CN108503697B

CN108503697B - Zika virus subunit vaccine expressed by drosophila cells

Info

Publication number: CN108503697B
Application number: CN201710108921.3A
Authority: CN
Inventors: 黄忠; 屈攀科; 张伟; 刘庆伟
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences
Priority date: 2017-02-27
Filing date: 2017-02-27
Publication date: 2023-03-31
Anticipated expiration: 2037-02-27
Also published as: CN108503697A

Abstract

The invention provides a Zika virus subunit vaccine expressed by drosophila cells, and particularly discloses a subunit Zika virus vaccine developed by utilizing drosophila S2 cells, which has the advantages of high yield, high purity, good stability and easiness in purification, and simultaneously has no possibility of restoring mutation because of no virus nucleic acid component, and is high in safety.

Description

Zika virus subunit vaccine expressed by drosophila cells

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a Zika virus subunit vaccine expressed by drosophila cells.

Background

Zika virus belongs to the Flaviviridae family (Flaviviridae) genus (Flavivirus) on the biological classification, and the genus virus is a single-positive-strand RNA virus. Zika virus was first isolated from the Kiwi of Wugandazhai Kasanlin in 1947 and was not noticed until 2013, where it was found that outbreaks of Guillain Barre syndrome were associated with the prevalence of the French Ponciri Zika virus. Zika virus is transmitted mainly by the bite of aedes mosquitoes, although most infections are asymptomatic, more and more cases of microcephaly are found to be related to the infection of Zika virus during pregnancy of mothers, and the relationship between Zika virus isolated from amniotic fluid and brain tissue of the microcephaly fetus is further confirmed. Outbreaks of Zika virus pose a serious threat to global public health.

Therefore, there is a strong need in the art to develop Zika virus vaccines and suitable methods for their production in order to effectively and specifically prevent and/or treat Zika virus infection.

Disclosure of Invention

The invention aims to provide a Zika virus subunit vaccine, a preparation method and application thereof.

In a first aspect of the present invention, there is provided an antigenic peptide derived from envelope protein of Zika virus and selected from the group consisting of:

(1) Amino acid sequences shown in SEQ ID NO.2 and 3;

(2) And (b) a derivative polypeptide formed by substituting, deleting or adding one or more (less than or equal to 20, such as 2-10, preferably 2-5) amino acid residues in the amino acid sequences shown in SEQ ID NO.2 and 3, wherein the derivative polypeptide has the function of inhibiting Zika virus infected cells and/or the function of inducing an immune response against Zika virus.

In another preferred embodiment, the antigenic peptide is a recombinant protein expressed by drosophila S2 cells.

In a second aspect of the invention, there is provided an isolated polynucleotide encoding the antigenic peptide of the first aspect of the invention.

In another preferred embodiment, the polynucleotide is selected from the group consisting of:

(a) Polynucleotides encoding the polypeptides as shown in SEQ ID NO.2 and 3;

(b) Polynucleotides with sequences shown as SEQ ID NO.5 and 6;

(c) Polynucleotide whose nucleotide sequence has homology of 95% or more (preferably 98% or more) with the sequence shown in SEQ ID NO.5, 6;

(d) Polynucleotides having 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added at the 5 'end and/or 3' end of the polynucleotides as shown in SEQ ID NO.5, 6;

(e) A polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In a third aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a host cell comprising an expression vector according to the third aspect of the invention or having integrated into its genome a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the host cell includes prokaryotic cells and eukaryotic cells.

In another preferred embodiment, the host cell includes Drosophila S2 cells, yeast, E.coli, CHO cells, DC cells, etc.

In a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising the antigenic peptide of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention or the expression vector of the third aspect of the present invention or the host cell of the fourth aspect of the present invention, and a pharmaceutically acceptable carrier and/or adjuvant.

In another preferred embodiment, the composition is a vaccine.

In another preferred embodiment, the vaccine is a therapeutic vaccine and/or a prophylactic vaccine.

In a sixth aspect of the invention, there is provided a vaccine composition comprising an antigenic peptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention or an expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention, and an immunologically acceptable carrier and/or adjuvant.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the adjuvant comprises alumina, saponin, quil A, muramyl dipeptide, mineral or vegetable oil, vesicle-based adjuvant, nonionic block copolymer or DEAE dextran, cytokines (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG).

In a seventh aspect of the invention, there is provided the use of an antigenic peptide according to the first aspect of the invention, (a) for the preparation of an antibody against Zika virus; and/or (b) is used for preparing a medicament for treating and/or preventing diseases related to the Zika virus.

In another preferred embodiment, said diseases associated with Zika virus comprise: zika virus infection, guillain Barre syndrome, microcephaly disease, etc.

In an eighth aspect of the present invention, there is provided a method for preparing the antigenic peptide of the first aspect of the present invention, comprising the steps of:

(i) Culturing the host cell of the fourth aspect of the invention under suitable conditions to express the antigenic peptide of the first aspect of the invention;

(ii) Purifying the antigenic peptide.

In another preferred embodiment, the cell density in step (i) of the method is 2-4x10 ⁶ At cell/ml, expression was induced by addition of chromium chloride at a final concentration of 5 uM.

In a ninth aspect of the invention, there is provided a method of treatment comprising administering to a subject in need thereof an antigenic peptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention or an expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention or a vaccine composition according to the sixth aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the construction of recombinant plasmids pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII.

FIG. 2 shows the expression and purification of the proteins of interest ZIKV E80 and ZIKV EDIII. (A) The purified ZIKV E80 was separated by 15% polyacrylamide gel electrophoresis and stained with coomassie brilliant blue R-250; (B) Analyzing the purified ZIKV E80 by Western blot, and detecting by using a monoclonal antibody resisting His-tag; (C) The purified ZIKV EDIII was separated by 15% polyacrylamide gel electrophoresis and stained with coomassie brilliant blue R-250; (D) Western blot analysis of purified ZIKV EDIII, detection with anti-His-tag monoclonal antibody.

