二、 构建重组质粒 pHexon -L1/L2-M
以冷适应、 减毒流感病毒株 A/AA/6/60 的 M基因片段为插入 HAdV-Hexon-Ll/L2 优势抗原表位基因的靶点， 利用分子生物学方法构建重组质粒 pHex0n-Ll/L2-M， 具体 策略如图 1B所示。
具体是将 HAdV- Hexon -L1/L2优势抗原表位基因插入到冷适应、 减毒流感病毒株 A/AA/6/60的 M基因开放阅读框（0RF) 的前 222位核苷酸和后 222位核苷酸之间， 将 构建好的重组质粒命名为 pHexon- L1/L2- M。
步骤如下：
(一） 合成 SEQ ID No. 3所示的 DNA分子， SEQ ID No. 3中自 5 ' 末端起第 1位至 第 15位为酶切位点， 第 16位至第 40位为 M基因 3 ' NCR, 第 40位至第 261位为冷适 应、 减毒流感病毒株 A/AA/6/60 M基因开放读码框的自 5 ' 末端起从第 1位到第 222 位核苷酸，第 262位至第 537位为 HAdV-Hexon-Ll/L2优势抗原表位基因，第 538位至 第 759位为冷适应、 减毒流感病毒株 A/AA/6/60 M基因开放读码框的自 5 ' 末端起从 第 761位到第 982位核苷酸，第 760位至第 782位为 M基因 5 ' NCR,第 783位至第 794 位为酶切位点。
(二、Bsi 酶切 SEQ ID No. 3所示的 DNA分子，得到基因片段； BsnS 酶切 pAD3000， 得到载体大片段； 将基因片段与载体大片段连接， 得到重组质粒， 将其命名为 pHexon-Ll/L2-M, 将重组质粒送测序结果正确。 三、 将重组质粒 pHexon-Ll/L2-M， 与冷适应、 减毒流感病毒株 A/AA/6/60的内部 病毒基因骨架 PB2、 PB1、 PA、 NP和 NS分别构建的重组质粒 pD-PB2、 pD-PBK pD-PA、 pD- NP、 pD-NS, 及流感病毒当年流行株 A/Cal if ornia/07/2009 (H1N1 ) 的 HA、 NA基 因构建的重组质粒 pD-HA、pD-NA，按照实施例 1中步骤三的方法将 8种质粒共转染 MDCK 细胞， 37°C， 5%C02，培养 48-60h， 得细胞悬液，将细胞悬液接种 9日龄 SPF鸡胚， 35 °C 培养 72h， 收获鸡胚尿囊液获得嵌合 HAdV-Hexon-Ll/L2优势抗原表位的 HAdV嵌合疫 苗株 （rFLU- HAdV/M)。
四、 对步骤三获得的疫苗株进行鉴定， 电镜观察病毒形态， 结果表明该疫苗株符 合流感病毒典型形态特征。
五、将 rFLU-HAdV/M接种 9-11日龄 SPF鸡胚（购自北京实验动物研究中心）传代， 取第二代鸡胚尿囊液提取病毒 RNA，经过 RT-PCR, 扩增出 PB2、 PB1、 PA、 NP、 HA、 NA、 M和 NS共 8个基因片段， 分别将基因片段送公司测序， 结果与预期基因序列一致。
六、将 rFLU-HAdV/M经鸡胚大量培养，超滤浓缩、蔗糖梯度离心纯化后，跑 SDS-PAGE 电泳， 抗原的主要成分存在。
七、 rFLU-HAdV/M 的温度每夂感 ( temperature sensitive, ts )、 冷适应 ( cold adapted, ca) 和减毒 (attenuated, att ) 表型检测
将 rFLU-HAdV/M接种 MDCK细胞或 9-11 日龄鸡胚， 分别于 25、 33、 37和 39°C条 件下培养 3天， 收集细胞上清或鸡胚尿囊液测定病毒滴度。 结果表明， 该疫苗株具有 ts、 ca表型。 同时， 该疫苗株在 BALB/c小鼠和雪貂动物上表现减毒表型。
八、 rFLU- HAdV/M在小鼠体内的免疫效果
将 rFLU-HAdV/M经超滤浓缩、 蔗糖密度梯度离心纯化后， 制备该疫苗的减毒活疫 苗剂型。
选用 6-8周龄 BALB/c小鼠， 分为疫苗组和对照组， 每组 20只。
