CN111363728A

CN111363728A - Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method and application thereof

Info

Publication number: CN111363728A
Application number: CN202010065431.1A
Authority: CN
Inventors: 朱应; 黄玙; 佘应龙; 刘实
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2020-07-03

Abstract

The invention discloses a recombinant influenza A virus carrying a hepatitis B virus gene, a host cell, a preparation method and application thereof. The recombinant influenza A virus of the invention takes the influenza A virus as a vector, integrates the hepatitis B virus gene to the influenza virus genome by utilizing the reverse genetics technology, can ensure that the recombinant influenza virus can be stably passaged in host cells or chick embryos, and can be used for developing hepatitis B vaccines and related medicaments and producing hepatitis B related proteins by utilizing the chick embryos or the cells as a bioreactor.

Description

Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a recombinant influenza A virus carrying a hepatitis B virus gene, a host cell, and a preparation method and application thereof.

Background

A segmented single-stranded negative-strand RNA virus of the genus Orthomyxoviridae, of the genus influenza, HAs a viral genome of about 13.6kb, divided into 8 separate segments of varying sizes, encoding structural proteins (PB1, PB2, PA, HA, NP, NA, M1, M2) and non-structural proteins (NS1, NS2) of the influenza virus. Wherein PB1, PB2 and PA constitute the viral polymerase and the nucleoprotein NP acts as a scaffold for binding to vRNA. Influenza viruses encode two surface glycoproteins: hemagglutinin HA and neuraminidase NA. Sialic acid variants of HA that can adsorb to the surface of host cells, facilitating viral infection of cells; NA facilitates release of the virus from infected cells after viral packaging is complete. Researchers have classified the discovered influenza a viruses based on differences in the antigenicity of HA and NA. Matrix protein 2(M2) is also a surface protein that constitutes the proton channel of the virus. Matrix protein 1(M1) and nonstructural protein 2(NS1) are involved in the export of the viral vRNP complex. The non-structural protein (NS1) is an antagonist of the host innate immune response encoded by the virus.

The reverse genetics of the influenza virus is characterized in that the reverse genetics of the RNA virus utilizes the genetic material of the virus to rescue virus-like particles or live viruses in susceptible cells or hosts again, the technology carries out in vitro operation on the RNA virus on the DNA molecule level by constructing infectious cDNA molecular clone of the RNA virus, researches on the structure and function of the virus, a replication regulation mechanism, a pathogenesis and the interrelation of the virus and the hosts and the like, in 1978, the 1 st example RNA virus Q β phage is successfully rescued by an RNA virus reverse genetics system, the RNA virus reverse genetics system is established and developed, and the molecular biology research of various RNA viruses is greatly advanced.

Currently, about 2.57 billion chronic HBV infected persons are around the world, mainly concentrated in southeast Asia and tropical regions of Africa, and have become public health problems (who. hepatitis B skins [ EB/OL ] (2019-07-19) [2019-07-01] with wide attention worldwide.

Http// www.who.Int/news-room/fact-sheets/detail/hepatitis-B.), about 9000 ten thousand of chronic HBV infectors exist in China at present, among which about 2000 to 3000 ten thousand of Chronic Hepatitis B (CHB) patients (Razavi-Shearer D, Gamkrelidid I, Nguyen M H, et al. Global prediction, treamtent, and prediction of hepatitis B virus infection 2016: a modelling study [ J ]. The Lancet Gastroenterology & Hepatology,2018,3(6): 383-.

Vaccination is the most effective measure for the prevention and control of infectious diseases in humans. In the past 50 years, hepatitis B vaccines have been developed through blood-borne vaccines, genetic engineering vaccines, synthetic peptide subunit vaccines, DNA vaccines, and have made important contributions to human health.