FIG. 3 shows the inhibition of ZiKV E80 and ZIKV EDIII proteins against Zika virus infection. After mixing the ZIKV E80, ZIKV EDIII or BSA diluted in gradient with 100PFU Zika virus, adding the mixture to Vero cells paved in advance, incubating at 37 ℃ for 1 hour, and then sucking off and changing the Vero cells to overlay culture medium, wherein BSA is used as a negative control. (A) Compared with the control group, the condition of the cell plaque reduction treated by ZIKV E80 and ZIKVEDIII is reduced; (B) The number of plaque reduction was counted and expressed as inhibition (%) which is the percentage of plaque reduction of treated cells relative to the number of plaques of untreated cells.

FIG. 4 shows the determination of serum titers after immunization of BALB/c mice with ZIKV E80, ZIKV EDIII. BALB/c mice were immunized three times with ZIKV E80, ZIKV EDIII or PBS plus aluminum adjuvant, two weeks after each immunization were bled, and the antibody titer was measured by ELISA. The endpoint titer was defined as the dilution of the highest positive result (OD 450 higher than preimmune serum titer 0.1). (A) Two-week post-two-and three-immunization serum end-point titers of PBS and ZIKV E80 groups; (B) Two-week post-secondary-and tertiary-immunization serum endpoint titers of PBS and ZIKV EDIII groups; horizontal bars represent geometric means of endpoint titers for each group of sera.

FIG. 5 shows the measurement of neutralizing antibodies in serum after ZIKV E80, ZIKV EDIII triaimmune BALB/c mice. Neutralizing capacity of ZIKV E80, ZIKV EDIII and PBS group antiserum is measured after three-immunity and is measured by a plaque reduction neutralization analysis method. The geometric mean and P value are shown.

FIG. 6 shows the cellular immune response induced by ZIKV E80, ZIKV EDIII in mice. Four weeks after three immunizations, splenocytes from each group of mice were isolated and analyzed by ELISA blots for IFN-. Gamma.and IL-4 producing cells. (a and B) ZIKV E80 group splenocytes were stimulated with medium as control; (C and D) ZIKV EDIII group splenocytes were stimulated with medium as a negative control. Mean ± SEM are shown.

Fig. 7 shows a passive protection experiment. Serum 100ul two weeks after PBS and ZIKV EDIII triatomization was incubated with 5PFU zika virus at 37 ℃ for 1 hour and then intraperitoneally injected into 5-week-old AG6 mice. Then, 2 weeks were observed, and the survival status (a) and the body weight change (B) of each group of mice were recorded daily.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found that a subunit zika virus vaccine developed using drosophila S2 cells has advantages of high yield, high purity, good stability, and easy purification, and that since it does not contain a viral nucleic acid component, there is no possibility of a recovery mutation, and it is highly safe. Moreover, after the antigenic peptides E80 and EDIII of the invention are combined with aluminum adjuvant, the immunized mice generate stronger immune response to induce the organism to generate high-titer antibodies with neutralizing activity, and the antibodies induced by the low-dose immunogen ZIKV EDIII can sufficiently protect AG6 mice from the attack of lethal dose of Zika virus. Experimental results show that the Zika virus subunit vaccines ZIKV EDIII and E80 provided by the invention have obvious protection effect on Zika virus infection prevention.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Zika virus envelope protein

The envelope protein (E protein) of zika virus is the primary target for neutralizing antibodies, while the E protein is divided into three regions, EDI, EDII, EDIII, most specific antibodies or partially cross-reactive neutralizing antibodies, primarily recognizing epitopes on EDIII. The E80 protein of the invention is 80% of the N-terminal region of the envelope protein of Zika virus, is an extracellular segment of the E protein, is responsible for binding with a cell receptor, and is a main epitope of zika virus. The main object of the present invention is to develop a vaccine capable of inducing organisms to produce neutralizing antibodies against target E proteins (E80 and EDIII) for preventing Zika virus infection.

The present invention provides an antigenic peptide derived from the envelope protein of Zika virus, preferably, the envelope protein of Zika virus used in the present invention is derived from strain Z1106033 (viral amino acid Genbank: ALX35659, strain nucleotide Genbank: KU 312312) circulating in south America of Asia type 2015 of Zika virus.

In a preferred embodiment of the present invention, the amino acid sequence of the envelope protein (E protein) is as follows:

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENS KMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNAKNGSISLMCLALGGVLIFLSTAVSA，SEQ ID NO.1。

in a preferred embodiment of the present invention, the antigenic peptide comprises a ZIKV E80 protein, the amino acid sequence of which is as follows:

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENS KMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGK，SEQ ID NO.2。

in another preferred embodiment of the present invention, the antigenic peptide comprises a ZIKV EDIII protein, the amino acid sequence of which is as follows:

KLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGST，SEQ ID NO.3。

optimization of gene sequences encoding antigenic peptides

In the present invention, nucleic acid coding sequences are provided that are optimized for the antigenic peptides of the invention that are particularly suitable for expression in Drosophila S2 cells.

The present inventors optimized the DNA sequence encoding the E protein according to the codon preference without changing the amino acid sequence. However, the inventors have found that optimized sequences obtained solely on the basis of codon frequency are not entirely suitable for expression in a host cell. Therefore, the inventor carries out secondary optimization, wherein adjustment and optimization are carried out on the GC content of the sequence, and a region with higher GC content in the original sequence is eliminated; optimizing complex structures (GGGGGG, GGTAAG) such as a repetitive sequence, cis-acting factors and the like in an original sequence; the rare codons (AGG, CGG, GGG, ACG) present in the prokaryotic nucleotides were optimized according to preference.

After extensive testing and screening, the inventors obtained a particularly optimized E protein coding sequence among the many optimized sequences, the polynucleotide sequence of which is shown below:

<xnotran> atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctggagcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcgaacatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaacccagggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaatggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagtcgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaacgataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccctgggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgaccatgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacaccggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggtgctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggacgcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaaggccttcgaggccaccgtgcgcggagccaagcgcatggccgtgctgggcgataccgcctgggacttcggaagcgtgggaggagccctgaacagcctgggcaagggcatccaccagatcttcggagccgccttcaagtccctgttcggaggcatgtcgtggttcagccagatcctgatcggcaccctgctgatgtggctgggcctgaacgccaagaatggctccatctcgctgatgtgcctggccctgggaggagtgctgatcttcctgagcaccgccgtgtccgcctaa, SEQ ID NO.4; </xnotran> The sequence codes the E protein shown in SEQ ID NO. 1.