按照实施例 1的步骤八的方法对小鼠进行 rFLU-HAdV/M的免疫， 同时设置对照组。 将疫苗组和对照组分别于初次免疫后、 加强免疫后 2周， 经小鼠尾静脉采血， 离 心收集各组小鼠的血清。
采用 HI方法测定血清中针对野生型流感病毒 A/California/07/2009 (H1N1 ) 的 抗体效价， 同时采用微量中和方法检测血清中针对 HAdV-3病毒株 VR-3的特异性抗体 产生情况， 结果如表 2所示。
表 2 rFLU-HAdV/M的免疫效果检测 流感病毒 HAdV
组别
(HI效价） (NT效价）
第一剂 160. 00 32. 00
疫苗组
第二剂 640. 00 240. 00
第一剂 <10 <10
对照组
第二剂 <10 <10 表 2表明， 以流感病毒为载体的 HAdV嵌合疫苗 （rFLU-HAdV/M) 免疫动物后， 血 清中可检测到针对 HAdV和流感病毒的抗体效价，表明该疫苗免疫动物后可诱导机体产 生针对 HAdV和流感病毒的双重免疫应答。 实施例 3、 流感病毒为载体的嵌合 HAdV-Hexon-Ll/L2优势抗原表位的 HAdV嵌合 疫苗 rFLU-HAdV/NA的制备
一、 3型和 7型的 HAdV中 HAdV-Hexon-Ll/L2优势抗原表位序列如 SEQ ID No. 1 所示， 按照实施例 1的步骤一的方法验证， 符合下一步实验要求。
二、 构建重组质粒 pHexon- L1/L2- NA
以 A/Cal if ornia/07/2009 (H1N1 ) 的 NA基因片段为插入 HAdV- Hexon - L1/L2优 势抗原表位基因的靶点， 利用分子生物学方法构建重组质粒 pHex0n -Ll/L2-NA， 具体 策略如图 1C所示。
具体是将 HAdV- Hexon -L1/L2优势抗原表位基因插入到当年流行流感病毒株 H1N1 亚型 A/Cal if ornia/07/2009的 NA基因开放阅读框（0RF)的前 183位核苷酸和后 157 位核苷酸之间， 将构建好的重组质粒命名为 pHexon-Ll/L2-NA。
步骤如下：
(一） 合成 SEQ ID No. 4所示的 DNA分子， SEQ ID No. 4中自 5 ' 端起第 1位至第 15位为酶切位点，第 16位至第 35位为 NA基因 3 ' NCR,第 35位至第 217位为流感病 毒当年流行株 A/Cal if ornia/07/2009 (H1N1 )的 NA基因开放读码框的自 5 ' 末端起第 1位至第 183位核苷酸，第 218位至第 493位为 HAdV-Hexon-Ll/L2优势抗原表位基因， 第 494位至第 650位为 NA基因开放读码框的自 5 ' 末端起第 1254位至第 1410位核苷 酸， 第 651位至第 681位为 NA基因 5 ' NCR，第 682位至第 693位为酶切位点。
d al酶切 SEQ ID No. 4所示的 DNA分子，得到基因片段； BsnBl酶切 pAD3000， 得到载体大片段； 将基因片段与载体大片段连接， 得到重组质粒， 将其命名为 PHexon-Ll/L2-NA, 将重组质粒送测序结果正确。
三、 将重组质粒 pHexon-Ll/L2-NA， 与冷适应、 减毒流感病毒株 A/AA/6/60的内 部病毒基因骨架 PB2、 PB1、 PA、 NP、 M和 NS分别构建的重组质粒 pD-PB2、 pD-PB pD-PA、 pD- NP、 pD-M和 pD-NS，及流感病毒当年流行株 A/Calif ornia/07/2009 (HlNl ) 的 HA基因构建的重组质粒 pD-HA，按照实施例 1中步骤三的方法将 8种质粒共转染 MDCK 细胞， 37°C， 5%C02，培养 48-60h， 得细胞悬液，将细胞悬液接种 9日龄 SPF鸡胚， 33 °C 培养 72h，收获鸡胚尿囊液获得嵌合 HAdV-Hexon-Ll/L2优势抗原表位的 HAdV嵌合疫苗 株 （rFLU- HAdV/NA)。
四、 对步骤三获得的疫苗株进行鉴定， 电镜观察病毒形态， 结果表明该疫苗株符 合流感病毒典型形态特征。
