CN107344969B

CN107344969B - Nano influenza vaccine, construction method and application

Info

Publication number: CN107344969B
Application number: CN201610292382.9A
Authority: CN
Inventors: 崔宗强; 陈建军; 齐咪; 张先恩
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2016-05-05
Filing date: 2016-05-05
Publication date: 2021-03-05
Anticipated expiration: 2036-05-05
Also published as: CN107344969A

Abstract

The invention discloses a nano influenza vaccine, a construction method and application thereof, wherein the nano influenza vaccine is 3M2e-rHF recombinant protein, and the sequence of the nano influenza vaccine is shown in SEQ ID NO. 2. The recombinant protein of the invention displays 3M2e on the surface of a ferritin cage structure, and obviously improves the immunogenicity of M2e, thereby constructing a novel influenza vaccine: 3M2e-rHF nanoparticles. According to the invention, a prokaryotic expression system escherichia coli is used for expressing the recombinant protein vaccine, the preparation process of the vaccine does not involve live viruses, and compared with the traditional method for preparing the influenza vaccine by using chick embryos, the method is safer, simpler and more convenient to operate and is suitable for rapid large-scale production; by utilizing the high sequence conservation of M2e in different subtype influenza viruses, experiments prove that 3M2e-rHF protects mice from infection of homotypic and heterotypic influenza viruses, and is expected to be developed into a universal influenza vaccine with cross protection efficacy.

Description

Nano influenza vaccine, construction method and application

Technical Field

The invention relates to the technical field of vaccine manufacture, in particular to a nano influenza vaccine, which is prepared by displaying triple tandem repeat influenza virus M2e polypeptide on the surface of a human ferritin cage structure and can be used for remarkably enhancing the immunogenicity of M2e and has cross protection efficacy.

Background

Influenza virus is a relatively common respiratory virus which is harmful to human health, and seasonal influenza causes 300-500 million people to be sick and 25-50 million people to die every year around the world. The emergence and prevalence of potentially new influenza virus strains pose a significant threat to human health.

Currently, vaccination is the most effective way to prevent influenza. The influenza vaccine in use is mainly inactivated, attenuated or recombined trivalent or quadrivalent influenza vaccine, and the ability of the body to resist the infection of the influenza virus is improved by inducing and generating specific antibodies aiming at the main antigen HA or NA of the influenza virus. Vaccines need to be renewed every year due to the continuous antigenic drift of HA, NA of influenza viruses and recombination between different influenza virus subtypes. It is difficult to produce sufficient vaccine in time during seasonal influenza epidemics or during major influenza outbreaks. Furthermore, cross-species transmission of animal host derived influenza viruses, such as avian influenza virus H5N1, H7N9 and swine influenza virus H1N1, makes the development of universal influenza vaccines with broad spectrum protective efficacy urgent.

The study of cross-protective influenza vaccines has focused mainly on two conserved epitopes of influenza a virus: the extracellular domain of matrix protein 2 (M2e) and hemagglutinin 2 glycan peptide (HA2 gp). M2 is a non-glycosylated homotetrameric transmembrane protein that functions as a proton channel in influenza a and B viruses. In influenza a viruses, the M2 protein is composed of three segments: the extracellular N-terminal region (M2e, amino acid residues 1-23), the transmembrane α -helix and the intracellular C-terminal region. Compared with HA and NA, M2e is highly conserved among different influenza a viruses, and thus is a potential target for developing universal influenza vaccines.

Nanotechnology provides a new strategy for vaccine preparation. A variety of nanoscale materials, such as polymers, liposomes, nanoclusters, virus-like particles, ferritin, are used to modify antigens to optimize their properties. Among them, ferritin is an octahedral symmetrical protein cage structure consisting of 24-mer, which is widely present in living bodies. Human ferritin consists of two subunits: h chain (rHF, 21kDa) and L chain (19 kDa). The 24 rHF subunits can also self-assemble in vitro to form a protein cage structure, with an outer diameter of about 12nm and an inner cavity of 6-8 nm. The N-terminal of ferritin extends to the outer surface, and is easy to perform gene modification, fusion protein and polypeptide. When the highly ordered repeated antigens are distributed on the surface of the microorganism at intervals of 5-10 nm, strong T cell-dependent antibody reaction is easily induced. The distance between adjacent amino termini on the surface of ferritin is in this rangeIt is theoretically advantageous to show that antigen induces a strong antibody response. Ferritin itself may also act as an adjuvant, human lymphocytes such as B cells, CD4⁺T lymphocytes, CD8⁺The surface of T lymphocytes possesses rHF specific binding sites, which may be more favorable for displaying antigens to be recognized by immune cells. Moreover, ferritin is highly resistant to heat, pH changes, and chemical denaturation, and is highly stable. Ferritin is therefore a stable, biocompatible nanocarrier, suitable for antigen display. The invention utilizes ferritin as a carrier to display multi-copy M2e polypeptide, and is expected to develop a novel nano influenza vaccine with cross protection efficacy.