The DNA sequence encoding E80 was as follows, based on the optimized DNA sequence described above:

atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctggagcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcgaacatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaacccagggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaatggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagtcgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaacgataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccctgggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgaccatgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacaccggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggtgctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggacgcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaag，SEQ ID NO.5；

the DNA sequence encoding the EDIII protein is as follows:

aagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcacc，SEQ ID NO.6。

vectors and host cells

The invention also provides a vector comprising the optimized antigenic peptide coding sequence of the invention, and a host cell comprising the vector.

In a preferred embodiment of the present invention, the vector has an expression cassette for expressing the antigenic peptide gene, and the expression cassette has the following elements in order from 5 'to 3': a promoter, an antigenic peptide gene, and a terminator.

The above optimized gene sequence of the antigenic peptide can be obtained by a conventional method used by those skilled in the art, such as total artificial synthesis or PCR synthesis. One preferred synthesis method is the asymmetric PCR method. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The polynucleotide sequences of the present invention may be used to express or produce a protein of interest (antigenic peptide) by conventional recombinant DNA techniques, including the steps of:

(1) Transforming or transducing a suitable host cell, preferably a Drosophila S2 cell, with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Culturing the host cell in a suitable medium;

(3) Separating and purifying protein from culture medium or cell.

Methods well known to those skilled in the art can be used to construct expression vectors comprising a DNA sequence encoding a protein of the invention and appropriate transcription/translation control signals, preferably commercially available vectors such as pMT/BiP/V5-HisA. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

Vectors containing the above DNA sequences and appropriate promoter or control sequences can be used to transform appropriate host cells and express the desired protein. Host cells capable of expressing the antigenic peptides of the invention may be prokaryotic cells, such as E.coli; or lower eukaryotic cells, such as yeast cells (pichia, saccharomyces cerevisiae); or higher eukaryotic cells, such as insect cells; preferably Drosophila S2 cells. Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. The engineered cells may be of the rapid methanol-utilizing type (Mut) ⁺ ) Or using methanol at a slow rate (Mut) ^s )。

Culture of engineered cells and fermentative production of target proteins

After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the invention. The medium used in the culture may be selected from various conventional media, depending on the host cell, and the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

In the present invention, conventional fermentation conditions may be employed. Representative conditions include (but are not limited to):

(a) In terms of temperature, the fermentation and induction temperatures of the antigenic peptides of the invention are maintained at 28-30 ℃;

(b) The pH value of the induction phase is controlled to be 3-9;

(c) For Dissolved Oxygen (DO), DO is controlled at 20-90%, and the maintenance of dissolved oxygen can be solved by introducing oxygen/air mixed gas;

(d) As for the feeding, the kind of the feeding may preferably include carbon sources such as glycerol, methanol, glucose and the like, and may be fed alone or in combination.

The target protein expressed by the engineering cells can be purified by adopting a chromatographic technique. The chromatography includes cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. Common chromatographic methods include:

1. anion exchange chromatography:

anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is higher, affecting binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography is performed. The sample can be replaced by means of dilution, ultrafiltration, dialysis, gel filtration chromatography and the like until the sample is similar to a corresponding ion exchange column equilibrium liquid system, and then the sample is loaded and subjected to gradient elution of salt concentration or pH.

2. Hydrophobic chromatography:

hydrophobic chromatographic media include (but are not limited to): phenyl-Sepharose, butyl-Sepharose, octyl-Sepharose. Samples were prepared by adding NaCl, (NH) ₄ ) ₂ SO ₄ And increasing the salt concentration, loading, and eluting by decreasing the salt concentration. The hetero-proteins with large differences in hydrophobicity were removed by hydrophobic chromatography.

3. Gel filtration chromatography

Hydrophobic chromatographic media include (but are not limited to): sephacryl, superdex, sephadex types. Replacing buffer system by gel filtration chromatography, or further purifying.

4. Affinity chromatography

Affinity chromatography media include (but are not limited to): hiTrap ^TM Heparin HP Columns。

Preparation of vaccine composition

The invention also provides a method for preparing the vaccine composition, which specifically comprises the following steps:

the antigenic peptides prepared by the present invention are mixed with pharmaceutically acceptable vaccine adjuvants to form vaccine compositions.

In another preferred embodiment, the adjuvant is aluminum adjuvant, GLA adjuvant, preferably GLA adjuvant.

Compositions and methods of administration

The present invention also provides a composition comprising: (i) A recombinant antigenic peptide prepared by the method of the invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant. In the present invention, the term "comprising" means that various ingredients can be applied or present together in the composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention may be monovalent or multivalent.

The pharmaceutical or vaccine composition of the present invention can be prepared in various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

(i) Pharmaceutical composition

The pharmaceutical compositions of the invention comprise an effective amount of an antigenic peptide prepared by the methods of the invention, which may be monovalent or multivalent.

The term "effective amount" as used herein refers to an amount of a therapeutic agent that treats, ameliorates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. The effect can be detected, for example, by antigen levels. Therapeutic effects also include reduction of physiological symptoms. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given situation, routine experimentation may be used to determine the effective amount.

For the purposes of the present invention, an effective dose is about 0.2 to 2 μ g/kg administered to an individual.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent (e.g., an antigenic peptide or other therapeutic agent). The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acid (polylactic acid), polyglycolic acid and the like. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991).

Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, the compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; it can also be made into solid form suitable for being mixed with solution or suspension, or liquid excipient before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

(ii) Vaccine composition

The vaccine compositions of the present invention may be prophylactic (i.e., to prevent infection) or therapeutic. The vaccine compositions comprise an immunizing antigen (comprising a protein of the invention or a self-assembled virus-like particle), and are typically combined with a "pharmaceutically acceptable carrier", including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants"). Alternatively, the antigen may be conjugated to a bacterial toxoid such as a toxoid from a pathogen such as diphtheria, tetanus, cholera, helicobacter pylori, and the like.