五、 将 rFLU-HAdV/NA接种 9-11 日龄 SPF鸡胚 （购自北京实验动物研究中心） 传 代， 取第二代鸡胚尿囊液提取病毒 RNA，经过 RT-PCR, 扩增出 PB2、 PB1、 PA、 NP、 HA、 NA、 M和NS共 8个基因片段，分别将基因片段送公司测序，结果与预期基因序列一致。
六、 将 rFLU-HAdV/NA 经鸡胚大量培养， 超滤浓缩、 蔗糖梯度离心纯化后， 跑 SDS-PAGE电泳， 抗原的主要成分存在。
七、 rFLU-HAdV/NA 的温度每夂感 ( temperature sensitive, ts )、 冷适应 ( cold adapted, ca) 和减毒 (attenuated, att ) 表型检测
将 rFLU-HAdV/NA接种 MDCK细胞或 9-11日龄鸡胚， 分别于 25、 33、 37和 39°C条 件下培养 3天， 收集细胞上清或鸡胚尿囊液测定病毒滴度。 结果表明， 该疫苗株具有 ts、 ca表型。 同时， 该疫苗株在 BALB/c小鼠和雪貂动物上表现减毒表型。
八、 rFLU-HAdV/NA在小鼠体内的免疫效果
将 rFLU-HAdV/NA经超滤浓缩、蔗糖密度梯度离心纯化后， 制备该疫苗的减毒活疫 苗剂型。
选用 6-8周龄 BALB/c小鼠， 分为疫苗组和对照组， 每组 20只。
按照实施例 1中步骤八的方法对小鼠进行 rFLU-HAdV/NA的免疫，同时设置对照组。 将疫苗组和对照组分别于初次免疫后、 加强免疫后 2周， 经小鼠尾静脉采血， 离 心收集各组小鼠的血清。
采用 HI方法测定血清中针对野生型流感病毒 A/California/07/2009 (HlNl ) 的 抗体效价， 同时采用微量中和方法检测血清中针对 HAdV-3病毒株 VR-3的特异性抗体 产生情况， 结果如表 3所示。
表 3 rFLU-HAdV/NA的免疫效果检测 流感病毒 HAdV
组别
(HI效价） (NT效价）
第一剂 120. 00 30. 00
疫苗组
第二剂 1024. 00 320. 00
第一剂 <10 <10
对照组
第二剂 <10 <10 表 3表明， 流感病毒为载体的 HAdV嵌合疫苗（rFLU-HAdV/NA) 免疫动物后， 血清 中可检测到针对 HAdV和流感病毒的抗体效价，表明该疫苗免疫动物后可诱导机体产生 针对 HAdV和流感病毒的双重免疫应答。 工业应用
本发明的 HAdV嵌合疫苗可以覆盖 HAdV传染病病原体， 保护更多人群免受流感病 毒和 HAdV感染， 为实现 "一苗两用"或 "一苗多用"奠定了基础。

Claims

权利要求
1、 SEQ ID No. 1所示的 DNA分子。
2、 一种重组病毒， 该病毒按照如下方法制备：
将分别含有冷适应、 减毒流感病毒的 PB2基因、 冷适应、 减毒流感病毒的 PB1基 因、 冷适应、 减毒流感病毒的 PA基因、 冷适应、 减毒流感病毒的 NP基因、 冷适应、 减毒流感病毒的 M基因和冷适应、减毒流感病毒的 NS基因的表达质粒， 以及分别含有 靶标流感病毒的 HA基因和靶标流感病毒的 NA基因的表达质粒共转染宿主细胞， 培养 得到重组流感病毒；
在如下所述基因中的至少一种基因的开放读码框中的任意位置插入或替换为 HAdV的结构蛋白基因：
( 1 ) 冷适应、 减毒流感病毒的 PB2基因;
( 2 ) 冷适应、 减毒流感病毒的 PB1基因;
( 3 ) 冷适应、 减毒流感病毒的 PA基因；
( 4) 冷适应、 减毒流感病毒的 NP基因；
( 5 ) 冷适应、 减毒流感病毒的 M基因；
( 6 ) 冷适应、 减毒流感病毒的 NS基因；
( 7 ) 靶标流感病毒的 HA基因；
( 8 ) 靶标流感病毒的 NA基因；
所述 HAdV的结构蛋白基因为 HAdV的六邻体蛋白全基因和 /或 HAdV的六邻体蛋白 的优势抗原表位基因。
3、根据权利要求 2所述的病毒，其特征在于：所述宿主细胞为 MDCK、 Vero、 293T、 COS细胞或 MDCK/293T、 MDCK/COS共培养的细胞；