Disclosure of Invention

The invention aims to provide a nano influenza vaccine which is 3M2e-rHF recombinant protein, the protein sequence of the nano influenza vaccine is shown in SEQ ID NO.2, and the gene sequence of the encoded 3M2e-rHF recombinant protein is shown in SEQ ID NO. 1.

Another objective of the present invention is to provide a method for preparing a nano influenza vaccine, wherein three segments of M2e polypeptides (3M2e-rHF) connected in series are connected to the amino terminal of a human ferritin H chain, and an escherichia coli expression system is used to express recombinant proteins, and ferritin nanoparticles displaying 3M2e polypeptides are obtained after purification.

The last purpose of the invention is to provide the application of the nano influenza vaccine in preparing the influenza virus vaccine.

In order to achieve the purpose, the invention adopts the following technical measures:

a nano influenza vaccine has a protein sequence shown in SEQ ID NO. 2.

A nano influenza vaccine is prepared by the following steps:

(1) inserting the sequence shown in SEQ ID NO.1 into a plasmid pET28a to obtain a recombinant plasmid pET28a/3M2 e-rHF.

(2) And the recombinant plasmid pET28a/3M2e-rHF is expressed in escherichia coli, and the protein is purified by a molecular sieve column and sucrose density gradient centrifugation.

The application of the nano influenza vaccine in preparing the influenza virus vaccine comprises the step of preparing the influenza virus vaccine by combining the 3M2e-rHF recombinant protein and other adjuvants, or preparing the concatenated vaccine by combining the 3M2e-rHF recombinant protein and other effective components.

Compared with the prior art, the invention has the following advantages and effects:

A. according to the invention, a prokaryotic expression system escherichia coli is used for expressing the recombinant protein vaccine, the preparation process of the vaccine does not involve live viruses, and compared with the traditional method for preparing the influenza vaccine by using chick embryos, the method is safer, simpler and more convenient to operate and is suitable for rapid large-scale production;

B. the human ferritin is used as an antigen display carrier, so that the biocompatibility is good, the ferritin can play an adjuvant effect, and the immunogenicity of the M2e antigen is obviously improved. The nano vaccine is used without adding an adjuvant, so that the preparation process of the vaccine is simplified;

C. the vaccine adopts a nasal drip way for immunization, compared with a common intramuscular injection immunization way, the vaccine is safer and more convenient to use, has small damage, is closer to a way of naturally invading hosts by influenza viruses, and is favorable for inducing the generation of cross protection effect;

D. the invention utilizes the high conservation of M2e sequences in different subtype influenza viruses, and is expected to be developed into a universal influenza vaccine with cross protection efficacy.

E. 3M2e-rHF nanoparticles. Under physiological conditions, the 3M2e-rHF nanoparticles are self-assembled by 24-mer to form a hollow cage structure with an octahedral symmetrical system. The 3M2e-rHF nanoparticles immunize Balb/c mice through a nasal drip way, and induce to generate a large amount of IgG antibody specific to M2 e. Furthermore, 3M2e-rHF protected mice against infection by both homotypic and heterotypic influenza viruses. The self-assembled ferritin shows the conservative antigenic peptide and provides a new strategy for developing a novel vaccine.

Drawings

FIG. 1 is a schematic diagram of the construction of a 3M2e-rHF nanoparticle vaccine;

wherein, a is a schematic diagram of construction of a 3M2e-rHF protein expression sequence, and b is a schematic diagram of assembly of 3M2e-rHF nanoparticles.

FIG. 2 is an identification diagram of the purified 3M2e-rHF protein.

In FIG. 2, a is the SDS-PAGE electrophoresis of purified 3M2e-rHF protein.