Preferred adjuvants to enhance the effect of the immunological composition include, but are not limited to: (1) Aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) Oil-in-water emulsion formulations, e.g., (a) MF59 (see WO 90/14837), (b) SAF, and (c) Ribi ^TM Adjuvant System (RAS) (Ribi Immunochem, hamilton, MT), (3) saponin adjuvant; (4) Freund complete adjuvant (CFA) and Freund incomplete adjuvant (IFA); (5) Cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CFS), tumor Necrosis Factor (TNF), etc.; (6) Detoxified variants of bacterial ADP-ribosylating toxins, such as cholera toxin CT, pertussis toxin PT or E.coli heat-labile toxin LT, see, e.g., WO93/13302 and WO92/19265; and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition.

Vaccine compositions, including immunogenic compositions (e.g., which may include an antigen, a pharmaceutically acceptable carrier, and an adjuvant), typically contain diluents such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.

More particularly, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as the other desired components described above. An "immunologically effective amount" refers to an amount that is therapeutically or prophylactically effective for administration to an individual as part of a single dose or a continuous dose. The amount will depend upon the health and physiological condition of the individual being treated, the class of individual being treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. It is expected that the amount will be within a relatively wide range and can be determined by routine experimentation.

Typically, the vaccine composition or immunogenic composition can be prepared as an injectable formulation, such as a liquid solution or suspension; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

(iii) Route of administration and dosage

The composition may be administered directly to a subject. The subject may be a human or non-human mammal, preferably a human. When used as a vaccine, the virus-like particles of the present invention can be administered directly to an individual using known methods. These vaccines are typically administered using the same route of administration as conventional vaccines and/or mimicking the route of pathogen infection.

Routes of administration of the pharmaceutical or vaccine compositions of the invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, intravaginal, oral, or other parenteral routes of administration. If desired, the routes of administration may be combined, or adjusted according to the disease condition. The vaccine composition may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

The virus-like particle vaccine should be administered in an "effective amount," i.e., an amount of virus-like particles sufficient to elicit an immune response in the chosen route of administration effective to promote protection of the host against Zika virus infection.

The amount of virus-like particles selected in each vaccine dose is that which elicits an immunoprotective response without significant side effects. Typically, each dose of vaccine is sufficient to contain about 1 μ g to 1000 μ g, preferably 1 μ g to 100 μ g, more preferably 10 μ g to 50 μ g of protein or VLP upon infection of the host cell. Standard research methods including observing antibody titers and other responses in a subject can be used to determine the optimal amount of a particular vaccine. The need for booster doses can be determined by monitoring the level of immunity provided by the vaccine. After the antibody titer in serum is assessed, booster doses of immunization may be selected. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention. The preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.

The main advantages of the invention are:

(1) The antigen peptide can be expressed in a large amount in Drosophila S2 cells, so that the preparation cost is low, and the antigen peptide is suitable for industrial application;

(2) The invention redesigns and optimizes the sequence of the gene of the Zika virus E protein, and the optimized gene sequence has high expression amount and good stability in host cells and is suitable for high-density fermentation;

(3) After the antigen peptides E80 and EDIII are used and combined with an aluminum adjuvant, after the mice are immunized, the immunized mice generate stronger immune response, an organism is induced to generate high-titer antibodies with neutralizing activity, the antibodies induced by the immunogen ZIKV EDIII with low dose can sufficiently protect AG6 mice from being attacked by the lethal dose of Zika virus, and the ZiKV EDIII and E80 subunit vaccines of the Zika virus are candidate vaccines for preventing Zika virus infection and have obvious protection effect.

(3) Compared with the traditional attenuated live vaccine, DNA vaccine and inactivated vaccine, the candidate vaccine is very safe because of no virus nucleic acid.

The present invention will be described in further detail with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and methods

1. Cells

Drosophila Schneider 2 (S2) cells were purchased from Invitrogen, cultured in Schneider' S Drosophila Media (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% diabody (Gibco) or Express supplemented with 1% L-glutamine (Gibco), 1% diabody (Gibco)

SFM medium (Gibco) was cultured in an incubator at 28 ℃.

2. Virus

The Zika strain used in this study was ZIKV/SZ-WIV01 strain (GenBank: KU 963796) and originated from the culture Collection of microorganisms and viruses of the Wuhan institute of Virus, academy of sciences of China (Virus accession number: IVCAS 6.6110).

3. Antibodies

Murine anti-His-tag monoclonal antibody (10E 2) was purchased from Abmart (cat # 264160), horseradish peroxidase (HRP) -labeled goat anti-mouse IgG was purchased from Sigma, and Alkaline Phosphatase (AP) -labeled goat anti-mouse IgG was purchased from Promega (cat # S3728).

4. Plasmid construction

Drosophila cell expression vector pMT/BiP/V5-HisA, selection plasmid pCoblast and calcium phosphate transfection kit were purchased from Invitrogen. Based on the E protein coding sequence of the Z1106033 strain (viral nucleotide GenBank: KU312312, amino acid GenBank: ALX 35659) which is prevalent in south America of Asia type 2015 of Zika virus, codon optimization and gene synthesis (GenScript Co., ltd.) were performed, and further cloned into a vector pUC57 (purchased from GenScript USA Inc.) to obtain a plasmid pZIKV-E. pZIKV-E is taken as a template, after PCR amplification is carried out by a specific primer, bgl II and Xba I enzyme cutting sites are arranged at two ends of the template, and the template is connected to an insect expression vector pMT/Bip/V5-His A (containing His labels and beneficial to detection and purification of target protein) containing the Bgl II and Xba I enzyme cutting sites to obtain recombinant plasmids pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII carrying target gene fragments of 80% of N end region (ZIKV E80) and III (ZIKV EDIII) of envelope protein regions of Zika viruses.