With the development of genetic engineering technologies such as DNA recombination technology, virus reverse genetics technology, gene editing and the like, the development of recombinant live vector vaccines is attracting more and more attention. The recombinant live vector vaccine is a live vaccine expressed by inserting exogenous protective antigen genes into a live vector genome by using a genetic engineering technology, and mainly comprises a recombinant virus vector live vaccine and a recombinant bacterial vector live vaccine (DRAPER S, HEENEY J. Virus as vaccine vectors for interacting diseases and cancer [ J ] Nat Rev Microbiol,2010,8(1): 62-73.). The vaccine obtained by the method can express target protein in a body and induce to generate corresponding antibody, thereby achieving the aim of immunization. Live vector vaccines have many advantages: can induce various immunity, including humoral immunity, cellular immunity and mucosal immunity; the vector has rich sources, can insert a plurality of antigen genes and express simultaneously, provides possibility for the development of multivalent vaccines (JORGES, DELLAGASTIN O. the depth of viral vaccines: A review of radial methods and model biotechnology prophaches [ J ]. Biotechnology Res In-nov, 2017,1(1):6-13.)

Bacterial vaccine vectors currently used for research are salmonella, lactobacillus, escherichia coli, bacillus calmette-guerin, and the like, and viral vaccine vectors used for research include poxvirus, herpesvirus, adenovirus, influenza virus, and the like (ERTL H C J. viral vectors as vaccines carriers [ J ] Curr Opin Virol,2016,21:1-8.) (Ian r. humphreys, Sarah sebastian. novel viral vectors in infectious diseases [ J ] Immunology,2017,153(1): 1-9.). Among them, the use of influenza virus as a vector has a significant advantage. First, the reverse genetics operating system for influenza viruses is efficient and mature. The influenza virus 8 plasmid system established by Hoffmann et al is currently the most commonly used influenza virus rescue system. The system utilizes RNA polymerase I and RNA polymerase II to produce mRNA on the same template and express corresponding proteins, which can not only improve the production efficiency of recombinant influenza virus, but also reduce the operation difficulty of plasmid transfection (Hoffmann E, Neumann G, Kawaoka Y, et al. A DNA transfer system for generation of influenza A viruses from human plasmids [ J ]. Proceedings of the national Academy of Sciences,2000,97(11): 6108-. Secondly, the genome structure of the influenza virus determines that each segment can be subjected to gene manipulation respectively without affecting the function of the segment; compared with other vectors such as adenovirus, poxvirus and other DNA viruses, influenza virus does not form DNA intermediate products during replication and is not integrated into the chromosome of a host, so that the influenza virus has higher safety. Recombinant chimeric Vaccine candidate strains of Influenza Virus as viral Vaccine vectors (Influenza chimeric Virus (A/B), parainfluenza, Respiratory Syncytial Virus (RSV), chlamydia, tubercle bacillus, malaria, HIV, etc.) have been successfully prepared (Chengnong B, Na L, Li X, et al. charateristic and immunoprotection infection of recombinant Influenza Virus as a Vector expressing Influenza protein epitope of recombinant synthetic Virus [ J ]. Immunolologic Journal,2013.), (Vector S T, Watanabe S, Katsura H, et al. A reproduction-content PB 2-Knockluenfluence A Virus Vaccine Vector [ J ]. J.J. J.J.of viral Vaccine Vector, V.J. J.9. of viral Vaccine, V.8. Vaccine strain [ G.41J. 418, V.41J.9. of viral Vaccine J.9, V.9, V.A. Vaccine of viral Vaccine strain [ 9, V.A. 41J.418, V.41J., 2009,27(42):0-5739.). These vaccine candidate strains broaden the development space for the study of multivalent vaccines using influenza viruses as vectors. However, the influenza virus is not reported at home and abroad as a vector for developing HBV vaccine.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a recombinant influenza A virus carrying a hepatitis B virus gene, a host cell, a preparation method and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a recombinant influenza a virus carrying a hepatitis b virus gene, characterized in that:

the recombinant influenza A virus is an influenza virus strain A/wsn/1933(H1N1) or A/Puerto Rico/8/1934(H1N 1);

the recombinant influenza A virus can save recombinant influenza virus carrying hepatitis B virus genes in host cells through an influenza virus reverse genetics technology; the recombinant influenza A virus can be stably passaged and amplified in MDCK, A549, Vero cells or chick embryos; the hepatitis B virus gene is located in the open reading frame of the modified influenza A virus NS segment.