In FIG. 2, b is a western blot identification chart of the purified 3M2e-rHF protein.

FIG. 3 is a transmission electron microscope characterization of 3M2e-rHF assembled nanoparticles.

FIG. 4 is a statistical graph showing the variation of the titer of M2 e-specific IgG antibodies in immune sera.

FIG. 5a is a histogram of pulmonary virus titers after infection with A/Puerto Rico/8/34(H1N1) influenza virus.

FIG. 5b is a statistical graph of the change in body weight of mice after infection with A/Puerto Rico/8/34(H1N1) influenza virus.

FIG. 5c is a graph of the survival rate of mice after infection with A/Puerto Rico/8/34(H1N1) influenza virus.

FIG. 6a is a histogram of pulmonary virus titers after infection with influenza A/Chiken/Jiangsu/7/2002 (H9N 2).

FIG. 6b is a statistical graph of the body weight changes of mice after influenza A/Chiken/Jiangsu/7/2002 (H9N2) infection.

FIG. 6c is a statistical plot of survival rates of mice after infection with influenza A/Chicken/Jiangsu/7/2002(H9N 2).

Detailed Description

The embodiment of the invention particularly discloses a preparation method of a novel nano influenza vaccine and a verification process of an immune effect of the novel nano influenza vaccine in a mouse body. The technical solutions described in the embodiments of the present invention are all conventional solutions in the art, unless otherwise specified.

Example 1:

the preparation method of the 3M2e-rHF nano influenza vaccine comprises the following steps:

(1) 3M2e-rHF protein expression plasmid pET28a/3M2e-rHF is constructed:

PCR amplification rHF sequence: the plasmid pET-rHF (gifted by Dr Paolo Santambrogio (Milan, Italy)) encoding the H chain sequence of human ferritin (rHF) was used as template (Nanoscale,2012,4,188), sense primer: 5'CCGGTGTAATGGAAGCTCCGACGGAGGAGGAGGATCTATGACGACCGCGTCCACCTC 3', reverse primer: 5'CCGCTCGAGTTAGCTTTCATTATCACTGTC 3';

3M2e sequence: the nucleic acid sequence of M2e was found in NCBI (Gene ID:956528), and the 3M2e sequence was chemically synthesized. Using the synthesized 3M2e nucleic acid sequence as a template, sense primer: 5'GGGAATTCCATATGATGTCTCTGCTGACCGAGG 3', reverse primer: 5'GAGGTGGACGCGGTCGTCATAGATCCTCCTCCTCCGTCGGAGCTTCCATTACACC 3', M2e has the amino acid sequence MSLLTEVETPIRNEWGCRCNGSSD, which is identical to the M2e sequence in A/Puerto Rico/8/34(H1N1) influenza virus.

And 3M2e-rHF sequences are amplified by Overlap-PCR by taking the PCR products rHF and 3M2e as templates, sense primer: 5'GGGAATTCCATATGATGTCTCTGCTGACCGAGG 3', reverse primer: 5'CCGCTCGAGTTAGCTTTCATTATCACTGTC 3', denaturation at 98 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1min for 30s, and amplification for 30 cycles to obtain the nucleotide sequence 3M2e-rHF shown in SEQ ID NO. 1. The amplified product is subjected to Nde1 and Xho1 double enzyme digestion, and the 3M2e-rHF sequence is inserted into an expression vector pET28a to construct a 3M2e-rHF protein expression plasmid pET28a/3M2 e-rHF. The sequence of the target gene is verified to be correct through sequencing.

(2) The plasmid pET28a/3M2e-rHF is replaced by CaCl₂The method comprises the steps of transforming the strain into Escherichia coli E.coli BL21 (lambda DE3), culturing the strain with LB culture medium at 37 ℃ and 180r/min until OD600 is between 0.4 and 0.6, adding IPTG until the final concentration is 1mM, continuing to induce at 20 ℃ for 16h, centrifuging at 4 ℃ and 6000g for 10min to collect the strain, suspending the strain in assembly buffer solution (20mM Tris-HCl, 50mM NaCl and pH 8.0), carrying out ultrasonic crushing for 40min, centrifuging at 10,000g for 30min, collecting supernatant, putting the supernatant in a 70 ℃ water bath for 10min, and centrifuging at 10,000g for 30min to remove precipitates. Collecting supernatant, concentrating with ultrafiltration tube with cut-off of 100KDa, collecting target protein elution peak with Superose 610/300 GL (from GE) molecular sieve column, centrifuging the concentrated protein with sucrose density gradient, sucking out target protein layer, dialyzing, concentrating and purifying to obtain 3M2e-rHF protein nanoparticles. 1L of initial OD600 is in a bacterial solution of 0.4-0.6, and after induction and purification, 0.5mg of 3M2e-rHF protein can be approximately purified, wherein the amino acid sequence of the 3M2e-rHF protein is shown in SEQ ID NO. 2.