Gene amplification specific primer

Primer name	Primer sequences	SEQ ID NO.
			ZIKV E80-F	CCAAGATCTATCCGCTGCATCGGCGTGTCGAA	7
ZIKV E80-R	AAATCTAGACTTGCCGATGGTGCTTCCCGAGCG					8
			ZIKV EDIII-F	AAAAGATCTAAGCTGCGCCTGAAGGGCGTGAGC	9
ZIKV EDIII-R	AAATCTAGAGGTGCTTCCCGAGCGGTGCCAGTG					10

5. Polyacrylamide gel electrophoresis and Western blot analysis

The expressed protein solution samples were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled at 100 ℃ for 5 minutes, then electrophoretically separated on a 15% polyacrylamide gel, the proteins were visualized by Coomassie blue staining or transferred to PVDF for Western Blotting (Western Blotting) analysis, western Blotting was performed using a monoclonal antibody against His-tag, followed by incubation with a secondary antibody of HRP-conjugated goat-anti-mouse, and finally developed to detect the position of specific bands.

6. Expression of target proteins ZIKV E80 and ZIKV EDIII in Drosophila S2 cells

1) Transient transfection assay for expression of target genes

The recombinant plasmid was transfected into S2 cells using calcium transfer. Wild type S2 cells were first plated in six-well plates, 3X10 ⁶ Culturing the cells/hole at 28 deg.C for 6-16 hr until the cell density reaches 2-4x10 ⁶ Cells were transfected at cell/ml. 36ul 2M CaCl ₂ 19ug heavyThe plasmid and sterile water were added to 300ul to prepare solution A, 300ul 2XHEPES (50mM HEPES,1.5mM Na ₂ HPO4,280mM NaCl, pH 7.1) was prepared as solution B. Placing the solution B on a shaking instrument, slowly dripping the solution A on the solution B, standing the mixed solution at room temperature for 30 minutes, uniformly dripping the mixed solution on cells in a 6-well plate, culturing at 28 ℃ for 16-24 hours, centrifuging the cells at 800rpm for 5 minutes, and washing the cells for three times by using a complete culture medium containing 10% serum so as to remove calcium particles and reduce damage to the cells. The cells were incubated at 28 ℃ for 72 hours, 5uM chromium chloride was added to induce the expression of the target gene, and the expression of the target protein was detected by Western blotting.

2) Screening of cells of stable line and expression of large amount of target protein

If transient transfection allows the detection of expression of the protein of interest, oryza sativa nemulin is used to screen for stable transgenic cell lines expressing the protein of interest. The 1ug pCoblast selection plasmid and recombinant plasmid were transfected transiently (36ul 2M CaCl) ₂ 19ug recombinant plasmid and sterile water added to 300ul to make solution A) were co-transformed into S2 cells when the cell density reached 2-4x10 ⁶ The cells/ml, positive cells were screened at 25ug/ml for nemacyclin, and the expression of the target gene was examined.

If the target protein is detected, continuously expanding and culturing the stable transformation line cells, and replacing the cells with serum-free medium Express

SFM (Gibco) (addition of 10ug/ml of nemacyclin). Expanding from a T25 flash to a T75 flash, and transferring into a rotary bottle for culturing until the cell density reaches 2-4x10 ⁶ At cell/ml, expression was induced by addition of chromium chloride at a final concentration of 5 uM.

After inducing for about 7 days, the supernatant was collected by centrifugation and filtered through a 0.45um filter membrane. Then, the mixture was concentrated to about 1/20 of the original volume by using a 3kDa ultrafiltration centrifuge tube (Millipore). Then, the protein was purified by a nickel column (Novagen) which was previously bound to a site having poor specificity by a binding buffer (0.5M NaCl,20mM Tris,10mM imidazole, pH 7.9) and then loaded, followed by elution of the target protein by a binding buffer and a washing buffer (0.5M NaCl,20mM Tris,40mM imidazole, pH 7.9) after washing off the foreign proteins. Finally, the target protein was quantified by SDS-PAGE and Brandford.

7. Plaque assay of viruses

The virus titer was analyzed by plaque assay. Briefly, vero-E6 monolayers of cells were plated on 24-well plates, incubated overnight at 37 ℃, 10-fold dilutions of Zika virus were incubated on the cells for 1 hour at 37 ℃, followed by removal of the virus sample and addition of overlay medium (0.2% agarose and 2% FBS-DMEM with 1. The cells were transferred to 4 ℃ and left for 15 minutes. Followed by incubation at 37 ℃ for about 80 hours. Finally fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Plaques were counted and virus titers were calculated.

Inhibition of Zika virus infection by ZIKV E80 and ZIKV EDIII proteins

Vero-E6 cells were plated one night in advance on 24-well plates, 10 ⁵ Cells/well, next day, ZIKV E80, ZIKV EDIII gradient diluted, incubated with 100PFU zika virus for one hour at 37 ℃ respectively, followed by virus sample removal and addition of overlay medium (0.2% agalose with 1 mix and 2% fbs-DMEM. The cells were transferred to 4 ℃ and left for 15 minutes. Followed by incubation at 37 ℃ for about 80 hours. Finally fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Plaques were counted and the inhibition rate of ZIKV E80 and ZIKV EDIII proteins against zika virus infection was calculated, with wells without target protein added as reference (0%).

9. Animal immunization

6-week old BALB/c female mice (6 mice per group) were each intraperitoneally injected with 10ug of immunogen using ZIKV E80 and ZIKV EDIII as immunogens and 500ug of aluminum hydroxide (Alhydrogel) (purchased from Invivogen) as adjuvant, and PBS group as a control. Immunization was performed once at week 0, week 2, and week 4, respectively, and retroorbital venous blood collection was performed before immunization at week two and week four. The collected blood is placed at room temperature for 1-3 hours, then 4 ℃ overnight, centrifuged at 4 ℃ the next day, centrifuged at 3000rpm for 30 minutes, and the separated serum is treated at 56 ℃ for 30 minutes. The serum is then tested for antibody titer as well as neutralizing antibody titer.

10. Determination of serum antibody titres

1) Determination of ZIKV E80 group serum antibody titer

Coating a 96-well plate with purified ZIKV E80, 50 ng/well, standing overnight at 4 ℃, blocking 5% skimmed milk at 37 ℃ for 1 hour, diluting the treated serum in 2-fold gradient, incubating the diluted serum with the coated antigen at 37 ℃ for 2 hours, washing the serum with PBST for 5 times, incubating the serum with horseradish peroxidase-conjugated goat anti-mouse secondary antibody at 37 ℃ for 1 hour, developing the color with TMB, keeping the room temperature away from light, terminating the reaction with 1M phosphoric acid, and measuring the absorbance of the sample at A450 by using a Thermo Scientific Varioskan Flash multifunctional reader.