所述 HAdV的结构蛋白基因为 HAdV的六邻体蛋白塔区 Loopl和 Loop2的优势抗原 表位基因的融合基因。
4、 根据权利要求 2或 3所述的病毒， 其特征在于： 所述 HAdV为 3型 HAdV或 7 型 HAdV;
所述 3型 HAdV具体为 HAdV-3病毒株 VR-3;
所述 Ί型 HAdV具体为 HAdV- 7病毒株 GZ08， GenBank accession No. GQ47834L
5、 根据权利要求 2-4任一所述的病毒， 其特征在于： 所述 HAdV的六邻体蛋白塔 区 Loopl和 Loop2的优势抗原表位基因的融合基因的核苷酸序列如 SEQ ID No. 1所示。
6、 根据权利要求 2-5任一所述的病毒， 其特征在于： 所述 HAdV的结构蛋白基因 插在所述冷适应、减毒流感病毒的 NS基因的开放读码框的自 5 ' 末端起第 375位至第 376位核苷酸之间；
所述 HAdV的结构蛋白基因与所述冷适应、 减毒流感病毒的 NS基因的开放读码框 的自 5 ' 末端起第 375位核苷酸之间连有 5 ' - UAAUG -3 ' 序列；
和 /或，
所述 HAdV的结构蛋白基因是将所述冷适应、减毒流感病毒的 M基因的开放读码框 的自 5 ' 末端起第 223位核苷酸至第 760位核苷酸替换；
和 /或，
所述 HAdV 的结构蛋白基因是将所述靶标流感病毒的 NA基因的开放读码框的自 5 ' 末端起第 184位核苷酸至第 1253位核苷酸替换。
7、 根据权利要求 2-6任一所述的病毒， 其特征在于： 所述冷适应、 减毒流感病毒 为冷适应、 减毒 A型流感病毒或冷适应、 减毒 B型流感病毒；
所述冷适应、 减毒 A型流感病毒具体为羅亚型冷适应、 减毒流感病毒， 再具 体为冷适应、 减毒流感病毒株 A/Ann Arbor/6/60 (H2N2)；
所述靶标流感病毒为 A型流感病毒或 B型流感病毒，所述 A型流感病毒具体为 HI 亚型 -H16 亚型中的任意一种， 再具体为 H1N1 亚型流感病毒， 再具体为流感病毒株 A/Cal ifornia/07/2009 (H1N1 )。
8、 根据权利要求 2-7任一所述的病毒， 其特征在于： 所述 HAdV的结构蛋白基因 插在所述冷适应、减毒流感病毒的 NS基因的开放读码框的自 5 ' 末端起第 375位至第 376位核苷酸之间所得序列为 SEQ ID No. 2中第 41-1162位核苷酸所示；
所述 HAdV的结构蛋白基因是将所述冷适应、 减毒流感病毒的 M基因的开放读码 框的自 5 ' 末端起第 223位核苷酸至第 760位核苷酸替换所得序列为 SEQ ID No. 3中 第 40-759位核苷酸所示；
所述 HAdV的结构蛋白基因将所述靶标流感病毒的 NA基因的开放读码框的自 5 ' 末端起第 184位核苷酸至第 1253位核苷酸替换所得序列为 SEQ ID No. 4中第 35_650 位核苷酸所示。
9、 由权利要求 2-8任一所述的病毒制备得到的嵌合疫苗。
10、 权利要求 1所述的 DNA分子在制备预防和 /或治疗流感病毒和 /或 HAdV引起 的疾病的产品中的应用；
和 /或，
权利要求 2-8任一所述的病毒在制备预防和 /或治疗流感病毒和 /或 HAdV引起的 疾病的产品中的应用；
和 /或，
权利要求 9所述的嵌合疫苗在制备预防和 /或治疗流感病毒和 /或 HAdV引起的疾 病的产品中的应用；
所述流感病毒为 A型流感病毒或 B型流感病毒， 所述 A型流感病毒具体为 HI亚 型 -H16亚型中的任意一种；
所述 HAdV为 3型 HAdV或 7型 HAdV。
PCT/CN2014/000581 2014-01-28 2014-06-13 流感病毒为载体的HAdV嵌合疫苗的制备及其应用 WO2015113188A1 (zh)

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