(3) The purified 3M2e-rHF protein was characterized. The purified protein was subjected to SDS-PAGE, and a 3M2e-rHF protein monomer band was observed between 35-40 kDa in comparison with purified rHF (21kDa) (FIG. 2 a). Western blot was used to further verify the expression of 3M2e-rHF (FIG. 2b) using anti-ferriti n heavy chain antibody (purchased from Abcam Inc., Cambridge, Mass.) as a primary antibody. Transmission electron microscopy (FIG. 3) characterization was performed on the assembled 3M2e-rHF nanoparticles. The preparation method of the transmission electron microscope sample comprises the following steps: the copper mesh was adsorbed on a drop containing 0.5mg/ml 3M2e-rHF nanoparticles for 2min, the nickel mesh edge solution was gently blotted with filter paper, and then stained with 2% phosphotungstic acid for 10 min. And (4) placing the copper mesh in the air for natural drying, and detecting by using a 200kV transmission electron microscope.

Example 2:

the 3M2e-rHF nanoparticle influenza vaccine immunization effect is verified in a mouse body, and the method comprises the following steps:

(1) balb/c mice 6-8 weeks old are randomly grouped, 10 mu g of 3M2e-rHF nanoparticles, 2.6 mu g of 3M2e synthetic polypeptide (containing M2e in equimolar amount compared with 3M2e-rHF nanoparticles), 6.3 mu g of rHF nanoparticles (containing rHF in equimolar amount compared with 3M2e-rHF nanoparticles) and PBS are respectively used for immunizing the Balb/c mice through a nasal drip route for three times, and the interval between two adjacent immunizations is three weeks. Two weeks after each immunization, mouse sera were collected and stored at-20 ℃ for detection of M2 e-specific antibodies by ELISA. Antibody detection results show that after three times of immunization, the 3M2e polypeptide and rHF nanoparticle immunized mice have no M2e specific IgG antibody detected in serum, and 3M2e-rHF nanoparticles induce a large amount of M2e specific IgG antibody (FIG. 4), which proves that 3M2e-rHF nanoparticles induce a strong humoral immune response.

(2) After mice were immunized three times for one month, 20. mu.l of 10LD was used₅₀The homologous human influenza virus strain A/Puerto Rico/8/34(H1N1) and the heterologous avian influenza virus strain A/Chiken/Jiangsu/7/2002 (H9N2) respectively infect the immunized mice, and the weight change and the survival rate of the mice are observed for two consecutive weeks, and the mice are considered to die when the weight is reduced by more than 25 percent. Lungs from 4 mice were harvested on day four of viral infection for pneumoviral titer detection.

Lung virus titer determination using histiocyte median infectivity (TCID)₅₀) The method is carried out. The lung homogenate was centrifuged at 1000rpm for 10min and the supernatant was collected. Adding 2X 10 to each well of 96-well plate⁴After MDCK cells are cultured in an incubator at 37 ℃ for 12 hours,lung homogenate supernatants diluted with 10-fold gradients were used to infect MDCK cells, 100 μ l per well, 4 replicates per concentration gradient per sample. After incubation for 1h in a 37 ℃ incubator, the cell culture supernatant was replaced with 200. mu.l of serum-free medium DMEM supplemented with antibiotics, and the cells were further placed in the incubator for three days and observed for cytopathic conditions daily. Three days later, the presence of influenza virus was determined by hemagglutination assay, Reed&The Muench method calculates the virus titer. The A/Puerto Rico/8/34(H1N1) infected pneumovirus titer determination result shows that the pneumovirus titer of the mice in the 3M2e-rHF nanoparticle immune group is remarkably reduced, while the pneumovirus titer of the mice in the 3M2e polypeptide and rHF nanoparticle immune group is not remarkably different from that of the PBS group (figure 5a), which shows that the 3M2e-rHF nanoparticles inhibit the amplification of the virus in the lung. After infection with A/Puerto Rico/8/34(H1N1) virus, mice in 3M2e-rHF nanoparticle immunization group started to rise again after slight weight reduction (FIG. 5b), the survival rate was finally 100% (FIG. 5c), and all mice in other immunization groups died in one week after virus infection, which indicates that 3M2e-rHF nanoparticles protect mice against the lethal infection of homotype influenza virus. The results of the A/Chiken/Jiangsu/7/2002 (H9N2) infection pneumovirus titer assay showed a reduction in pneumovirus titer in mice from the 3M2e-rHF nanoparticle immunized group, although there was no significant difference compared to the control immunized group (FIG. 6 a). After viral infection, mice in the 3M2e-rHF nanoparticle immunized group began to regain weight at day eight of infection (fig. 6b), with a final survival rate of 100% (fig. 6c), while mice in other immunized groups continued to lose weight, all died all one week after viral infection, indicating that the 3M2e-rHF nanoparticles protected the mice against the lethal infection with the heterotypic avian influenza virus.