2) Determination of ZIKV EDIII group serum antibody titer

The ZIKV E80 group was also tested, but the plates were packed with ZIKV EDIII.

11. Determination of serum neutralizing antibody titer

Vero-E6 cells were plated at 10 ⁵ The cells/well were plated in 24-well plates and cultured overnight, after cell coverage of about 90%, the inactivated serum was diluted to 100ul in serum-free DMEM medium in a gradient, incubated with 100PFU Zika virus at 37 ℃ for 1 hour, 200ul of the mixture was applied to Vero-E6 cells, cultured at 37 ℃ for 1 hour, the cell supernatant was discarded, 700ul of overlay medium was applied to each well, and cultured at 37 ℃ for about 72 hours. The medium was then discarded, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and UV-irradiated for 30 minutes. The paraformaldehyde was discarded, stained with 0.1% crystal violet for 30 minutes at room temperature, washed twice with distilled water, air dried and counted. After fitting the curve with GraphPad Prism software, the titer at which the plaques were reduced by half (PRNT) was counted ₅₀ )。

12. Enzyme linked immunospot assay

1) 96-well plates (Mi llipore MultiScreen 96-well ELISPOT plates) were wetted in advance with 35% ethanol, then coated with diluted human-derived IFN-. Gamma.capture antibody (10 ug/ml) and IL-4 capture antibody (4 ug/ml), 50 ul/well, and incubated overnight at 4 ℃;

2) Discarding the coating solution, washing with PBS 4 times, and blocking with RPMI-1640 culture medium for 3 hr;

3) Adding diluted ZIKV E80, ZIKV EDIII and positive control (Concanavalin a), negative control (RPMI-1640) to the respective wells;

4) Splenocytes were isolated and counted, then added to the corresponding wells and cultured at 37 ℃ for 24-48 hours;

5) Discard cells and use the cold ddH ₂ O washing, and then washing 5 times by PBST;

6) Adding diluted mouse anti-IFN-gamma (2 ug/ml) and IL-4 (4 ug/ml) detection antibody, and incubating at room temperature for 3 hr; washed three times with PBST; AP-conjugated Streptavidin (1 ₂ O, terminating;

7) After drying, the cells were counted by an ELISPOT plate reader.

13. Passive protection experiment

100ul of sera from the three-immune mice (the sera from each mouse group were mixed together) and 5PFU of Zika virus were incubated at 37 ℃ for 1 hour, and then were injected intraperitoneally into 5-week-old AG6 mice (type I, type II interferon receptor knock-outs); the condition of the mice was observed for 2 consecutive weeks and weighed; and finally judging the protective effect of the vaccine according to the survival condition and the weight change condition.

14. Statistical analysis

All data analyses were analyzed using GraphPad Prism software v 5.0. The Kaplan-Meier survival curve comparison was performed using log-rank test. Other results were analyzed using Student's 2-tailed t test. The data significance difference is expressed as: ns (no significant difference, P ≧ 0.05); * P is more than or equal to 0.01 and less than 0.05; * P <0.01; * P <0.001.

The experimental materials used in the examples of the present invention were obtained from commercial sources unless otherwise specified.

Example 1 expression of proteins of interest ZIKV E80 and ZIKV EDIII in Drosophila S2 cells

The constructed recombinant plasmids pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII (as shown in figure 1) are transiently transfected into drosophila cells, and are induced by chromium chloride to be subjected to western blot detection culture medium supernatant, so that the target protein is detected. And then co-transforming the recombinant plasmid and the pCoBlast screening plasmid, screening stable line cells, performing induced expression, obtaining cell supernatant, and purifying, wherein the size of ZIKV E80 protein is 54KD (shown in figure 2A) by SDS-PAGE, a mouse anti-His-tag antibody is used as a primary antibody, and the size of ZIKV E80 (shown in figure 2B) detected by western blot is consistent with the result of SDS-PAGE. Similarly, the ZIKV EDIII size was 15kD (FIG. 2C), consistent with the band detected for western blots (FIG. 2D). The results indicate that the target proteins E80 and EDIII are expressed, and the expression yields of the ZIKV E80 and ZIKV EDIII antigen peptides are calculated to reach 10mg/l and 2.6mg/l respectively after purification.

The yeast expression experiment result shows that the target protein ZIKV E80 can not be expressed in yeast cells, but can be efficiently expressed in drosophila S2 cells, and the yield reaches 10mg/l.

Example 2 inhibition of Zikv E80 and ZIkv EDIII proteins against Zika Virus infection

To preliminarily identify the biological functions of ZIKV E80 and ZIKV EDIII, we performed inhibition analysis of ZIKV E80 and ZIKV EDIII proteins against zika virus infection. As a result, as shown in FIG. 3, infection of Vero cells with ZiKV E80 (IC) virus was observed ₅₀ ＝54.63ug/ml)、ZIKV EDIII(IC ₅₀ =71.85 ug/ml) and in a dose-dependent manner, whereas the control protein BSA does not inhibit infection of cells by the zika virus. The inhibitory activity of the ZIKV E80 antigen peptide on Zika virus infection is obviously superior to that of the ZIKV EDIII protein, and the IC of the ZIKV E80 antigen peptide is ₅₀ Compared with the ZIKV EDIII, the value is reduced by more than 30%.

The results of this example show that ZIKV E80, ZIKV EDIII can compete with ZIKV virus for entry into cells and therefore have the potential to induce neutralizing antibodies in animals.

Example 3 specific antibody response induced in vivo by ZIKV E80 and ZIKV EDIII in mice

To evaluate the immunogenicity of ZIKV E80, ZIKV EDIII, three groups of BALB/c were immunized three times intraperitoneally, ZIKV E80+ Alum, ZIKV EDIII + Alum, PBS + Alum, respectively. Mouse antiserum induced by ZIKV E80 and ZIKV EDIII is detected by ELISA. As shown in fig. 4A, ZIKV E80 induced specific antibody titers of the diabodies and triales geometric means of 252.0, 1007.9, respectively; as shown in fig. 4B, ZIKV EDIII induced specific antibody titers of the geometric mean of the diabodies and triales, 178.2, 5701.8, respectively, whereas control sera showed no binding capacity.