Preferably, the hepatitis B virus gene in the recombinant influenza A virus is Pres1 whole gene; the sequence is shown as SEQ ID NO. 1; the recombinant influenza A virus strain is now preserved in China center for type culture Collection with the preservation number: CCTCC NO: V202009. (preservation Unit code: CCTCC; preservation date: 2019, 12, 24 days; preservation Unit Address: Wuhan, Wuhan university, China; Classification nomenclature: influenza A Virus IAV-HBV-1; survival)

As another preferred scheme, the hepatitis B virus gene in the recombinant influenza A virus is the combination of Pres2 whole gene and A dominant antigen epitope gene of sHBs, the two genes are connected in a flexible joint mode, and the total gene sequence is shown as SEQ ID NO. 2; the recombinant influenza A virus strain is now preserved in China center for type culture Collection with the preservation number: CCTCC NO: V202010. (preservation Unit code: CCTCC preservation date: 24 months 12 in 2019; preservation Unit Address: Wuhan, China university, Classification nomenclature: influenza A Virus IAV-HBV-2; survival)

In a second aspect, the present invention provides a host cell of the recombinant influenza a virus carrying the hepatitis b virus gene, wherein: the host cell is a 293T, 293T and MDCK co-culture cell line, and COS and MDCK co-culture cell line; the recombinant influenza A virus can be stably passaged in 9-11 day old chick embryos, MDCK, Vero and A549 cells, and host cells can generate anti-HBV antibodies by expressing carried HBV gene proteins.

In a third aspect, the present invention provides a method for producing the recombinant influenza a virus carrying the hepatitis b virus gene, comprising: comprises the following steps:

(1) the NS fragment was engineered to be incapable of autonomous splicing:

performing synonymous point mutation on the RNA splice sites in the NS fragment so as to obtain a brand-new open reading frame of NS1 and NEP; the RNA splice site of the NS segment of the recombinant influenza A virus is subjected to synonymous mutation and can not be spliced autonomously; the hepatitis B virus gene is integrated into any position of an open reading frame of an influenza virus NS gene segment, or replaces part of an influenza virus gene sequence;

(2) the hepatitis B virus gene is inserted between NS1 and the open reading frame of NEP by connecting short peptide linker1 and linker2, and an influenza A virus NS segment carrying the hepatitis B virus gene is constructed:

a hepatitis B virus gene is inserted between the open reading frames of the recombinant influenza A virus; the open reading frames of the hepatitis B virus gene and the influenza A virus gene are connected by self-sheared Linker1 and Linker 2;

the hepatitis B virus gene may be Pres1 whole gene; alternatively, the hepatitis B virus gene may be a combination of Pres2 whole gene and A dominant antigen epitope gene of sHBs, which are connected in the form of flexible linker;

(3) and (3) carrying out rescue on the recombinant influenza A virus by cotransfecting the host cell with the recombinant plasmid obtained in the step (2) and the rest wild-type plasmid.

In a fourth aspect, the present invention provides an application of the recombinant influenza a virus carrying the hepatitis b virus gene in the preparation of an HBV vaccine, wherein the application is characterized in that: the HBV vaccine is a monogenic and multivalent chimeric vaccine; the recombinant influenza a virus can be processed into preparations for clinical use by using a general technology, and the preparations comprise liquid preparations, freeze-dried preparations, capsule preparations, tablets and pills.

Preferably, the single-gene and multi-valent chimeric vaccine is administered by intramuscular injection, subcutaneous injection, oral administration, nasal cavity, oral cavity, anus and vaginal mucosa.

In a fifth aspect, the invention provides a method for producing hepatitis B virus protein by using the recombinant influenza A virus carrying the hepatitis B virus gene as a bioreactor by using chick embryos.