SEQUENCE LISTING

<110> Wuhan Virus institute of Chinese academy of sciences

<120> nano influenza vaccine, construction method and application

<130> nano influenza vaccine, construction method and application

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<170> PatentIn version 3.1

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atgtctctgc tgaccgaggt ggaaacaccc atcaggaacg agtggggctg cagatgtaat 60

gggagctccg acggatctgg aggaggagga atgagtctgc tgactgaggt ggaaacccct 120

attcggaacg aatggggctg ccgctgtaat ggatctagtg atggaagcgg aggaggagga 180

atgtccctgc tgacagaggt ggaaacccca attagaaacg agtggggctg ccggtgtaat 240

ggaagctccg acggaggagg aggatctatg acgaccgcgt ccacctcgca ggtgcgccag 300

aactaccacc aggactcaga ggccgccatc aaccgccaga tcaacctgga gctctacgcc 360

tcctacgttt acctgtccat gtcttactac tttgaccgcg atgatgtggc cttgaagaac 420

tttgccaaat actttcttca ccaatctcat gaggagaggg aacatgctga gaaactgatg 480

aagctgcaga accaacgagg tggccgaatc ttccttcagg atatcaagaa accagactgt 540

gatgactggg agagcgggct gaatgcgatg gagtgtgcat tacatttgga aaaaaatgtg 600

aatcagtcac tactggaact gcacaaactg gccactgaca aaaatgaccc ccatttgtgt 660

gacttcattg agacacatta cctgaatgag caggtgaaag ccatcaaaga attgggtgac 720

cacgtgacca acttgcgcaa gatgggagcg cccgaatccg gcttggcgga atatctcttt 780

gacaagcaca ccctgggaga cagtgataat gaaagctaa 819

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<211> 272

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<213> Artificial sequence

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Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly

1 5 10 15

Cys Arg Cys Asn Gly Ser Ser Asp Gly Ser Gly Gly Gly Gly Met Ser

20 25 30

Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg

35 40 45

Cys Asn Gly Ser Ser Asp Gly Ser Gly Gly Gly Gly Met Ser Leu Leu

50 55 60

Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn

65 70 75 80

Gly Ser Ser Asp Gly Gly Gly Gly Ser Met Thr Thr Ala Ser Thr Ser

85 90 95

Gln Val Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg

100 105 110

Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser

115 120 125

Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr

130 135 140

Phe Leu His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met

145 150 155 160

Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys

165 170 175

Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys

180 185 190

Ala Leu His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His

195 200 205

Lys Leu Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu

210 215 220

Thr His Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp

225 230 235 240

His Val Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala

245 250 255

Glu Tyr Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser

260 265 270

Claims

1. The preparation method of the artificially constructed recombinant protein 3M2e-rHF comprises the following steps:

(1) inserting the sequence shown in SEQ ID NO.1 into a plasmid pET28a to obtain a recombinant plasmid pET28a/3M2 e-rHF;

(2) and expressing the recombinant plasmid pET28a/3M2e-rHF in escherichia coli, and purifying the escherichia coli through a molecular sieve column and sucrose density gradient centrifugation to obtain the recombinant plasmid pET 28/3M 2 e-rHF.

2. Use of the recombinant protein of claim 1 for the preparation of a nano influenza vaccine for immunization via the nasal drip route.