Example 4 determination of serum neutralizing antibodies after ZIKV E80, ZIKV EDIII triabodies BALB/c mice

To evaluate the neutralizing capacity of mouse antisera against zika virus, we used a plaque reduction neutralization assay (PRNT). As shown in FIG. 5, ZIKV E80 group sera neutralized Zika virus, PRNT ₅₀ 365.5, while ZIKV EDIII group has stronger serum neutralizing capacity, PRNT ₅₀ 1633.8, while control serum had little neutralizing capacity, PRNT ₅₀ It was 49.1.

Example 5 cellular immune response induced in mice by ZIKVE80 and ZIKV EDIII

To investigate whether the immunogen could induce a specific T cell response, splenocytes from mice were isolated four weeks after three immunizations and cells producing IFN-. Gamma.and IL-4 were analyzed by ELISA spots. As shown in FIG. 6, both the ZIKV E80 group and the ZIKV EDIII group induced a greater number of cells secreting IFN-. Gamma.and IL-4, whereas the PBS group did not induce a greater number of cells secreting IFN-. Gamma.and IL-4. Therefore, ZIKV E80 and ZIKV EDIII can induce and generate antigen-specific IFN-gamma and IL-4 memory T cells in mice.

Example 6 Passive protection experiment

According to the result of the serum neutralization test, the serum subjected to ZIKV EDIII triallent immunization is adopted for a passive protection test. After incubation of serum and Zika virus, the mice were intraperitoneally injected, observed for two weeks and weighed daily. The results showed (fig. 7) that control AG6 mice started to lose weight on day 5 after challenge and died completely on day 9, whereas ZIKV EDIII group AG6 mice survived all but one on

day

12 and 4 on day 14. These results indicate that antisera raised against ZIKV EDIII are able to protect against challenge with lethal doses of ZIKV zika virus after incubation with ZIKV EDIII.

Conclusion

The inventor adopts a drosophila S2 system to obtain a stable cell line and expresses envelope proteins E80 and EDIII truncated by Zika virus. The target proteins ZIKV E80 and ZIKV EDIII obtained by the invention have better inhibition effect in the experiment of inhibiting the Zika virus from infecting cells. After the third immunization of BALB/c mice, the mice immunized with both antigenic peptides produced stronger immune responses, both in terms of serum antibody titers and specific T cell responses. Most importantly, antibodies induced with the low dose of immunogen ZIKV EDIII, in combination with aluminum adjuvant, were sufficient to protect AG6 mice from challenge with lethal doses of zika virus. In the aspect of preparation of target proteins, the applicant finds in research that expression is carried out by adopting different host cells, under the condition that the gene sequences are completely the same, the expression quantity of antigen peptides is greatly different, and the difference is also large in the aspect of protein activity, for example, the yeast cells cannot be used for successfully preparing the active ZIKV E80 antigen peptides in the invention, the fruit fly-expressed ZIKA E80 antigen peptides not only have high yield, but also show good antiviral effects on the inhibiting activity of ZIKA viruses and the neutralizing level of induced antibodies, and the fruit fly-expressed ZIKA E80 antigen peptides are ideal ZIKA virus candidate vaccines.

Compared with the traditional attenuated live vaccine, DNA vaccine and inactivated vaccine, the candidate vaccine has no virus nucleic acid, so the candidate vaccine is very safe. In addition, the method is convenient to purify, does not need complex technology, is simple to operate and has relatively large development potential.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe

1 5 10 15

Thr Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr Val Thr Val

20 25 30

Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln

35 40 45

Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly Arg Leu Ile Thr

50 55 60

Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys Met Met Leu

65 70 75 80

Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly

85 90 95

Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser Thr

100 105 110

<210> 4

<211> 1515

<212> DNA

<213> Artificial sequence

<400> 4

atccgctgca tcggcgtgtc gaatcgcgat ttcgtggagg gaatgagcgg aggaacctgg 60

gtggacgtgg tgctggagca cggaggatgc gtgaccgtga tggcccagga taagccgacc 120

gtggacatcg agctggtgac caccaccgtg tcgaacatgg ccgaggtgcg cagctactgc 180

tacgaggcct cgatcagcga tatggcctcc gactcgcgct gcccaaccca gggcgaggcc 240

tacctggata agcagagcga cacccagtac gtgtgcaagc gcaccctggt ggatcgcgga 300

tggggaaatg gatgcggact gttcggcaag ggatccctgg tgacctgcgc caagttcgcc 360

tgctccaaga agatgaccgg caagtcgatc cagccagaga acctggagta ccgcatcatg 420

ctgtcggtgc acggaagcca gcactccggc atgatcgtga acgataccgg ccacgagacc 480

gacgagaatc gcgccaaggt ggagatcacc ccgaactccc cacgcgccga ggccaccctg 540

ggaggattcg gatcgctggg cctggattgc gagccacgca ccggcctgga tttctccgac 600

ctgtactacc tgaccatgaa caataagcac tggctggtgc acaaggagtg gttccacgat 660

atcccactgc cctggcacgc cggagccgac accggaaccc cacactggaa caataaggag 720

gccctggtgg agttcaagga cgcccacgcc aagcgccaga ccgtggtggt gctgggaagc 780

caggagggag ccgtgcacac cgccctggcc ggagccctgg aggccgagat ggatggagcc 840

aagggacgcc tgagctccgg acacctgaag tgccgcctga agatggacaa gctgcgcctg 900

aagggcgtga gctactccct gtgcaccgcc gccttcacct tcaccaagat cccagccgag 960

accctgcacg gaaccgtgac cgtggaggtg cagtacgccg gaaccgatgg accatgcaag 1020

gtgccagccc agatggccgt ggacatgcag accctgaccc cagtgggacg cctgatcacc 1080

gccaatcccg tgatcaccga gtccaccgag aactcgaaga tgatgctgga gctggatccc 1140

ccgttcggcg acagctacat cgtgatcggc gtgggcgaga agaagatcac ccaccactgg 1200

caccgctcgg gaagcaccat cggcaaggcc ttcgaggcca ccgtgcgcgg agccaagcgc 1260

atggccgtgc tgggcgatac cgcctgggac ttcggaagcg tgggaggagc cctgaacagc 1320

ctgggcaagg gcatccacca gatcttcgga gccgccttca agtccctgtt cggaggcatg 1380

tcgtggttca gccagatcct gatcggcacc ctgctgatgt ggctgggcct gaacgccaag 1440

aatggctcca tctcgctgat gtgcctggcc ctgggaggag tgctgatctt cctgagcacc 1500

gccgtgtccg cctaa 1515

<210> 5

<211> 1227

<212> DNA

<213> Artificial sequence

<400> 5

atccgctgca tcggcgtgtc gaatcgcgat ttcgtggagg gaatgagcgg aggaacctgg 60

gtggacgtgg tgctggagca cggaggatgc gtgaccgtga tggcccagga taagccgacc 120

gtggacatcg agctggtgac caccaccgtg tcgaacatgg ccgaggtgcg cagctactgc 180

tacgaggcct cgatcagcga tatggcctcc gactcgcgct gcccaaccca gggcgaggcc 240

tacctggata agcagagcga cacccagtac gtgtgcaagc gcaccctggt ggatcgcgga 300

tggggaaatg gatgcggact gttcggcaag ggatccctgg tgacctgcgc caagttcgcc 360

tgctccaaga agatgaccgg caagtcgatc cagccagaga acctggagta ccgcatcatg 420

ctgtcggtgc acggaagcca gcactccggc atgatcgtga acgataccgg ccacgagacc 480

gacgagaatc gcgccaaggt ggagatcacc ccgaactccc cacgcgccga ggccaccctg 540

ggaggattcg gatcgctggg cctggattgc gagccacgca ccggcctgga tttctccgac 600

ctgtactacc tgaccatgaa caataagcac tggctggtgc acaaggagtg gttccacgat 660

atcccactgc cctggcacgc cggagccgac accggaaccc cacactggaa caataaggag 720

gccctggtgg agttcaagga cgcccacgcc aagcgccaga ccgtggtggt gctgggaagc 780

caggagggag ccgtgcacac cgccctggcc ggagccctgg aggccgagat ggatggagcc 840

aagggacgcc tgagctccgg acacctgaag tgccgcctga agatggacaa gctgcgcctg 900

aagggcgtga gctactccct gtgcaccgcc gccttcacct tcaccaagat cccagccgag 960

accctgcacg gaaccgtgac cgtggaggtg cagtacgccg gaaccgatgg accatgcaag 1020

gtgccagccc agatggccgt ggacatgcag accctgaccc cagtgggacg cctgatcacc 1080

gccaatcccg tgatcaccga gtccaccgag aactcgaaga tgatgctgga gctggatccc 1140

ccgttcggcg acagctacat cgtgatcggc gtgggcgaga agaagatcac ccaccactgg 1200

caccgctcgg gaagcaccat cggcaag 1227

<210> 6

<211> 330

<212> DNA

<213> Artificial sequence

<400> 6

aagctgcgcc tgaagggcgt gagctactcc ctgtgcaccg ccgccttcac cttcaccaag 60

atcccagccg agaccctgca cggaaccgtg accgtggagg tgcagtacgc cggaaccgat 120

ggaccatgca aggtgccagc ccagatggcc gtggacatgc agaccctgac cccagtggga 180

cgcctgatca ccgccaatcc cgtgatcacc gagtccaccg agaactcgaa gatgatgctg 240

gagctggatc ccccgttcgg cgacagctac atcgtgatcg gcgtgggcga gaagaagatc 300

acccaccact ggcaccgctc gggaagcacc 330

<210> 7

<211> 32

<212> DNA

<213> Artificial sequence

<400> 7

ccaagatcta tccgctgcat cggcgtgtcg aa 32

<210> 8

<211> 33

<212> DNA

<213> Artificial sequence

<400> 8

aaatctagac ttgccgatgg tgcttcccga gcg 33

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence

<400> 9

aaaagatcta agctgcgcct gaagggcgtg agc 33

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence

<400> 10

aaatctagag gtgcttcccg agcggtgcca gtg 33

Claims

1. An antigenic peptide, which is derived from envelope protein of Zika virus, has amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3, and is a recombinant protein expressed by Drosophila S2 cells.

2. An isolated polynucleotide encoding the antigenic peptide of claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide is selected from the group consisting of:

(a) Polynucleotides encoding the polypeptides as shown in SEQ ID NO.2 and 3;

(b) The sequence is shown as the polynucleotide of SEQ ID NO.5 and 6.

4. An expression vector comprising the polynucleotide of claim 2.

5. A host cell comprising the expression vector of claim 4 or having the polynucleotide of claim 2 integrated into its genome, wherein said host cell is a Drosophila S2 cell.

6. A pharmaceutical composition comprising the antigenic peptide of claim 1, the polynucleotide of claim 2, or the expression vector of claim 4, or the host cell of claim 5, and a pharmaceutically acceptable carrier and/or adjuvant.

7. The pharmaceutical composition of claim 6, wherein the composition is a vaccine.

8. A vaccine composition comprising the antigenic peptide of claim 1, the polynucleotide of claim 2 or the expression vector of claim 4 or the host cell of claim 5, and an immunologically acceptable carrier and/or adjuvant.

9. The vaccine composition of claim 8, further comprising an adjuvant.

10. Use of the antigenic peptide of claim 1, (a) for the preparation of antibodies against Zika virus; and/or (b) is used for preparing a medicament for treating and/or preventing diseases related to the Zika virus.

11. The use of claim 10, wherein said disease associated with Zika virus comprises: zika virus infection, guillain Barre syndrome, and microcephaly.

12. A method of preparing the antigenic peptide of claim 1, comprising the steps of:

(i) Culturing the host cell of claim 5 under suitable conditions to express the antigenic peptide of claim 1;

(ii) Purifying the antigenic peptide.

13. The method of claim 12, wherein in step (i) of the method the cell density is 2-4x10 ⁶ At cell/ml, expression was induced by addition of chromium chloride at a final concentration of 5 uM.