The recombinant virus can be stably passaged in 9-11 day old chick embryos, MDCK, Vero and A549 cells.

The single-gene and multi-valence chimeric vaccine can be processed into various preparations for clinical use by using a general technology, wherein the preparation is selected from one of a liquid preparation, a freeze-dried preparation, a capsule preparation, a tablet and a pill, the preferable preparation is a liquid preparation, an animal-dried preparation and a capsule preparation, the liquid preparation and the freeze-dried preparation are more preferable, and the liquid preparation is most preferable.

The single-gene and multivalent chimeric vaccine inoculation routes comprise intramuscular injection, subcutaneous injection and oral administration, and also comprise nasal cavity, oral cavity, anus and vaginal mucosa routes.

The recombinant influenza A virus carrying the hepatitis B virus gene can be used for preparing HBV vaccine. (II) function research of HBV protein (III) production of hepatitis B virus protein by using chick embryo as bioreactor.

The invention has the following advantages and beneficial effects:

1. in the construction of the recombinant NS plasmid, a different self-cleaving 2A peptide was used as a linker. During protein translation, self-cleaving Linker terminates the synthesis of the previous protein by ribosome skipping and begins the translational synthesis of the next protein. The shearing mode can enable the recombinant proteins to form correct spatial structures and exert the biological activity of the recombinant proteins. Meanwhile, the 2A peptide selected in the invention has high shearing efficiency, thereby ensuring the effective shearing of each part of protein.

2. The recombinant virus of the invention uses influenza A virus as a vector to express hepatitis B virus protein, so that a host generates anti-HBV antibody, and the recombinant virus can be used for developing hepatitis B vaccine. When the recombinant virus is used as a vaccine, the recombinant virus can cover influenza virus and HBV infection at the same time, and lays a foundation for realizing 'one-vaccine dual-purpose'.

3. The influenza virus is used as a carrier for developing hepatitis B virus vaccines, and has not been reported at home and abroad. Influenza viruses have many advantages as viral vaccine vectors, and candidate strains of related recombinant chimeric vaccines have been successfully prepared, and these results lay a solid foundation for the study of multivalent vaccines using influenza viruses as vectors. The research of HBV candidate vaccine strains taking influenza virus as a vector is a new milestone in the research field of HBV vaccines.

4. The nasopharynx part is a good immune organ, has rich antigen presenting cells, and can effectively present and process antigens to generate mucosal immunity and systemic immune response. Compared with injection administration, the nasal cavity administration is simple and easy to implement, can avoid injection pain, and is easier to be accepted by patients.

5. The recombinant influenza A virus can be produced in large scale by using the chick embryo as a bioreactor, and the production cost is saved.

Drawings

FIG. 1: the left figure is a schematic diagram of influenza a virus structure. The right panel is a schematic representation of the structure of the recombinant influenza virus after engineering the NS segment.

FIG. 2: schematic representation of the engineered influenza a virus genome. An exogenous fragment is inserted between open reading frames of NS1 and NS2 through two connecting short peptides (linker1 and linker2) to construct an influenza A virus NS fragment carrying a hepatitis B virus gene.

FIG. 3: specific embodiments of the engineered NS. SD is the splice donor site and SA is the splice acceptor site. The 6 bases-CCAGGA-at the 525-position 530 of the NS fragment is mutated into-CCCGGG-so as to destroy the splicing acceptor site on the NS fragment and lead the NS not to naturally generate alternative splicing. The Linker1 fragment was ligated after the NS1 open reading frame, the Linker2 fragment was introduced before the NEP fragment and the exogenous fragment was inserted between Linker1 and Linker2 and NS1, Linker1, exogenous fragment, Linker2 were in the same open reading frame.

FIG. 4: the recombinant influenza virus infects MDCK cells, and the expressions of NP, NS and the insert fragment are respectively detected. Wild-type influenza virus (wtWSN) and recombinant NS expression plasmid were used as control groups.

FIG. 5: ten generations of genetic stability of recombinant influenza viruses. Control is an NS recombinant expression plasmid.

FIG. 6: therapeutic effects of recombinant influenza viruses in mice.

Detailed Description

In order to better understand the present invention, the following description illustrates the present invention through several optimization schemes.

Example 1: construction of recombinant NS fragments

1. The RNA splice sites in the NS segment are subjected to synonymous point mutation by using a molecular biological means, and the 525-CCAGGA-530 mutation is 525-CCCGGG-530.

2. The hepatitis B virus gene was obtained from a plasmid stored in this laboratory by PCR.

3. As shown in the figure, the mutated NS fragment is connected with the exogenous hepatitis B virus gene through two short peptides, Linker1 and Linker2 according to the open reading frames of NS1 and NS2, thereby obtaining the recombinant NS fragment. Connecting the target gene sequence with the bidirectional transcription expression vector pHW2000 by using a molecular biological method, and selecting a positive clone. The constructed recombinant plasmid is identified by sequencing, the fragment size is completely consistent with the expected fragment size, and no gene mutation exists.

Example 2: rescue of recombinant influenza A virus strains carrying hepatitis B virus

Plasmids carrying influenza virus wild type PB2, PB1, PA, HA, NP, NA and M fragments and recombinant plasmids carrying NS and target fragments are co-transfected into 293T or COS cells, or co-culture cell lines of 293T or COS and MDCK are transfected, and after 6h, DMEM medium containing TPCK pancreatin is replaced. The final concentration of TPCK pancreatin is 1 ug/ml. Culturing at 37 deg.C in 5% C02 cell culture box for 48h, and collecting supernatant. The collected supernatant was freed of cell debris and infected with MDCK cells. Collecting the supernatant of MDCK cells after 48-72h, carrying out plaque purification, carrying out three rounds of plaque purification, amplifying viruses in MDCK, and finally obtaining the recombinant influenza A virus strain carrying hepatitis B virus.

Example 3: plaque purification of recombinant influenza A virus strains carrying hepatitis B virus

Plate paving: the MDCK cells in the cell vial were digested with pancreatin and plated in 6-well plates with 10 cells per well⁶. After MDCK in the six-well plate adhered to the wall and grown into a monolayer of cells, the medium was aspirated and washed twice with PBS.

Adsorption: the collected virus-containing supernatants were diluted 10-fold with PBS containing 0.3% BSA and added to six well plates at a dose of 400ul per well, with 2-3 secondary wells per gradient. The adsorption time was 1h, and the plate was shaken every 15 min during the adsorption period to evenly distribute the supernatant and prevent dehydration of the cells.

Covering, washing residual supernatant with PBS after adsorption, mixing 2 × DMEM with melted 0.6% low melting point agarose 1:1, adding TPCK pancreatin with a final concentration of 1ug/ml, adding 2ml of the mixture into each well, and culturing in an incubator at 37 ℃ after the mixture is cooled and solidified.

Selecting a single clone: after obvious plaques grow in the cell plate, the front section of the gun head of 1ml is cut off by scissors, and the front end of the gun head is kept consistent with the size of the formed plaques. Sucking the plaque and the covering material by using a gun head, eluting by using a proper amount of serum-free DMEM medium, then infecting MDCK cells by using the elution medium, and observing whether cytopathic effect exists.

Example 4: fragment identification of recombinant influenza A virus strains carrying hepatitis B virus

The amplified virus strain re-infects MDCK cells, and after the cytopathic effect, the supernatant was aspirated with a pipette gun and the residual cell debris was washed away with PBS. Appropriate quantities of rnaasso Plus were added to the six-well plates and the cells lysed. Extraction of RNA from cells was performed according to the instructions. The extracted RNA is subjected to reverse transcription by using a universal primer and a random primer. The obtained cDNA was subjected to PCR identification. The NP, NS, and foreign fragments of the recombinant virus were identified using different primers, respectively, and the results are shown in FIG. 4. 4A, detecting NP fragments; 4B, detecting the NS fragment; 4C, detecting the exogenous insert. Wherein the wild type WSN (wtWSN) and the recombinant plasmid are positive controls.

Example 5: genetic stability characterization of recombinant influenza A virus strains carrying hepatitis B virus

The amplified recombinant virus strain infected MDCK cells (6-well plate format, 10)⁶Cells/well) (MOI 0.01), incubate until 70% cytopathic effect (CPE) is observed. The supernatant was then harvested and the ratio 1: 100 dilutions and reinfection with fresh MDCK cells for a total of 10 passages. During each virus passage, supernatants were harvested and each generation of recombinant virus was tested for NP, NS and foreign fragments. The results are shown in FIG. 5. Wherein, the control is a recombinant NS expression plasmid.

Example 6: effect of recombinant influenza A Virus strains carrying hepatitis B Virus on the mouse immune System

BALB/C mice of 6 weeks of age were selected and divided into three groups of 5 mice, a Pres1 group, a Pres2+ HBs (A) group and a PBS blank control group. Establishing a mouse HBV chronic infection model, namely establishing a transfection and expression model of HBV plasmid by injecting pAAV/HBV 1.2 plasmid through a high-pressure tail vein. After 1 week, taking purified recombinant influenza virus liquid and PBS to carry out nasal drip immunization on each group; the immunization was boosted again after 4 weeks. And (3) collecting mouse serum regularly, and detecting the HBV antigen, antibody and HBVDNA levels in the mouse serum.

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Claims

1. A recombinant influenza A virus carrying a hepatitis B virus gene, characterized in that:

2. The recombinant influenza a virus carrying a hepatitis b virus gene according to claim 1, wherein: the hepatitis B virus gene in the recombinant influenza A virus is a Pres1 whole gene; the sequence is shown as SEQ ID NO. 1; the recombinant influenza A virus strain is now preserved in China center for type culture Collection with the preservation number: CCTCC NO: V202009.

3. The recombinant influenza a virus carrying a hepatitis b virus gene according to claim 1, wherein: the hepatitis B virus gene in the recombinant influenza A virus is the combination of a Pres2 whole gene and an A dominant antigen epitope gene of sHBs, the two genes are connected in a flexible joint mode, and the total gene sequence is shown as SEQ ID NO. 2; the recombinant influenza A virus strain is now preserved in China center for type culture Collection with the preservation number: CCTCC NO: V202010.

4. A host cell of a recombinant influenza a virus carrying a hepatitis b virus gene according to any one of claims 1 to 3, characterized in that: the host cell is a 293T, 293T and MDCK co-culture cell line, and COS and MDCK co-culture cell line; the recombinant influenza A virus can be stably passaged in 9-11 day old chick embryos, MDCK, Vero and A549 cells, and host cells can generate anti-HBV antibodies by expressing carried HBV gene proteins.

5. A method for producing a recombinant influenza a virus carrying a hepatitis b virus gene according to any one of claims 1 to 3, characterized in that: comprises the following steps:

(1) the NS fragment was engineered to be incapable of autonomous splicing:

6. Use of a recombinant influenza a virus carrying a hepatitis b virus gene as defined in any one of claims 1 to 3 for the preparation of an HBV vaccine wherein: the HBV vaccine is a monogenic and multivalent chimeric vaccine; the recombinant influenza a virus can be processed into preparations for clinical use by using a general technology, and the preparations comprise liquid preparations, freeze-dried preparations, capsule preparations, tablets and pills.

7. The use of the recombinant influenza a virus carrying a hepatitis b virus gene according to claim 6 in the preparation of an HBV vaccine, wherein: the single-gene and multivalent chimeric vaccine inoculation routes comprise intramuscular injection, subcutaneous injection and oral administration, and also comprise nasal cavity, oral cavity, anus and vaginal mucosa routes.

8. A method for producing hepatitis B virus protein by using chicken embryo as bioreactor, wherein the recombinant influenza A virus carrying hepatitis B virus gene as defined in any one of claims 1 to 3 is used.